RU2804853C1 - Method of identification of species of predatory mites of amblyseius genus - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method of identifying species of predatory mites of Amblyseius genus is proposed. DNA is isolated from pre-assembled biological material, PCR of a ribosomal DNA section is carried out using specific primers. Restriction analysis of the amplicon and visualization of restriction products in agarose gel are carried out. AccB1I, AccB7I, DseDI and HaeIII restriction endonucleases are used for restriction analysis. The presence of DNA fragments 510 and 109 bp long when treated with AccB1I restriction enzyme indicates belonging to A. cucumeris species, the presence of DNA fragments 514 and 93 bp long when treated with AccB7I restriction enzyme indicates belonging to A. swirskii species, the presence of DNA fragments 451 and 171 bp long after restriction analysis with DseDI enzyme indicates belonging to A. montdorensis species, and the presence of DNA fragments 496 and 120 bp long after treatment with HaeIII restriction enzyme indicates belonging to A. califomicus species, while other restriction enzymes have no restriction sites.

EFFECT: method enables faster and more accurate species identification when identifying four commercially available species of predatory mites of genus Amblyseius genus using the PCR-RFLP method.

1 cl, 2 dwg, 1 tbl, 2 ex

Description

The invention relates to the field of biochemistry, namely to molecular genetic methods for identifying organisms - mites of the genus Amblyseius, and can be used for the correct diagnosis of farmed mites.

The use of predatory mites for biological protection of crops is becoming increasingly popular in agriculture and horticulture. Nowadays, predatory mites of the families Amblyseius, Phytoseiidae, Laelapidae, Macrochelidae, Parasitidae, Tydeidae, Cheyletidae, Cunaxidae, Erythraeidae, Stigmaeidae are used or proposed to control pests such as herbivorous mites, thrips and whiteflies. A prerequisite for the commercial use of predatory mites as biological pest control agents is their availability at reasonable prices.

Ticks of the genus Amblyseius (Amblyseius swirskii, Amblyseius cucumeris, Amblyseius montdorensis and Amblyseius californicus) are the most important commercially significant entomophages, used as a biological control of thrips and other pests that damage vegetable crops in greenhouses.

These types of predatory mites are the most common and difficult to differentiate from each other. The importance of differentiating these mites is due to the fact that they are grown on similar nutrient substrates, having almost identical morphological features, unlike other mites of the same genus, and there is a high probability of incorrect species identification. Meanwhile, this is an important issue, because these species have varying commercial values. A. swirskii is highly effective against whiteflies and thrips (Bolckmans K. et al. Biological control of whiteflies and western flower thrips in greenhouse sweet peppers with the phytoseiid predatory mite Amblyseius swirskii Athiashenriot (Acari: Phytoseiidae). Second International Symposium on Biological Control of Arthropods, Davos, Switzerland, 12-16 September, p 555-565), A. cucumeris is used for protection against various types of thrips (Gillespie D. R. Biological control of thrips [Thysanoptera: Thripidae] on greenhouse cucumber by Amblyseius cucumeris //Entomophaga - 1989 - vol 34 - No. 2. - p 185-192), A. californicus is used for protection against various types of herbivorous mites (Weintraub P., Palevsky E. Evaluation of the predatory mite, Neoseiulus californicus, for spider mite control on greenhouse sweet pepper under hot arid field conditions //Experimental and Applied Acarolog. - 2008 - vol. 45 - No. 1 - p 29-37), A. montdorensis against thrips and other arthropod pests (Holmes N. D. et al. Control of whitefly (Trialeurodes vaporariorum (Westwood)) and thrips (Thrips tabaci Lindeman) with the predatory Phytoseiid mite Typhlodromips montdorensis (Schicha) on cucumber plants //Control of whitefly (Trialeurodes vaporariorum (Westwood)) and thrips (Thrips tabaci Lindeman) with the predatory Phytoseiid mite Typhlodromips montdorensis (Schicha) on cucumber plants - 2011. - vol. 68 - p. 55-58).

There is a known method for differentiating mites of the genus Amblyseius by morphological characteristics, which is a difficult task that only a highly qualified specialist can solve. It is often necessary to quickly identify the species in order to evaluate the batch of purchased products, as well as the species identity of the cultivated entomophages with a view to replacing the crop with mites of another species or genus that do not have useful characteristics. Also, it is often important to identify mites living in greenhouses in order to adjust the predator application rates (Anisimov A.I., Dobrokhotov S.A. Morphological features of predatory mites Amblyseius mckenziei and A. cucumeris. Plant protection and quarantine. No. 1, 2008).

The closest to the claimed method - the prototype - is a method for differentiating commercially significant mites of the genus Amblyseius based on restriction analysis, including DNA extraction from previously collected biological material, subsequent amplification of a section of mitochondrial DNA using PCR, restriction amplicon analysis, electrophoresis in an agarose gel, in this case, when carrying out PCR, primers are used: direct ITS1 reverse ITS4 and for restriction analysis, restriction enzymes AccB1I, AspLE, SspI are used, and the presence of DNA fragments with a length of 509 and 129 bp. when treated with the restriction enzyme AccB1I, it indicates that it belongs to the species A. cucumeris, the presence of DNA fragments of 381 and 260 bp in length. when treated with restriction enzyme Ssp I indicates belonging to the species A. barkeri, and the presence of DNA fragments 405 and 227 bp in length. when processed with a restriction enzyme, AspLE indicates that it belongs to the species A. swirskii, while the other two restriction enzymes do not have restriction sites (Patent RU 2631933 C1, published on September 28, 2017).

The disadvantages of the prototype are limited functionality, since it provides identification of only three species of predatory mites of the genus Amblyseius, namely Amblyseius swirskii, A. cucumeris and A. barkeri using the restriction fragment length polymorphism method after PCR (PCR-RFLP) using AccB1I restriction enzymes, AspLE and SspI.

The objective of the invention is to provide the possibility of faster and more accurate species identification of four commercially available species of predatory mites of the genus Amblyseius using the PCR-RFLP method, which does not require the involvement of highly qualified acarologists.

Technical result: expansion of the functionality of the method.

The goal is achieved by the proposed method, which is as follows.

A preliminary analysis of the nucleotide sequence of a DNA region including the following genes was carried out: 18S rRNA, ITS1, 5.8S rRNA, ITS2, 28S rRNA. Differences in the nucleotide sequence were established between four species of predatory mites (Amblyseius swirskii, A. cucumeris, A. montdorensis, A. californicus), which made it possible to develop a method for identifying these mite species based on restriction analysis (PCR-RFLP).

Biological material is collected and, if necessary, samples are preserved in 96% ethyl alcohol. DNA is isolated from the obtained samples using commercial kits or classical methods (phenol-chloroform extraction). In this case, the step of extracting DNA from the tick can be omitted and the collected sample (egg, nymph or adult of any sex) can be used directly as a template in the amplification reaction. PCR is carried out using the isolated genomic DNA as a template. The following primers are used for amplification: forward 1TS1 GAATATGAGCCGGAATAGTAGGA, reverse ITS4 ATGTGTTGAAGTTACGGTCA, which are universal for ticks of this genus.

Amplification program: primary denaturation at 94°C for 3 min, 35 cycles of sequential denaturation at 94°C for 30 sec, primer annealing at 51°C for 30 sec and elongation at 72°C for 45 sec. Final elongation at 72°C for 10 minutes. The resulting PCR products are purified from the reaction mixture and subjected to restriction enzymes AccB1I, AccB7I, DseDI and HaeIII. The reaction mixture consists of the following components: 200ng of PCR product, 1 µl of 10x buffer solution, 0.5 µl of restriction endonuclease, 1 µl of bovine serum albumin (BSA) with a concentration of 1 mg/ml (in the case of endonucleases AccB1I and DseDI), double-distilled water to volume 10 µl. The reaction is incubated at 37°C for 1 hour and then the enzymes are inactivated at 65°C (in the case of endonucleases AccB1I and AccB7I) or 80°C (in the case of endonucleases DseDI and HaeIII) for 20 minutes. Restriction products are visualized in a 1-2% agarose gel in 1x Tris-acetate buffer (1x TAE).

In fig. Figure 1 shows an electropherogram of the restriction products of the PCR product obtained by amplification of a rDNA fragment using the restriction enzymes AccB1I, AccB7I and HaeIII, where: 1) A.cucumeris, 2) A.swirskii, 3) A.montdorensis, 4) A.californicus. M - DNA molecular weight marker Step 100 (Biolabmix, Novosibirsk). In fig. Figure 2 shows an electropherogram of the restriction products of the PCR product obtained by amplification of a rDNA fragment using the DseDI restriction enzyme 1) A.cucumeris, 2) A.swirskii, 3) A.montdorensis, 4) A.californicus. M - DNA molecular weight marker Step 100 (Biolabmix, Novosibirsk).

Presence of DNA fragments 510 and 109 bp long. when treated with the restriction enzyme AccB1I, it indicates that it belongs to the species A. cucumeris, the presence of DNA fragments of 514 and 93 bp in length. when treated with the restriction enzyme AccB7I, it indicates that it belongs to the species A. swirskii, the presence of DNA fragments of 451 and 171 bp in length. after restriction analysis with the DseDI enzyme, it indicates that it belongs to the species A. montdorensis, and the presence of DNA fragments of 496 and 120 bp in length. after treatment with restriction enzyme HaeIII - to determine whether it belongs to the species A. californicus.

Table 1 shows data on the length of the expected restriction fragments of the PCR product obtained during amplification using primers ITS1 and ITS4 in these ticks.

The proposed method for differentiating predatory mites is highly sensitive, highly reproducible and economical. Depending on the method of DNA extraction from biological samples, the analysis takes from 4.5 to 6 hours. The analysis can be carried out in laboratories that have a DNA amplifier, centrifuge, thermostat, electrophoresis chamber and associated reagents and consumables.

The defining difference of the proposed method, in comparison with the prototype, is that at the stage of restriction analysis, experimentally selected restriction endonucleases are used - AccB1I, AccB7I, DseDI and HaeIII, which makes it possible to identify 4 predatory species of mites of the genus Amblyseius: Amblyseius swirskii, A. cucumeris, A . montdorensis and A. californicus.

The invention is illustrated by the following examples of specific implementation.

Example 1

Biological material is collected and, if necessary, samples are preserved in 96% ethyl alcohol. DNA is isolated from the obtained samples using commercial kits or classical methods (phenol-chloroform extraction). PCR is carried out using the isolated genomic DNA as a template. The following primers are used for amplification: forward ITS1 reverse ITS4 Amplification program: primary denaturation at 94°C for 3 minutes, 35 cycles of sequential denaturation at 94°C for 30 sec, primer annealing at 51°C for 30 sec and elongation at 72°C for 45 sec. Final elongation at 72°C for 10 minutes.

The resulting PCR products are purified from the reaction mixture and subjected to restriction enzymes AccB1I, AccB7I, DseDI and HaeIII. The reaction mixture consists of the following components: 200ng of PCR product, 1 µl of 10x buffer solution, 0.5 µl of restriction endonuclease, 1 µl of bovine serum albumin (BSA) with a concentration of 1 mg/ml (in the case of endonucleases AccB1I and DseDI), double-distilled water to volume 10 µl. The reaction is incubated at 37°C for 1 hour and then the enzymes are inactivated at 65°C (in the case of endonucleases AccB1I and AccB7I) or 80°C (in the case of endonucleases DseDI and HaeIII) for 20 minutes. Restriction products are visualized in a 1-2% agarose gel in 1x Tris-acetate buffer (1x TAE).

When the A. cucumeris DNA sample PCR product was treated with the AccB1I restriction enzyme, DNA fragments of 510 and 109 bp in length were formed; the AccB7I, DseDI and HaeIII restriction enzymes did not have restriction sites. When the PCR product of the A. swirskii DNA sample was treated with the AccB7I restriction enzyme, DNA fragments of 514 and 93 bp in length were formed; the AccB1I, DseDI and HaeIII restriction enzymes did not have restriction sites. When the PCR product of the A. montdorensis DNA sample was treated with DseDI restriction enzyme, DNA fragments of 451 and 171 bp in length were formed; the restriction enzymes AccB1I, AccB7I and HaeIII did not have restriction sites. When the PCR product of the A. californicus DNA sample was treated with HaeIII restriction enzyme, DNA fragments of 496 and 120 bp in length were formed; the restriction enzymes AccB1I, AccB7I and DseDI did not have restriction sites.

Example 2

Identification of ticks was carried out similarly to example 1, except that genomic DNA was not isolated before PCR, and a whole tick at any stage of the life cycle (egg, nymph, adult) was used as a template in the PCR reaction.

The resulting PCR products were purified from the reaction mixture and subjected to hydrolysis with the enzymes AccB1I, AccB7I, DseDI and HaeIII. The reaction mixture consists of the following components: 200ng of PCR product, 1 µl of 10x buffer solution, 0.5 µl of restriction endonuclease, 1 µl of bovine serum albumin (BSA) with a concentration of 1 mg/ml (in the case of endonucleases AccB1I and DseDI), double-distilled water to volume 10 µl. The reaction is incubated at 37°C for 1 hour and then the enzymes are inactivated at 65°C (in the case of endonucleases AccB1I and AccB7I) or 80°C (in the case of endonucleases DseDI and HaeIII) for 20 minutes. Restriction products are visualized in a 1-2% agarose gel in 1x Tris-acetate buffer (1x TAE).

When the A. cucumeris DNA sample PCR product was treated with the AccB1I restriction enzyme, DNA fragments of 510 and 109 bp in length were formed; the AccB7I, DseDI and HaeIII restriction enzymes did not have restriction sites. When the PCR product of the A. swirskii DNA sample was treated with the AccB7I restriction enzyme, DNA fragments of 514 and 93 bp in length were formed; the AccB1I, DseDI and HaeIII restriction enzymes did not have restriction sites. When the PCR product of the A. montdorensis DNA sample was treated with DseDI restriction enzyme, DNA fragments of 451 and 171 bp in length were formed; the restriction enzymes AccB1I, AccB7I and HaeIII did not have restriction sites. When the PCR product of the A. californicus DNA sample was treated with HaeIII restriction enzyme, DNA fragments of 496 and 120 bp in length were formed; the restriction enzymes AccB1I, AccB7I and DseDI did not have restriction sites.

The use of the proposed invention will allow you to quickly and accurately identify 4 species of predatory mites of the genus Amblyseius: Amblyseius swirskii, A. cucumeris, A. montdorensis and A. californicus. The invention expands the arsenal of methods for identifying commercially important predatory mites using restriction analysis.

Claims (1)

A method for identifying species of predatory mites of the genus Amblyseius, including isolating DNA from previously collected biological material, performing PCR of a section of ribosomal DNA using primers: forward ITS1 GAATATGAGCCGGAATAGTAGGA, reverse ITS4 ATGTGTTGAAGTTACGGTCA, restriction analysis of the amplicon using restriction endonuclease AccB1I, electrophoresis in an agarose gel, different the fact that for restriction analysis, along with the AccB1I restriction endonuclease, the AccB7I, DseDI and HaeIII restriction endonucleases are used, and the presence of DNA fragments of 510 and 109 bp in length. when treated with the restriction enzyme AccB1I, it indicates that it belongs to the species A. cucumeris, the presence of DNA fragments of 514 and 93 bp in length. when treated with the restriction enzyme AccB7I, it indicates that it belongs to the species A. swirskii, the presence of DNA fragments of 451 and 171 bp in length. after restriction analysis with the DseDI enzyme, it indicates that it belongs to the species A. montdorensis, and the presence of DNA fragments of 496 and 120 bp in length. after treatment with the HaeIII restriction enzyme, it is determined that it belongs to the species A. californicus, while other restriction enzymes do not have restriction sites.