RU2735911C1 - Method of identifying heterobasidion annosum fungus - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: invention represents a method for identifying Heterobasidion annosum fungus, based on PCR, involving DNA recovery from a plant substrate or fungal mycelium, real-time PCR amplification involves DNA denaturation, primer attachment and DNA chain elongation. PCR amplification involves using primers: forward SEQ ID NO: 1 GAATATCGTGCAAGGTTG and reverse SEQ ID NO: 2 CGAAGAGTTTGTGAGAAG, and also TaqMan probe SEQ ID NO: 3 FAM-CGCGGCTCGGAAGGGGTCAAAAGAACCC-BHQ1. Following temperature cycles are used: 1 cycle – temperature 94 °C for 5 minutes and 44 cycles – alternately temperature 94 °C for 20 s and temperature of 59 °C for 60 s. Logarithmic increase of the fluorescence level up to 38 cycle in the FAM channel of the thermocycler testifies to the presence of fungus Heterobasidion annosum in the analyzed sample.EFFECT: invention enables fast identification of fungus Heterobasidion annosum.1 cl, 1 dwg, 1 tbl, 1 ex

Description

The invention relates to the field of forestry, in particular, to measurement methods using microorganisms, namely nucleic acids, and can be used in the forestry industry to identify the pathogenic fungus Heterobasidion annosum.

Heterobasidion annosum (root sponge) is a species complex of pathogenic white rotting woody fungi that cause root and root rot in conifers and deciduous trees in the Northern Hemisphere. Annual losses to forest managers are estimated at billions of dollars due to tree death, yield decline and wood decay (Pathogens, 2019, V. 8, No. 3, P. 156).

The disease of conifers is characterized by root rot and progressive drying out of trees. The causative agent of the disease lives everywhere in coniferous and deciduous plantations, regardless of their condition. The fungus spreads freely to living trees only in case of serious disturbances in the biogeocenosis resulting from the influence of natural and anthropogenic factors, including errors in forestry activities (often when growing monocultures on non-forest lands) (Recommendations for the protection of forests from root sponges in the forests of the European part Russian Ministry of Natural Resources, VNIILM. Pushkino, 2001.9 p.). With the intensification of anthropogenic influence on ecosystems, the rate of development of the root sponge increases.

At present, the principles and methods of prevention and treatment of plant diseases caused by this pathogen are being developed. Financial analyzes have shown the effectiveness of stump treatment in relatively healthy stands with a high content of Heterobasidion annosum spores throughout Finland. In addition, the quality of stump processing has a significant impact on profitability, together with the price paid for rotten wood (Forest Policy and Economics, 2019, V. 105, P. 1-9). These methods are also carried out in Russia (Recommendations for the protection of forests from root sponges in the forests of the European part of Russia. Ministry of Natural Resources of the Russian Federation, VNIILM. Pushkino, 2001. 9 p.). There is a need to monitor and predict the spatio-temporal risks of infection with high-quality processing of the stump.

A known method for determining the virulence, aggressiveness and pathogenicity of the root sponge (Application for the invention of the Russian Federation No. 96115783; IPC A01G 7/00; publ. 27.01.1999) and a method for assessing the sanitary and forest pathological state of forest areas for the presence of root pathogens (Pat.RF No. 2619987; IPC A01G 23/00; publ. 04/28/2017), which are based on an indirect analysis of the morphological manifestations of plant infection with a pathogenic fungus - taking into account the survival rate of cuttings, preservation, the appearance of mycelium (rhizomorphs) and (or) the fruiting bodies of the fungus. To analyze the spread of the root sponge, it is proposed to use the calculation formula, which takes into account the total conditional faulty per 1 tree, the coefficient of the categories of foci of drying out, the actual damp growing stock of the susceptible species, and other coefficients.

The disadvantage of these methods is the impossibility of species identification of the pathogen, in particular the fungus Heterobasidion annosum, in plants.

A known method for determining fungal pathogens using polymerase chain reaction (PCR) (Pat.RF No. 2161196; IPC C12N 1/14, C12Q 1/68, C12N 15/80, A01N 63/04, C12P 19/34; publ. 27.12. 2000), which makes it possible to identify a wide range of fungal infections and includes the isolation of DNA from the plant substrate, real-time PCR amplification, which consists in DNA denaturation, primer attachment and DNA strand elongation. For this, primers specific to various pathogenic fungi are designed on the basis of DNA in the region of the internal transcribing spacer (ITS) of the ribosomal RNA gene. The ITS DNA sequences from different pathotypes of pathogen species or genera may be different for different representatives of the species or genera. If the ITS sequences of the pathogen are determined, these sequences can be compared with other ITS sequences. Thus, primers can be obtained from the ITS sequences. Primers based on these sequences can be used to identify specific pathogens during the polymerase chain reaction in plant samples. Taken as a prototype.

The objective of the present invention is to develop a highly specific and operational method for identifying the fungus Heterobasidion annosum.

The technical result consists in the development of a highly sensitive (the sensitivity of the method is at least 1 pg of Heterobasidion annosum DNA) and fast (within 2-3 hours, depending on the method for isolating DNA from the plant substrate or fungal mycelium) method for analyzing the presence of the Heterobasidion annosum fungus.

The technical result is achieved by the fact that in a method for identifying the fungus Heterobasidion annosum, based on PCR, including the isolation of DNA from a plant substrate or fungal mycelium, carrying out amplification of PCR in real time, which consists in DNA denaturation, attachment of primers and elongation of the DNA chain, according to the invention, when For PCR amplification, primers are used: forward SEQ ID NO: 1 GAATATCGTGCAAGGTTG and reverse SEQ ID NO: 2 CGAAGAGTTTGTGAGAAG, as well as TaqMan probe SEQ ID NO: 3 FAM-CGCGGCTCGGAAGGGGTCA ° BAHQ1ACCC-94 C for 5 min. and 44 cycles - alternately a temperature of 94 ° C for 20 s and a temperature of 59 ° C for 60 s, and the logarithmic increase in the fluorescence level to 38 cycles in the FAM channel of the amplifier indicates the presence of the Heterobasidion annosum fungus in the analyzed sample.

FIG. 1 shows the amplification curves (FAM channel) of real-time PCR.

A preliminary analysis of the nucleotide sequence of a DNA region containing genes 18S rRNA, 5.8S rRNA, and 28S rRNA and intergenic regions ITS1 and ITS2 for the fungus Heterobasidion annosum was performed. Conservative regions for this taxonomic group have been established, which make it possible to develop a method for fungal identification based on real-time PCR using a TaqMan probe, which is an oligonucleotide to which a fluorophore molecule and a fluorescence quencher molecule are attached. During PCR, the probe hybridized with DNA is cleaved by DNA polymerase (due to its exonuclease activity), and a fluorescent label is released. Thus, an increase in the level of fluorescence is observed.

The method for identifying the fungus Heterobasidion annosum using real-time PCR using the TaqMan probe is implemented as follows.

1. Collect biological material (plants, plant residues, fungal mycelium). No more than 1 g of biological material is used for analysis.

2. Produce DNA extraction from biological material. DNA isolation can be carried out using commercial kits (Bio-Silica, DNA Technologies, Qiagen, Zymo Research), etc.), and by organic extraction methods using CTAB buffer.

3. Real-time PCR amplification is performed, which consists in DNA denaturation, primer attachment and DNA strand elongation. In this case, the following temperature cycles are used: 1 cycle - temperature 94 ° C for 5 minutes. and 44 cycles - alternately a temperature of 94 ° C for 20 s and a temperature of 59 ° C for 60 s.

The following primers and probe are used:

forward primer SEQ ID NO: 1 GAATATCGTGCAAGGTTG;

reverse primer SEQ ID NO: 2 CGAAGAGTTTGTGAGAAG;

TaqMan probe SEQ ID NO: 3 FAM-CGCGGCTCGGAAGGGGTCAAAAGAACCC-BHQ1.

These primers amplify the PCR product, which contains nucleotide polymorphisms characteristic only of the fungus Heterobasidion annosum, with which the TaqMan probe interacts.

4. If there is a logarithmic increase in the fluorescence level up to 38 cycles in the FAM channel of the amplifier, this means that the analyzed sample contains the Heterobasidion annosum fungus.

Example.

After preliminary isolation of DNA from 12 plant samples (Table 1), real-time PCR amplification was carried out (Fig. 1).

In two samples (No. 1 and No. 5) there was a logarithmic increase in the fluorescence level in the FAM channel of the amplifier up to cycle 38, while in other samples (No. 2-4 and No. 6-12) no increase in fluorescence was observed, on the basis of which it was concluded that samples # 1 and # 5 contained the Heterobasidion annosum fungus.

The proposed method for identifying the fungus Heterobasidion annosum is highly sensitive, well reproducible and economical. The analysis can be carried out in laboratories that have a real-time amplifier, centrifuge and associated reagents and consumables.

Claims (1)

A method for identifying the fungus Heterobasidion annosum, based on PCR, including isolation of DNA from a plant substrate or fungal mycelium, carrying out real-time PCR amplification, which consists in DNA denaturation, attachment of primers and DNA strand elongation, characterized in that primers are used during PCR amplification: forward SEQ ID NO: 1 GAATATCGTGCAAGGTTG and reverse SEQ ID NO: 2 CGAAGAGTTTGTGAGAAG, as well as TaqMan probe SEQ ID NO: 3 FAM-CGCGGCTCGGAAGGGGTCAAAAGAACCC-BHQ1, with 1 cycle using the following temperature cycles: 44 cycles - alternately a temperature of 94 ° C for 20 s and a temperature of 59 ° C for 60 s, and a logarithmic increase in the level of fluorescence to 38 cycles in the FAM channel of the amplifier indicates the presence of the Heterobasidion annosum fungus in the analyzed sample.

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