CN108102992B - Microbacterium aurantiacus and application thereof in prevention and treatment of tomato root-knot nematodes - Google Patents

Abstract

The invention discloses a Microbacterium aurantiacus and application thereof in preventing and treating tomato root-knot nematodes. The strain is a Microbacterium aurantiacum (Exiguobacterium aurantiacaum) strain 8220605, is separated from lake mud of the south Sihu of Jining City in Shandong province, is preserved in the China general microbiological culture Collection center in 2017, 9-26 months, and has the preservation number of CGMCC No. 14676. The test result shows that: the microbial prevention and control preparation of the Microbacterium aurantiacus strain 8220605 has remarkable effect of preventing and controlling tomato root-knot nematodes, the prevention and control effect reaches 74.25%, and tomatoes have good growth vigor and no phytotoxicity.

Description

Microbacterium aurantiacus and application thereof in prevention and treatment of tomato root-knot nematodes

Technical Field

The present invention belongs to the field of biological plant disease preventing and controlling technology. Specifically, the invention relates to a Microbacterium aurantiacus and application thereof in prevention and treatment of tomato root-knot nematodes, and a biological prevention and treatment preparation prepared from the Microbacterium aurantiacus.

Background

The root-knot nematode damage is one of the main damages in the production process of tomato crops, and can cause the yield of tomatoes to be reduced by more than 30 percent, even the tomatoes are not harvested completely. In recent years, the planting area of vegetables in protected areas such as solar greenhouses and the like in China is continuously enlarged, the temperature and the humidity of the solar greenhouses are proper, and the root-knot nematodes are aggravated year by year due to continuous cropping all year round. To date, more than 80 species of root-knot nematodes have been reported, of which the southern root-knot nematode (melodogyne incognita), root-knot nematode javanica (m.javanica), root-knot nematode northern (m.hapla) and root-knot nematode arachidis (m.arenaria) are the most important species of plant-parasitic nematodes due to their wide distribution and multi-parasitism. In China, the four root-knot nematodes occur in most provinces (districts), and infect more than one hundred species of crops, and almost every kind of crops such as field crops, vegetables, fruit trees, flowers and medicinal plants can be infected. Vegetable root knot nematode disease is one of the most serious plant diseases at home and abroad. Plant diseases caused by the invasion and parasitism of plant parasitic nematodes, and normal growth and development of damaged plants can be influenced by the invasion of nematodes to absorb nutrition in vivo; the secretion in the metabolic process of the nematode can also stimulate cells and tissues of host plants, so that the plants are deformed, the yield of agricultural products is reduced, and the quality of the agricultural products is reduced; in addition, other diseases can be aggravated by the harm of nematode, particularly for vegetables in a protected area, the yield of hosts is reduced by 15-5% all the year round, sometimes, the yield is more than 70%, production and export foreign exchange of crops such as greenhouse vegetables, fruit trees, flowers and the like are severely restricted, and some of the plants even reach the land where the plants cannot be planted. It is estimated that the economic loss of crops in China caused by the damage of root-knot nematodes reaches more than one hundred million yuan each year.

Because the root-knot nematode disease belongs to soil-borne diseases, the prevention and the treatment are difficult. At present, people mainly adopt organic phosphorus chemical nematocides, select disease-resistant crop varieties, implement crop rotation, establish disease-free seedling bases and carry out strict quarantine on imported and exported crops to control the occurrence of root-knot nematode diseases. Although the chemical nematicide has obvious prevention effect, the selectivity is poor, and the ecological environment of crops is damaged; is not easy to degrade, has high residue and is extremely unsafe for people and livestock; the soil structure is damaged, and the soil is desertified; the problem of drug resistance is increasingly prominent. These factors have severely limited the scope of use of chemical pesticides. Therefore, non-chemical control measures have become the core of crop root knot nematode disease control, and biological control is the most promising method.

The microbacterium (Exiguobacterium spp.) has wide distribution range, various living environments and various unique properties including cold resistance, thermophilic property, alkalophilicity and halophilicity, and has the functions of decomposing complex organic pollutants, converting heavy metals, promoting growth and the like with high practical value. In addition, the genus microbacterium contains a variety of heat-resistant, cold-resistant and alkali-resistant proteins and active enzymes, and can be applied to a variety of fields such as biotechnology, bioremediation, environmental monitoring, industry, agricultural production and the like, and further exploration and utilization of these abundant and precious biological resources are urgently needed.

Disclosure of Invention

The invention aims to provide a novel microorganism, namely Microbacterium aurantiacum 8220605, for efficiently preventing and treating tomato root-knot nematodes. The micro-bacterium aurantiacus strain has the characteristics of high growth speed, strong stress resistance, capability of quickly and massively colonizing on the surface of a plant (especially a tomato) and the like, and has a good application prospect.

The above purpose of the invention is realized by the following technical scheme: the strain provided by the invention is a Microbacterium aurantiacum (Exiguobacterium aurantiacaum) strain 8220605 which is separated from lake mud of the four lakes of south China lake of Jining City in Shandong, and is preserved in the general microbiological center of China Committee for culture Collection of microorganisms (CGMCC for short, the address: No. 3 of West Siro 1 of the sunward area in Beijing, institute of microbiology in China institute of academy of sciences) in 26 months of 2017, and the preservation number is CGMCC No. 14676. It has the following biological properties: on a beef extract peptone agar culture medium or an LB (Luria-Bertani) culture medium, the strain is a punctate microcolony, is round and flat, has a smooth surface, and is neat in edge, golden orange and non-transparent; the thalli at the early stage is in a slender and irregular rod shape, the rod shape at the later stage is shortened, the thalli are in a spherical shape, and no obvious rod-to-ball conversion period exists; no spore formation. After culturing for two days at 28 ℃ on a beef extract peptone agar medium, the somatic cells are rod-shaped and have no motility by microscopic examination. Gram-positive (E.coli control).

The culture method or propagation method of the Microbacterium aurantiacaum (E.aurantiacaum) strain 8220605 comprises the following steps:

(1) an LB culture medium is adopted for ordinary culture and preservation, and the formula is as follows: 10g of tryptone, 5g of yeast extract, 10g of sodium chloride, 15-20 g of agar and 1000mL of distilled water, and the pH is adjusted to 7.1-7.3.

(2) The laboratory liquid culture adopts an LB liquid culture medium, and the formula is as follows: 10g of tryptone, 5g of yeast extract, 10g of sodium chloride and 1000mL of distilled water, and adjusting the pH value to 7.1-7.3.

(3) The solid culture medium formula comprises: comprises a solid material and inorganic salt, wherein the mass ratio of the solid material to the inorganic salt is 98.5: 0.5; the solid materials are rice hulls, corn flour, soybean meal and bran in a mass ratio of 55: 25: 12: 8; the inorganic salt comprises the following components in percentage by mass: 25% of monopotassium phosphate, 5% of magnesium sulfate, 5% of ammonium sulfate and 65% of light calcium carbonate.

(4) The formula of the mass fermentation culture comprises: the formula of the solid culture medium in the step (3).

The invention also provides a biological control preparation for controlling the tomato root-knot nematode, which comprises the Microbacterium aurantiacaum (E.aurantiacaum) strain 8220605.

Also, the present invention provides a biocontrol method for controlling tomato root-knot nematodes, comprising applying the above-mentioned Microbacterium aurantiacaum (E.aurantiacaum) strain 8220605 or the above-mentioned biocontrol agent to tomato plants having root-knot nematodes.

The invention also provides application of the micro-bacterium aurantiacus strain or the biological control preparation in controlling tomato root-knot nematodes.

The preparation method of the biological control preparation comprises the following steps:

(1) preparing a seed solution of the micro-organism aurantiamarus strain 8220605;

(2) inoculating the seed liquid prepared in the step (1) into a solid culture medium, and culturing at a constant temperature of 25-27 ℃;

(3) and (3) adding sterile water into the culture cultured in the step (2), mixing, filtering, inoculating the filtrate into a large amount of fermentation medium, and performing fermentation culture in a fermentation chamber with the room temperature of 25-27 ℃ and the relative humidity of more than 85%.

In one embodiment of the present invention, the preparation method comprises the steps of:

(1) transplanting spores of the Microbacterium aurantiacus strain 8220605 into an LB liquid culture medium, and performing shake culture on a shaker at 25-27 ℃ for 3-5 days to obtain a seed solution;

(2) inoculating the seed solution prepared in the step (1) into a solid culture medium according to the mass ratio of 10%, and performing shake culture at 25-27 ℃ for 3-5 days;

(3) mixing the culture cultured in the step (2) with sterile water according to the mass ratio of 1: 15, filtering with sterile gauze, inoculating the filtrate to a large amount of fermentation medium according to the volume ratio of 1: 6, and performing fermentation culture in a fermentation chamber at the room temperature of 25-27 ℃ and the relative humidity of more than 85% for 8-9 days.

Wherein the LB liquid culture medium in the step (1) has the formula: 10g of tryptone, 5g of yeast extract, 10g of sodium chloride and 1000mL of distilled water, and adjusting the pH value to 7.1-7.3.

The solid culture medium in the step (2) comprises a solid material and inorganic salt, wherein the mass ratio of the solid material to the inorganic salt is 98.5: 0.5; the solid materials are rice hulls, corn flour, soybean meal and bran in a mass ratio of 55: 25: 12: 8; the inorganic salt comprises the following components in percentage by mass: 25% of monopotassium phosphate, 5% of magnesium sulfate, 5% of ammonium sulfate and 65% of light calcium carbonate.

The mass fermentation medium in the step (3) is synchronous with the solid medium in the step (2).

Experiments show that the Microbacterium aurantiacus strain has the characteristics of high growth speed, strong stress resistance, capability of fast and massively colonizing on the surface of a plant and the like, and therefore, the Microbacterium aurantiacus strain has a good application prospect. The biological control preparation prepared from the Microbacterium aurantiacus not only can efficiently control tomato root-knot line insect pests, but also can effectively promote tomato growth, and is a biological control preparation with a great application prospect. The microbial preparation can be used as a biological pesticide or a biological fertilizer for preventing and treating tomato root-knot nematodes. The test result shows that: the solid fermentation culture of the Microbacterium aurantiacum (E.aurantiacaum) strain 8220605 has remarkable effect of preventing and treating tomato root-knot nematodes, the prevention and treatment effect reaches 74.25%, and the tomato has good growth vigor and no phytotoxicity.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications are within the scope of the invention.

Example 1

1. Isolation and purification of Microbacterium aurantiacaum (E.aurantiacaum) 8220605

The invention relates to a Microbacterium aurantium (E.aurantiacam) 8220605 which is obtained by separating from lake mud by a dilution plate method and a plate marking method, wherein the separation method comprises the following steps:

(1) isolation of Microbacterium

Samples of lake mud were collected from south four lakes of Jining, Shandong province on days 4, 15, 2014. Weighing 1g of lake mud in 100mL of sterile water, placing in a shaking table at 30 ℃ and shaking at 150rpm for 10min, then placing in a water bath kettle at 60 ℃ and incubating for 30min, and taking 100 mu L of 10 ^-2、10 ^-3、10 ^-4And coating the diluent on an LB medium plate, coating three layers of the diluent in parallel in each gradient, culturing at 30 ℃ for 2d, picking microbial colonies with different forms on the LB medium, streaking the microbial colonies on the LB medium plate, and observing the growth condition of the colonies regularly. And then, purifying the micro bacillus strain by adopting a plate marking method, numbering and storing.

(2) Screening of high-efficiency antagonistic microbacterium for tomato root-knot nematode

① preliminary screening

A. Preparation of root-knot nematode second-instar larva suspension

Selecting fresh egg blocks from the roots of the tomatoes, placing the fresh egg blocks in a self-made hatching device for hatching, collecting larvae 24 hours later, collecting the larvae once a day, and collecting the larvae for a week. Diluting the nematode solution to 100 per mL, and storing at 4 ℃ for later use.

B. Action of Microbacterium aurantiacus on second-instar larvae of Meloidogyne incognita

Sucking 2mL of the bacterial suspension, adding into a sterilized culture dish with the diameter of 7.5cm, adding 2mL of the second-instar larva suspension, using clear water as a control, repeating each treatment for 3 times, and culturing at 25 ℃. The activity of the nematodes was observed after 12h, 24h, 36h, 48h and 60h, respectively.

C. Function of Microbacterium aurantiaca on hatching of meloidogyne incognita egg mass

Sucking 2mL of the bacterial suspension, adding into a sterilized culture dish with the diameter of 7.5cm, adding 10 egg blocks, using clear water as a control, repeating each treatment for 3 times, and culturing at 25 ℃. The hatching of the larvae was observed after 24h, 48h and 60h, respectively.

② rescreening

The screened microbacterium strain with high-efficiency antagonistic activity is subjected to secondary screening, the microbacterium strain with better tolerance is screened mainly through temperature resistance, acid and alkali resistance and drug resistance tests, a pot culture control test and a field test are carried out, and the screened strain 8220605 is identified.

The inventor obtains a Microbacterium aurantium (E.aurantiacaum) 8220605 capable of efficiently preventing and treating tomato root knot nematode disease through a large amount of screening work. Experiments prove that the micro-organism aurantiacus solid fermentation culture shows very high-efficiency control effect in controlling tomato root knot nematode disease, so that tomatoes grow well. Therefore, the micro-bacterium aurantiacus of the invention is a new strain of micro-bacterium aurantiacus with wide application prospect, and can be used for preparing a biological control preparation for preventing and treating tomato root knot nematode disease.

2. Identification of strains

(1) Microbiological characteristics: on a beef extract peptone agar culture medium or an LB (Luria-Bertani) culture medium, the strain is a punctate microcolony, is round and flat, has a smooth surface, and is neat in edge, golden orange and non-transparent; the thalli at the early stage is in a slender and irregular rod shape, the rod shape at the later stage is shortened, the thalli are in a spherical shape, and no obvious rod-to-ball conversion period exists; no spore formation. After culturing for two days at 28 ℃ on a beef extract peptone agar medium, the somatic cells are rod-shaped and have no motility by microscopic examination. Gram-positive (E.coli control).

(2) Molecular biological Properties

The sequence determination result of the 16s rDNA gene of the strain is as follows (SEQ-1):

CCGACTTCGGGTGTTGCAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGACCCGGGAACGTATTCACCGCAGTATGCTGACCTGCGATTACTAGCGATTCCGACTTCATGCAGGCGAGTTGCAGCCTGCAATCCGAACTGAGAACGGCTTTCTGGGATTGGCTCCACCTCGCGGCTTCGCTGCCCTTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAACTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCCCTAGAGTGCCCAACCAAATGCTGGCAACTAAGGACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACCCCTGCCCCCGAAGGGGAAGGTACATCTCTGTACCGGTCAGGGGGATGTCAAGAGTTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGTCCCCGTCAATTCCTTTGAGTTTCAGCCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTTCAGCACTGAAGGGCGGAAACCCTCCAACACCTAGCACTCATCGTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCGCCTCAGCGTCAGTTATAGGCCAGAGAGTCGCTTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTACACTCTTCTCTCCTATACTCAAGCCTCCCAGTTTCCAATGGCCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAGGCCGCCTGCGCGCGCTTTACGCCCAATAATTCCGGACAACGCTTGCCACATACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTCGCAAGGTACCGTCAAGGTGCCGCCATCGCCTGCGGCACTTGTTCTTCCCTTACAACAGAACTTTACGACCCGAAAGCTTTCATCGTTCACGGGGCGTTGCTCCATCAGACTTTCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTATGCATTGTCGCCTTGGTGGGCCGTTACCCCACCAACTAGCTAATGCACCGCAAAGCCATCCATGGGCGACGCCGGAGCGCCTTTCATCAGCGGACCATGCGGTCCGATGACACATCCGGTATTAGCCCCGATTTCTCGTGGTTACCCCAGACCTATGGGCAGGTTCTTTACGTGTTACTCACCCGTCCGCCGCTCATTCCGCCGTCGTCCCCCCGAAGGGTTCAGACTGGTTCCTGCGCTCGACTGCATGTAA。

(3) results of cell morphology and physicochemical experiments

TABLE 1 physiological and biochemical experimental results of Microbacterium aurantiacus (E.aurantiacam) 8220605

The strain 8220605 is identified as Microbacterium aurantium (Exiguobacterium aurantium), and has been deposited in China general microbiological culture Collection center in 2017 at 9 and 26 months with the preservation number of CGMCCNo.14676.

Example 2

1. Fermentation process of Microbacterium aurantiacus strain

The LB liquid culture medium formula is: 10g of tryptone, 5g of yeast extract, 10g of sodium chloride and 1000mL of distilled water, and adjusting the pH value to 7.1-7.3.

The solid culture medium consists of a solid material, inorganic salt and water, wherein the mass ratio of the solid material to the inorganic salt is 98.5: 0.5; the solid materials are rice hulls, corn flour, soybean meal and bran in a mass ratio of 55: 25: 12: 8; the inorganic salt comprises the following components in percentage by mass: 25% of monopotassium phosphate, 5% of magnesium sulfate, 5% of ammonium sulfate and 65% of light calcium carbonate. The preparation method comprises the following steps: adding a proper amount of water (10-15 times of the mass) to dissolve inorganic salt to obtain an inorganic salt solution; mixing inorganic salt solution and solid material, adding water to adjust water content of the solid culture medium to 40-60%, and grasping the solid culture medium in hand with water in finger joint without dripping.

The mass fermentation culture medium is the same as the solid culture medium.

A mass solid fermentation process of a micro-bacterium aurantiacaum (e.aurantiacam) strain 8220605:

① culture of strain seed liquid

A small amount of spores are picked from a test tube inclined plane by a micro-organism aurantiacaum (E.aurantiacam) 8220605, the spores are transferred to an LB liquid culture medium, and shaking culture is carried out on a shaking table at 26 ℃ for 3-5 days, and the culture medium is a seed solution.

② cultivation of solid producer strains

Inoculating the seed solution into a solid culture medium (500mL triangular flask) according to the proportion of 10%, culturing at the constant temperature of 26 ℃ for 3-5 days, and shaking for multiple times in the middle.

③ fermentation of large amount of solid

Diluting a culture obtained by solid culture in ② with sterile water according to a ratio of 1: 15, filtering with sterile gauze, removing coarse residues to obtain a production bacterial liquid, inoculating the production bacterial liquid into a large amount of fermentation medium according to an inoculation ratio of 1: 6, placing the inoculated raw materials into a fermentation chamber (26 ℃ and relative humidity of more than 85%), and fermenting and culturing for 8-9 days, wherein the active bacteria are 8 hundred million/g.

In order to determine the control effect of the Microbacterium aurantiacaum (E.aurantiacaum) strain 8220605 on the tomato root-knot nematode, a basis is provided for the popularization and the application of the tomato root-knot nematode. We tested in 2016 at 10-12 months in Shandong province, chat county, and the results are reported as follows:

1 test materials and methods

1.1 test agent

Microbacterium aurantiacaum (E.aurantiacaum) strain 8220605 solid fermentation culture (8 hundred million/g) produced by the fermentation method of example 2 of the present invention; 1.8 percent of abamectin emulsifiable concentrate (produced by Henan Lu Ye star crop protection Co., Ltd., sold on the market).

1.2 test materials and control objects

The test material is tomato, and the variety is West powder No. 3; the control object is tomato root knot nematode disease (Meloidogynespp.).

1.3 conditions of the test

The experimental plot is arranged in winter-warm greenhouse in Shenzhou village, eighteen miles and Zhengtang village. Due to continuous planting of tomatoes in the area, the tomato root-knot nematode disease is increasingly serious, particularly, the tomato root-knot nematode disease continuously occurs in four seasons under the greenhouse condition, seedling damage events frequently occur, the yield loss reaches 20-40%, and the tomato root-knot nematode disease is serious and even cannot be harvested. The test soil type is moist soil, the organic matter content is 1.21%, the pH value is 7.8, the cultivation conditions and management measures of all test districts are consistent, and the tomato root-knot nematode is in the early stage of disease development when the pesticide is applied.

1.4 test design and arrangement

The experiment has 3 treatments, namely 20 times of solid fermentation culture of Microbacterium aurantium (E.aurantiacaum) strain 8220605, 1000 times of 1.8% abamectin emulsifiable solution and no application of clear water as controls, wherein each treatment is repeated for 4 times, and each treatment is 60 strains. Irrigating roots of the diseased plants and the plants nearby the diseased plants at the early stage of disease for 2 times, and irrigating 250ml of liquid medicine for each diseased plant. The roots were irrigated once each at 2016, 10, 16 and 15 months.

1.5 test investigation and calculation method

1.5.1 weather conditions

The medicine for the 1 st application (10 months and 16 days) is cloudy in the day, the wind power is 3 grades, the highest air temperature is 22 ℃, the lowest air temperature is 13 ℃, and the relative humidity is 70%. The medicine is applied for the 2 nd time (12 months and 15 days), the day is sunny, the wind power is 3 grades, the highest air temperature is 7 ℃, the lowest air temperature is-4 ℃, and the relative humidity is 69%.

1.5.2 drug efficacy and safety investigation

And (3) drug effect investigation: investigation was performed 14d after the last application. Investigating 30 plants, investigating the infection degree of diseased plants and roots, recording the disease progression and calculating the disease index.

Safety investigation: and (4) observing the safety of the tomatoes at 7d and 14d after the first pesticide application, describing the phytotoxicity symptoms in detail and determining the phytotoxicity degree according to phytotoxicity degree grading standards if phytotoxicity occurs.

Grading standard of tomato root-knot nematode:

level 0: the root system has no root knot.

Level 1: mild infection, only a few small root nodules, with a root nodule rate below 3%.

And 2, stage: the root-knot rate is 3-25%.

And 3, level: the root knot is more, and the root knot rate is 25-50%.

4, level: secondary root knots are formed on the root nodules, and 50% -75% of root systems have root knots.

And 5, stage: the root knots are mutually connected into root knot blocks, and over 75 percent of root systems have roots.

1.5.3 method for calculating drug effect

The preventing and treating effect is calculated according to the formulas (1) and (2):

in the formula: CK (CK) ₀-pre-drug condition index in the placebo zone;

CK ₁-disease index after drug administration in the placebo zone;

PT _{0————}pre-drug disease index prior to drug treatment;

PT _{1————}disease index after drug application after drug treatment.

1.5.4 direct effects on tomatoes

Observing whether the pesticide has phytotoxicity on the tomatoes, and recording the type and the degree of the phytotoxicity. In addition, the effect on tomato growth was also recorded.

Phytotoxicity was recorded in the following manner:

(a) if the phytotoxicity can be measured or calculated, it is expressed in absolute terms, for example, as plant height.

(b) In other cases, the extent and frequency of phytotoxicity can be estimated in two ways:

① the phytotoxicity degree of each cell is recorded according to phytotoxicity grading method, and is represented by-, + + + + + + +, +.

The phytotoxicity grading method comprises the following steps:

and (2) preparing: no chemical injury;

+: mild phytotoxicity, no influence on plant growth;

++: moderate phytotoxicity, can be recovered, and does not reduce the biomass of plants;

+++: moderate phytotoxicity affects the normal growth of plants and reduces the biomass of the plants to a certain extent;

++++: serious phytotoxicity, plant growth retardation and serious reduction of plant biomass.

② the percentage of phytotoxicity is evaluated by comparing the chemical treatment area with the blank control area, and the phytotoxicity symptoms (dwarfing, chlorosis, malformation, etc.) of the plants are accurately described.

2 results

2.1 prevention and treatment Effect of the test agent on tomato root-knot nematode

The control effect of the test agent on the tomato root-knot nematode is shown in tables 2-3. The results in table 3 show that the solid fermentation culture of the Microbacterium aurantium (E.aurantiacaum) strain 8220605 and the 1.8% avermectin emulsifiable solution have better control effect on the tomato root-knot nematode disease, wherein the control effect of the former reaches more than 70%.

From the disease index of the field test of the tomato root-knot nematode (table 3), 14 days after the last application, the control effect of the Microbacterium aurantium (E.aurantiacam) 8220605 is 74.25 percent and is obviously higher than that of 1.8 percent of abamectin emulsifiable solution 52.72 percent.

TABLE 2 three Agents before treatment of tomato root knot nematode disease

TABLE 3 control of tomato root-knot nematode by three agent treatments

2.2 tomato safety survey

Through the observation of the application of the pesticide for 7d and 14d, compared with the control area, the tomato growth is normal and no phytotoxicity is generated in each medicament treatment area, which indicates that the micro-bacillus aurantiacus solid fermentation culture is safe to the tomato.

3 small knot

3.1 from the view of disease index and prevention and treatment effect, the Microbacterium aurantiacum (E.aurantiacam) strain 8220605 (with the preservation number of CGMCC No.14676) has better prevention and treatment effect on the tomato root-knot nematode, and the prevention and treatment effect reaches more than 70% in 14 days after the last application.

3.2 from the tomato growth vigor, the Microbacterium aurantiacum (E.aurantiacaum) strain 8220605 has the effect of promoting the growth of tomatoes, has no phytotoxicity, and is safe and reliable.

SEQUENCE LISTING

<110> Shandong Xinhe Feng crop Nutrition Co Ltd

<120> Microbacterium aurantiacus and application thereof in prevention and treatment of tomato root-knot nematodes

<130>0

<160>1

<170>PatentIn version 3.3

<210>1

<211>1407

<212>DNA

<213> 16s rDNA Gene sequence of Microbacterium aurantiacus (Exiguobacterium aurantiacaum) Strain 8220605

<400>1

ccgacttcgg gtgttgcaaa ctctcgtggt gtgacgggcg gtgtgtacaa gacccgggaa 60

cgtattcacc gcagtatgct gacctgcgat tactagcgat tccgacttca tgcaggcgag 120

ttgcagcctg caatccgaac tgagaacggc tttctgggat tggctccacc tcgcggcttc 180

gctgcccttt gtaccgtcca ttgtagcacg tgtgtagccc aactcataag gggcatgatg 240

atttgacgtc atccccacct tcctccggtt tgtcaccggc agtctcccta gagtgcccaa 300

ccaaatgctg gcaactaagg acaagggttg cgctcgttgc gggacttaac ccaacatctc 360

acgacacgag ctgacgacaa ccatgcacca cctgtcaccc ctgcccccga aggggaaggt 420

acatctctgt accggtcagg gggatgtcaa gagttggtaa ggttcttcgc gttgcttcga 480

attaaaccac atgctccacc gcttgtgcgg gtccccgtca attcctttga gtttcagcct 540

tgcgaccgta ctccccaggc ggagtgctta atgcgttagc ttcagcactg aagggcggaa 600

accctccaac acctagcact catcgttacg gcgtggacta ccagggtatc taatcctgtt 660

tgctccccac gctttcgcgc ctcagcgtca gttataggcc agagagtcgc tttcgccact 720

ggtgttcctc cacatctcta cgcatttcac cgctacacgt ggaattacac tcttctctcc 780

tatactcaag cctcccagtt tccaatggcc ctccccggtt gagccggggg ctttcacatc 840

agacttaaga ggccgcctgc gcgcgcttta cgcccaataa ttccggacaa cgcttgccac 900

atacgtatta ccgcggctgc tggcacgtag ttagccgtgg ctttctcgca aggtaccgtc 960

aaggtgccgc catcgcctgc ggcacttgtt cttcccttac aacagaactt tacgacccga 1020

aagctttcat cgttcacggg gcgttgctcc atcagacttt cgtccattgt ggaagattcc 1080

ctactgctgc ctcccgtagg agtctgggcc gtgtctcagt cccagtgtgg ccgatcaccc 1140

tctcaggtcg gctatgcatt gtcgccttgg tgggccgtta ccccaccaac tagctaatgc 1200

accgcaaagc catccatggg cgacgccgga gcgcctttca tcagcggacc atgcggtccg 1260

atgacacatc cggtattagc cccgatttct cgtggttacc ccagacctat gggcaggttc 1320

tttacgtgtt actcacccgt ccgccgctca ttccgccgtc gtccccccga agggttcaga 1380

ctggttcctg cgctcgactg catgtaa 1407

Claims (7)

1. Microbacterium aurantiacum (Exiguobacterium aurantiacaum) strain 8220605, wherein the preservation number of the strain is CGMCC No. 14676.

2. Use of the micro organism bacterium aurantiacus strain 8220605 of claim 1 for controlling tomato root knot nematode.

3. A biocontrol formulation comprising the micro organism bacillus aurantiacus strain 8220605 of claim 1.

4. A process for the preparation of a biocontrol formulation as defined in claim 3 comprising the steps of:

(1) preparing a seed solution of the Microbacterium aurantiacus strain 8220605;

(2) inoculating the seed liquid prepared in the step (1) into a solid culture medium, and culturing at a constant temperature of 25-27 ℃;

(3) and (3) adding sterile water into the culture cultured in the step (2), mixing, filtering, inoculating the filtrate into a large amount of fermentation medium, and performing fermentation culture in a fermentation chamber with the room temperature of 25-27 ℃ and the relative humidity of more than 85%.

5. The process for the preparation of a biocontrol agent as claimed in claim 4, characterized in that,

the solid culture medium in the step (2) comprises a solid material and inorganic salt, wherein the mass ratio of the solid material to the inorganic salt is 98.5: 0.5; the solid materials are rice hulls, corn flour, soybean meal and bran in a mass ratio of 55: 25: 12: 8; the inorganic salt comprises the following components in percentage by mass: 25% of monopotassium phosphate, 5% of magnesium sulfate, 5% of ammonium sulfate and 65% of light calcium carbonate;

the mass fermentation medium in the step (3) is synchronous with the solid medium in the step (2).

6. Use of the biocontrol formulation of claim 3 for controlling tomato root knot nematode.

7. A biocontrol method characterized in that the micro organism aureoflavus strain 8220605 of claim 1 or the biocontrol formulation of claim 3 is applied to tomato plants having root knot nematodes.