LU500639B1

LU500639B1 - METHOD FOR SCREENING ENDOPHYTIC BACTERIA RESISTANT TO GARLIC ROOT-KNOT NEMATODES

Info

Publication number: LU500639B1
Application number: LU500639A
Authority: LU
Inventors: Qianqian Shi; Ziyi He; Xiao Li
Original assignee: Univ Qingdao Agricultural
Priority date: 2021-09-09
Filing date: 2021-09-09
Publication date: 2022-03-09

Abstract

Disclosed is a method for screening endophytic bacteria resistant to garlic root-knot nematodes, which belongs to the technical field of microorganisms. The method of the present invention includes separating single colonies with different morphologies from garlic root hair damaged by root-knot nematodes and purifying same; inoculating obtained single colonies into a lysogeny broth (LB) liquid culture medium for fermentation, to obtain fermentation supernatants; inoculating live root-knot nematodes into the fermentation supernatants; counting the number of dead nematodes after 24 h; calculating corrected death rates of the nematodes; and screening strains corresponding to a supernatant with a corrected death rate over 70%, to serve as screened endophytic bacteria resistant to the garlic root-knot nematodes. The method of the present invention is simple and high in efficiency and can screen biological control bacteria with a desirable direct contact killing effect on Meloidogyne incognita and provide a biological control resource for biological control of the root-knot nematodes.Disclosed is a method for screening endophytic bacteria resistant to garlic root-knot nematodes, which belongs to the technical field of microorganisms. The method of the present invention includes separating single colonies with different morphologies from garlic root hair damaged by root-knot nematodes and purifying same; inoculating obtained single colonies into a lysogeny broth (LB) liquid culture medium for fermentation, to obtain fermentation supernatants; inoculating live root-knot nematodes into the fermentation supernatants; counting the number of dead nematodes after 24 h; calculating corrected death rates of the nematodes; and screening strains corresponding to a supernatant with a corrected death rate over 70%, to serve as screened endophytic bacteria resistant to the garlic root-knot nematodes. The method of the present invention is simple and high in efficiency and can screen biological control bacteria with a desirable direct contact killing effect on Meloidogyne incognita and provide a biological control resource for biological control of the root-knot nematodes.

Description

METHOD FOR SCREENING ENDOPHYTIC BACTERIA RESISTANT TO LU500639 GARLIC ROOT-KNOT NEMATODESMETHOD FOR SCREENING ENDOPHYTIC BACTERIA RESISTANT TO LU500639 GARLIC ROOT-KNOT NEMATODES

BACKGROUND OF THE INVENTIONBACKGROUND OF THE INVENTION

[0001] 1. Technical Field[0001] 1.Technical Field

[0002] The present invention belongs to the technical field of microorganisms and particularly relates to a method for screening endophytic bacteria resistant to garlic root-knot nematodes.The present invention belongs to the technical field of microorganisms and particularly relates to a method for screening endophytic bacteria resistant to garlic root-knot nematodes.

[0003] 2. Description of Related Art[0003] 2. Description of Related Art

[0004] As a principal soil-borne disease in facility agriculture, the root-knot nematode disease makes a loss up to 100 billion US dollars per year in the world. The root-knot nematodes have extensive hosts, and most crops can be hardly immune from it, which can bring about a serious result that the yield of the crops is reduced by over 75%. At present, chemical pesticides are generally used to control the root-knot nematodes in production. Although the chemical control is convenient to use and quick in effect, in consideration of the particularity of a body wall structure of the nematode, numerous highly toxic nematicides are available in general. Consequently, the pollution to the environment and water is prone to be caused, in addition to that, the human health faces potential safety hazards. Therefore, a variety of extremely and highly toxic nematicides are banned or restricted one after another due to the environmental safety issues. Whereas, as an environmentally friendly and effective method, the biological control draws increasing attention.[0004] As a principal soil-borne disease in facility agriculture, the root-knot nematode disease makes a loss up to 100 billion US dollars per year in the world. The root-knot nematodes have extensive hosts, and most crops can be hardly immune from it, which can bring about a serious result that the yield of the crops is reduced by over 75%. At present, chemical pesticides are generally used to control the root-knot nematodes in production. Although the chemical control is convenient to use and quick in effect, in consideration of the particularity of a body wall structure of the nematode, numerous highly toxic nematicides are available in general. Consequently, the pollution to the environment and water is prone to be caused, in addition to that, the human health faces potential safety hazards. Therefore, a variety of extremely and highly toxic nematicides are banned or restricted one after another due to the environmental safety issues. Whereas, as an environmentally friendly and effective method, the biological control draws increasing attention.

BRIEF SUMMARY OF THE INVENTIONBRIEF SUMMARY OF THE INVENTION

[0005] An objective of the present invention is to provide a method for screening endophytic bacteria resistant to garlic root-knot nematodes. LU500639[0005] An objective of the present invention is to provide a method for screening endophytic bacteria resistant to garlic root-knot nematodes. LU500639

[0006] In order to achieve the objective mentioned above, the present invention uses the following technical solution:[0006] In order to achieve the objective mentioned above, the present invention uses the following technical solution:

[0007] A method for screening endophytic bacteria resistant to garlic root-knot nematodes includes separating single colonies with different morphologies from garlic root hair damaged by root-knot nematodes and purifying same; inoculating obtained single colonies into a lysogeny broth (LB) liquid culture medium for fermentation, to obtain fermentation supernatants, inoculating live root-knot nematodes into the fermentation supernatants, counting the number of dead nematodes after 24 h; calculating corrected death rates of the nematodes; and screening strains corresponding to a supernatant with a corrected death rate over 70%, to serve as screened endophytic bacteria resistant to the garlic root-knot nematodes.[0007] A method for screening endophytic bacteria resistant to garlic root-knot nematodes includes separating single colonies with different morphologies from garlic root hair damaged by root-knot nematodes and purifying same; inoculating obtained single colonies into a lysogeny broth (LB) liquid culture medium for fermentation, to obtain fermentation supernatants, inoculating live root-knot nematodes into the fermentation supernatants, counting the number of dead nematodes after 24 h; calculating correct death rates of the nematodes; and screening strains corresponding to a supernatant with a corrected death rate over 70%, to serve as screened endophytic bacteria resistant to the garlic root-knot nematodes.

[0008] In one specific example, a method for separating single colonies with different morphologies from garlic root hair damaged by root-knot nematodes and purifying same includes:[0008] In one specific example, a method for separating single colonies with different morphologies from garlic root hair damaged by root-knot nematodes and purifying same includes:

[0009] sterilizing and rinsing the garlic root hair damaged by the root-knot nematodes, then grinding same into a paste, and adding sterilized water; fully and uniformly mixing same and then leaving same for standing; sucking a diluent, uniformly coating a lysogeny broth ampicillin (LBA) culture medium with same, and culturing the same at a constant temperature of 37°C for 2-3 days; and selecting the single colonies with different morphologies, separating and purifying same through a streak plate method, transferring obtained strains to a slant of the LBA culture medium for culture, and storing same at 4°C.[0009] sterilizing and rinsing the garlic root hair damaged by the root-knot nematodes, then grinding the same into a paste, and adding sterilized water; fully and uniformly mixing same and then leaving same for standing; sucking a diluent, uniformly coating a lysogeny broth ampicillin (LBA) culture medium with the same, and culturing the same at a constant temperature of 37°C for 2-3 days; and selecting the single colonies with different morphologies, separating and purifying same through a streak plate method, transferring obtained strains to a slant of the LBA culture medium for culture, and storing same at 4°C.

[0010] In one specific example, a method for obtaining the live root-knot nematodes includes:[0010] In one specific example, a method for obtaining the live root-knot nematodes includes:

[0011] selecting egg masses from Meloidogyne incognita, thoroughly LU500639 sterilizing and washing same, then placing the same in a sterilized culture dish, adding a small amount of sterilized water, incubating the same at a constant temperature of 28°C, and collecting the live root-knot nematodes after incubation.[0011] selecting egg masses from Meloidogyne incognita, thoroughly LU500639 sterilizing and washing same, then placing the same in a sterilized culture dish, adding a small amount of sterilized water, incubating the same at a constant temperature of 28°C, and collecting the live root-knot nematodes after incubation.

[0012] In one specific example, the fermentation supernatant is obtained by transferring the single colony into the LB liquid culture medium to be cultured at 37°C and 200 r for 48 h; and after fermentation, centrifuging a fermentation broth at 9000 rmp for 10 min.In one specific example, the fermentation supernatant is obtained by transferring the single colony into the LB liquid culture medium to be cultured at 37°C and 200 r for 48 h; and after fermentation, centrifuging a fermentation broth at 9000 rmp for 10 min.

[0013] In one specific example, the inoculation amount of the live root-knot nematodes is 50 pieces/mL fermentation supernatant.[0013] In one specific example, the inoculation amount of the live root-knot nematodes is 50 pieces/mL fermentation supernatant.

[0014] In one specific example, a formula for calculating the corrected death rate is:[0014] In one specific example, a formula for calculating the corrected death rate is:

[0015] corrected death rate = (survival rate of control group - survival rate of treatment group) x 100 / survival rate of control group,[0015] corrected death rate = (survival rate of control group - survival rate of treatment group) x 100 / survival rate of control group,

[0016] where the control group is treated with sterilized water, and the treatment group is treated with the fermentation supernatant.[0016] where the control group is treated with sterilized water, and the treatment group is treated with the fermentation supernatant.

[0017] In one specific example, strains corresponding to a supernatant with a corrected death rate over 80% are selected; and more preferably, strains corresponding to a supernatant with a corrected death rate over 90% are selected.[0017] In one specific example, strains corresponding to a supernatant with a corrected death rate over 80% are selected; and more preferably, strains corresponding to a supernatant with a corrected death rate over 90% are selected.

[0018] The technical solution of the present invention has the advantages:[0018] The technical solution of the present invention has the advantages:

[0019] According to the method of the present invention, bacteria for biologically controlling the root-knot nematodes are screened, through a spread plate method, from a grinding liquid of garlic root hair tissue damaged by the root-knot nematodes, and total four biological control bacterium strains with a desirable direct contact killing effect on Meloidogyne incognita are separated from the garlic root hair damaged by the root-knot nematodes, where a corrected death rate of Meloidogyne LU500639 incognita is up to 90.8% when a strain S1-1 is applied; and a pot experiment has proved a control effect of the biological control strain, a control effect of the strain S1-1 being 74.86%.According to the method of the present invention, bacteria for biologically controlling the root-knot nematodes are screened, through a spread plate method, from a grinding liquid of garlic root hair tissue damaged by the root-knot nematodes, and total four biological control bacterium strains with a desirable direct contact killing effect on Meloidogyne incognita are separated from the garlic root hair damaged by the root-knot nematodes, where a corrected death rate of Meloidogyne LU500639 incognita is up to 90.8% when a strain S1-1 is applied; and a pot experiment has proved a control effect of the biological control strain, a control effect of the strain S1-1 being 74.86%.

[0020] The method of the present invention is simple and high in efficiency and may screen biological control bacteria with a desirable direct contact killing effect on Meloidogyne incognita and provide a biological control resource for biological control of the root-knot nematodes.The method of the present invention is simple and high in efficiency and may screen biological control bacteria with a desirable direct contact killing effect on Meloidogyne incognita and provide a biological control resource for biological control of the root-knot nematodes.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGSBRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0021] Fig. 1 is a phylogenetic tree of a strain S1-1.[0021] Fig. 1 is a phylogenetic tree of a strain S1-1.

DETAILED DESCRIPTION OF THE INVENTIONDETAILED DESCRIPTION OF THE INVENTION

[0022] The terms used in the present invention, unless otherwise indicated, generally have the meanings generally understood by those of ordinary skill in the art.The terms used in the present invention, unless otherwise indicated, generally have the meanings generally understood by those of ordinary skill in the art.

[0023] The present invention is described in further detail below with reference to the specific examples and data. The following examples are merely to illustrate the present invention, instead of limiting the scope of the present invention in any way.The present invention is described in further detail below with reference to the specific examples and data. The following examples are merely to illustrate the present invention, instead of limiting the scope of the present invention in any way.

[0024] Example 1[0024] Example 1

[0025] A method for screening endophytic bacteria resistant to garlic root-knot nematodes includes:[0025] A method for screening endophytic bacteria resistant to garlic root-knot nematodes includes:

[0026] (1) 10 g of garlic root hair damaged by root-knot nematodes (a garlic sample damaged by the root-knot nematodes and collected from Qingdao, Shandong) was weighed, sterilized with 1% sodium hypochlorite for 5 min, rinsed with sterilized water three times, put into a sterilized mortar, and ground into a paste, and then the paste was washed with a small amount of sterilized water into a triangular flask filled LU500639 with 90 mL of sterilized water. The paste and the sterilized water were fully and uniformly mixed and then left for standing for 10 min. 100 pL of diluent was sucked, uniformly coated a lysogeny broth ampicillin (LBA) culture medium, and was 5 cultured at a constant temperature of 37°C for 2-3 days. Single colonies with different morphologies were selected, separated and purified through a streak plate method, and obtained strains were transferred to a slant of the LBA culture medium to be cultured for 24 h, and then stored at 4°C.[0026] (1) 10 g of garlic root hair damaged by root-knot nematodes (a garlic sample damaged by the root-knot nematodes and collected from Qingdao, Shandong) was weighed, sterilized with 1% sodium hypochlorite for 5 min, rinsed with sterilized water three times, put into a sterilized mortar, and ground into a paste, and then the paste was washed with a small amount of sterilized water into a triangular flask filled LU500639 with 90 mL of sterilized water. The paste and the sterilized water were fully and uniformly mixed and then left for standing for 10 min. 100 µL of diluent was sucked, uniformly coated a lysogeny broth ampicillin (LBA) culture medium, and was 5 cultured at a constant temperature of 37°C for 2-3 days. Single colonies with different morphologies were selected, separated and purified through a streak plate method, and obtained strains were transferred to a slant of the LBA culture medium to be cultured for 24 h, and then stored at 4°C.

[0027] (2) A mature nematode egg mass (from a nematode laboratory of the College of Plant Health & Medicine, Qingdao Agricultural University) was selected from water spinach used to culture Meloidogyne incognita, sterilized with 0.5% sodium hypochlorite for 1 min, and thoroughly washed with sterilized water. The egg mass was collected into a sterilized culture dish, added with a small amount of sterilized water, and incubated at a constant temperature of 28°C, and after 48 h, incubated nematodes were collected for later use.[0027] (2) A mature nematode egg mass (from a nematode laboratory of the College of Plant Health & Medicine, Qingdao Agricultural University) was selected from water spinach used to culture Meloidogyne incognita, sterilized with 0.5% sodium hypochlorite for 1 min, and thoroughly washed with sterilized water. The egg mass was collected into a sterilized culture dish, added with a small amount of sterilized water, and incubated at a constant temperature of 28°C, and after 48 h, incubated nematodes were collected for later use.

[0028] (3) The strains stored on the slant of the LBA culture medium at 4°C were selected and activated on an LBA plate through streaking, and after one day, single colonies were selected, transferred to a triangular flask filled with 100 mL of lysogeny broth (LB) liquid culture medium, and cultured at 37°C and 200 r for 48 h.[0028] (3) The strains stored on the slant of the LBA culture medium at 4°C were selected and activated on an LBA plate through streaking, and after one day, single colonies were selected, transferred to a triangular flask filled with 100 mL of lysogeny broth (LB) liquid culture medium, and cultured at 37°C and 200 r for 48 h.

After fermentation, a fermentation broth was centrifuged at 9000 rmp for 10 min, and a supernatant was taken for later use. 1 mL of supernatant was added into a 24-well plate, an equivalent amount of sterilized water was added into a control group, 50 or so nematodes were selected, added and cultured at a constant temperature of 28°C, the number of dead nematodes was recorded after 24 h, and corrected death rates of the nematodes were calculated, whereAfter fermentation, a fermentation broth was centrifuged at 9000 rmp for 10 min, and a supernatant was taken for later use. 1 mL of supernatant was added into a 24-well plate, an equivalent amount of sterilized water was added into a control group, 50 or so nematodes were selected, added and cultured at a constant temperature of 28°C, the number of dead nematodes was recorded after 24 h, and corrected death rates of the nematodes were calculated, where

[0029] corrected death rate = (survival rate of control group - survival rate of LU500639 treatment group) x 100 / survival rate of control group[0029] corrected death rate = (survival rate of control group - survival rate of LU500639 treatment group) x 100 / survival rate of control group

[0030] Strains corresponding to a supernatant with a corrected death rate over 70% were screened.[0030] Strains corresponding to a supernatant with a corrected death rate over 70% were screened.

[0031] An LB solid (LBA) culture medium was preparing by adding distilled water into a mixture containing 5 g of tryptone, 2.5 g of yeast extracts, 5 g of NaCl, and 7.5 g of agar powder till a constant volume of 500 mL is reached, where a PH was[0031] An LB solid (LBA) culture medium was prepared by adding distilled water into a mixture containing 5 g of tryptone, 2.5 g of yeast extracts, 5 g of NaCl, and 7.5 g of agar powder till a constant volume of 500 mL is reached, where a PH was

7.0.7.0.

[0032] 102 bacterium strains were primarily separated through a spread plate method, and then 4 bacterium strains with a desirable biological control effect on Meloidogyne incognita were secondarily screened through a direct contact killing experiment on Meloidogyne incognita, where a death rate of Meloidogyne incognita was up to 90.08% when a fermentation broth of a strain S1-1 which was cultured at 37°C and 200 rpm for 48 h was applied (Table 1).[0032] 102 bacterium strains were primarily separated through a spread plate method, and then 4 bacterium strains with a desirable biological control effect on Meloidogyne incognita were secondarily screened through a direct contact killing experiment on Meloidogyne incognita, where a death rate of Meloidogyne incognita was up to 90.08% when a fermentation broth of a strain S1-1 which was cultured at 37°C and 200 rpm for 48 h was applied (Table 1).

[0033] Table 1 Biological control bacterium fermentation broth’s contact killing control effect on Meloidogyne incognita Nematode corrected death Number of strain Nematode death rate % rate % S1-1 90.96 90.08 S1-5 78.87 78.43 S1-8 85.83 85.55 S1-21 85.17 84.88[0033] Table 1 Biological control bacterium fermentation broth’s contact killing control effect on Meloidogyne incognita Nematode corrected death Number of strain Nematode death rate % rate % S1-1 90.96 90.08 S1-5 78.87 78.43 S1-8 85.83 85.55 S1-21 85.17 84.88

[0034] Example 2 Experiment of biological control bacterium pot control effect on root-knot nematodes[0034] Example 2 Experiment of biological control bacterium pot control effect on root-knot nematodes

[0035] After experimental tomato seedlings (Lichun breed) grew the fourth LU500639 real leaves, the strain S1-1 screened in Example 1 was prepared into a bacterial fermentation broth, and a cell concentration was finally adjusted to 10’ CFU/mL. 20 mL of biological control bacterium fermentation broth was poured into roots of tomato seedlings in a treatment group, and only sterilized water was poured into roots of tomato seedlings in a control group. After 3 days, the roots of the tomato seedlings were uniformly inoculated with second instar larvae of Meloidogyne incognita (1000 larvae/plant). 20 tomato seedlings were treated for each group. An incidence of the tomato seedlings was investigated 37 days later after inoculation. Root knots were graded with reference to a Bridge method and a Page method (Table 2). The numbers of the root knots and disease indexes of the tomato seedlings were counted to calculate a control effect.[0035] After experimental tomato seedlings (Lichun breed) grew the fourth LU500639 real leaves, the strain S1-1 screened in Example 1 was prepared into a bacterial fermentation broth, and a cell concentration was finally adjusted to 10’ CFU/mL. 20 mL of biological control bacterium fermentation broth was poured into roots of tomato seedlings in a treatment group, and only sterilized water was poured into roots of tomato seedlings in a control group. After 3 days, the roots of the tomato seedlings were uniformly inoculated with second instar larvae of Meloidogyne incognita (1000 larvae/plant). 20 tomato seedlings were treated for each group. An incidence of the tomato seedlings was investigated 37 days later after inoculation. Root knots were graded with reference to a Bridge method and a Page method (Table 2). The numbers of the root knots and disease indexes of the tomato seedlings were counted to calculate a control effect.

[0036] Root disease index (DI) = X (number of gall plants of a certain grade x gall grade) / (total number of investigated plants x 5) x 100[0036] Root disease index (DI) = X (number of gall plants of a certain grade x gall grade) / (total number of investigated plants x 5) x 100

[0037] Table 2 Determination on the root-knot nematodes Gall grade Root knot 0 No knot on main root and hair root 1 Some tiny knots on hair root 2 Small obvious knots on hair root and no knot on main root 3 Large obvious knot on hair root and no knot on main root 4 Large knots as majority of knots on hair root 5 Knots on 50% hair root and few knots on main root 6 Knots on over 50% hair root and large obvious knot on main root[0037] Table 2 Determination on the root-knot nematodes Gall grade Root knot 0 No knot on main root and hair root 1 Some tiny knots on hair root 2 Small obvious knots on hair root and no knot on main root 3 Large obvious knot on hair root and no knot on main root 4 Large knots as majority of knots on hair root 5 Knots on 50% hair root and few knots on main root 6 Knots on over 50% hair root and large obvious knot on main root

PS LU500639 7 Knots on over 50% main root 8 Knots on all main roots 9 Knots on all main roots and all hair roots 10 Serious secondary knot on main root and no hair rootPS LU500639 7 Knots on over 50% main root 8 Knots on all main roots 9 Knots on all main roots and all hair roots 10 Serious secondary knot on main root and no hair root

[0038] A strain S1-1 control effect on Meloidogyne incognita was determined through a pot experiment. A result showed that the number of root knots and a disease index of the tomato seedlings in the treatment group were significantly lower than those in the control group after the fermentation broth of the strain S1-1 was applied, and the control effect reached 74.86% (Table 3), which indicated that the strain S1-1 had a desirable control effect on Meloidogyne incognita.A strain S1-1 control effect on Meloidogyne incognita was determined through a pot experiment. A result showed that the number of root knots and a disease index of the tomato seedlings in the treatment group were significantly lower than those in the control group after the fermentation broth of the strain S1-1 was applied, and the control effect reached 74.86% (Table 3), which indicated that the strain S1-1 had a desirable control effect on Meloidogyne incognita.

[0039] Table 3 Pot experiment on Meloidogyne incognita CK 366a 5.071a 0 S1-1 92b 2.533b 74.86 Note: in each column, the same letter indicated that there was no significant difference (P > 0.05), but different letters indicated that there was a significant difference (P > 0.05).[0039] Table 3 Pot experiment on Meloidogyne incognita CK 366a 5.071a 0 S1-1 92b 2.533b 74.86 Note: in each column, the same letter indicated that there was no significant difference (P > 0.05), but different letters indicated that there was a significant difference (P > 0.05).

[0040] Example 3 Determination of strain S1-1[0040] Example 3 Determination of strain S1-1

[0041] (1) morphologic observation on biological control bacterium[0041] (1) morphological observation on biological control bacteria

[0042] A strain S1-1, which had a desirable direct contact killing effect on root-knot nematodes, was streaked on an LBA culture medium plate, and cultured at a constant temperature of 37°C for 24 h, a morphology of a single colony of the bacterium was observed, and then the strain was subjected to gram staining and microscopic examination.A strain S1-1, which had a desirable direct contact killing effect on root-knot nematodes, was streaked on an LBA culture medium plate, and cultured at a constant temperature of 37°C for 24 h, a morphology of a single colony of the bacterium was observed, and then the strain was subjected to gram staining and microscopic examination.

[0043] A result showed that S1-1 had a misty-white colony on an LB culture medium and a scattered colony morphology like a weed, particularly, a round colony morphology on a 10% NaCl culture medium, which had a neat edge and a smooth LU500639 surface, the colony was neither raised nor transparent, and a color of the culture medium was not changed significantly. After gram staining, the strain is rod-like and purple-red, which showed that S1-1 was a gram-positive bacterium.A result showed that S1-1 had a misty-white colony on an LB culture medium and a scattered colony morphology like a weed, particularly, a round colony morphology on a 10% NaCl culture medium, which had a neat edge and a smooth LU500639 surface, the colony was neither raised nor transparent, and a color of the culture medium was not changed significantly. After gram staining, the strain is rod-like and purple-red, which showed that S1-1 was a gram-positive bacterium.

[0044] (2) physiological and biochemical determination[0044] (2) physiological and biochemical determination

[0045] According to “Bergey's Manual of Determinative Bacteriology” and “Manual of Determinative Common Bacterium System”, the strain S1-1 was subjected to physiological and biochemical determination such as a Voges-Proskauer experiment and a citrate utilization experiment, and results were shown in Table 4.[0045] According to “Bergey's Manual of Determinative Bacteriology” and “Manual of Determinative Common Bacterium System”, the strain S1-1 was subjected to physiological and biochemical determination such as a Voges-Proskauer experiment and a citrate utilization experiment, and results were shown in Table 4.

[0046] Table 4 Physiological and biochemical characteristics of S1-1 Voges-Proskauer experiment + Methylred experiment + Citrate + Mycoderm formation + Amylolysis - Gelatin liquefaction - Salt tolerance 1% + 5% + 10% +[0046] Table 4 Physiological and biochemical characteristics of S1-1 Voges-Proskauer experiment + Methylred experiment + Citrate + Mycoderm formation + Amylolysis - Gelatin liquefaction - Salt tolerance 1% + 5% + 10% +

[0047] (3) extraction of bacterial deoxyribonucleic acid (DNA) and analysis of 16S rDNA gene sequence[0047] (3) extraction of bacterial deoxyribonucleic acid (DNA) and analysis of 16S rDNA gene sequence

[0048] DNA of the strain S1-1 was extracted by utilizing an OmegaBio-Tek kit. 16S rDNA bacterial universal primers, 27F/1492R, were used for polymerase chain reaction (PCR) amplification.[0048] DNA of the strain S1-1 was extracted by utilizing an OmegaBio-Tek kit. 16S rDNA bacterial universal primers, 27F/1492R, were used for polymerase chain reaction (PCR) amplification.

[0049] A PCR reaction system (25 pL) included: 2.5 uL of 10X Buffer, Zu | -U500639 of deoxy-ribonucleoside triphosphate (dNTP), 1 uL of each of the primers, 27F/1492R,[0049] A PCR reaction system (25 pL) included: 2.5 uL of 10X Buffer, Zu | -U500639 of deoxy-ribonucleoside triphosphate (dNTP), 1 µL of each of the primers, 27F/1492R,

0.5 ul of recombinant thermus aquaticus (rTaq), 16 uL of double distilled (dd) H20 and 2 pL of template DNA; and0.5 µl of recombinant thermus aquaticus (rTaq), 16 µL of double distilled (dd) H20 and 2 µL of template DNA; and

[0050] reaction parameters included: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s; annealing at 63°C for 30 s, extension at 72°C for 90 s; 35 cycles in total; overall extension at 72°C for 7 min; and storage at 4°C.[0050] reaction parameters included: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s; annealing at 63°C for 30 s, extension at 72°C for 90 s; 35 cycles in total; overall extension at 72°C for 7 min; and storage at 4°C.

[0051] After the PCR amplification, 1% agarose gel electrophoresis was used to detect a PCR product, an amplified product was sent to Beijing Tsingke Biotechnology Co., Ltd. for sequencing, and then subjected to basic local alignment search tool (BLAST) comparative analysis based on a national center for bioinformation (NCBI) database, to create a phylogenetic tree.[0051] After the PCR amplification, 1% agarose gel electrophoresis was used to detect a PCR product, an amplified product was sent to Beijing Tsingke Biotechnology Co., Ltd. for sequencing, and then subjected to basic local alignment search tool (BLAST) comparative analysis based on a national center for bioinformation (NCBI) database, to create a phylogenetic tree.

[0052] An obtained 16S rDNA gene sequence of the strain S1-1 was shown as SEQ ID NO: 1.[0052] An obtained 16S rDNA gene sequence of the strain S1-1 was shown as SEQ ID NO: 1.

[0053] SEQ ID NO:1 attctatatg cagtcgagcg gacagaaggg agcettgetee cggatgttag cggeggacgg 60 gtgagtaaca cgtgggtaac ctgectgtaa gactgggata actccgggaa accggagcta 120 ataccggata gttccttgaa ccgeatggtt caaggatgaa agacggtttc gectatcact 180 tacagatgga cecgcgecec attagctagt tggtgaggta acgectcacc aaggegacga 240 tgcgtageeg acctgagagg gtgatcggec acactgggac tgagacacgg cecagactec 300 tacgggaggc agcagtaggg aatcttccge aatggacgaa agtctgacgg agcaacgecg 360 cgtgagtgat gaaggttttc ggatcgtaaa getctgttgt tagggaagaa caagtgcaag 420 agtaactgct tgcaccttga cggtacctaa ccagaaagec acggctaact acgtgecage 480 agccgeggta atacgtaggt ggcaagcegtt gtceggaatt attgggcgta aaggectcgc 540 aggcggtttc ttaagtctga tgtgaaagee cecgectcaa ccggggaggg tcattggaaa 600 ctgggaaact tgagtgcaga agaggagagt ggaattccac gtgtageggt gaaatgcgta 660 LU500639 gagatgtgga ggaacaccag tggcgaagge gactetetgg tetgtaactg acgctgagga 720 gcgaaagcet ggggagcgaa caggattaga taccctggta gtccacgecg taaacgatga 780 gtectaagtg ttaggggett tccgcccctt agtgetgeag ctaacgcatt aagcactecg 840 cctggggagt acgetcgcaa gactgaaact caaaggaatt gacgggggec cgcacaageg 900 gtggagcatg tggtttaatt cgaagcaacg cgaagaacct taccaggtct tgacatectc 960 tgacaaccct agagataggg ctttcectte ggggacagag tgacaggtgg tgcatgattg 1020 tcgtcagcete gtgtecgteag atattggatt aagteccgca acgagegeaa cecttgatet 1080 tagttgccag cattcagttg ggcactctaa ggtgactgee ggtgacaaac cggaggaagg 1140 tggggatgac gtcaaatcat catgececctt atgacctggg ctacacacgt gctacaatgg 1200 acagaacaaa gggctgcgag accgcaaggt ttagccaate ccacaaatct gttctcagtt 1260 cggatcgceag tetgcaacte gactgegtga agctggaate gctagtaatc geggatcage 1320 atgccgcggt gaatacgttc ccgggcecttg tacacaccge cecgtcacace acgagagttt 1380 gcaacacccg aagtcggtga ggtaaccttt atggagccag ccgegaag 1428[0053] SEQ ID NO: 1 attctatatg cagtcgagcg gacagaaggg agcettgetee cggatgttag cggeggacgg 60 gtgagtaaca cgtgggtaac ctgectgtaa gactgggata actccgggaa accggagcta 120 ataccggata gttccttgaa ccgeatggtt caaggatgaa agacggtttc gectatcact 180 tacagatgga cecgcgecec attagctagt tggtgaggta acgectcacc aaggegacga 240 tgcgtageeg acctgagagg gtgatcggec acactgggac tgagacacgg cecagactec 300 tacgggaggc agcagtaggg aatcttccge aatggacgaa agtctgacgg agcaacgecg 360 cgtgagtgat gaaggttttc ggatcgtaaa getctgttgt tagggaagaa caagtgcaag 420 agtaactgct tgcaccttga cggtacctaa ccagaaagec acggctaact acgtgecage 480 agccgeggta atacgtaggt ggcaagcegtt gtceggaatt attgggcgta aaggectcgc 540 aggcggtttc ttaagtctga tgtgaaagee cecgectcaa ccggggaggg tcattggaaa 600 ctgggaaact tgagtgcaga agaggagagt ggaattccac gtgtageggt gaaatgcgta 660 LU500639 gagatgtgga ggaacaccag tggcgaagge gactetetgg tetgtaactg acgctgagga 720 gcgaaagcet ggggagcgaa caggattaga taccctggta gtccacgecg taaacgatga 780 gtectaagtg ttaggggett tccgcccctt agtgetgeag ctaacgcatt aagcac tecg 840 cctggggagt acgetcgcaa gactgaaact caaaggaatt gacgggggec cgcacaageg 900 gtggagcatg tggtttaatt cgaagcaacg cgaagaacct taccaggtct tgacatectc 960 tgacaaccct agagataggg ctttcectte ggggacagag tgacaggtgg tgcatgattg 1020 tcgtcagcete gtgtecgteag atattggatt aagteccgca acgagegeaa cecttgatet 1080 tagttgccag cattcagttg ggcactctaa ggtgactgee ggtgacaaac cggaggaagg 1140 tggggatgac gtcaaatcat catgececctt atgacctggg ctacacacgt gctacaatgg 1200 acagaacaaa gggctgcgag accgcaaggt ttagccaate ccacaaatct gttctcagtt 1428

[0054] The 16S rDNA gene sequence of the strain S1-1 was comparatively analyzed with the Blast, to create the phylogenetic tree. According to the phylogenetic tree, the strain S1-1 and Bacillus pumilus belong to the same branch, and the strain may be determined as Bacillus pumilus with reference to morphological characteristics and physiological and biochemical determination results of the strain (Fig. 1).[0054] The 16S rDNA gene sequence of the strain S1-1 was comparatively analyzed with the Blast, to create the phylogenetic tree. According to the phylogenetic tree, the strain S1-1 and Bacillus pumilus belong to the same branch, and the strain may be determined as Bacillus pumilus with reference to morphological characteristics and physiological and biochemical determination results of the strain (Fig. 1).

[0055] The above are only the preferred embodiments of the present invention, and are not intended to limit the present invention in other forms. Any skilled in the art may make changes of variations on the technical content disclosed above to become equivalent embodiments with the same changes. However, any simple modification, equivalent change and variation made on the above embodiments by LU500639 using the contents without departing from the technical solution of the present invention based on the technical essence of the present invention are all still within the protection scope of the present invention.The above are only the preferred embodiments of the present invention, and are not intended to limit the present invention in other forms. Any skilled in the art may make changes of variations on the technical content disclosed above to become equivalent embodiments with the same changes. However, any simple modification, equivalent change and variation made on the above embodiments by LU500639 using the contents without departing from the technical solution of the present invention based on the technical essence of the present invention are all still within the protection scope of the present invention .

Sequence List LU500639 <110> QINGDAO AGRICULTURAL UNIVERSITY <120> METHOD FOR SCREENING ENDOPHYTIC BACTERIA RESISTANT TO GARLIC ROOT-KNOTSequence List LU500639 <110> QINGDAO AGRICULTURAL UNIVERSITY <120> METHOD FOR SCREENING ENDOPHYTIC BACTERIA RESISTANT TO GARLIC ROOT-KNOT

NEMATODES <160> 1 <170> SIPOSequenceListing 1.0 <218> 1 <211> 1428 <212> DNA <213> Bacillus pumilus <400> 1 attctatatg cagtcgagcg gacagaaggg agcttgctcc cggatgttag cggcggacgg 60 gtgagtaaca cgtgggtaac ctgcctgtaa gactgggata actccgggaa accggagcta 120 ataccggata gttccttgaa ccgcatggtt caaggatgaa agacggtttc ggctgtcact 180 tacagatgga cccgcggcgc attagctagt tggtgaggta acggctcacc aaggcgacga 240 tgcgtagccg acctgagagg gtgatcggcc acactgggac tgagacacgg cccagactcc 300 tacgggaggc agcagtaggg aatcttccgec aatggacgaa agtctgacgg agcaacgccg 360 cgtgagtgat gaaggttttc ggatcgtaaa gctctgttgt tagggaagaa caagtgcaag 420 agtaactgct tgcaccttga cggtacctaa ccagaaagcc acggctaact acgtgccagc 480 agccgcggta atacgtaggt ggcaagcgtt gtccggaatt attgggcgta aagggctcgc 540 aggcggtttc ttaagtctga tgtgaaagcc cccggctcaa ccggggaggg tcattggaaa 600 ctgggaaact tgagtgcaga agaggagagt ggaattccac gtgtagcggt gaaatgcgta 660 gagatgtgga ggaacaccag tggcgaaggc gactctctgg tctgtaactg acgctgagga 720 gcgaaagcet ggggagcgaa caggattaga taccctggta gtccacgccg taaacgatga 780 gtgctaagtg ttagggggtt tccgcccctt agtgctgcag ctaacgcatt aagcactccg 840 cctggggagt acggtcgcaa gactgaaact caaaggaatt gacgggggcc cgcacaagceg 900 gtggagcatg tggtttaatt cgaagcaacg cgaagaacct taccaggtct tgacatcctc 960 tgacaaccct agagataggg ctttcccttc ggggacagag tgacaggtgg tgcatggttg 1020 tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa cccttgatct 1080 tagttgccag cattcagttg ggcactctaa ggtgactgcc ggtgacaaac cggaggaagg 1140 tggggatgac gtcaaatcat catgcccctt atgacctggg ctacacacgt gctacaatgg 1200 acagaacaaa gggctgcgag accgcaaggt ttagccaatc ccacaaatct gttctcagtt 1260 cggatcgcag tctgcaactc gactgcgtga agctggaatc gctagtaatc gcggatcagc 1320 atgccgcggt gaatacgttc ccgggccttg tacacaccgc ccgtcacacc acgagagttt 1380 gcaacacccg aagtcggtga ggtaaccttt atggagccag ccgcgaag 1428NEMATODES <160> 1 <170> SIPOSequenceListing 1.0 <218> 1 <211> 1428 <212> DNA <213> Bacillus pumilus <400> 1 attctatatg cagtcgagcg gacagaaggg agcttgctcc cggatgttag cggcggacgg 60 gtgagtaaca cgtgggtaac ctgcctgtaa gactgggata actccgggaa accggagcta 120 ataccggata gttccttgaa ccgcatggtt caaggatgaa agacggtttc ggctgtcact 180 tacagatgga cccgcggcgc attagctagt tggtgaggta acggctcacc aaggcgacga 240 tgcgtagccg acctgagagg gtgatcggcc acactgggac tgagacacgg cccagactcc 300 tacgggaggc agcagtaggg aatcttccgec aatggacgaa agtctgacgg agcaacgccg 360 cgtgagtgat gaaggttttc ggatcgtaaa gctctgttgt tagggaagaa caagtgcaag 420 agtaactgct tgcaccttga cggtacctaa ccagaaagcc acggctaact acgtgccagc 480 agccgcggta atacgtaggt ggcaagcgtt gtccggaatt attgggcgta aagggctcgc 540 aggcggtttc ttaagtctga tgtgaaagcc cccggctcaa ccggggaggg tcattggaaa 600 ctgggaaact tgagtgcaga agaggagagt ggaattccac gtgtagcggt gaaatgcgta 660 gagatgtgga ggaacaccag tggcgaaggc gactctctgg tctgtaactg acgctgagga 720 gcgaaagcet ggggagcgaa caggattaga taccctggta gtccacgc cg taaacgatga 780 gtgctaagtg ttagggggtt tccgcccctt agtgctgcag ctaacgcatt aagcactccg 840 cctggggagt acggtcgcaa gactgaaact caaaggaatt gacgggggcc cgcacaagceg 900 gtggagcatg tggtttaatt cgaagcaacg cgaagaacct taccaggtct tgacatcctc 960 tgacaaccct agagataggg ctttcccttc ggggacagag tgacaggtgg tgcatggttg 1020 tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa cccttgatct 1080 tagttgccag cattcagttg ggcactctaa ggtgactgcc ggtgacaaac cggaggaagg 1140 tggggatgac gtcaaatcat catgcccctt atgacctggg ctacacacgt gctacaatgg 1200 acagaacaaa gggctgcgag accgcaaggt ttagccaatc ccacaaatct gttctcagtt 1260 cggatcgcag tctgcaactc gactgcgtga agctggaatc gctagtaatc gcggatcagc 1320 atgccgcggt gaatacgttc ccgggccttg tacacaccgc ccgtcacacc acgagagttt 1380 gcaacacccg aagtcggtga ggtaaccttt atggagccag ccgcgaag 1428

Claims

CLAIMS LU500639CLAIMS LU500639

1.A method for screening endophytic bacteria resistant to garlic root-knot nematodes, including separating single colonies with different morphologies from garlic root hair damaged by root-knot nematodes and purifying same; inoculating obtained single colonies into a lysogeny broth (LB) liquid culture medium for fermentation, to obtain fermentation supernatants; inoculating live root-knot nematodes into the fermentation supernatants; counting the number of dead nematodes after 24 h; calculating corrected death rates of the nematodes; and screening strains corresponding to a supernatant with a corrected death rate over 70%, to serve as screened endophytic bacteria resistant to the garlic root-knot nematodes.1.A method for screening endophytic bacteria resistant to garlic root-knot nematodes, including separating single colonies with different morphologies from garlic root hair damaged by root-knot nematodes and purifying same; inoculating obtained single colonies into a lysogeny broth (LB) liquid culture medium for fermentation, to obtain fermentation supernatants; inoculating live root-knot nematodes into the fermentation supernatants; counting the number of dead nematodes after 24 h; calculating correct death rates of the nematodes; and screening strains corresponding to a supernatant with a corrected death rate over 70%, to serve as screened endophytic bacteria resistant to the garlic root-knot nematodes.

2. The method for screening endophytic bacteria resistant to garlic root-knot nematodes according to claim 1, a method for separating single colonies with different morphologies from garlic root hair damaged by root-knot nematodes and purifying same includes: sterilizing and rinsing the garlic root hair damaged by the root-knot nematodes, then grinding same into a paste, and adding sterilized water; fully and uniformly mixing same and then leaving same for standing; sucking a diluent, uniformly coating a lysogeny broth ampicillin (LBA) culture medium with same, and culturing the same at a constant temperature of 37°C for 2-3 days; and selecting the single colonies with different morphologies, separating and purifying same through a streak plate method, transferring obtained strains to a slant of the LBA culture medium for culture, and storing same at 4°C.2. The method for screening endophytic bacteria resistant to garlic root-knot nematodes according to claim 1, a method for separating single colonies with different morphologies from garlic root hair damaged by root-knot nematodes and purifying same includes: sterilizing and rinsing the garlic root hair damaged by the root-knot nematodes, then grinding the same into a paste, and adding sterilized water; fully and uniformly mixing same and then leaving same for standing; sucking a diluent, uniformly coating a lysogeny broth ampicillin (LBA) culture medium with the same, and culturing the same at a constant temperature of 37°C for 2-3 days; and selecting the single colonies with different morphologies, separating and purifying same through a streak plate method, transferring obtained strains to a slant of the LBA culture medium for culture, and storing same at 4°C.

3. The method for screening endophytic bacteria resistant to garlic root-knot nematodes according to claim 1, a method for obtaining the live root-knot nematodes includes:3. The method for screening endophytic bacteria resistant to garlic root-knot nematodes according to claim 1, a method for obtaining the live root-knot nematodes includes:

selecting egg masses from Meloidogyne incognita, thoroughly sterilizing and LU500639 washing same, then placing the same in a sterilized culture dish, adding a small amount of sterilized water, incubating the same at a constant temperature of 28°C, and collecting the live root-knot nematodes after incubation.selecting egg masses from Meloidogyne incognita, thoroughly sterilizing and LU500639 washing same, then placing the same in a sterilized culture dish, adding a small amount of sterilized water, incubating the same at a constant temperature of 28°C, and collecting the live root- knot nematodes after incubation.

4.The method for screening endophytic bacteria resistant to garlic root-knot nematodes according to claim 1, the fermentation supernatant is obtained by transferring the single colony into the LB liquid culture medium to be cultured at 37°C and 200 r for 48 h; and after fermentation, centrifuging a fermentation broth at 9000 rmp for 10 min.4.The method for screening endophytic bacteria resistant to garlic root-knot nematodes according to claim 1, the fermentation supernatant is obtained by transferring the single colony into the LB liquid culture medium to be cultured at 37°C and 200 r for 48 h; and after fermentation, centrifuging a fermentation broth at 9000 rmp for 10 min.

S. The method for screening endophytic bacteria resistant to garlic root-knot nematodes according to claim 1, the inoculation amount of the live root-knot nematodes is 50 pieces/mL fermentation supernatant.S. The method for screening endophytic bacteria resistant to garlic root-knot nematodes according to claim 1, the inoculation amount of the live root-knot nematodes is 50 pieces/mL fermentation supernatant.

6. The method for screening endophytic bacteria resistant to garlic root-knot nematodes according to claim 1, a formula for calculating the corrected death rate is: corrected death rate = (survival rate of control group - survival rate of treatment group) x 100 / survival rate of control group, where the control group is treated with sterilized water, and the treatment group is treated with the fermentation supernatant.6. The method for screening endophytic bacteria resistant to garlic root-knot nematodes according to claim 1, a formula for calculating the corrected death rate is: corrected death rate = (survival rate of control group - survival rate of treatment group) x 100 / survival rate of control group, where the control group is treated with sterilized water, and the treatment group is treated with the fermentation supernatant.

7. The method for screening endophytic bacteria resistant to garlic root-knot nematodes according to claim 1, strains corresponding to a supernatant with a corrected death rate over 80% are selected;7. The method for screening endophytic bacteria resistant to garlic root-knot nematodes according to claim 1, strains corresponding to a supernatant with a corrected death rate over 80% are selected;

8. The method for screening endophytic bacteria resistant to garlic root-knot nematodes according to claim 1, strains corresponding to a supernatant with a corrected death rate over 90% are selected.8. The method for screening endophytic bacteria resistant to garlic root-knot nematodes according to claim 1, strains corresponding to a supernatant with a corrected death rate over 90% are selected.

REVENDICATIONS 7500699CLAIMS 7500699

1. Procédé de criblage de bactéries endophytes résistante aux nematodes à galles de l'ail, caractérisé en ce qu’il est, des colonies uniques de différentes morphologies sont obtenues par isolement et purification à partir de verticilles de racines d'ail infestés de nématodes à galles, les colonies uniques obtenues sont inoculées dans un milieu liquide LB pour la fermentation, le surnageant de fermentation est obtenu, des nématodes à galles vivants sont inoculés dans le surnageant de fermentation, le nombre de décès de nématodes est compté après 24 h, le taux de mortalité corrigé des nématodes est calculé, et la souche correspondant au surnageant avec un taux de mortalité corrigé de >70% a été sélectionnée comme la bactérie endophyte résistante aux nématodes à galles de l'ail.1. A method for screening endophytic bacteria resistant to garlic root-knot nematodes, characterized in that it is, single colonies of different morphologies are obtained by isolation and purification from root whorls of garlic infested with nematodes root-knot nematodes, the single colonies obtained are inoculated into LB liquid medium for fermentation, the fermentation supernatant is obtained, live root-knot nematodes are inoculated into the fermentation supernatant, the number of nematode deaths is counted after 24 h, the corrected nematode death rate is calculated, and the strain corresponding to the supernatant with a corrected death rate of >70% was selected as the endophytic bacterium resistant to garlic root-knot nematodes.

2. Procédé de criblage de bactéries endophytes résistante aux nématodes à galles de l'ail selon la revendication 1, caractérisé en ce qu’il est, le procédé d'isolement et de purification de colonies uniques de bactéries morphologiquement distinctes à partir de verticilles de racines d'ail infestés de nématodes à galles consiste à : les perles de racine d'ail affectées par les nématodes des racines ont été désinfectées, rincées, broyées en une pâte et ajoutées à de l'eau stérile ; elles ont été mélangées soigneusement et laissées au repos ; la solution diluée a été appliquée uniformément sur le milieu LBA et incubée à 37°C pendant 2-3 jours ; des colonies uniques avec une morphologie différente ont été isolées et purifiées par marquage de la plaque et les souches obtenues ont été transférées sur le milieu LBA pour la culture en pente et stockées à 4°C.2. Method for screening endophytic bacteria resistant to garlic gall nematodes according to claim 1, characterized in that it is the method for isolating and purifying single colonies of morphologically distinct bacteria from whorls of garlic roots infested with root-knot nematodes consists of: garlic root beads affected by root-knot nematodes were disinfected, rinsed, ground into a paste and added to sterile water; they were mixed carefully and left to stand; the diluted solution was applied evenly on the LBA medium and incubated at 37°C for 2-3 days; single colonies with different morphology were isolated and purified by staining the plate, and the resulting strains were transferred to LBA medium for slant culture and stored at 4°C.

3. Procédé de criblage de bactéries endophytes résistante aux nématodes à galles de l'ail selon la revendication 1, caractérisé en ce qu’il est, lesdits nématodes à galles vivants sont obtenus par : Les masses d'œufs matures de nématodes à galles du sud ont été prélevées, désinfectées et rincées, puis incubées dans des boîtes de pétri stérilisées avec une petite quantité d'eau stérile à une température constante de 28°C, les nématodes à galles vivants ont été collectés après l'éclosion.3. A method of screening for endophytic bacteria resistant to garlic root-knot nematodes according to claim 1, characterized in that it is, said living root-knot nematodes are obtained by: The masses of mature eggs of root-knot nematodes south were collected, disinfected and rinsed, then incubated in sterilized petri dishes with a small amount of sterile water at a constant temperature of 28°C, live root-knot nematodes were collected after hatching.

4. Procédé de criblage de bactéries endophytes résistante aux nématodes à galles de l'ail selon la revendication 1, caractérisé en ce qu’il est, ledit surnageant de fermentation est obtenu en transférant une seule colonie sur un milieu liquide LB à 37°C, 200r pendant 48h ; après fermentation, le bouillon de fermentation est prélevé et centrifugé à 9000 rmp pendant 10min pour obtenir le surnageant de fermentation.4. Method for screening endophytic bacteria resistant to garlic root-knot nematodes according to claim 1, characterized in that it is, said fermentation supernatant is obtained by transferring a single colony on an LB liquid medium at 37°C , 200r for 48h; after fermentation, the fermentation broth is removed and centrifuged at 9000 rmp for 10 min to obtain the fermentation supernatant.

5. Procédé de criblage de bactéries endophytes résistante aux nématodes à galles de l'ail selon la revendication 1, caractérisé en ce que lesdits nématodes à galles vivants sont inoculés avec 50 bandes/mL de surnageant de fermentation.5. Method for screening endophytic bacteria resistant to garlic root-knot nematodes according to claim 1, characterized in that said live root-knot nematodes are inoculated with 50 bands/mL of fermentation supernatant.

6. Procédé de criblage de bactéries endophytes résistante aux nématodes à galles de l'ail selon la revendication 1, caractérisé en ce qu’il est, ledit taux de mortalité corrigé est calculé par la formule suivante : taux de mortalité corrigé = (taux de survie du témoin - taux de survie du traitement) x 100/taux de survie du témoin.6. Method for screening endophytic bacteria resistant to garlic root-knot nematodes according to claim 1, characterized in that it is, said corrected mortality rate is calculated by the following formula: corrected mortality rate = (rate of control survival - treatment survival rate) x 100/control survival rate.

dans lequel ledit groupe témoin est un traitement à l'eau stérile et ledit groupe de traitement est un traitement au surnageant de fermentation.wherein said control group is a sterile water treatment and said treatment group is a fermentation supernatant treatment.

7. Procédé de criblage de bactéries endophytes résistante aux nématodes à galles de l'ail selon l'une quelconque des revendications 1 à 6, caractérisé en ce qu’il est, l'on crible les souches correspondant aux surnageants ayant un taux de mortalité corrigé >80%.7. Method for screening endophyte bacteria resistant to garlic gall nematodes according to any one of claims 1 to 6, characterized in that it is, one screens the strains corresponding to the supernatants having a mortality rate corrected >80%.

8. Procédé de criblage de bactéries endophytes résistante aux nématodes à galles de l'ail selon la revendication 7, caractérisé en ce qu’il est, les souches correspondant aux surnageants avec un taux de mortalité corrigé >90% sont criblées.8. Method for screening endophyte bacteria resistant to garlic gall nematodes according to claim 7, characterized in that it is, the strains corresponding to the supernatants with a corrected mortality rate >90% are screened.