KR101938047B1 - Monitoring apparatus for fruit flies, diagnostic primer sets for 5 quarantine fruit fly species of Korea, and molecular diagnostic techniques using them - Google Patents

Abstract

The present invention relates to an apparatus for monitoring infestation of a fruit fly of Malaysia, a oriental fruit fly, a cucumber fruit fly, a Queensland fruit fly and a mediterranean fruit fly, a species-specific diagnostic primer set capable of diagnosing these collected fruit flies, The present invention relates to a method for diagnosing fruit flies using the same. After the occurrence of the disease, it is diagnosed by using the diagnostic primer set, and it is possible to perform early prevention treatment by checking whether five fruit flies have not yet flown into the domestic area.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a primer set for diagnosing fruit flies and a molecular diagnostic method using the same,

The present invention relates to an apparatus for attracting and monitoring males of fruit flies, a diagnostic primer set for diagnosing attracted fruit fly species, and a diagnostic method for diagnosing five prohibited fruit fly species using such a diagnostic primer.

Fruit fly (Tephritoidea) is a large taxa with 9 families and about 7,300 species richness (Korneyev, 1999). It is known that the taxa belonging to fruit-bearing flies are Tephritidae, which has the greatest species abundance among the fruit flies, and that more than 4,400 species are contained in the fruit flies (White and Elson- Harris, 1992; Norrbom et al., 1999).

At the animal and plant quarantine inspection headquarters, 42 kinds of fruit flies are classified as insect pests that can not enter the country (http://www.qia.go.kr/plant/imQua/plant_no_imp.jsp). Prohibited levels are divided into four risk categories according to the pest risk, host range, invasiveness and frequency of use. Stage I (Class I) is distributed in a wide area and includes highly invasive, high-risk or high-severity single fruit flies. Stage 2 (Class II) includes single fruit varieties with intrinsic and multi-species or high severity in relatively restricted areas. The third stage (Class III) is either intrusive or includes some fruit or vegetable fruit flies in the family. Level 4 (Class IV) includes fruit flies that occasionally occur in edible fruit or bark.

Of these, Oriental fruit flies ( Bactrocera dorsalis ), Malaysian fruit flies ( Egg fruit flies, Bactrocera latifrons ), cucumber fruit flies ( Bactrocera cucurbitae , Queensland fruit bactrocera tryoni ), Mediterranean fruit fly ( Ceratitis capitata ) are high - risk gold - filled fruit flies.

Representatively, the oriental fruit fly ( Bactrocera dorsalis) is composed of a composite of the error is classified as tanks (Tephritidae), and in large and onion riahgwa (Dacinae) to the variation of about 70 kinds (Drew and Hancock, 2000). Oriental fruit flies are classified as Class I prohibited fruit flies which have the highest risk due to their high pest resistance and invasiveness (Kim and Kim, 2016).

To identify the origins of Oriental fruit flies, Wan et al. (2012) analyzed the populations of Oriental fruit flies in various regions based on the sequence of cytochrome oxidase I in the mitochondrial genome. As a result, The origin of fruit fly was estimated to be southeast of China. According to this study, Oriental fruit flies are believed to have migrated from the southeastern part of China to the central region of China, to Pakistan to the west, to Southeast Asia to the south, and to Taiwan and the High South Island to the east. Oriental fruit flies found in remote Hawaii were also regarded as bottleneck populations, possibly due to the initial settlement of some individuals.

The distribution of oriental fruit flies in large areas is believed to be due to their wide host range, mobility and ability to adapt to various environments (Vargas et al., 2015). In particular, the migration ability of oriental fruit flies is related to the input of oriental fruit flies. Generally, the distance of oriental fruit flies is within 50 km. However, in southern China where varietal varieties are high, oriental fruit flies can travel 100 to 250 km (Chen et al., 2007; Liu et al., 2007). Due to recent climate warming and increased human and physical exchanges, there is a problem that distribution areas of oriental fruit flies can be widened further.

Methods to control fruit flies include (1) control with chemical insecticides, (2) control with male annihilation technique (MAT) using inducers, (3) sterile insect release technology Techniques: SIT).

The control method using chemical insecticide is the most representative and continuously used control method. This control method has a problem that the animals other than the control object are highly toxic, and individuals having resistance to chemical insecticides are generated, which makes control difficult. Regarding oriental fruit flies, malachite organophosphate insecticides were first added to protein baits to control oriental fruit flies, low in mammalian toxicity, low in cost of control and low in fruit flies, Fruit flies were controlled (Steiner, 1952; Roessler, 1989).

The control method by infertile insect spinning technology (SIT) is a genetic control technology that sterilizes males by irradiating them to radiation and prevents infertile males from mating with wild females to produce offspring (Knipling, 1955 ).

The control method using the male eradication technique (MAT) is to mix the pesticide with the insecticide to induce the male of the fruit flies, to make the bait by impregnating the emitter, and then repeatedly treating it with aerial drop or ground installation, It is a method of controlling. Methyl eugenol, raspberry ketone and the like are typically used as attractants for attracting males of fruit fly.

As a device for monitoring while controlling fruit flies, a monitoring device in which 5 ml of the attractant and 1 ml of a cotton plug containing the pesticide (organophosphorus pesticide) are put into the steamer trap has been used. In this case, the attractant contained in the cotton seeds is easily volatilized into a liquid phase, so it is troublesome to change the attractant every 5 to 10 days.

Conventionally, a steamer trap is used for monitoring and controlling the fruit flies. The steamer trap is a structure trap which prevents the insects from entering the trap with a narrow passage to catch the smell of the handicap material. In addition, in the case of spinosad, an insecticide commonly used for the control of fruit flies, which has an insecticidal effect specifically on the flyworm, the stunning effect is low. In other words, some insects feeding on spinosad may fall off, but many individuals will fly to other places after eating, so they can be effective in controlling. However, trapping density trapped in traps is not desirable for monitoring .

 Chen, P., Ye, H., Mu, Q.A., 2007. Migration and dispersal of the oriental fruit fly, Bactrocera dorsalis, in regions of Nujiang River based on fluorescence mark. Acta Ecol. Sin. 27, 24682476.  Drew, R. A. I., Hancock, D. L., 2000. Phylogeny of the Tribe Dacinia (Dacinae) based on morphological, distributional, and biological data, in: Aluja, M., Norrbom A.L. (Eds.), Fruit flies (Tephritidae): phylogeny and evolution of behavior. CRC, Boca Raton, FL, pp. 491-504.  Kim, E., Kim, Y., 2014. A report on mixed occurrence of tobacco whitefly (Bemisia tabaci) biotypes B and Q in Oriental melon farms in Kyungpook province, Korea. Korean J. Appl. Entomol. 53, 465-472.  Kim, K., Kim, Kwon, G., Kim, Y., 2017. Technologies required for development of trap-based MAT control against the striped fruit fly, Bactrocera scutellata. Korean J. Appl. Entomol. 56, in press.  Kim, Y., Kim, D., 2016. Integrated pest management against Bactrocera fruit flies. Korean J. Appl. Entomol. 55, 359-376.  Liu, J., Shi, W., Ye, H., 2007. Population genetics analysis of the Oriental fruit fly, Bactrocera dorsalis Hendel (Diptera: Tephritidae), in northern Yunnan Province, China. Entomol. Sci. 10, 1119.  Schutze, MK, Krosch, MN, Armstrong, KF, Chapman, TA, Englezou, A., Chomic, A., Cameron, S., Hailstones, D., Clarke, AR, 2012. Population structure of Bactrocera dorsalis ss, B papayae and B. philippinensis (Diaptera: Tephritidae) in southeast Asia: evidence for a single species hypothesis using mitochondrial DNA and wing-shape data. BMC Evol. Biol. 12, 130.  Vargas, R. I., Pinero, J. C., Leblanc L., 2015. An overview of pest species of Bactrocera fruit flies (Diptera: Tephritidae) and their integration with biopesticides. Insects 6, 297-318.

Currently, domestic fruit fly insect pests are pumpkin fruit flies ( Bactrocera depressus ) and bactrocera ( Bactrocera scutellata ). Oriental fruit flies, Malaysian fruit flies, Cucumber fruit flies, Queensland fruit flies, and Mediterranean fruit flies were not introduced into the country. When they enter the country, they are dangerous and seriously harmful insects. There is a need for a method of diagnosis.

The present invention relates to a device for monitoring the occurrence of malaria fruit flies, oriental fruit flies, cucumber fruit flies, Queensland fruit flies and Mediterranean fruit flies, which are highly dangerous and which are prohibited from entering the country, and species-specific diagnoses Primer set, and a method for diagnosing these fruit flies using the primer set.

In order to achieve the above object, the present invention provides a primer set for diagnosing fruit fly comprising one or more selected from the following primer sets:

1 Malaysia error of the sequence SEQ ID NO: 1 and SEQ ID NO: 2 in the list fly (Bactrocera latifrons) diagnostic primer set, (2) SEQ ID NO: 3 in the Sequence Listing as SEQ ID NO: 4, Oriental fruit fly (Bactrocera of dorsalis ) diagnostic primer set, (3) cucumber fruit flies of SEQ ID NO: 5 and SEQ ID NO: 6 ( Bactrocera cucurbitae diagnostic primer set, (4) a Queensland Bactrocera tryoni diagnostic primer set of SEQ ID NO: 7 and SEQ ID NO: 8, and (5) a primer set of SEQ ID NO: 9 and SEQ ID NO: 10 Ceratitis capitata ) diagnostic primer set.

Further, the present invention provides a diagnostic method for fruit flies characterized by diagnosing at least one of malaysia fruit flies, oriental fruit flies, cucumber fruit flies, Queensland fruit flies, and Mediterranean fruit flies by PCR using the primer set .

In the above diagnostic method, it is preferable that the PCR is repeated at 35-40 times for 30 seconds at 94 ° C, annealing at 45-63 ° C for 30 seconds, and extension at 72 ° C for 30 seconds.

In a preferred embodiment of the present invention, the diagnostic method comprises the steps of: 30 sec denaturation at 94 deg. C, 30 sec annealing at 53 deg. C, and 72 deg. C at 94 deg. C using the Malaysian fruit flies diagnostic primer set of SEQ ID NO: In which a 30 second elongation PCR cycle is repeated 35 times to diagnose malaysian fruit flies.

In a preferred embodiment of the present invention, the diagnostic method comprises the steps of: 30 sec denaturation at 94 deg. C, 30 sec annealing at 60 deg. C, and 72 deg. C using an oriental fruit flies diagnostic primer set of SEQ ID NO: 3 and SEQ ID NO: In which a PCR cycle of 30 seconds elongation is repeated 35 times to diagnose an oriental fruit fly.

In a preferred embodiment of the present invention, the diagnostic method comprises the steps of: 30 sec denaturation at 94 < 0 > C, 30 sec annealing at 45 [deg.] C and annealing at 72 [ , The PCR cycle of 30 sec elongation was repeated 10 times, and PCR cycles of 30 sec denaturation at 94 ° C, annealing at 55 ° C for 30 sec, and extension at 72 ° C for 30 sec were repeated 30 times to diagnose cucumber fruit fly .

In a preferred embodiment of the present invention, the diagnostic method comprises using a Queensland fruit flies diagnostic primer set of SEQ ID NO: 7 and SEQ ID NO: 8 of the Sequence Listing and carrying out 30 sec denaturation at 94 DEG C, 30 sec annealing at 63 DEG C and 72 DEG C And the PCR cycle of 30 seconds elongation was repeated 35 times to diagnose Queensland fruit flies.

In a preferred embodiment of the present invention, the diagnostic method comprises using a set of primers set forth in SEQ ID NO: 9 and SEQ ID NO: 10 of the Sequence Listing for a Mediterranean fruit fly, In which a 30 second elongation PCR cycle is repeated 35 times to diagnose the Mediterranean fruit fly.

Also,

A delta trap and a wax-like emitter installed in the delta trap,

The wax-like emitter can be an emulsifier such as methyl eugenol or raspberry ketone; And a monitoring device for monitoring one or more of Oriental fruit flies, Malaysian fruit flies, cucumber fruit flies, Queensland fruit flies, and Mediterranean fruit flies, characterized by comprising a pesticide with effective stunning effect on the fruit flies as an insecticide. to provide.

In the monitoring apparatus, the biological pesticide may be selected from the group consisting of gosam extract ( Sophora flavescens extract).

In the monitoring apparatus, it is preferable that the attractant and the pesticide are mixed at a ratio (w / v) of 40 to 60: 1.

In a preferred embodiment of the present invention, the monitoring device monitors oriental fruit flies using a mammalian eugenol as an inducer.

Further, the present invention provides a fruit fly monitoring method for monitoring at least one of a fruit fly of Malaysia, an oriental fruit fly, a cucumber fruit fly, a Queensland fruit fly, and a Mediterranean fruit fly using the fruit fly monitoring device.

In a preferred embodiment of the present invention, the monitoring method uses a monitoring device to monitor oriental fruit flies using a methyl eugenol as an inducer.

The present invention was made to diagnose the occurrence of five kinds of fruit flies of Malaysian fruit flies, Oriental fruit flies, Cucumber fruit flies, Queensland fruit flies, and Mediterranean fruit flies, which are designated as major quarantine targets, The diagnostic primer set can be used to diagnose the occurrence of five fruit flies that have not yet flown into Korea.

Further, the present invention relates to an attractant for attracting the five kinds of pests, a wax-like emitter using the pesticide having a stunning effect effective for the insect pests, and a monitoring device installed in the delta trap, Can be monitored with high sensitivity by detecting the fruit flies falling on the sticky plate in the delta trap by being stunned or killed by biological pesticides, thereby detecting the occurrence of Malayia fruit flies, Oriental fruit flies, Cucumber flies, Queensland fruit flies and Mediterranean fruit flies .

The fruit flies collected by the monitoring device can be diagnosed by the diagnostic method using the above diagnostic primer set, and it can be confirmed whether or not the fruit flies of Malaysia, Oriental fruit flies, Cucumber fruit flies, Queensland fruit flies and Mediterranean fruit flies are generated. It is very useful to prevent the infestation of these pests.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing the results of a study on the growth of B. licheniformis (B.lat), oriental fruit fly (B.dor), cucumber B.cuc, Queensland fruit tree (B.try), mediterranean fruit fly (C.cap) This shows the process of designing the diagnostic primer of B.dep and B.scu. Cytochrome oxidase I (COI) sequences were collected from GenBank, and the numbers indicate gene sequence numbers in each species.
Fig. 2 shows a variable region for designing a forward primer (FP) and a reverse primer (RF) for each species, and bar indicates consensus residues between species.
FIG. 3 is a species-specific primer sequence for seven fruit flies designed by the above process.
FIG. 4 shows the results of diagnosing seven different fruit flies using a diagnostic primer. 'DI' indicates a diagnostic.
Figure 5 shows a delta trap comprising a wax-like emitter containing the methyl eugenol of the present invention in a tree in the Kota Kinabalu area.
Fig. 6 is a photograph of flies collected in the trap of Fig. 5, and the insect photograph is a photograph of flies taken from the back. Fig.
Figure 7 is a photograph of flies falling on a sticky plate after ingesting the attractant contained in the wax-like emitter of the present invention.
Figure 8 is a photograph of fruit fly larvae found in mangosteen fruit purchased in the Kota Kinabalu region. (a) shows larvae in mangosteen fruit. (b) is the head region showing the cephalo-pharyngeal skeleton (CPS) and the buccal carinae (BC) region, (c) is the abdominal end showing the metapneustic spiracles (posterior end) region.
Figure 9 shows molecular diagnostics results for 5 individuals randomly selected from collected adult and larvae.

The present invention provides a primer set for diagnosing fruit fly comprising one or more selected from the following primer sets:

(1) a primer set for diagnostic use of Bactrocera latifrons of SEQ ID NO: 1 and SEQ ID NO: 2,

(2) a primer set for diagnosis of Oriental fruit flies ( Bactrocera dorsalis ) of SEQ ID NO: 3 and SEQ ID NO: 4,

(3) a primer set for diagnosis of cucumber fruit bactrocera ( Bactrocera cucurbitae ) of SEQ ID NO: 5 and SEQ ID NO: 6,

(4) a Queensland Bactrocera tryoni diagnostic primer set of SEQ ID NO: 7 and SEQ ID NO: 8,

(5) A set of diagnostic primers for the Mediterranean Sea fruit fly ( Ceratitis capitata ) of SEQ ID NO: 9 and SEQ ID NO: 10 of the Sequence Listing.

The five types of primer sets for diagnosing fruit flies of the present invention are shown in Table 1 below. Table 1 also shows the diagnostic primer sets of pumpkin fruit flies and amber fruit flies present in Korea.

Types of fruit flies order
number direction The primer (5 '- > 3') Amplicon
size (bp) Malaysia Fruit Paris
(B.lat) One Forward TACACTATTATTAGTGAGAAGC 463 2 Reverse GATAGGATCTCCTCCTCCAGCG Oriental fruit flies
(B.dor) 3 Forward CCCATATTATTAGTCAAGAATCAGGAAAG 452 4 Reverse GATTTAATACTAGCCCTGTAAATAGGGGG Cucumber fruit
(B.cuc) 5 Forward ATTAGGATTTATTGTTTGAGCA 325 6 Reverse TCAATGAACAAATCCTGCTATG Queensland Fruit Paris
(B.try) 7 Forward CTACCCCCTTCCCTTACACTAC 369 8 Reverse GTCTCCTCCCCCAGCAGGGTCAAAAAAG Mediterranean Fruit Paris
(C.cap) 9 Forward GGATTATTAGGATTTATTGTTTGAGCA 398 10 Reverse GGATATCAATGAACAAATCCTGCTATG Pumpkin fruit flies
(B.dep) 11 Forward ATAGTAGAAAATGGGGCTGGAACAGGC 260 12 Reverse GCCAATACTGGTAGTGATAACAATAAC Amber fruit
(B.scu) 13 Forward ATTTTGACTACTGCCCCCTTCA 395 14 Reverse ATTTCGATCTGTTAATAGCATT

Oriental fruit flies Diagnostic primers can also detect papaya fruit flies that have been identified as belonging to Oriental fruit flies s.s. as a result of reclassification by gene distance.

PCR is performed using the diagnostic primer set to diagnose malaysia fruit flies, oriental fruit flies, cucumber fruit flies, Queensland fruit flies and Mediterranean fruit flies.

PCR for the diagnosis is preferably repeated 35 to 40 times at 94 ° C for 30 seconds denaturation, 45 to 63 ° C for 30 seconds annealing and 72 ° C for 30 seconds extension.

In the case of the fruit fly of Malaysia, it is more preferable to diagnose the PCR cycle by repeating 35 cycles of 30 sec denaturation at 94 ° C, 30 sec annealing at 53 ° C and 30 sec elongation at 72 ° C.

In the case of an oriental fruit fly, it is more preferable to diagnose the PCR cycle by repeating 35 cycles of 30 sec denaturation at 94 캜, 30 sec annealing at 60 캜 and 30 sec elongation at 72 캜.

In the case of the fruit fly of Queensland, it is more preferable to diagnose 35 cycles of the PCR cycle of 30 sec denaturation at 94 캜, 30 sec annealing at 63 캜 and 30 sec elongation at 72 캜.

In the case of the medicinal fruit fly, it is preferable to repeat the PCR cycles of 30 sec denaturation at 94 ° C, 30 sec annealing at 55 ° C and 30 sec elongation at 72 ° C for 35 times.

In the case of cucumber fruit flies, the PCR was repeated 10 cycles of 30 sec denaturation at 94 ° C, 30 sec anneal at 45 ° C and 30 sec elongation at 72 ° C, followed by 30 sec denaturation at 94 ° C, 30 sec at 55 ° C Annealing and a PCR cycle of 30 seconds elongation at 72 캜 are repeated 30 times to diagnose.

Monitoring devices for monitoring the oriental fruit flies, Malaysian fruit flies, cucumber fruit flies, Queensland fruit flies, and Mediterranean fruit flies of the present invention include delta traps and wax-like emitters installed in delta traps.

In the present invention, a delta trap is used as a trap, and a delta trap has a sticky plate, so that the insect that has taken the insecticide together with the attractant is caught on the sticky plate and is easy to use and is suitable for monitoring.

The wax-like emitter includes an attractant and a biocidal pesticide with effective knockdown effect on the fruit fly as an insecticide.

It is preferable to use methyl eugenol (ME) or raspberry ketone (RK) as the attractant. These materials are effective in attracting fruit flies and have a relatively low volatility and a long duration in the open air. Typically, in the case of oriental fruit flies, it is preferable to use methyl eugenol as an inducing agent.

With the biopesticide is to use a biological pesticide which is effective to stun fruit flies, preferably from Sophora flavescens extract (Sophora flavescens extract). A representative example is 'Cochiri (Korea Biotechnology, Korea)'. The biocidal pesticide is excellent in stunning effect and is stunned immediately after ingesting it and is attached to the sticky plate of the delta trap, so that it is preferable for monitoring. The conventional pesticide or pesticide used in the fruit fly trap of the present invention has a strong insecticidal effect. For example, in the case of spinosad, a part of the pesticide or pesticide may be dropped on the spot. However, since many individuals die after being fed and die, Were not desirable for critical monitoring. In the present invention, a biological pesticide is selected considering stunning effect as the most important factor. Those skilled in the art can appropriately select and use the biocidal pesticides having effective stunning effects on fruit flies among known biocidal pesticides in the present invention.

The attractant and the insecticide are formulated into a wax type to prepare a wax-like emitter.

The attractants and pesticides are preferably mixed at a ratio of 40 to 60: 1 (w / v), particularly preferably at a ratio of 50: 1 (w / v). The wax-like emitters may contain conventional ingredients used in wax-form preparations, and those not specified in the present invention are prepared according to conventional methods.

Since the wax-like emitter of the present invention uses a solidified attractant, it usually has an effective period of 2 to 4 months. Therefore, the monitoring device of the present invention increases the outdoor effective duration, so that the labor force required for frequent replacement of the conventional attractant can be greatly reduced.

Hereinafter, the present invention will be described in more detail with reference to examples. These examples illustrate the present invention and the scope of the present invention is not limited thereto.

< Example  1>

Diagnosis Primer  making

Oriental fruit flies, malaysia fruit flies, cucumber fruit flies, Queensland fruit flies and Mediterranean fruit flies, and pumpkin fruit flies and amber fruit flies which are present in Korea. A total of seven fruit flies, cytochrome oxidase I (CO-I) gene was collected from the gene bank, GenBank (www.ncbi.nlm.nih.gov).

There are 37 sequences from the fruit fly from Malaysia, 1,099 sequences from Oriental fruit flies, 71 sequences from cucumber fruit flies, 78 sequences from Queensland fruit flies, 82 sequences from the Mediterranean fruit flies, 25 sequences, and 43 sequences from amber fruit flies, respectively. This process is shown in Fig.

Using SeqMag among the Lasergene programs developed by DNAStar, the CO-I sequences of seven fruit flies were arranged based on mutual homology, resulting in a consensus sequence of each species. As a result, seven common sequences representing each species obtained were rearranged using SeqMag, and regions in which species differences were observed were selected. This is shown in FIG.

The forward primer was searched for the 770th to 1235th sequences based on the CO-I sequence number of the oriental fruit fly. The reverse primer was searched for sequences 918 to 1383 based on the CO-I sequence number of the oriental fruit fly. In particular, we sought to increase the specific PCR efficiency by looking specifically for the 3 'terminal bases of the primers. With this approach, diagnostic primers for seven fruit flies were designed. This is shown in FIG.

< Example  2>

Diagnosis Primer  Diagnosis using

PCR analysis of fruit flies was carried out using the species specific diagnostic primers of Example 1 above.

Oriental fruit flies, Malaysian fruit flies, Cucumber fruit flies, Mediterranean fruit flies were sold from internally mass-proliferating individuals from the United States USDA (Hilo, Hawaii). Queensland Fruit Paris has been sold infertile from the New Zealand Food and Plant Research Institute. Pumpkin fruit flies and pumpkin fruit flies were used for analysis in the Andong area.

Whole genomic DNA was extracted using Chelex method (Kim and Kim, 2014). First, one fruit fly adults were filled with 500 의 of 20% Chelex and heated at 95 캜 for 20 minutes. After warming, the mixture was stored on ice for 2 minutes to stop the reaction. The mixture was centrifuged at 14,000 × g for 3 minutes, and the supernatant was extracted and used for PCR.

The PCR reaction solution contained 2.5 μl each of the substrate dNTPs (10 mM each), 2.5 μl each of the primers (10 pmol / μl), 1 μl of the Taq polymerase, and 1 μl of the Taq polymerase Ion distilled water.

Table 2 and Table 3 show the temperature and time conditions of the PCR reaction performed for each species.

step Malaysia Fruit Paris Oriental
Fruit flies Queensland
Fruit flies Mediterranean
Fruit flies pumpkin
Fruit flies Amber
Fruit flies D 94 ° C 30s 94 ° C 30s 94 ° C 30s 94 ° C 30s 94 ° C 30s 94 ° C 30s A 53 ℃ 30s 60 ° C 30s 63 ° C 30s 55 ° C 30s 55 ° C 30s 48 ° C 30s E 72 ℃ 30s 72 ℃ 30s 72 ℃ 30s 72 ℃ 30s 72 ℃ 30s 72 ℃ 30s

step Cucumber fruit D 94 ° C 30s 10 cycles A 45 ° C 30s E 72 ℃ 30s D 94 ° C 30s 30 cycles A 55 ° C 30s E 72 ℃ 30s

In this step, denaturation in step D, annealing in step A, extension in step E, and 35 cycles in fruit flies in Table 2 were used . In the case of cucumber fruit flies using touchdown PCR, PCR was carried out under the conditions shown in Table 3.

As a result of PCR under the above conditions, a single band of amplified product was obtained. The results are shown in FIG. At this time, CO-I common primer (5'-CCCGGTAAAATTAAAATATAAACTTC-3 ', 5'-GGATCACCTGATATAGCATTCCC-3') was used for positive control of total DNA amplification.

< Example  3>

Fruit flies Recovered wax type Emitter  Produce

A wax-like emitter for use in a fruit-fly monitoring apparatus, an oriental fruit fly attracting wax emitter including methyl eugenol was prepared as follows.

The composition of the wax-type emitter for oriental fruit flies attracted is shown in Table 4 below.

ingredient Weight (g) producer Paraffin wax 300 EMD Millipore Corporation, MA, USA Emulsifier (Almax 3600) 60 Illshinwells, Seoul, Korea alpha -tocopherol 20 Sigma, St Louis, MO, USA Jojoba oil 20 Sigma, St Louis, MO, USA Biological pesticides (Cochili) 2 KoreaBio, Inc., Korea Methyl eugenol 100 Sigma, St Louis, MO, USA Distilled water 498 Sum 1,000

First, 15 g of paraffin wax (EMD Millipore Corporation, MA, USA) was dissolved in a heated magnetic stirrer at about 70 ° C., and 3 g of an emulsifier (Almax 3600, Illshinwells, Seoul, Korea) The reactants were mixed. When the emulsifier was evenly mixed, 0.5 g of α-tocopherol (Sigma-Aldrich Korea) and 0.5 g of Jojoba oil (Sigma-Aldrich Korea) were added, respectively. To this was added 5 g of methyl eugenol dissolved in 10 ml of dichloromethane. Lastly, 0.1 ml of an environmentally friendly antiseptic agent (Coqi, Koryo Bio) was added as a product standard. After all components were dissolved, 24.5 ml of deionized distilled water was added, and the mixture was stirred at 1,000 rpm for 10 minutes. This was slowly cooled at room temperature and stored at 4 ° C until use.

< Example  4>

Fruit flies monitoring  Manufacturing of devices

The wax-like emitter prepared in Example 3 was installed in a delta trap (GreenAgrotech, Kyunsan, Korea) to produce a fruit fly monitoring device.

< Experimental Example  1>

Field test

Oriental fruit flies were not inhabited domestically, so they conducted outdoor field tests in Kota Kinabalu (N 5.98, E 116.07), Malaysia. This area is home to a variety of fruit flies, including Oriental fruit flies (Schutze et al., 2012).

A wax-like emitter for attracting oriental fruit flies was used as prepared in Example 3 above.

On Jan. 23, 2017, a wax-type emitter containing the methyl eugenol prepared in Example 3 was installed in a delta trap (GreenAgrotech, Kyunsan, Korea) at 4 pm (Fig. 5) Lt; / RTI &gt;

As a result, a total of 37 males were captured, and the flies captured in the traps and the photographs taken from the back of the captured males are shown in Fig. The caught males had yellowish scutellum and yellow vertical lines on both sides of the scutum. There are two black horizontal lines in the abdominal tergum. In the third node of abdomen, the vertical line begins to extend to the last abdominal segment, forming a horizontal line and a T-shaped pattern. Based on these morphological characteristics, the collected fruit flies were estimated as oriental fruit flies.

In Fig. 7, it can be seen that a large number of oriental fruit flies are attached to the wax-like emitter, feeding the attractant, and falling below the emitter and sticking to the sticky plate. It is presumed that they were attracted to the incentive, fed the incentive, fed the insecticide contained in the incentive, and fainted into the sticky plate.

Meanwhile, Mangosteen sold in the region ( Garciana Fruit fly larvae were also found in mangostana fruit (Fig. 8). Twenty birds were detected in one fruit. The characteristic morphological traits of Oriental fruit flies were observed in the external form of the larvae. At the head, cephalo-pharyngeal skeleton (CPS) and buccal carinae (BC) were observed. At the abdominal end, three pairs of posterior gingiva were observed with hydrophobic hair.

< Experimental Example  2>

Molecular identification of collected fruit flies

In order to confirm whether the fruit flies collected in Experimental Example 1 were oriental fruit flies, molecular identification was performed using a diagnostic primer specific to oriental fruit flies. Adults and larvae were randomly selected and analyzed. The results are shown in FIG.

As can be seen from Fig. 9, both adults and larvae showed a single band in the region of 452 bp. That is, all fruit flies attracted to methylene glycol in the Kota Kinabalu region of Malaysia were determined to be oriental fruit flies.

<110> andong national university industry-Academic cooperation foundation <120> Monitoring apparatus for fruit flies, diagnostic primer sets for          5 quarantine fruit fly species of Korea, and molecular diagnostic          techniques using them <130> KP3276 <160> 14 <170> Kopatentin 2.0 <210> 1 <211> 22 <212> DNA <213> Bactrocera latifrons <400> 1 tacactatta ttagtgagaa gc 22 <210> 2 <211> 22 <212> DNA <213> Bactrocera latifrons <400> 2 gataggatct cctcctccag cg 22 <210> 3 <211> 29 <212> DNA <213> Bactrocera dorsalis <400> 3 cccatattat tagtcaagaa tcaggaaag 29 <210> 4 <211> 29 <212> DNA <213> Bactrocera dorsalis <400> 4 gatttaatac tagccctgta aataggggg 29 <210> 5 <211> 22 <212> DNA <213> Bactrocera cucurbitae <400> 5 attaggattt attgtttgag ca 22 <210> 6 <211> 22 <212> DNA <213> Bactrocera cucurbitae <400> 6 tcaatgaaca aatcctgcta tg 22 <210> 7 <211> 22 <212> DNA <213> Bactrocera tryoni <400> 7 ctaccccctt cccttacact ac 22 <210> 8 <211> 28 <212> DNA <213> Bactrocera tryoni <400> 8 gtctcctccc ccagcagggt caaaaaag 28 <210> 9 <211> 27 <212> DNA <213> Ceratitis capitata <400> 9 ggattattag gatttattgt ttgagca 27 <210> 10 <211> 27 <212> DNA <213> Ceratitis capitata <400> 10 ggatatcaat gaacaaatcc tgctatg 27 <210> 11 <211> 27 <212> DNA <213> Bactrocera depressus <400> 11 atagtagaaa atggggctgg aacaggc 27 <210> 12 <211> 27 <212> DNA <213> Bactrocera depressus <400> 12 gccaatactg gtagtgataa caataac 27 <210> 13 <211> 22 <212> DNA <213> Bactrocera scutellata <400> 13 attttgacta ctgccccctt ca 22 <210> 14 <211> 22 <212> DNA <213> Bactrocera scutellata <400> 14 atttcgatct gttaatagca tt 22

Claims (14)

A delta trap and a wax-like emitter installed in the delta trap,
The wax-like emitter can be an emulsifier such as methyl eugenol; And a biological pesticide containing Sophora flavescens extract having an effective stunning effect on the fruit fly as an insecticide, wherein the attractant and the pesticide are mixed at a ratio (w / v) of 40 to 60: 1 It monitors the fruit fly using the characteristic fruit fly monitoring device,
Using a primer set for diagnosis of Bactrocera dorsalis of SEQ ID NO: 3 and SEQ ID NO: 4 in SEQ ID NO: 3, 30 sec denaturation at 94 DEG C, 30 sec annealing at 60 DEG C and 30 sec elongation at 72 DEG C were repeated 35 times A method for monitoring and diagnosing an oriental fruit fly, characterized in that it is diagnosed whether or not the fruit fly trapped in the device is an oriental fruit fly by repeated PCR. delete delete delete delete delete delete delete delete delete delete delete delete delete

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