CN112553114B - Bacillus aryabhattai 1K02966 and uses thereof - Google Patents
Bacillus aryabhattai 1K02966 and uses thereof Download PDFInfo
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Abstract
The invention discloses a bacillus aryabhattai MCCC1K02966 and application thereof. The fermentation supernatant of the Bacillus aryabhattai MCCC1K02966 provided by the invention has good insecticidal effect on plant pathogenic nematodes, namely, meloidogyne incognita; meanwhile, the volatile substance methyl thioacetate produced by the strain also has good contact killing, fumigating killing, egg hatching inhibiting and avoidance effects on Meloidogyne incognita; the application of the fermentation supernatant of the Bacillus aryabhattai MCCC1K02966 and the insecticidal activity of the produced volatile substance methyl thioacetate on the root-knot nematodes has great significance and application prospect for the control of plant root-knot nematode diseases and the development of novel nematode control strategies.
Description
Technical Field
The invention belongs to the technical field of agricultural microbiology, and particularly relates to Bacillus aryabhattai 1K02966 and application thereof.
Technical Field
Root-knot nematodes (Meloidogynespp.) are obligate root parasites that are widespread worldwide, causing crop losses of up to billions of dollars each year, and are among the most serious pests responsible for agricultural economic losses. In recent years, with the rapid development of agricultural industry and the continuous adjustment of industrial structure in China, particularly the rapid development of vegetables in protected areas, the continuous planting of the same kind of vegetable crops in greenhouses, the multiple cropping index is high, and root-knot nematode diseases are increasingly serious due to human factors. Moreover, because root-knot nematodes mainly damage roots, the onset symptoms are not easy to be detected by people; root knot nematodes are mainly spread through disease seedlings, disease soil, irrigation and the like, and are difficult to control, so that the control of root knot nematode diseases is an important and very difficult agricultural production task.
The prevention and control method for root-knot nematodes mainly uses chemical nematocides historically, and has quick response and obvious effect. However, many chemical nematicides have been or will be banned due to the increasing prominence in the problems of poor selectivity, high residue, difficulty in degradation, environmental pollution, ecological balance damage and social public nuisance. In recent years, with the progress of science and technology and the enhancement of environmental awareness of people, biological control methods attract great attention of researchers, and will gradually become one of important means for comprehensively controlling nematodes.
Bacillus aryabhattai (Bacillus aryabhattai), short for Bacillus aryabhattai, belongs to gram-positive bacteria; bacillus aryabhattai is a bacterium discovered in the beginning of the 21 st century, has wide distribution and very strong stress resistance, and is found in animal intestinal tracts, plant rhizosphere, deep sea and plateau. Bacillus aryabhattai has various biological activities: the Bacillus aryabhattai can protect plants, prevent the toxic action of arsenic, copper, lead and nitrite, and promote the conversion of zinc into an ionic state so as to facilitate the absorption of the plants; the bacillus aryabhattai can promote the absorption of zinc elements, simultaneously inhibit the damage of rice blast bacteria (Pyricularia oryzae) and Fusarium moniliforme (Fusarium moniliforme) to rice, and simultaneously play a role in increasing the yield, the zeitai delphinium and the like disclose the bacillus aryabhattai J5 and an invention patent of the application thereof, the bacillus aryabhattai J5 can not only prevent and control plant diseases, but also improve soil nutrients and promote the growth of plants; can synthesize biological hormone or active organic matter to promote plant growth or plant in plant root system to symbiotic with plant, Bacillus aryabhattai AB211 is proved to dissolve inorganic phosphate, synthesize iron carrier and produce hormone such as indoleacetic acid (IAA).
There are a number of reports in the literature on the nematicidal activity of Bacillus species. The deep sea monarda bracteata bacillus has the nematicidal effect found by the Huangdian and the like. Chengwanli et al found that Bacillus polymyxa KM2501-1 can produce 8 volatile nematicidal substances. Bacillus thuringiensis (Bt) is well known for its production of specific insecticidal crystallins, and Bt produces other virulence factors with nematicidal activity in addition to the well known varieties of Bt parasporal crystallins with nematicidal activity. However, many bacilli have no insecticidal effect in screening, and Songmin and the like are separated from 187 bacilli in the soil of Yongxing island to carry out nematicidal activity screening, and the result shows that only 15 bacilli have nematicidal activity; moghaddam and the like isolate 52 strains of Bacillus from the Iran tomato field, and consequently 8 strains of Bacillus pumilus ToIr-MA, Bacillus ToIr-10 and the like which have nematicidal activity are screened out.
The invention discovers for the first time that the fermentation supernatant of the bacillus aryabhattai MCCC1K02966 has the effect of killing nematodes, and the produced volatile substance methyl thioacetate has the effects of killing by contact, fumigating, inhibiting egg hatching and avoiding nematodes.
Disclosure of Invention
The invention aims to provide the application of the Bacillus aryabhattai or the fermentation supernatant thereof in preparing nematode insecticides; the Bacillus aryabhattai is MCCC1K 02966.
It is another object of the present invention to provide the use of the volatile substance methyl thioacetate produced by bacillus aryabhattai 1K02966 in the preparation of a nematode insecticide.
In order to achieve the above purpose, the following technical measures are adopted in the research:
the application of the Bacillus aryabhattai or the fermentation supernatant thereof in preparing the nematode insecticide comprises preparing the plant nematode insecticide by utilizing the Bacillus aryabhattai or the fermentation liquor of the Bacillus aryabhattai, wherein the Bacillus aryabhattai is MCCC1K 02966.
In the application, the effective component in the fermentation liquor of the Bacillus aryabhattai is methyl thioacetate, the molecular weight of the methyl thioacetate is 90.14, and the molecular formula of the methyl thioacetate is C 3 H 6 OS, chemical formula as follows:
the application of methyl thioacetate in preparing nematode pesticide also belongs to the protection scope of said invention, including that it can be made into nematode contact insecticide, fumigant, egg hatching inhibitor or repellent.
Compared with the prior art, the invention has the following advantages:
1. the invention reports that the fermentation supernatant of the Bacillus aryabhattai 1K02966 has good effect of killing the plant root-knot nematodes in a contact manner for the first time, and provides a new choice for preparing a novel plant nematode prevention and control preparation.
2. A headspace solid-phase microextraction and gas mass spectrometry combined method (SPME-GC-MS) is utilized to identify and obtain a volatile substance methyl thioacetate from Bacillus aryabjeldae 1K02966, the volatile substance methyl thioacetate has good effects of killing and fumigating plant root-knot nematodes, inhibiting egg hatching and avoiding nematodes, and the four characteristics are comprehensively utilized, so that the effect of the nematicide can be improved, and a basis is provided for a novel nematicide.
Drawings
FIG. 1 is a graph showing the effect of Bacillus aryabhattai 1K02966 on killing Meloidogyne incognita at 0h (left panel) and 48h (right panel).
FIG. 2 is a schematic diagram of a method for determining the chemotaxis of Meloidogyne incognita towards volatile substances.
Detailed Description
The experimental procedures in the following examples are reported as conventional procedures in microbiology unless otherwise specified. The reagents or materials, unless otherwise specified, are commercially available. Bacillus aryabhattai (Bacillus aryabhattai) MCCC1K02966 of the present invention is disclosed in the 1 st edition of 7 months of 2010, catalog of marine microorganism strains in China.
Example 1:
preparation of a Bacillus aryabhattai (Bacillus aryabhattai)1K02966 fermentation supernatant:
the Bacillus aryabhattai 1K02966 is streaked and activated on a plate, and a single colony on the plate is picked and inoculated into a triangular flask filled with 100mL of 2216E liquid culture medium, wherein the culture temperature is 28 ℃; the rotating speed of the shaking table is 180 rpm; cultivation time 48h (OD) 600 1.85); centrifuging at 12000rpm for 10min, and filtering the supernatant with 0.22 μm sterile filter to obtain fermented supernatant of Bacillus aryabhattai MCCC1K02966 (hereinafter referred to as 1K002966 fermented supernatant).
Example 2:
the application of the fermented supernatant of Bacillus aryabhattai (Bacillus aryabhattai)1K02966 in the preparation of plant meloidogyne pesticide:
this example illustrates meloidogyne incognita, other plant meloidogyne incognita, for example: the meloidogyne hapla, the meloidogyne arachidicola and the meloidogyne javanica have insecticidal effects;
determination of virulence of meloidogyne incognita:
1) carrying out virulence determination by using a 96-well plate, and adding 30-50 heads of meloidogyne incognita into each well;
2) add 120. mu.L of filter sterilized 1K02966 fermentation supernatant (prepared in example 1) per well;
3) experimental setup 2 groups:
1K02966 fermentation supernatant group (CF): 1K02966 fermenting the supernatant plus Meloidogyne incognita;
control group (CK): 2216E Medium + Meloidogyne incognita.
4) Incubating at 20 ℃ for 48h to count the death rate;
5) the number of dead insects was determined by visual observation or staining observation, and the results are shown in Table 1.
Nematode mortality rate ═ nematode death number/nematode to be tested × (100)%
Nematode corrected mortality (treatment group nematode mortality-control group nematode mortality)/(1-control group nematode mortality) × 100%
TABLE 11 in vitro experimental Effect of K02966 fermentation supernatants on Meloidogyne incognita
Results show that compared with a control, the 1K02966 fermentation supernatant has a better in vitro insecticidal effect on meloidogyne incognita.
Example 3:
identification of volatile substances from the Yerballowsonia 1K02966 fermentation broth:
1) taking 3-20 mL of 1K02966 fermentation liquid in a headspace bottle, extracting for 60min by using a 75-micrometer CAR/PDMS solid-phase microextraction fiber head under the condition of water bath at 60 ℃, and using a 2216E culture medium as a control;
2) after extraction, 7890GC/5975MSD GC-MS is used for mass spectrometry to obtain a result, and the experiment is repeated three times.
3) Identifying to obtain volatile substance methyl thioacetate, wherein the molecular weight of the methyl thioacetate is 90.14, and the molecular formula is C 3 H 6 OS, chemical formula as follows:
example 4:
virulence effects of methyl thioglycolate on root-knot nematodes:
1) and (3) picking nematodes from tomato roots to infect root knots, placing the egg masses in a 96-well plate for incubation at 20 ℃, and observing the incubation condition of the root-knot nematodes. The nematodes were rinsed with sterile water, 200. mu.L of methyl thioacetate of various concentrations were added to 96-well plates, 3. mu.L of chloramphenicol (30mg/mL) and 30-40 head nematodes were added, 3 replicates per group, using solvent as a blank. Culturing in an incubator at 20 ℃, counting once every 24h, and observing and counting for 72 h; the number of dead insects was determined by visual observation or staining observation, and the results are shown in Table 2.
TABLE 2 direct contact killing activity of the volatile substance methyl thioacetate against nematodes
The results show that methyl thioacetate with different concentrations has good direct contact nematicidal activity on meloidogyne incognita (figure 1).
2) Adding 200 μ L of methyl thioacetate with different concentrations into a 96-well plate, measuring the fumigating activity of the methyl thioacetate on the nematodes, adding 30-40 head nematodes and 200 μ L of sterile water into four holes around the methyl thioacetate addition hole, and using a solvent as a blank control, wherein each group is repeated for 3 times. Culturing in an incubator at 20 ℃, counting every 24h, and observing and counting for 72h (the counting method is the same as the above); the number of dead insects was determined by visual observation or staining observation, and the results are shown in Table 3.
TABLE 3 Fumigation Activity of the volatile substance methyl Thioacetate against nematodes
The results show that methyl thioacetate with different concentrations has good fumigating and killing activity on Meloidogyne incognita.
Example 5:
effect of volatile substance methyl thioacetate on hatching of nematode eggs:
picking the egg blocks on the tomato roots, collecting the egg blocks together, and washing the egg blocks with sterile water for later use; mu.L of methyl thioacetate at various concentrations was added to 96-well plates, 3. mu.L of chloramphenicol (30mg/mL) and 1 egg mass were added, 3 replicates per group, using solvent as a blank. Culturing in 20 deg.C incubator for 7 days, and counting the number of nematodes hatched in each well, the results are shown in Table 4.
Inhibition of egg hatching (%) (control group egg hatching-treatment group egg hatching)/control group egg hatching
TABLE 4 inhibition of meloidogyne egg hatching by the volatile substance methyl thioacetate
The result shows that the methyl thioacetate has good effect of inhibiting the hatching of the root knot nematode eggs.
Example 6:
effect of volatile substance methyl thioacetate on nematode chemotaxis:
1) preparing a drug sensitive paper sheet: the filter paper sheet was punched with a punch to a radius of 5.6mm (area 1 cm) 2 ) The round pieces of (A) were placed in clean and dry PA bottles, respectively, sealed with caps, and autoclaved at 121 ℃ for 20 min.
2) Preparation of chemotactic plate: a disposable 90mm diameter cell culture dish was placed on a clean bench, and about 10mL of 2% (w/v) water agar solution was poured in uniformly and coagulated at room temperature. 2 marking points are made on each agar chemotaxis plate at equal intervals at the symmetrical position 2.0cm away from the center of the plate, 1 piece of each piece of filter paper is arranged at the marking points, 30 mu L of methyl thioacetate with different dilution degrees is added into one filter paper sheet, the other filter paper sheet is added with solvent with the same volume, and the same plate is divided into two areas (an experimental area and a control area) by two different drug sensitive paper sheets. The distance between the edge of each drug sensitive paper sheet and the release point of the central larva is 2.0cm, and the agar plate is kept complete and smooth in operation. In addition, the range of 0.4cm to the left and right of the center line is a buffer area (fig. 2).
3) Implementation of chemotaxis experiments: hatched larvae of meloidogyne incognita J2 (5 μ L, around 150) were released at the central larval release point of the plate. After incubation for 8h in the dark at room temperature (nematodes need to disperse freely for 2-3 h), the number of nematodes in the experimental area and the control area is counted under an inverted microscope (no larvae in the buffer area are counted).
4) Calculation of chemotaxis Index (chemostatic Index, c.i.): chemotactic index is a commonly used measure describing the degree of trending activity of nematodes under the influence of different external environmental factors.
C.i. ═ number of nematodes in experimental zone-number of nematodes in control zone)/(number of nematodes in experimental zone + number of nematodes in control zone)
If c.i. epsilon (0,1], indicates that methyl thioacetate has an attractive effect on larvae of meloidogyne incognita stage J2 compared to the solvent;
if C.I. epsilon < -1,0), indicating that methyl thioacetate has a repellent effect on larvae in the J2 stage of Meloidogyne incognita compared with a solvent;
if c.i. ═ 0, it means that the meloidogyne incognita larvae are in random free motion, the substance has no specific effect on the chemotaxis of the meloidogyne incognita.
The chemotaxis index was calculated for the number of nematodes per treatment and the number of control nematodes using the above formula to determine the activity trend of meloidogyne incognita in methyl thioacetate at different concentrations, and the experiment was repeated 3 times with the results shown in table 5.
TABLE 5 Effect of the volatile substance methyl thioacetate on nematode chemotaxis
The results show that methyl thioacetate has the evasion activity to the root-knot nematode under different concentrations.
Claims (1)
1. Bacillus aryabhattai (B.aryabhattai)Bacillus aryabhattai) The application of the Bacillus aryabhattai is in preparing the medicine for treating or preventing plant meloidogyne, and the Bacillus aryabhattai is Bacillus aryabhattai MCCC1K 02966.
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