2-nonanone for use in attracting parasitic wasps of drosophila

Abstract

This invention discloses the application of 2-nonanone in attracting parasitic wasps of the oriental fruit fly. The invention utilizes 2-nonanone, a volatile compound produced by the symbiotic bacteria of the oriental fruit fly, as a specific attractant signal to directionally activate the host-localizing behavior of the parasitic wasp *Protospalum notatum*. Experimental results show that 2-nonanone can still attract >64% of parasitic wasps even without a host substrate, and increases the directional selection rate of parasitic wasps to 72.4% in guava carriers. Therefore, 2-nonanone can be used to prepare an attractant for *Protospalum notatum*, effectively solving the bottleneck of insufficient field efficiency in existing biocontrol systems, possessing both environmental compatibility and targeted specificity, and showing good application prospects.

Description

Technical Field

[0001] This invention belongs to the field of agricultural pest control technology, specifically relating to the application of 2-nonanone in attracting parasitic wasps of fruit flies. Background Technology

[0002] The orchid fruit fly ( Bactrocera dorsalis The oriental fruit fly (Hendel) is omnivorous and has a wide host range (over 350 host species, including mangoes, guavas, and other economic crops). Its damage primarily stems from the female laying eggs inside the fruit; the larvae hatch and feed and develop within the fruit, directly causing fruit damage. To combat this pest, chemical pesticides are widely used, but these easily lead to pesticide resistance in the oriental fruit fly. Therefore, improving the control effectiveness of natural enemies (especially parasitic natural enemies) has become an important direction for solving this problem. In the integrated management of the oriental fruit fly, biological control using parasitic wasps is a crucial component. Statistics show that there are over 70 species of parasitic natural enemies of the oriental fruit fly, among which the *Hendel* sp. (*Hendel* sp.) Diachasmimorpha longicaudata This is the most widely used and successful method. As an important natural enemy of fruit flies, the long-tubed wasp can parasitize a variety of economically important fruit flies, and it shows a high degree of parasitism preference for the older larvae of the citrus fruit fly.

[0003] Currently, although the parasitic wasp biological control system with the *Cyclophorus sparganum* as its core has achieved some success, there is still considerable room for improvement in its actual pest control efficiency in the field. Summary of the Invention

[0004] To overcome the shortcomings and disadvantages of the prior art, this invention provides the application of 2-nonanone in attracting parasitic wasps in fruit fly and a method for controlling fruit flies by identifying the volatiles of symbiotic bacteria on fruit fly eggs and verifying their attraction to parasitic wasps.

[0005] The first objective of this invention is to provide the use of 2-nonanone in attracting parasitic wasps in fruit flies.

[0006] Preferably, the parasitic wasp is *Paraceras buergerianus* (also known as the long-tubed parasitic wasp). Diachasmimorpha longicaudata ).

[0007] Preferably, the *Paraceras antepenaeus* is a female *Paraceras antepenaeus*.

[0008] The second objective of this invention is to provide a method for controlling fruit flies, comprising the following steps: using 2-nonanone to attract the parasitic natural enemy of fruit flies, *Pteris vittata*, thereby enhancing the targeting efficiency of *Pteris vittata* against fruit flies, and using *Pteris vittata* for biological control of fruit flies.

[0009] Preferably, the fruit fly is the oriental fruit fly (Bactrocera dorsalis).

[0010] Preferably, 2-nonanone is used as the active ingredient of the attractant, and the concentration of 2-nonanone in the attractant is 0.1 μg / μL or higher.

[0011] Preferably, the concentration of 2-nonanone in the attractant is 0.1-1 μg / μL.

[0012] Preferably, an attractant containing 2-nonanone is applied to the surface of the fruit to be controlled by fruit flies.

[0013] Previous studies have generally suggested that the strong host-finding ability of the parasitic wasp *Bacteroides proximate* in the field mainly relies on volatile signals released by the affected fruit. This invention utilizes 2-nonanone, a volatile compound produced by the symbiotic fungus *Bacteroides citrinum*, as a specific attractant signal to directionally activate the host-localizing behavior of the parasitic wasp *Bacteroides proximate*. Experimental results show that 2-nonanone can still attract >64% of the parasitic wasps even without a host substrate, and in guava carriers, it increases the directional selection rate of the parasitic wasps to 72.4% (compared to 27.6% in the control). Therefore, 2-nonanone can be used to prepare an attractant for *Bacteroides proximate*, effectively addressing the bottleneck of insufficient field efficiency in existing biocontrol systems. It possesses both environmental compatibility and targeted specificity, showing promising application prospects.

[0014] The present invention has the following advantages and beneficial effects compared with the prior art:

[0015] (1) Strong targeting: The symbiotic bacteria involved are all isolated from the body of the oriental fruit fly and have a natural symbiotic relationship with the target pest;

[0016] (2) High environmental compatibility: The key attractant 2-nonanone is a compound that is widely found in nature, is easily degraded and has no ecological risk;

[0017] (3) Wide range of control: As a parasitic natural enemy of many fruit fly pests, the long-tubed parasitic wasp can work with symbiotic bacteria to improve the control efficiency of multiple target pests. Attached Figure Description

[0018] Figure 1 This image shows the results of screening for symbiotic bacteria strains of the oriental fruit fly that exhibit attraction activity to the long-tubed parasitic wasp.

[0019] Figure 2 The diagram shows the identification results of the common volatile chemical signal substances of the three symbiotic bacteria; (a) is the volatile substance identification of the selected strain 1, (b) is the volatile substance identification of the selected strain 2, and (c) is the volatile substance identification of the selected strain 3.

[0020] Figure 3The following are the antennal potential responses and behavioral selection analysis of *Protopterus longtuberis* to 2-nonanone; (a) is the antennal potential diagram, where A is air, B is anhydrous ethanol, C is 0.1 μg / μL 2-nonanone, and D is 1 µg / μL 2-nonanone; (b) is the relative potential difference of the EAG response of *Protopterus longtuberis* to the standard, where A is anhydrous ethanol, B is 0.1 µg / μL 2-nonanone, and C is 1 µg / μL 2-nonanone; (c) is the selection rate result of the regulation of host localization behavior of female *Protopterus longtuberis* by 2-nonanone. Detailed Implementation

[0021] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0022] The 2-nonanone (CAS: 821-55-6) used in Example 3, with a purity of GC ≥ 98%, was purchased from Chengdu Biyang Biotechnology Co., Ltd.

[0023] Our preliminary observations revealed an important phenomenon: the long-tubed wasp can clearly distinguish between guava pulp consumed by sterile oriental fruit fly larvae and those carrying symbiotic bacteria (i.e., bacteria-carrying larvae), and shows a significant preference for guava pulp consumed by bacteria-carrying larvae. Further research indicates that the symbiotic bacteria carried by oriental fruit fly larvae play a crucial role in enhancing the long-tubed wasp's localization efficiency.

[0024] Example 1: Screening for symbiotic bacteria of the oriental fruit fly that attract the prostaglandin wasp *Bacillus citrinum*.

[0025] After homogenizing and filtering the pulp of guava fruit damaged by the oriental fruit fly, the pulp was serially diluted (10... 4 -10 6 The bacterial culture was spread on LB agar plates and incubated at 28°C for 24-48 h. Morphologically different colonies were screened and purified through three generations. After DNA extraction, the 16S rRNA gene was amplified using 27F / 1492R primers and sequenced. The bacterial species were identified by NCBI alignment and MEGA phylogenetic tree analysis. The identified strains were inoculated into LB liquid medium and cultured (28°C, 200 rpm) until the logarithmic growth phase. 200 μL of the culture was then transferred to 150 mL of LB liquid medium and cultured for 48 h. In a dual-selection apparatus, the treatment group (50 mL bacterial culture) and the control group (sterile LB liquid medium) were placed 50 cm apart. Twenty mated but non-oviparous female *Pterygospermum jasminoides* (6-8 days old) were introduced at the midpoint. The frequency of oviposition was observed for 5 min and recorded.

[0026] The test results are as follows:

[0027] The results showed that eight bacterial strains were isolated from guava pulp treated with arboreal fruit fly larvae, of which only three bacteria induced female wasps to stay and probe. Figure 1 The oviposition selection rate of female *Bacteroides protosporum* in the three bacterial cultures was significantly higher than that in the control group (sterile LB liquid medium). These results indicate that the three symbiotic bacteria of *Bacteroides protosporum* have a significant attraction effect on *Bacteroides protosporum*. P <0.05), indicating good application prospects.

[0028] Example 2: Identification of common chemical signaling substances in three symbiotic bacteria of the oriental fruit fly

[0029] Three symbiotic bacterial strains that significantly attracted the long-tube wasp, obtained from screening in Example 1, were cultured at 28°C and 180 rpm for 48 h with shaking. The volatiles were analyzed by SPME-GC / MS (HP-5MS column), and the compounds were verified by the NIST23.0 database (matching degree >90%) and standards.

[0030] The test results are as follows:

[0031] The volatile substances of three symbiotic bacteria were determined and analyzed, and a total of 23 volatile compounds were identified, among which 2-nonanone (CAS: 821-55-6) was a common component of the three strains. Figure 2 Meanwhile, 2-nonanone was not detected in uninoculated LB liquid medium, fresh artificial feed (used to feed oriental fruit fly larvae), and guava control samples, but the compound showed significant enrichment in the artificial feed and guava after oriental fruit fly larvae had consumed it. P <0.05). The above results indicate that the symbiotic bacteria of the oriental fruit fly synthesize and release 2-nonanone during the feeding process of its larvae.

[0032] Example 3: Determination of antennal potential response and behavioral selection of the prostaglandin worm *Bretschneidera sinensis* to 2-nonanone

[0033] Antennae of female Braconid wasps were cut off for EAG detection. Stimulants were 2-nonanone (0.1 μg / μL and 1 μg / μL), solvent ethanol, and air control (flow rate 4 mL / s, pulse 0.5 s). Each treatment was repeated ≥8 times and the amplitude was recorded.

[0034] Based on the quantitative results of GC-MS (standard curve method), 2 mL of 0.1 μg / μL 2-nonanone was applied to: (1) a 1 / 4 guava fruit piece (about 80 g), (2) a mixture of 2 g of artificial feed, and (3) an empty cup carrier, with ethanol as a control, to evaluate the differences in parasitic wasp attraction.

[0035] The test results are as follows:

[0036] Antennae potential (EAG) measurements showed that the EAG response of the female antennae of the proximal braconid wasp to 2-nonanone was as follows: the response amplitude was 0.89 ± 0.06 mV at a concentration of 0.1 μg / μL, and significantly increased to 1.18 ± 0.05 mV at 1 μg / μL. P <0.001), and all were significantly higher than the ethanol control (0.52±0.07 mV). P <0.01)( Figure 3 (a) and (b) in the text.

[0037] Based on the EAG results, the regulatory effect of 2-nonanone on the host-localizing behavior of female Brachydius robinis was further quantified through behavioral selection experiments. In natural fruit, guava segments coated with 0.1 μg / μL 2-nonanone attracted 72.4% of female wasps (vs. 27.6% for the ethanol control). P <0.001); In artificial feed, the feed selectivity rate of adding 0.1 μg / μL concentration of 2-nonanone reached 68.4% (vs. control 31.6%). P <0.001). It is noteworthy that in a matrix-free empty cup, 2-nonanone still attracted 64.86% of the individuals ( Figure 3 (c)). The above data together verify that 2-nonanone can specifically activate the electrophysiological response of *Bacillus proximalus* and independently drive its strong directional behavior, and that 2-nonanone has the effect of attracting *Bacillus proximalus* parasitic wasp.

Claims

The application of 1,2-nonanone in attracting parasitic wasps in fruit flies is characterized by, The parasitic wasp is *Elaphe septum*. Diachasmimorpha longicaudata .

2. The application according to claim 1, characterized in that, The aforementioned *Pterygodium septum* is a female *Pterygodium septum*.

3. A method for controlling fruit flies, characterized in that, Includes the following steps: Using 2-nonanone to attract Braconid wasps, parasitic natural enemies of fruit flies, enhances the wasps' ability to locate fruit flies, allowing for biological control of fruit flies using Braconid wasps.

4. The method according to claim 3, characterized in that, The fruit fly mentioned is the orange fruit fly (Bactrocera dorsalis). Bactrocera dorsalis Hendel.

5. The method according to claim 4, characterized in that, 2-Nonone is used as the active ingredient of the attractant, and the concentration of 2-nonone in the attractant is above 0.1 μg / μL.

6. The method according to claim 5, characterized in that, The concentration of 2-nonanone in the attractant is 0.1-1 μg / μL.

7. The method according to claim 5, characterized in that, Apply an attractant containing 2-nonanone to the surface of the fruit flies to be controlled.

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