Proper sampling is the key to successful identification of a phytopathogenic organism. For the analysis, one should take the organ of the plant in which the maximum amount of the desired organism is located. So, in identifying the causative agents root rot , the affected roots and the root part of the stem should be taken. In leaves, flowers, etc. the causative agent of root rot, most likely, will not be. Even if the lesions of other organs are observed, they are highly likely to be caused by very different pathogens.
When sampling, the life cycle of the pathogen should also be considered. The causative agent of a dusty bunt in latent form is present in the stalk of a young plant. However, if the plant is infected in the summer through a flower, then it is useless to look for the causative agent in the stalk - it is not there. Success can only come from the study of the flower. In all cases of sampling, the best diagnostic results are obtained by analyzing tissues from the border of the affected and healthy parts of the plant.
Special care should be taken to store the affected material. It is best to isolate DNA from the sample as quickly as possible (within a few hours) after taking the sample. With prolonged storage in warm and humid conditions, a saprotrophic microbiota will develop on the sample, which may make it difficult to identify the cause of the disease. Therefore, when sampling for analysis by PCR, the sample should be preserved as much as possible. For this, the sample can be frozen immediately after sampling at -20 ° C, or placed in a special solution that prevents the growth of the microbiota and the destruction of the DNA molecule. As such a solution, 70% ethyl alcohol is often used. The canned sample can be stored for a long time before starting laboratory tests.
For analysis using PCR, or DNA-DNA hybridization according to E. Southern (see paragraph 5.5.), it is necessary to extract DNA from the sample under investigation (in the analysis of the material for the presence of viruses, RNA is isolated). Currently, there are many methods of DNA isolation, kits for isolation from various substrates, there are special automatic DNA isolation stations. In Fig. В.7 one of the sets for DNA extraction is shown.
The essence of all isolation methods is to extract DNA from the sample and remove or inactivate impurities that interfere with PCR. In order for DNA to enter the solution, it is necessary, first of all, to destroy the cells. In some cases (for example, in the analysis of bacteria or animal cells) simple boiling with alkali is sufficient, as a result of which cell walls are destroyed.
However, when working with fungi, especially when extracting DNA directly from the affected organ of the plant, this may not be enough, since fungal hyphae and, in particular, spores can have a very strong membrane. In this case, the sample is ground with a pestle in a pre-sterilized porcelain mortar.
For even greater reliability and increasing the yield of DNA (which is especially important if you need to isolate the DNA of a pathogen that is in a diseased organ in a very small concentration) use liquid nitrogen. The sample is pre-frozen and ground in a chilled mortar.
To isolate DNA from the soil, special devices are used that grind biological soil material in a rapidly rotating head. Glass beads or quartz sand are often used for better destruction of DNA bearing objects.
Further from the mixture of DNA with shell residues, inorganic components (sand, soil residues), proteins, etc. it is necessary to obtain a purified DNA preparation. All modern methods of purification of nucleic acids can be divided into two groups:
- methods with a phased removal of impurities from the aqueous solution;
methods based on the sorption of nucleic acids on the solid phase.
Of the variants of the first method, extraction with chloroform is most known. The method is applicable for virtually any type of fungus, can be used to isolate the affected plant tissue.
However, if there are many PCR inhibitors in the sample, then the DNA must be further purified on the column, i.e. apply the method of the second group - to sorb DNA on a solid phase. Usually, the purification is carried out in disposable plastic micro-columns with the sorbent packed in them (silicon oxide or polyvinyl-lipipyrrolidone (PVPP) is often used). Washing columns with solutions of high ionic strength removes proteins and low-molecular compounds, leaving pure DNA on the sorbent. DNA is easily washed off from the sorbent by solutions with a low ionic strength, for example, distilled water.
DNA Extraction Protocol for PCR
Fill a sample of frozen mycelium with liquid nitrogen and grind in a pre-soaked mortar mortar. About 0.25 ml of mashed mycelium is transferred to a microtube Eppendorph for 2 ml. Add 800 μl of lysis buffer (STDB buffer) to it. Stir on the vortex.
Preparation of the STAB -buffer (on 1l):
TRIS pH 8.0 100 mmoles;
NaCl 1.4 mol;
EDTA pH 8.0 20 mmol;
CTAB (Hexadecyltnmethylammonium bromide) solid 2% (W/V).
After mixing the microtubes with mycelium, place on a water bath or in a solid-state thermostat (65X) for 1 hour; stir vortex every 20 minutes. After 1 hour, add 500 μl of chloroform (also a mixture of chloroform and isoamyl or octyl alcohol may be used in a 24: 1 ratio), shake, centrifuge for 10 minutes and transfer 700 μl of the supernatant top phase to a new 1.5 ml clean microtube. Add 400 μl (0.6 volume) of isopropanol and potassium acetate (CH 3 COOK, 1/10 volume, 5 moles, pH = 4.6) to a microtube and mix by hand and centrifuge for 10 minutes. The supernatant is carefully drained and the precipitate is washed with 70% ethanol cooled. Centrifuge for 5 minutes, drain off alcohol, remove its residues with filter paper and dry the residue for 1-2 hours (until it becomes clear). Resuspend the resulting DNA in 100 μl of TE buffer or deionized water.
The isolation of DNA is usually carried out in the laboratory, but if necessary, it can be carried out in almost any room or in a mobile laboratory. The company "DNA-Theology", for example, produces portable laboratories that allow DNA isolation and PCR diagnostics, packed in a portable case (Figure B.8).
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