CN110628664A

CN110628664A - Pseudomonas eastern China for preventing and treating root-knot nematode as well as preparation method and application thereof

Info

Publication number: CN110628664A
Application number: CN201910752350.6A
Authority: CN
Inventors: 李林; 张润; 丁美娟; 孙孝文; 刘永轩
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2019-08-15
Filing date: 2019-08-15
Publication date: 2019-12-31
Anticipated expiration: 2039-08-15
Also published as: CN110628664B

Abstract

The invention belongs to the technical field of microbial pesticides, and particularly relates to pseudomonas far east for preventing and treating root-knot nematodes, and a preparation method and application thereof. The strain is pseudomonas far eastern (A), (B) and (C)Pseudomonas extremorientalis) MB751 is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M2019495 in 6 and 25 months in 2019, is separated from plant rhizosphere soil, has obvious control effect on Meloidogyne incognita, has growth promoting effect on plants, has wide salt tolerance range, is prepared by conventional liquid fermentation culture, has simple production process and does not pollute the environment.

Description

Pseudomonas eastern China for preventing and treating root-knot nematode as well as preparation method and application thereof

Technical Field

The invention belongs to the technical field of microbial pesticide, and particularly relates to pseudomonas far east for preventing and treating root-knot nematode, and a preparation method and application thereof.

Background

Food is essential for human survival. However, annual losses of food and vegetable crops caused by parasitic nematodes are very large in the whole agricultural production. Therefore, the prevention and treatment of root-knot nematodes is urgent. At present, chemical control is commonly applied to the control of root-knot nematodes. However, the chemical nematicide has serious environmental pollution, is harmful to human and animals in the using process, has the defects of high residual rate, damage to ecological balance and the like although playing a poisoning role on the nematodes, and is not beneficial to the development of agricultural production. Therefore, research efforts to address safe and efficient control methods have met the concept of sustainable agricultural development, such as biological control technologies.

At present, bacteria for biologically preventing and treating root-knot nematodes comprise rhizosphere bacteria, endophytic bacteria and obligate parasitic bacteria, the rhizosphere bacteria are relatively hot to research, the endophytic bacteria research is relatively few, the parasitic bacteria limit is large, the rhizosphere bacteria can promote plants to quickly absorb and utilize mineral nutrition, so that the growth of the plants is promoted, the cultivation is convenient, the colonization is easy, a strong lethal effect is provided for plant parasitic nematodes, and the method has good research application advantages and market development potential. The research is now more extensive in the genus Bacillus and Pseudomonas. The pseudomonas is an important component of soil, the microbial community of the pseudomonas plays a leading role in arable land, and the pseudomonas can dissolve minerals and provide other important nutrient substances for the root system of plants to absorb. On one hand, the pseudomonas can promote the seed germination and root growth of the plant so as to improve the absorption of the root to mineral nutrition, moisture and the like, and on the other hand, the secondary metabolite of the pseudomonas can inhibit the propagation of plant parasitic nematodes in soil so as to indirectly promote the plant growth. Therefore, the pseudomonas becomes a biological control resource with great application potential.

Disclosure of Invention

The invention aims to separate and obtain a new far east pseudomonad strain with the function of preventing and treating root-knot nematode, and the far east pseudomonad strain preparation for preventing and treating root-knot nematode is prepared by fermentation of the strain, the prevention and treatment effect can reach 80.43 percent, and the plant growth can be promoted.

The technical scheme of the invention is as follows:

the applicant obtains a far-east pseudomonad with high-efficiency control effect on root-knot nematodes by separating and screening in Kuerler orchard in Xinjiang in 2017, the strain is named as far-east pseudomonad (Pseudomonas extremomentalis) MB751, and the strain is delivered to a China center for type culture collection for collection in 2019 in 25 th month, wherein the collection address is Wuhan-Wuhan university, and the collection number is CCTCC NO: M2019495.

Pseudomonas eastern distal MB751 strain has the following distinct characteristics:

(1) the strain belongs to the pseudomonas, is gram-negative, rod-shaped and has no spore; on an LB solid culture medium, bacterial colonies are protruded, are in light yellow and are neat in edge; an obvious biological membrane can be formed after shaking culture for 8 hours in an LB liquid culture medium.

(2) The strain fermentation culture and the fermentation supernatant thereof have high-efficiency control effect on meloidogyne incognita and promote plant growth.

(3) The salinity tolerance range of the strain is wide, and the strain has high biomass between 1% and 7% of salinity.

The invention provides an industrial fermentation culture medium of pseudomonas far east MB751, the formula of which is glucose 12.46g/L, corn flour 15.13g/L, soybean cake flour 4.00g/L, MgSO₄ 0.30g/L、MnSO₄ 0.04g/L、NaH₂PO₄ 0.30g/L、Na₂HPO₄1.50g/L, pH 8.42. After shaking culture at 28 deg.C for 24 hr, the maximum viable count can reach 1.57 × 10¹¹CFU/mL, 3.5-fold higher than the initial fermentation medium activity.

Compared with the prior art, the invention has the following advantages and beneficial effects:

the pseudomonas far east MB751 disclosed by the invention is derived from plant rhizosphere soil, not only can kill root-knot nematodes, but also can effectively promote the growth of plants, has no adverse effect on the environment and all organisms such as plants, people and animals, and is wide in salt tolerance range, good in growth vigor, low in production cost, simple in production process, free of pollution to the environment and suitable for popularization and use.

Drawings

FIG. 1 is a microscopic morphology of Pseudomonas fardonae MB 751.

FIG. 2 shows the colony morphology of Pseudomonas fardonae MB 751.

FIG. 3 shows a phylogenetic tree of Pseudomonas fardonae MB 751.

Fig. 4 shows meloidogyne incognita after treatment with pseudomonas distans MB751 and e.coli OP 50.

FIG. 5 shows the determination of the semi-lethal concentration of M.farndodonicus MB751 on Meloidogyne incognita.

Fig. 6 shows tomato roots after root irrigation with pseudomonas MB 751.

FIG. 7 shows the effect of different salinity on the growth of M.distans MB 751.

Detailed Description

The experimental procedures used in the following examples are conventional unless otherwise specified.

Example 1 isolation and characterization of Pseudomonas distans MB751

1. Collecting soil 0-15cm away from the ground surface in a Kuerle orchard in Xinjiang in 2017, removing sundries such as dead branches and stones, drying at room temperature, grinding, crushing, sieving with a 40-mesh sieve, and separating plant rhizosphere soil bacteria by using a gradient dilution method. 10g of soil is taken and dissolved in 90mL of sterile water, and the mixture is shaken uniformly. Adding 100 μ L of the soil turbid solution into a centrifuge tube containing 900 μ L of sterile water, and uniformly oscillating (20sec) by using a rapid vortex oscillator; dilution 10 in sequence²，10³，10⁴，10⁵，10⁶，10⁷And (4) doubling. The diluted solution in the dilution tube was applied to beef extract peptone (NA) culture plates at 100. mu.L each, and incubated at 28 ℃ for 48 hours. The isolated single colonies were purified by streaking.

2. The separated and purified pseudomonas far east MB751 is streaked and transferred to an LB solid culture medium to be cultured for 24h, and the colony morphology is observed; then, a single colony was picked up and subjected to gram staining observation. As shown in FIG. 1, Pseudomonas fardonae MB751 was gram-negative and rod-shaped as observed by a microscope after gram staining. FIG. 2 is a morphological diagram of a colony of Pseudomonas MB751 cultured on LB solid medium for 24h, which is round, yellowish, smooth in surface, neat in edge, convex in middle, and easy to pick.

3. The 16S rRNA gene of MB751 strain was PCR amplified and sequenced, the resulting DNA fragment was ligated to T-vector using T-vector kit, positive clones were selected and sent to the Progesteron organism company for sequence sequencing, the sequence is shown in SEQ ID NO 1. The target fragment was cut out based on the primers, and the sequence similarity alignment was carried out in EzbioCloud, and the strain MB751 had 99.79% similarity to Pseudomonas aeruginosa (Pseudomonas exotriementalis). The isolated strain MB751 was identified as Pseudomonas aeruginosa (Pseudomonas extorementalis) based on morphological observation, partial physiological and biochemical determination, and 16S rRNA gene sequencing and sequence alignment analysis. The applicant has been deposited by China Center for Type Culture Collection (CCTCC) in 2019 at 25.6.M, and the deposited number is CCTCC NO: M2019495.

Example 2 preparation of Pseudomonas fardonae MB751

1. Activation of pseudomonas far east MB751 strain: 10.00g of sodium chloride, 10.00g of peptone, 5.00g of yeast powder, distilled water to a constant volume of 1.00L and a pH of 7.0, selecting a single colony from a solid plate for culturing MB751 to a PA bottle containing 5.0mL of the liquid culture medium under aseptic operation, and carrying out culture at 28 ℃ and 160 r.min^-1Culturing for about 8 h.

2. Liquid amplification culture: inoculating to a triangular flask containing 200mL LB liquid culture medium at 28 deg.C and 160 r.min in an amount of 1%^-1And carrying out amplification culture for 24 h.

3. Optimization test of industrial fermentation medium:

1) single factor experiments: MB751 strain was inoculated into a basal medium under initial conditions of medium initial pH 7.0, inoculum size 1%, culture temperature 28 ℃ and rotation speed 150 rpm. Glucose, yeast powder, soluble starch, sucrose, corn flour and lactose are respectively used as carbon sources in a basic culture medium, the influence of different carbon sources on the yield of MB751 is researched, and the optimal carbon source is selected.

Taking the yield of MB751 strain as a detection index, when the nitrogen source is soybean cake powder, soybean meal powder, soybean peptone, peanut cake powder, tryptone, (NH4)₂SO₄In time, different nitrogen sources were investigated for MB751 productionQuantity and selecting the most suitable nitrogen source.

The yield of MB751 strain is used as a detection index, and the optimal fermentation process parameters of the culture medium are selected. When the initial pH is 5, 6, 7, 8 and 9 respectively, investigating the change condition of viable count, and selecting the optimal pH; when the initial inoculation amount is 1%, 2%, 3%, 4% and 5%, the change condition of viable count is inspected, and the optimal viable count is selected. When the temperature was 16 ℃, 20 ℃, 28 ℃, 30 ℃ and 37 ℃, respectively, the change of viable cell count was examined and the optimum temperature was selected.

2) Plackett-Burman experiment: the biomass of MB751 was optimized using an eight-factor two-level design to yield three most significant factors, glucose, corn meal and pH.

3) Design of steepest climbing experiment: the Plackett-Burman design analysis was performed by SAS 9.2 analysis software, and 3 factors (glucose, corn meal, and pH) having the greatest influence were selected as the direction of greatest growth by the steepest hill path method. The biomass of MB751 was optimized using the steepest hill path test with the number of three-factor tests N-7.

4) Response surface Box-Behnken experiment: the optimal combination of 3 factors (glucose, corn flour and pH) with the largest influence is selected as 3 factors of the BOX-Behnken test through the result of the steepest climbing path test, each factor is respectively set to be low (-1), medium (0) and high (1)3 levels, and the BOX-Behnken test design with three factors 3 levels is adopted to optimize the biomass of MB 751. Finally, the optimal industrial fermentation medium formula is obtained: 12.46g/L glucose, 15.13g/L corn flour and 4.00g/L, MgSO g soybean cake flour₄ 0.30g/L、MnSO₄ 0.04g/L、NaH₂PO₄ 0.30g/L、Na₂HPO₄1.50 g/L. Fermentation process parameters: pH 8.42, inoculum size 2%, shaker temperature 28 ℃. The optimum fermentation medium activity is 1.57X 10¹¹CFU/mL, 3.52X 10 activity over the initial fermentation medium¹⁰CFU/mL increased by 3.5-fold.

4. Preparation of a bacterial suspension of Pseudomonas fardonae MB 751: after 24h of scale-up culture, 12000 r.min^-1Centrifuging for 2min, and obtaining the precipitate as thallus. The cells were washed four times with PBS and suspendedFloating thalli, namely the thalli sample required by the experiment bioassay.

5. Preparation of fermentation supernatant of Pseudomonas fardonae MB 751: the culture solution after 72h of amplification culture is subjected to 8000r min^-1Centrifuging for 10min, and collecting supernatant as fermentation supernatant sample.

6. And (3) measuring the concentration of the sample to be measured: OD values of pseudomonas MB751 strain and e.coli OP 50; the fermentation supernatant was assayed for protein concentration using the Coomassie Brilliant blue method.

Example 3 Pseudomonas fardonae MB751 Activity against Meloidogyne incognita

1. Preparing meloidogyne incognita eggs: collecting tomato root infected by root-knot nematode, washing surface silt with clear water, removing egg mass from root with fine needle, shaking with 1% sodium hypochlorite for 5min, centrifuging (8000 r.min)^-1And 1min), discarding the supernatant, and repeatedly washing for more than three times to obtain a large amount of worm eggs.

2. Preparing second-instar larvae of meloidogyne incognita: suspending the obtained ovum with sterile water, placing in 96-well plate, incubating at 25 deg.C for 4d with 200 μ L ovum suspension per well to obtain large amount of second instar larva, and determining toxicity.

3. Activity assay of MB751 against second instar larvae of root-knot nematodes: the assay was performed in 96 well plates with 50 instar larvae per well, 150. mu.L samples, 10mg/ml chloramphenicol 0.6. mu.L, 3 replicates per sample, and the control group used OP50 instead of samples. The method for identifying nematode death comprises the following steps: after 24 hours of action, the dead insects are treated by 2 percent sodium chloride solution for about 2min, and the dead insects are stiff and immobile, and the live insects curl or twist.

TABLE 1 poisoning Effect of Pseudomonas distans MB751 on Meloidogyne incognita

Sample (I)	Concentration of	Relative lethality of Meloidogyne incognita
			Coli OP50 strain control	OD_600nm＝0.60	2.37±1.38％
MB751 bacterial cell	OD_600nm＝0.60	77.98±1.73％
			MB751 fermentation supernatant	1800μg·mL^-1	79.94±7.69％

The results show that the pseudomonas far east MB751 thallus and the fermentation supernatant have better poisoning effect on the meloidogyne incognita. After the thalli is used for killing the meloidogyne incognita for 24 hours, the lethality rate reaches 77.98%. As shown in fig. 4, after treatment with pseudomonas far east MB751, both meloidogyne incognita died, while after treatment with control e.coli OP50, there was almost no mortality. As shown in FIG. 5, the semilethal concentration (LC) of MB751 was measured₅₀) 0.29mg/mL (0.228-0.349) (dry weight of the bacteria), linear regression equation: Y3.141X-7.741. The lethality of the fermentation supernatant reaches 79.94 percent, which indicates that the metabolite of the pseudomonas far east MB751 has better poisoning effect on meloidogyne incognita.

Example 4 potting application of Pseudomonas fardonae MB751 to control Meloidogyne incognita infection of tomato

Pot experiment: is carried out under the temperature condition of 25 +/-3 ℃. Culturing tomato in seedling tray to four-leaf stage, transferring to 13cm × 12cm black plastic pot containing sterile soil, culturing, and planting, irrigating tomato root system with MB751 fermentation broth 10m L (10 days)⁸CFU·m L^-1) And pouring the same volume of industrial fermentation culture medium by contrast treatmentAnd inoculating about 1000 southern root knot nematode second-instar larvae (RKN-J2) to the tomato root system after 3 days. Measuring the plant shape of the planted seedlings 40 days after the application of the bacteria: plant height, stem thickness, root length, fresh weight of aerial parts, fresh weight of root systems, dry weight of aerial parts and dry weight of root systems. And (5) counting the root knots to calculate the prevention and treatment effect. Each treatment was repeated 5 times, each was repeated 1 tomato, and the experiment was repeated 3 times.

TABLE 2 control Effect of Pseudomonas distans MB751 on Meloidogyne incognita

Treatment of	Index of disease condition	Controlling effect
			Media controls	61.33％	-
MB751 microbial inoculum	12.12％	80.43％

The result shows that the number of root knots on the tomato root system is obviously reduced after the fermentation liquor of the pseudomonas fardonalis MB751 is irrigated, and the prevention and treatment effect of the MB751 obtained by calculation reaches 80.43 percent.

TABLE 3 growth-promoting potting experiment results for Pseudomonas far east MB751

Through a pot experiment, the plant height, stem thickness, root length, overground fresh weight, root fresh weight, overground dry weight and root dry weight of the plant are measured 40 days after the application of the bacteria. The results show that the tomato irrigated by the pseudomonas far east MB751 has significant increase (p is less than 0.05) in various characters compared with the control group, the plant height is increased by 37.51%, the stem thickness is increased by 46.45%, the root length is increased by 13.88%, the fresh weight on the ground is increased by 66.67%, the fresh weight under the ground is increased by 27.42%, the dry weight on the ground is increased by 1.375 times, and the dry weight under the ground is increased by 2.21 times. FIG. 6 shows the roots of the control and far east Pseudomonas MB751 irrigated plants, which were more rooted, more damaged by nematode infestation, and more numerous eggs; in contrast, the tomato roots after being irrigated by the pseudomonas fardonae MB751 are rarely rooted and are slightly damaged by nematodes, which shows that the pseudomonas fardonae MB751 has an obvious control effect on the meloidogyne incognita.

Example 5 Pseudomonas far east MB751 salt tolerance test

In LB medium, the salinity (in NaCl) was designed to be 0, 1%, 3%, 5%, 7%, 8%, 10%, respectively, and the absorbance was measured by culturing for 48 hours with MB03 (Pseudomonas syringae) and OP50 (E.coli) as controls. Each experiment was repeated 3 times.

As shown in FIG. 7, by culturing the microorganism in a shaking table for 48 hours and measuring the absorbance, the Pseudomonas distans MB751 has the strongest strain growth in different salinity ranges and has a larger tolerance range to salinity, and can have a higher biomass in salinity of 1% -7% compared with the control. The OD of the most suitable growth salinity (NaCl is 3%) of the Pseudomonas far east MB751 cultured for 48h_600nmIs 1.9925. The results show that the bacterial agent prepared from MB751 has a large salt tolerance range and can be popularized and applied in actual production.

Sequence listing

<110> university of agriculture in Huazhong

<120> Pseudomonas fardongensis for preventing and treating root-knot nematode as well as preparation method and application thereof

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agagtttgat catggctcag agcgaacgct ggcggcaggc ctaacacatg caagtcgaac 60

ggacccttcg gggttagtgg cggacgggtg agtaacacgt gggaacgtgc ctttaggttc 120

ggaatagctc ctggaaacgg gtggtaatgc cgaatgtgcc cttcggggga aagatttatc 180

gcctttagag cggcccgcgt ctgattagct agttggtgag gtaatggctc accaaggcga 240

cgatcagtag ctggtctgag aggatgacca gccacattgg gactgagaca cggcccaaac 300

tcctacggga ggcagcagtg gggaatcttg cgcaatgggc gaaagcctga cgcagccatg 360

ccgcgtgaat gatgaaggtc ttaggattgt aaaattcttt caccggggac gataatgacg 420

gtacccggag aagaagcccc ggctaacttc gtgccagcag ccgcggtaat acgaaggggg 480

ctagcgttgc tcggaattac tgggcgtaaa gggcgcgtag gcggacattt aagtcagggg 540

tgaaatccca gagctcaact ctggaactgc ctttgatact gggtgtcttg agtgtgagag 600

aggtatgtgg aactccgagt gtagaggtga aattcgtaga tattcggaag aacaccagtg 660

gcgaaggcga catactggct cattactgac gctgaggcgc gaaagcgtgg ggagcaaaca 720

ggattagata ccctggtagt ccacgccgta aacgatgatt gctagttgtc gggctgcatg 780

cagttcggtg acgcagctaa cgcattaagc aatccgcctg gggagtacgg tcgcaagatt 840

aaaactcaaa ggaattgacg ggggcccgca caagcggtgg agcatgtggt ttaattcgaa 900

gcaacgcgca gaaccttacc accttttgac atgcctggac cgccagagag atctggcttt 960

ctcttcggag actaggacac aggtgctgca tggctgtcgt cagctcgtgt cgtgagatgt 1020

tgggttaagt cccgcaacga gcgcaaccct cgccattagt tgccatcatt tagttgggaa 1080

ctctaatggg actgccggtg ctaagccgga ggaaggtggg gatgacgtca agtcctcatg 1140

gcccttacag ggtgggctac acacgtgcta caatggcgac tacagagggt taatccttaa 1200

aagtcgtctc agttcggatt gtcctctgca actcgagggc atgaagttgg aatcgctagt 1260

aatcgcggat cagcatgccg cggtgaatac gttcccgggc cttgtacaca ccgcccgtca 1320

caccatggga gttggttcta cccgaaggcg atgcgctaac cagcaatgga ggcagtcgac 1380

cacggtaggg tcagcgactg gggtgaagtc gtaacaaggt aa 1422

Claims

1. The Pseudomonas far east for preventing and treating the root-knot nematode is characterized in that the strain is Pseudomonas far east (Pseudomonas extorementalis) MB751 with the preservation number of CCTCC NO: M2019495.

2. Use of pseudomonas eastern distal as claimed in claim 1 for the control of root-knot nematodes.

3. A rhizomatoid control inoculant produced by Pseudomonas distans according to claim 1.

4. The inoculant for controlling meloidogyne prepared by pseudomonas fardonae according to claim 3, wherein the active ingredient of the inoculant is a fermentation culture or fermentation supernatant of pseudomonas fardonae MB 751.

5. The fermentation culture medium of pseudomonas distal east of claim 1, wherein the culture medium formula comprises glucose 12.46g/L, corn flour 15.13g/L, soybean cake flour 4.00g/L, MgSO₄ 0.30g/L，MnSO₄ 0.04g/L，NaH₂PO₄0.30g/L，Na₂HPO₄ 1.50g/L。