CN110951656A - A strain of Exiguobacterium acetyl and its application in controlling plant parasitic nematodes - Google Patents
A strain of Exiguobacterium acetyl and its application in controlling plant parasitic nematodes Download PDFInfo
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Abstract
The invention discloses a micro-bacillus acetobacter and application thereof in preventing and treating root knot nematode disease, wherein the micro-bacillus acetobacter AMCC101217 has a preservation number of CGMCC No. 16305; the strain can effectively prevent and treat tomato root knot nematode disease, and the number of oocysts, the number of root knots and the density of insect population of the strain are respectively reduced by 84.69%, 65.86% and 73.24% compared with blank control; the strain also effectively reduces the harm of the root knot nematode disease to the tomatoes, and compared with a blank control, the difference between the dry and fresh weight of the overground part and the dry and fresh weight of the underground part is extremely obvious.
Description
Technical Field
The invention relates to a micro-bacillus acetobacter and application thereof in preventing and treating plant parasitic nematode diseases, belonging to the technical field of microorganisms.
Background
Plant parasitic nematodes are common soil-borne diseases, are one of the important factors for restricting the development of agriculture in the world at present, and are statistically lost to the worldwide annual dollar 1570 million due to the plant parasitic nematodes. Plant parasitic nematode disease is also called plant cancer because of its "easy infection, difficult control and great harm".
The chemical pesticide for preventing and controlling plant parasitic nematodes is called a nematicide, which is divided into two types of fumigants and non-fumigants, and has the characteristics of economy and quick effect in the past decades, so that the chemical nematicide is deeply welcomed by farmers. However, with the enhancement of environmental awareness, the national attention is paid to green agriculture and healthy agriculture, and the application of chemical nematocides is gradually limited due to the disadvantages of high residue, high toxicity, high risk and the like.
With the attention and research on root-knot nematode diseases, some chemical control substitution methods are continuously discovered, and biological control is one of the chemical control substitution methods, so that the chemical control substitution methods become a hot point for the research on plant parasitic nematode control. Biological control of plant parasitic nematodes refers to the use of natural enemies of root-knot nematodes or their metabolites to kill or inhibit nematodes, thereby reducing the occurrence of disease. Biological control is concerned in research and industry because of the characteristics of environmental protection, obvious effect, no residue, safety to people and livestock in the using process and the like. At present, although researches on biocontrol bacteria are more and more, the registered and actually applied biocontrol bacteria account for a small proportion in agricultural production, and the strains are single, wherein the important reason is that the biocontrol bacteria have unstable field control effect, and the development of biocontrol is greatly limited.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a strain of tiny acetobacter which can be applied to biological control of plant parasitic nematode diseases.
A strain of Microbacterium acetylicum (Exiguobacterium acetylicum) AMCC101217, called AMCC101217 strain for short, is separated from rhizosphere soil, is preserved in China general microbiological culture Collection center (CGMCC for short) in 2018, 08 and 16 months, and has the address: the No. 3 Xilu Beijing, Chaoyang, Beijing area has a preservation number of CGMCC No. 16305.
The main biological characteristics of the AMCC101217 strain are: the colony shape is irregular circle, and the edge is irregular, and is opaque, and the colony color is orange yellow, and deepens along with the culture time extension, and gram staining microscopy is positive, and cell culture is the shaft-like earlier stage, and the later stage is oval-shaped.
The strain, namely the Microbacterium acetylicum (Exiguobacterium acetylicum) AMCC101217, is separated from rhizosphere soil and is called AMCC101217 for short.
The preparation method of the AMCC101217 microbial agent comprises the following steps:
(1) activating strains: inoculating Acetobacter aceti (Exiguobacterium aceticum) AMCC101217 on LB solid medium plate, and culturing at 35-37 deg.C for 22-27 h;
(2) preparing a seed solution: inoculating the activated AMCC101217 into an LB liquid culture medium, and culturing at 35-37 ℃ and 220rpm for 18-22 h;
(3) seed liquid amplification culture: inoculating the seed liquid in the step (2) into a seed liquid amplification culture medium in an inoculation amount of 5-8%, wherein the amplification culture medium is an LB liquid culture medium, and culturing at 35-37 ℃ and 150-220rpm for 10-13 h;
(4) fermentation culture: inoculating the seed solution which is subjected to the expanded culture in the step (3) to an LANDY fermentation culture medium in an inoculation amount of 5-8%, and culturing at 35-37 ℃ and 220rpm for 22-27h to obtain an AMCC101217 liquid microbial inoculum;
the LANDY fermentation medium comprises the following components per liter: 18.0-20.0g of glucose, 4.0-5.0g of L-glutamic acid, 0.8-1.0g of monopotassium phosphate, 0.4-0.5g of magnesium sulfate heptahydrate, 0.4-0.5g of potassium chloride, 1.0-1.2g of yeast powder, and L-phenylalanine (1.8-2.4) multiplied by 10-3g, manganese sulfate (4.5-5). times.10-3g, copper sulfate (0.16-0).18)×10-3g, ferrous sulfate heptahydrate (0.15-0.17). times.10-3g, water in balance, pH 6.8-7.3.
Preferably, the LB solid medium plate after inoculation in step (1) is cultured at 37 ℃ for 24 h.
Preferably, the LB liquid culture medium after the inoculation in step (2) is cultured at 37 ℃ and 180rpm for 18 h.
According to the invention, the seed liquid after inoculation in the step (3) is expanded in culture medium and cultured for 10h at 37 ℃ and 200 rpm.
According to the invention, the fermentation culture in the step (4) is carried out at 37 ℃ and 200rpm for 24 h.
Preferably, the LANDY fermentation medium in step (4) comprises the following components per liter: 20.0g of glucose, 5.0g of L-glutamic acid, 1.0g of monopotassium phosphate, 0.5g of magnesium sulfate heptahydrate, 0.5g of potassium chloride, 1.0g of yeast powder and 2 x 10 of L-phenylalanine-3g, manganese sulfate 5X 10-3g, copper sulfate 0.16X 10-3g, ferrous sulfate heptahydrate 0.15X 10-3g, balance water, pH 7.0.
A liquid microbial agent contains AMCC101217 strain, and the concentration of the strain is 1.0 x 107-2.5×108CFU/mL。
The application of the micro acetobacter AMCC101217 strain in preventing and treating diseases caused by one or more nematodes.
Preferably according to the invention, the nematodes are: one or a combination of more than two of Meloidogyne incognita, Meloidogyne enterolobii and Bursaphelenchus xylophilus.
The application of the micro acetobacter AMCC101217 microbial inoculum in preventing and treating diseases caused by one or more nematodes.
Preferably according to the invention, the nematodes are: one or a combination of more than two of Meloidogyne incognita, Meloidogyne enterolobii and Bursaphelenchus xylophilus.
The AMCC101217 strain has an obvious killing effect on various plant parasitic nematodes, the fatality rate of the fermentation supernatant for 12 hours to second instar larvae of the meloidogyne incognita is 86.61%, and the incubation inhibition rate of the strain to meloidogyne incognita eggs can reach 92.56%; the mortality rate of the two-instar larvae of the enterolobium cyclocarpum meloidogyne is 81.36%, and the hatching inhibition rate of the enterolobium cyclocarpum meloidogyne eggs can reach 81.36%; the mortality rate to pine wood nematode larvae was 90.21%.
The invention has the advantages of
1. The AMCC101217 strain has an obvious killing effect on various plant parasitic nematodes, the fatality rate of the fermentation supernatant for 12 hours to second-instar larvae of the meloidogyne incognita is 86.61%, and the incubation inhibition rate to meloidogyne incognita eggs can reach 92.56%; the mortality rate of the two-instar larvae of the enterolobium cyclocarpum meloidogyne is 81.36%, and the hatching inhibition rate of the enterolobium cyclocarpum meloidogyne eggs can reach 81.36%; the mortality rate to pine wood nematode larvae was 90.21%.
2. In the potting experiment of the AMCC101217 strain, the strain can obviously inhibit root-knot nematodes, and the number of oocysts, the number of root knots and the population density of the strain are respectively reduced by 84.69%, 65.86% and 73.24% compared with those of a blank control.
Drawings
FIG. 1: colony morphology and microscopic morphology photographs of AMCC101217
In the figure, A is the colony morphology of AMCC101217 in LB solid medium, B is the electron microscope morphology of the bacteria cultured for 12h, and C is the electron microscope morphology of the bacteria cultured for 48 h.
FIG. 2: genome-wide map of AMCC101217
In the figure, COG annotations of GC skew, GC content, non-coding RNA and gene are shown in the order from the inside to the outside.
Detailed Description
The invention is further illustrated by the following specific examples, without limiting the scope of protection thereto.
The experimental methods in the following examples are all conventional methods unless otherwise specified; the test materials used in the following examples, unless otherwise specified, were all purchased from conventional biochemicals; the quantitative tests in the following examples, all set up three replicates and the results averaged.
Microbacterium acetobacter (Aliguobacterium acetylicum) AMCC10077 is purchased from Shandong agricultural microbial strain resource preservation and utilization center.
Example 1
The separation, identification and preservation of the AMCC101217 strain comprise the following specific steps:
(1) separation and screening process of AMCC101217 strain
Screening a nematicidal strain from plant rhizosphere soil, wherein the number of the nematicidal strain is AMCC 101217.
(2) Identification of the strain AMCC101217
① AMCC101217 strain has the main biological characteristics of irregular round colony shape, irregular edge, opacity, orange colony color, deepening with the culture time, positive gram staining microscopic examination, rod-shaped cell culture in the early stage and elliptic cell culture in the later stage.
② AMCC101217 Whole genome sequencing
Inoculating activated AMCC101217 preserved on the inclined plane into a LANDY liquid culture medium, performing shake culture at 200rpm and 37 ℃ for 16h, taking 10mL of bacterial liquid 12000rpm, centrifuging at 4 ℃ for 5min, and removing supernatant to obtain a large amount of AMCC101217 thalli; freezing the thallus in liquid nitrogen for 10min, and extracting bacterial genome; the whole genome sequence was determined using the PacBio RS II method. The results showed that the AMCC101217 genome was 3283403bp in size, 47.21% in GC content, and contained a 125000bp sized plasmid.
The LANDY culture medium comprises the following components per liter: 20.0g of glucose, 5.0g of L-glutamic acid, 1.0g of monopotassium phosphate, 0.5g of magnesium sulfate heptahydrate, 0.5g of potassium chloride, 1.0g of yeast powder and 2 x 10 of L-phenylalanine-3g, manganese sulfate 5X 10-3g, copper sulfate 0.16X 10-3g, ferrous sulfate heptahydrate 0.15X 10-3g, balance water, pH 7.0.
By combining the main biological characteristics of the AMCC101217 strain and the identification result of the genome sequence, the strain is determined to be the acetobacter xylinum (Exiguobacterium acetylicum) which is preserved in China general microbiological culture Collection center (CGMCC for short) in 2018, 08-month and 16-month, and the address is as follows: the No. 3 Xilu Beijing, Chaoyang, Beijing area has a preservation number of CGMCC No. 16305.
Example 2
Determination of nematicidal capacity of AMCC101217 strain
Selecting an AMCC101217 strain stored on an inclined plane by using an inoculating loop, and marking the AMCC101217 strain on an LB flat plate for activation; inoculating the activated strain into LANDY culture medium, shake culturing at 37 deg.C and 200rpm for 48 hr, centrifuging at 12000rpm for 10min, and collecting supernatant.
Experiments on meloidogyne incognita/meloidogyne enterolobii:
cleaning picked meloidogyne incognita/enterolobium rhizosphere oocysts, sterilizing in 0.5% sodium hypochlorite solution for 3min, repeatedly washing with sterile water for 3 times, picking oocysts, incubating in a Beeman funnel, and collecting second instar larvae every 2 d; adopting a 24-pore plate liquid immersion method to measure the activity of the nematode; respectively adding 100 mu L of meloidogyne incognita/meloidogyne enterolobii two-age larva suspension (about 200 pieces), 100 mu L of fermentation supernatant and 300 mu L of sterile water into a 24-well plate, gently blowing and beating the mixture by using a pipette, uniformly mixing the mixture, repeating each sample for 3 times, replacing fermentation liquor with blank culture medium as negative control, and standing the mixture at 28 ℃ for 12 hours to count the mortality of the nematodes.
Egg hatching inhibition experiment: cleaning the picked meloidogyne incognita/enterolobium rhizosphere oocysts, sterilizing in 0.5% sodium hypochlorite solution for 3min, repeatedly washing with sterile water for 3 times, and collecting eggs by grinding with forceps; carrying out egg hatching inhibition determination by adopting a 24-pore plate liquid immersion method; respectively adding 100 mu L of egg suspension (about 200 grains), 100 mu L of fermentation supernatant and 300 mu L of sterile water into a 24-well plate, gently blowing and uniformly mixing by using a pipette, repeating each sample for 3 times, replacing fermentation liquor with a blank culture medium as a negative control, and standing at 28 ℃ for 12h for counting the hatchability.
Pine wood nematode killing experiment: inoculating pine wood nematode with surface disinfection on a PDA plate full of botrytis cinerea, culturing at 28 ℃ for 10 days, washing the pine wood nematode with sterile water, and adjusting the concentration to 2000 strips/mL. And (3) adopting a 24-pore plate liquid immersion method to measure the nematode resistance activity. Respectively adding 100 mu L of pine wood nematode second-instar larva suspension (about 200 pieces), 100 mu L of fermentation supernatant and 300 mu L of sterile water into a 24-well plate, gently and uniformly blowing and beating by using a pipette, repeating each sample for 3 times, replacing fermentation liquor with a blank culture medium as a negative control, and standing at 28 ℃ for 12 hours to count the nematode death rate.
Corrected mortality (%) - (treatment mortality-control mortality)/(1-control mortality) × 100
Inhibition of egg hatching (%) - (control hatching rate-treatment hatching rate)/(1-treatment hatching rate) × 100
The results show that: the strain has an obvious killing effect on various plant parasitic nematodes, the fatality rate of the fermented supernatant for 12 hours to second-instar larvae of the meloidogyne incognita is 86.61%, and the incubation inhibition rate of the strain to meloidogyne incognita can reach 92.56%; the mortality rate of the two-instar larvae of the enterolobium cyclocarpum meloidogyne is 81.36%, and the hatching inhibition rate of the enterolobium cyclocarpum meloidogyne eggs can reach 81.36%; the mortality rate to pine wood nematode larvae was 90.21%.
Example 3
Preparation of AMCC101217 microbial inoculum
(1) Activating strains: a. acetylicum (Exiguobacterium acetylicum) AMCC101217 was streaked on LB solid medium plate and cultured at 37 ℃ for 24 hours.
(2) Preparing a seed solution: the activated AMCC101217 is inoculated into a triangular flask filled with LB liquid culture medium, and is subjected to shaking culture at the temperature of 37 ℃ and the speed of 180rpm for 18h in a constant temperature shaker.
(3) Seed liquid amplification culture: inoculating the seed liquid in the step (2) into a seed tank liquid culture medium with the inoculation amount of 7%, wherein the expanding culture medium is LB liquid culture medium, and culturing at 37 ℃ and 200rpm for 10 h.
(4) Fermentation culture: inoculating the seed solution which is expanded and cultured in the step (3) to a LANDY fermentation culture medium in an inoculation amount of 7%, and culturing at 37 ℃ for 24h to obtain an AMCC101217 liquid microbial inoculum with viable count of 1.5 multiplied by 108CFU/mL was filled into 1L plastic bottles with caps.
The LANDY culture medium comprises the following components per liter: 20.0g of glucose, 5.0g of L-glutamic acid, 1.0g of monopotassium phosphate, 0.5g of magnesium sulfate heptahydrate, 0.5g of potassium chloride, 1.0g of yeast powder and 2 x 10 of L-phenylalanine-3g, manganese sulfate 5X 10-3g, copper sulfate 0.16X 10-3g, ferrous sulfate heptahydrate 0.15X 10-3g, balance water, pH 7.0.
Comparative example
The difference from example 2 is that in example, the strain AMCC101217 was replaced by Microbacterium acetylicum (Exiguobacterium acetylicum) AMCC 10077.
The result shows that the acetobacter aceti (Exiguobacterium aceticum) AMCC10077 has no killing effect on three nematodes and has no inhibition effect on egg hatching.
Examples of effects
AMCC101217 bacterial agent pot culture experiment
(1) CK: 80mL LANDY medium;
(2) t1: 80mL of 2000-fold diluent of commercial abamectin;
(3) t2: 80mL of AMCC101217 (prepared in example 3) liquid inoculum.
Soil used for pot culture contains root-knot nematodes, the density of insect population is adjusted to be 15 per gram, the soil is collected from a greenhouse of diseased tomatoes, the tomato variety is a nematode-susceptible variety, ginkgo is strong and abundant, the size of the flowerpot is 10cm multiplied by 10cm, and the amount of the soil added is 800g per pot; the microbial inoculum, the abamectin and the LANDY culture medium are all used in a mode of irrigating roots in advance, 6 treatment steps are performed in parallel, strong tomato seedlings with four true leaves are transplanted after 3 days of treatment, and the tomato seedlings are cultured in a light culture box under the conditions of light irradiation, the light intensity is 66%, the culture time is 12 hours, and the culture time is 28 ℃; dark 12h, 24 ℃. And (3) normally watering, not applying other agents, cleaning the soil at the root of the plant after 30 days, reserving a sample of the separated soil, detecting, and counting indexes such as root knot number, oocyst number and insect population density.
The data is collated by using Excel software, data analysis is carried out by using SPSS 17.0, and statistical analysis is carried out on test data between a sample and a control group by using Duncan's test in one-way variance analysis.
The pot experiment results are shown in table 1: the AMCC101217 microbial inoculum can effectively prevent and treat the tomato root knot nematode disease, and the number of oocysts, the number of root knots and the density of insect population of the tomato root knot nematode disease are respectively reduced by 84.69%, 65.86% and 73.24% compared with a blank control; the strain also effectively reduces the harm of the root knot nematode disease to the tomatoes, and compared with a blank control, the difference between the dry and fresh weight of the overground part and the dry and fresh weight of the underground part is extremely obvious.
TABLE 1
Note: NG, root knot number; NEM, oocyst number; JD, population density; SFW, fresh weight of aerial parts; SDW, above ground dry weight; RFW, fresh weight underground; RDW, underground dry weight.
Claims (10)
1. A strain of Microbacterium acetylicum (Exiguobacterium acetylicum) AMCC101217 is preserved in China general microbiological culture Collection center at 16 th 08 th month in 2018, and the addresses are as follows: no. 3 Xilu No.1 Beijing, Chaoyang, and the preservation number of the strain is CGMCC No. 16305.
2. The method for preparing the microbial agent of the microbacterium acetylicum AMCC101217 as claimed in claim 1, comprising the following steps:
(1) activating strains: inoculating Acetobacter aceti (Exiguobacterium aceticum) AMCC101217 on LB solid medium plate, and culturing at 35-37 deg.C for 22-27 h;
(2) preparing a seed solution: inoculating the activated AMCC101217 into an LB liquid culture medium, and culturing at 35-37 ℃ and 220rpm for 18-22 h;
(3) seed liquid amplification culture: inoculating the seed liquid in the step (2) into a seed liquid amplification culture medium in an inoculation amount of 5-8%, wherein the amplification culture medium is an LB liquid culture medium, and culturing at 35-37 ℃ and 150-220rpm for 10-13 h;
(4) fermentation culture: inoculating the seed solution which is subjected to the expanded culture in the step (3) to an LANDY fermentation culture medium in an inoculation amount of 5-8%, and culturing at 35-37 ℃ and 220rpm for 22-27h to obtain an AMCC101217 liquid microbial inoculum;
the LANDY fermentation medium comprises the following components per liter: 18.0-20.0g of glucose, 4.0-5.0g of L-glutamic acid, 0.8-1.0g of monopotassium phosphate, 0.4-0.5g of magnesium sulfate heptahydrate, 0.4-0.5g of potassium chloride, 1.0-1.2g of yeast powder, and L-phenylalanine (1.8-2.4) multiplied by 10-3g, sulfuric acidManganese (4.5-5) x 10-3g, copper sulfate (0.16-0.18). times.10-3g, ferrous sulfate heptahydrate (0.15-0.17). times.10-3g, water in balance, pH 6.8-7.3.
3. The method for preparing the microbial agent of the microbacterium acetylicum AMCC101217 as claimed in claim 2, wherein the LB solid medium plate after inoculation in step (1) is cultured at 37 ℃ for 24 hours.
4. The method for preparing the microbial inoculum of the microbacterium acetylicum AMCC101217 of claim 2, wherein the LB liquid culture after the inoculation in the step (2) is performed at 37 ℃ and 180rpm for 18 hours.
5. The method for preparing the microbial agent of Microbacterium acetylicum AMCC101217 as claimed in claim 2, wherein the seed liquid after inoculation in step (3) is expanded in culture medium and cultured at 37 ℃ and 200rpm for 10 h.
6. The method for preparing the microbial agent of Microbacterium acetobacter AMCC101217 as claimed in claim 2, wherein the fermentation culture in step (4) is performed at 37 ℃ and 200rpm for 24 h.
7. The method for preparing the microbial agent of the micro acetobacter AMCC101217 in the claim 2, wherein the culture medium for fermentation in the step (4) contains the following components per liter: 20.0g of glucose, 5.0g of L-glutamic acid, 1.0g of monopotassium phosphate, 0.5g of magnesium sulfate heptahydrate, 0.5g of potassium chloride, 1.0g of yeast powder and 2 x 10 of L-phenylalanine-3g, manganese sulfate 5X 10-3g, copper sulfate 0.16X 10-3g, ferrous sulfate heptahydrate 0.15X 10-3g, balance water, pH 7.0.
8. A liquid microbial agent comprising the Acetobacter aceti AMCC101217 strain of claim 1, wherein the cell concentration is 1.0X 107-2.5×108CFU/mL。
9. Use of the strain of microminia acetobacter AMCC101217 according to claim 1 for controlling diseases caused by one or more nematodes;
preferably, the nematodes are: one or a combination of more than two of Meloidogyne incognita, Meloidogyne enterolobii and Bursaphelenchus xylophilus.
10. The use of the A.acetylicus AMCC101217 bacterial agent of claim 8 for controlling diseases caused by one or more nematodes;
preferably, the nematodes are: one or a combination of more than two of Meloidogyne incognita, Meloidogyne enterolobii and Bursaphelenchus xylophilus.
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