CN111579720B - Method for screening and killing meloidogyne incognita plants - Google Patents

Abstract

The invention relates to the technical field of botanical pesticides, and in particular relates to a method for screening and killing M. incognita plants. The method uses D. oatmeal instead of M. incognita as a test material, which can predict the effect of nematode plants on M. incognita. The killing effect of knot nematodes. The growth cycle and culture requirements of E. oats are far lower than those of M. incognita, which is more conducive to the further screening of nematicidal plants and to speed up the experimental process of nematode biological control.

Description

A kind of method for screening and killing M. incognita plants

technical field

The invention relates to the technical field of botanical pesticides, in particular to a method for screening and killing M. incognita plants.

Background technique

Plant-parasitic nematodes are a class of obligate plant-parasitic pathogenic nematodes, which cause huge economic losses to the production of agricultural commercial crops. The annual economic losses caused by plant-parasitic nematodes in the world are about 150 billion US dollars. Root-knot nematodes are considered the most serious phytopathogenic nematodes and can parasitize in more than 3,000 plants including wheat, tomato, cucumber, etc. They destroy the normal metabolic function of host cells and cause lesions, resulting in a 10% reduction in crop yield -20%, up to 30%-40% in severe cases, and even stop giving birth.

With the improvement of human awareness of environmental protection, biological control has become the dominant method for nematode control. The nematicidal potential of many plant extracts to plant nematodes has been widely evaluated, and most species with nematicidal activity are in Compositae and legumes. Although more than 300 kinds of nematicidal plants have been reported, the natural plant resources are extremely rich. There are more than 30,000 species of plants in my country, which are rich in resources, and many plants can be used as botanical pesticides. The book "Poisonous Plants in China" lists more than 1,300 poisonous plants, many of which have insecticidal activity. Therefore, full investigation and excavation of more and more effective new nematicidal plant species will become the direction of future development.

In the process of routine screening of nematicide plants, it is necessary to use M. incognita. The reason is: M. incognita is the most serious and destructive root-knot nematode in agricultural production. According to statistics, 51% of the The root-knot nematode species of the affected crops was M. incognita, which had the highest distribution ratio among the four root-knot nematodes. However, M. incognita has the following problems: (1) the growth cycle of M. incognita is long; (2) M. incognita can only reproduce on the roots of plants, and the growth environment is harsh and easily affected by the external environment. The second instar larvae of M. incognita are less mobile in vitro, and the worms often appear in a state of suspended animation when faced with adversity. The screening process for nematicide plants has been slow.

SUMMARY OF THE INVENTION

In view of this, the present invention provides a method for screening and killing M. incognita plants. The method adopts the fungus-eating nematode A. oatmeal to replace the M. incognita to initially screen the test plants, which accelerates the test process. , more nematode plants can be screened out in a shorter time.

In order to achieve the above object, the technical scheme of the present invention is as follows:

A method for screening and killing M. incognita plants, using the oat nematode to replace M. incognita as a test worm, specifically comprising the following steps:

S1. The nematicidal plants are cleaned, dried, pulverized and sieved, added with a solvent for extraction, filtered with gauze to obtain a plant extract, and diluted to a series of concentrations.

S2. Mix the hatched second-instar oat nematode with distilled water evenly, and inject it quantitatively into the cell culture plate, and then inject the series of plant extracts prepared in step S1 into the holes of the nematodes in the same amount, and use the same amount of distilled water to make Blank control; placed at 26°C, with a light cycle of 16:8 and a humidity of 70%, cultured at a constant temperature, observed and recorded the survival number of nematodes, and calculated the corrected mortality;

S3, predict according to the result of step S2 and determine the insecticidal effect of plant extract to Nematode incognita;

The corrected mortality calculation formula is:

Further, in the above technical solution, the extraction solvent in step S1 is distilled water.

Further, in the above technical solution, the preparation method of the plant extract is as follows: weighing the plant powder, adding an appropriate amount of distilled water, placing on a shaker for 12-24 hours, rotating speed 180-200r/min, temperature 32-38 ℃, filtering have to.

Beneficial effect: the present invention finds and confirms by experiment for the first time that E. oatmeal and S. incognita have basically the same response (corrected death rate and LC ₅₀ ) to the tested plants, so it can replace S. incognita and accurately preliminarily screen and kill nematodes plant. It only takes 11-12 days for E. oats to complete a life cycle at 20°C, which is 46-47 days shorter than that of M. incognita, more nematodes can be obtained in a shorter time, and the waiting time for the test is greatly shortened. ; The edible fungus of C. oatmeal can be propagated indoors, which is less affected by the external environment, the test operation process is simpler and the pollution to the soil is reduced; there are fewer suspended animations during the test, and the preliminary screening results more precise.

Description of drawings

Figure 1 is a comparison chart of the activity of different plants to two nematodes after treatment for 72h when the concentration is 0.78125g/L;

Figure 2 is a comparison diagram of the activity of different plants to two nematodes after treatment for 72h when the concentration is 1.5625g/L;

Figure 3 is a comparison diagram of the activity of different plants to two nematodes after treatment for 72h when the concentration is 3.125g/L;

Figure 4 is a comparison diagram of the activity of different plants on two nematodes after treatment for 72h when the concentration is 6.25g/L;

Figure 5 is a comparison diagram of the activity of different plants on two nematodes after treatment for 72h when the concentration is 12.5g/L;

Detailed ways

In order to better understand the model of the present invention, the present invention will be further described in detail below with reference to the embodiments.

Example 1

(1) Clean the roots of the nematode plant, dry them in a blast drying oven at 45°C to a constant weight, pulverize them with a multi-functional pulverizer, and pass through a 60-mesh sieve; weigh 5g of the root powder of the fish vines into a triangular flask, add 100 mL Distilled water, placed on a shaker for 24h, rotating speed 200r/min, temperature 35°C, filtered to obtain the extraction mother liquor of rhododendron root powder, the concentration was 50g/L, after 4 times of dilution, the concentration was 12.5g/L, and then continued to dilute twice in turn to obtain The extracts of fish vine powder with concentrations of 6.25g/L, 3.125g/L, 1.5625g/L and 0.78125g/L.

(2) Mix the hatched second-instar oatmeal nematodes with distilled water evenly, quantitatively inject them into a 48-well cell culture plate, and inject 500 μl of nematode solution into each well (20 μl of nematode solution contains 20 nematodes), and then put different nematodes into each well. Plant extracts with an equal concentration were injected into the holes of nematodes, five treatments and five replicates were set, and an equal volume of distilled water was used as a blank control; placed at 26°C, the light cycle was 16:8 (L:D), and the humidity was 70%. Cultivated in a constant temperature light incubator, observed and recorded the survival number of nematodes at 24h, 48h and 72h, and calculated the corrected mortality.

Example 2

The killing plant is the whole plant of the broken bowl flower, and the others are the same as in Example 1.

Example 3

The nematode plant is the chrysanthemum leaf, and the others are the same as those in Example 1.

Example 4

The nematode plant is the whole plant of the lice grass plant, and the others are the same as those in Example 1.

Example 5

The thread-killing plant is the whole plant of Houttuynia cordata, and others are the same as in Example 1.

Comparative Example 1

In step (2), the root knot nematode is used to replace the oat nematode, and the others are consistent with embodiment 1.

Comparative Example 2

In step (2), use Nematode incognita to replace oat nematode, other are consistent with embodiment 2

Comparative Example 3

In step (2), use Nematode incognita to replace oat nematode, other are consistent with embodiment 3

Comparative Example 4

In step (2), use Nematode incognita to replace C. oatmeal, other are consistent with embodiment 4

Comparative Example 5

In step (2), the root knot nematode was used to replace E. oatmeal, and the others were consistent with Example 5.

Analysis of the results: See Figures 1 to 5 and Tables 1 and 2 for the results of the comparative test of the nematicidal plants' activity against D. oatmeal and S. incognita.

Table 1 Corrected mortality of various plant extracts at different concentrations for two nematodes

Note: lowercase letters indicate the significance analysis of the same material under different concentrations at the level of P=0.05 Table 2 The results of toxicity analysis after 48h treatment

It can be seen from Figures 1 to 5 that when the concentration of the nematode plant extract is 0.78125g/L, 1.5625g/L, 3.125g/L, 6.25g/L, and 12.5g/L, when the nematodes were treated for 72h, the same plant extract was treated. There was no significant difference in the corrected mortality between D. oatmeal and R. incognita; especially when the extraction concentration was 3.125g/L and 6.25g/L, the difference was the smallest. It can be seen from Table 1 and Table 2 that the LC ₅₀ of the same plant is basically the same after the two nematodes are treated, and the nematicidal activity is also basically the same (the overall LC ₅₀ of M. incognita is too large, which may be caused by its strong resistance, but The difference was not significant). In conclusion, the data indicated that E. oatmeal could replace R. incognita in the primary screening of nematicide plants.

The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection of the present invention. within the range.

Claims (3)

1. A method for screening and killing meloidogyne incognita plants is characterized in that the meloidogyne incognita is replaced by true-root-knot nematode oat as a test insect body, and the method specifically comprises the following steps:

s1, cleaning nematicidal plants, drying, crushing, sieving, adding a solvent for extraction, filtering to obtain a plant extract, and diluting the plant extract into a series of concentrations;

in the step, the nematode killing plant is one of derris root, Chinese flowering cabbage, crowndaisy hemp leaf, lice grass and houttuynia cordata;

s2, mixing the hatched two-year-old real-leaf-worm-like oat with distilled water uniformly, quantitatively injecting the mixture into a cell culture plate, injecting the series of plant extract prepared in the step S1 into the holes with the nematodes in equal amount, and taking the distilled water in equal amount as a blank control; placing at 22-28 deg.C, lighting for 12-16h in daytime and 8-12 h at night, and keeping humidity at 65% -85%, culturing at constant temperature, observing and recording survival number of nematodes, and calculating corrected mortality;

s3, prejudging and determining the insecticidal effect of the plant extract on the meloidogyne incognita according to the result of the step S2;

the corrected mortality calculation formula is:

2. the method for screening plants for killing meloidogyne incognita according to claim 1, wherein the extraction solvent is distilled water in step S1.

3. The method for screening the plant for killing meloidogyne incognita according to claim 2, wherein the plant extract is prepared by: weighing plant powder, adding appropriate amount of distilled water, placing on a shaking table for 12-24h at a rotation speed of 180-.

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