CN111961618A

CN111961618A - Strain for antagonizing watermelon and muskmelon fruit blotch and application thereof

Info

Publication number: CN111961618A
Application number: CN202010819257.5A
Authority: CN
Inventors: 张建; 王朋成; 田红梅; 陶珍; 许钢; 王明霞; 王艳; 王家嘉; 朱培蕾; 赵贵云
Original assignee: Institute of Gardening of Anhui Academy Agricultural Sciences
Current assignee: Institute of Gardening of Anhui Academy Agricultural Sciences
Priority date: 2020-08-14
Filing date: 2020-08-14
Publication date: 2020-11-20
Anticipated expiration: 2040-08-14
Also published as: CN111961618B

Abstract

The invention discloses a muskmelon fruit blotch antagonistic strain and application thereof. Belongs to the technical field of biological control. Comprises Burkholderia plantarii N01(Burkholderia sp.N01) with the preservation number of CCTCC NO: m2020248, and provides a method for preparing the biocontrol bacterial agent and an application of the biocontrol bacterial agent in optimizing a seedling substrate by combining with a nutritional supplement. The strain provided by the invention has the characteristics of inhibiting the growth of the fruit blotch germs well and tolerating heavy metal cadmium ions. After the muskmelon fruit spot antagonistic bacteria are cultured and propagated, the biological control microbial inoculum can be prepared, a seedling culture substrate containing the strain or the biological control microbial inoculum is further prepared, and the components of the seedling culture substrate are optimized by adding a nutritional supplement, so that the muskmelon fruit spot disease is effectively reduced, and the nutrient requirement of muskmelon growth in the seedling stage is met.

Description

Strain for antagonizing watermelon and muskmelon fruit blotch and application thereof

Technical Field

The invention relates to the technical field of biological control, in particular to a muskmelon fruit blotch antagonistic strain and application thereof.

Background

Bacterial Fruit Blotch (BFB) of melons is the most important destructive seed-borne Bacterial quarantine disease on watermelons and melons, and the pathogenic bacterium is acidophilic bacteria (Acidovorax citrulli). The sigatoka of melon occurs in countries such as the united states, australia, korea, japan, turkey, and thailand. The planting area and the yield of watermelons and melons in China are not only in the front of the world, but also are important seed production bases of the watermelons and the melons in China. Since the disease is first reported in China in 1998, the disease has occurred in large area in China and is in an ascending trend, so that the yield of field watermelons and melons is reduced, even the field melons and melons are absolutely harvested, and huge loss is caused to the watermelon and melon planting industry. How to effectively reduce the incidence rate of fruit blotch and reduce the influence of the disease on the yield and the quality of the watermelon and melon becomes one of the important problems to be solved urgently in the production of the watermelon and melon planting industry in China at present. The disease can cause plant fruit spots and water-based diseases, the commodity attribute and the quality of the watermelon and melon are obviously reduced, the incidence rate of a seriously ill field reaches 80-98.5%, and the yield of the watermelon and melon fruits is seriously reduced. The disease not only causes great economic loss to watermelon and muskmelon farmers, but also seriously affects the healthy development of the watermelon and muskmelon industry.

At present, methods for preventing and treating the diseases mainly comprise breeding disease-resistant varieties, seed treatment, agricultural prevention and treatment, chemical prevention and treatment and the like.

However, the watermelon and muskmelon fruit blotch lacks effective chemical agents, the fruit blotch germs are easy to generate drug resistance to chemical pesticides, and the environmental pollution and the production cost increase are caused by using a large amount of chemical pesticides.

The biological control of crop diseases has the advantages of abundant available resources, low cost and capability of avoiding secondary environmental pollution, and is considered as a promising approach for reducing plant diseases.

Therefore, the problem to be solved by those skilled in the art is how to utilize pathogenic bacteria antagonistic microorganisms for biological control and further optimize antagonistic seedling substrate components.

Disclosure of Invention

In view of the above, the invention provides a muskmelon fruit blotch antagonistic strain and application thereof.

Burkholderia N01(Burkholderia sp. N01) isolated from soil samples of cucurbita pepo in each region of anhui in 2018 to 2020 in 5 months. The bacterium is Burkholderia sp (Burkholderia sp.) as determined by sequencing identification and final analysis. The new strain is preserved in China Center for Type Culture Collection (CCTCC) at 30/6/2020, with the preservation number being CCTCC NO: m2020248. The address of the collection center: china, wuhan university.

The invention utilizes the characteristic of good pathogenic bacterium antagonism of Burkholderia N01 for antagonizing watermelon and muskmelon fruit blotch to prepare the biocontrol microbial inoculum, is applied to the production of (antagonistic) seedling substrate, prepares the substrate for antagonizing the watermelon and muskmelon fruit blotch of plant diseases in a compounding way, perfects and optimizes the components of the seedling substrate, meets the antibacterial requirement and the nutrient absorption of plants, and has better effects of economy, environmental pollution prevention and plant disease prevention and control of watermelon and muskmelon fruit blotch. Is suitable for large-scale agricultural production and can improve the agricultural benefit.

In order to achieve the purpose, the invention adopts the following technical scheme:

burkholderia plantarii N01(Burkholderia sp.N01) with the preservation number of CCTCC NO: m2020248

The invention also provides a preparation method of the biocontrol microbial inoculum, which comprises the following steps:

(1) mixing 1-10% by mass volume of Burkholderia N01, a liquid culture medium, inorganic salt calcium, magnesium and phosphorus, performing liquid shake flask fermentation, culturing at 25-35 ℃ and rotating at 120-220 rpm to obtain a seed solution;

(2) and (3) amplification culture: the raw materials comprise 1-5 wt% of bran, 0-3 wt% of vegetable cake, 0-3 wt% of bean cake powder, inorganic salt calcium, magnesium and phosphorus, the water addition amount is 99-89%, and the raw materials are stirred and mixed to obtain a mixed solution; after the mixture is sterilized under high pressure at 115-120 ℃, when the temperature of the mixture is cooled to below 35 ℃, the seed solution is inoculated in an aseptic operation mode according to the inoculum size of 1-10% by mass volume, the positive pressure environment of more than 0.01MPa is always kept, the aerated culture is carried out for 24-96 hours at 25-35 ℃, and the number of fermentation bacteria reaches 10⁸More than cfu/ml, wherein in the steps (1) and (2), the final concentration of calcium is 0.9-1.5 g/L, the final concentration of magnesium is 0.5-1.3 g/L, and the final concentration of phosphorus is 0.8-2.5 g/L; and finally, aseptically packaging to obtain the microbial inoculum.

Liquid culture medium: 5.0g of glucose, 1.0g of casein hydrolysate, 10.0g of bacterial grade peptone, 5ml of glycerol and 1000ml of distilled water, wherein the pH value is 7.0-7.2. Sterilizing with high pressure and moist heat at 115.0 deg.C for 25 min.

Has the advantages that: the cost is reduced, and meanwhile, the antagonistic bacteria is propagated to the maximum extent.

The invention also provides an application of the seedling substrate and the nutritional supplement in the prevention and treatment of watermelon and melon fruit blotch, wherein the seedling substrate contains the Burkholderia strain N01 or the prepared biocontrol microbial inoculum.

Preferably: the preparation method of the seedling substrate comprises the following steps: respectively mixing mushroom residue clinker, turf, vermiculite and perlite according to the volume percentage of 45: 35: 13: 7, mixing uniformly according to a ratio of 3kg/m³Adding a biocontrol microbial inoculum containing the Burkholderia strain N01, uniformly mixing, and adjusting the water content of the substrate to 30-40% by using tap water to ensure that the number of Burkholderia contained in the substrate reaches 10⁷The seedling culture substrate is obtained above cfu/g.

The preparation method of the mushroom residue rotten clinker comprises the following steps:

(1) preparing mushroom residues: preparing 20% of sawdust, 70% of cottonseed hulls and 10% of auxiliary materials into a mushroom culture medium according to the mass percentage, planting mushrooms on the mushroom culture medium, and crushing the mushroom culture medium after the mushrooms are planted into particles of 2-3 cm, namely mushroom residues; the auxiliary materials are prepared from bran, lime and gypsum according to the mass percentage of 8:1: 1;

(2) harmless treatment of mushroom residues: adding a nitrogen source into the mushroom residues by using a composting principle, adjusting the carbon-nitrogen ratio, adding water to adjust the water content of the mushroom residues to be 55-65%, and adjusting the water content to be 2kg/m³Adding a fermentation inoculum for composting, piling, and turning and uniformly mixing 3-5 times during the high-temperature process of 55-65 ℃ for 30-35 days to obtain mushroom residue rotten materials; and keeping the water content of the mushroom residue decomposed material to be 55-60%;

wherein the mushroom residue is mushroom culture medium after planting Lentinus Edodes or needle mushroom.

Preferably: in combination with a nutritional supplement: according to the hole number of the hole tray used for planting the watermelon and muskmelon, a mixture of nitrogen, phosphorus and potassium is supplemented, and any one of the following schemes is adopted in sequence:

if the seedling raising substrate is a 32-hole plug tray, the mass ratio of the nitrogen, phosphorus and potassium mixture to the seedling raising substrate is 4.8-5.2 g/kg, wherein the mass ratio of nitrogen: phosphorus: the mass ratio of potassium is 2.1-2.3: 3-3.2: 4.1 to 4.3;

and if the number of the holes is 50, the mass ratio of the mixture of nitrogen, phosphorus and potassium to the seedling substrate is 5.8-6.2 g/kg, wherein the mass ratio of nitrogen: phosphorus: the mass ratio of potassium is 3.1-3.3: 2.1-2.3: 4.2 to 4.4;

and if the number of the holes is 60, the mass ratio of the mixture of nitrogen, phosphorus and potassium to the seedling substrate is 6.8-7.2 g/kg, wherein the mass ratio of nitrogen: phosphorus: the mass ratio of potassium is 2.4-2.6: 3.2-3.4: 4-4.2;

and if the seedling raising substrate is a 72-hole plug tray, the mass ratio of the mixture of nitrogen, phosphorus and potassium to the seedling raising substrate is 7.8-8.2 g/kg, wherein the mass ratio of nitrogen: phosphorus: the mass ratio of potassium is 3-3.2: 3.5-3.7: 4.1 to 4.3;

alternatively, the first and second electrodes may be,

the mass ratio of the nitrogen, phosphorus and potassium mixture to the seedling substrate is 8.8-9.2 g/kg, wherein the mass ratio of nitrogen: phosphorus: the mass ratio of potassium is 2.8-3: 3.4-3.6: 6 to 6.2.

Has the advantages that: the nutrient proportion in the matrix can be fully adjusted according to the planting condition, and the melon growth is facilitated.

The total nutrients are different, and the proportion of the three elements needs to be adjusted so as to change the nutrient absorption effect of the melons.

if the number of the holes is 32, the mass ratio of the nitrogen, phosphorus and potassium mixture to the seedling substrate is 5g/kg, wherein the mass ratio of nitrogen: phosphorus: the mass ratio of potassium is 2.2: 3.1: 4.2;

if the number of the holes is 50, the mass ratio of the mixture of nitrogen, phosphorus and potassium to the seedling substrate is 6g/kg, wherein the mass ratio of nitrogen: phosphorus: the mass ratio of potassium is 3.2: 2.2: 4.3;

if the number of the holes is 60, the mass ratio of the mixture of nitrogen, phosphorus and potassium to the seedling substrate is 7g/kg, wherein the mass ratio of nitrogen: phosphorus: the mass ratio of potassium is 2.5: 3.3: 4.1;

if the seedling raising substrate is a 72-hole plug tray, the mass ratio of the nitrogen, phosphorus and potassium mixture to the seedling raising substrate is 8g/kg, wherein the mass ratio of nitrogen: phosphorus: the mass ratio of potassium is 3.1: 3.6: 4.2;

alternatively, the first and second electrodes may be,

the mass ratio of the nitrogen, phosphorus and potassium mixture to the seedling substrate is 9g/kg, wherein the mass ratio of nitrogen: phosphorus: the mass ratio of potassium is 2.9: 3.5: 6.1.

preferably: the potassium dihydrogen phosphate is used for supplementing phosphorus and potassium, wherein the content of P is more than or equal to 52.0 percent, and the content of K is more than or equal to 34.0 percent; potassium sulfate is used for supplementing potassium, wherein the content of K is more than or equal to 51.0 percent; the nitrogen is supplemented by urea, wherein the content of N is more than or equal to 46.0 percent.

According to the technical scheme, compared with the prior art, the invention discloses and provides the muskmelon fruit blotch antagonism strain and the application thereof, and the technical effect is that Burkholderia N01(Burkholderia sp.N01) is bred from natural definition to separation, and the plate antagonism experiment shows that the strain has good capability of inhibiting the growth of the fruit blotch germs, and meanwhile, the strain can resist heavy metal cadmium ions (200 mu g/ml), which indicates that the strain can survive in the soil polluted by the cadmium ions. Further through culturing and breeding, preparing (biological control) microbial inoculum products, utilizing mushroom dregs after edible mushroom cultivation to prepare (antagonistic) seedling raising substrates with other substrates according to a certain proportion after fermentation and decomposition. The biocontrol microbial inoculum is directly and organically combined with the seedling culture substrate, has the advantages of abundant available resources, low cost, avoidance of secondary environmental pollution and the like, and is an effective way for solving the plant disease of the watermelon and muskmelon fruit blotch.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a diagram showing the pathogenic bacteria inhibiting effect of the screened strains.

FIG. 2 is a schematic diagram of the pathogenic bacterial strain provided by the invention, wherein the left diagram a is a control group, the middle diagram b is a pathogenic bacteria group, and the right diagram c is an antagonistic bacteria N01 group.

FIG. 3 is a comparison chart of the disease incidence provided by the invention, wherein a control group, a pathogenic bacteria group and an antibacterial N01 group are arranged from left to right.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The embodiment of the invention discloses a muskmelon fruit blotch antagonistic strain and application thereof.

In the examples, the experimental materials, raw materials and equipment (such as liquid fermenters) required are all obtained from commercial sources, the brands of the experimental materials and the equipment are not required, and the experimental methods which are not mentioned are all conventional experimental methods, which are not described herein again.

Example 1

Primary screening of Burkholderia (Burkholderia sp.) strains

1 culture Medium

Casein peptone agar medium: 5.0g of glucose, 1.0g of casein hydrolysate, 10.0g of bacterial grade peptone, 5ml of glycerol, 15.0-20.0 g of agar and 1000ml of distilled water, wherein the pH value is 7.0-7.2. Sterilizing with high pressure and moist heat at 115.0 deg.C for 25 min.

2 test procedure

2.1 preparation of the culture Medium

A bacterial slant medium was prepared by preparing a casein peptone agar medium as described in 1.

2.2 preparation of soil Diluent

(1) Weighing 10g of soil sample, adding into a triangular flask containing 90ml of sterile water and 250ml of sterile water, and oscillating for 15 minutes to obtain 10g of soil sample^-1The diluted soil solution A of (1);

(2) sucking diluted soil solution A1ml, adding into 9ml sterile water 50ml triangular flask to obtain 10^-2The diluted soil solution B;

(3) sucking the diluted soil solution B1ml, adding into 9ml sterile water 50ml triangular flask to obtain 10^-3Diluted soil solution C of (1);

(4)sucking diluted soil solution C1ml, adding into 9ml sterile water 50ml triangular flask to obtain 10^-4The diluted soil solution D of (1);

(5) sucking diluted soil solution D1ml, adding into 9ml sterile water 50ml triangular flask to obtain 10^-5The diluted soil solution E of (1);

(6) the diluted soil solution E1ml was aspirated and added to a 9ml sterile water 50ml Erlenmeyer flask to obtain 10^-6Diluted soil solution F.

2.3 bacterial isolation

(7) Respectively sucking 0.2ml of each of the diluted soil solution C, the diluted soil solution D and the diluted soil solution E, adding the diluted soil solution C, the diluted soil solution D and the diluted soil solution E into a casein peptone agar medium plate for coating culture, and culturing for 2-3 days at 28-30 ℃.

The above steps of bacterial isolation were repeated 3 times, the results were combined, and the strains were screened. To obtain more strains, and simultaneously verify whether the number of the strains obtained by screening in each experiment is the same or the difference, and finally, the strains with good screening effect and higher strain activity are selected as target strains.

(8) Selecting a culture dish with 20-200 colony numbers to pick out colonies for slant culture, wherein the culture condition is to culture for 2-3 days at 28-30 ℃;

2.4 multiple soil samples

(9) And (3) taking 10 soil samples in total, and repeating the steps 2.1-2.3 for each soil sample.

2.5 results

100 strains of the bacteria were isolated as described above.

Example 2

Plate antagonism test of isolated bacteria against protospecies of watermelon muskmelon fruit blotch solanaceous blight

1. Culture medium

1.1 solid Medium

Preparing a watermelon and muskmelon fruit blotch culture medium: 0.2g of monopotassium phosphate, 0.8g of dipotassium phosphate, 5.0g of yeast extract, 1.0g of casein hydrolysate, 5.0g of glucose, 15.0g of agar and 1000ml of distilled water, wherein the pH value is 7.0-7.2. Sterilizing with high pressure and moist heat at 115.0 deg.C for 25 min.

Preparation of a casein peptone agar culture medium: the same medium as casein peptone agar medium in example 1.

1.2 liquid Medium

The liquid medium is prepared by adopting casein peptone agar medium without adding agar, and the Burkholderia and melon fruit spot pathogenic bacteria can grow on the liquid medium.

2. Test procedure

2.1 preparation of the culture Medium

Respectively preparing a muskmelon fruit blotch culture medium and a liquid culture medium according to the formula, and preparing a pathogenic bacterium eggplant fruit blotch primary bacterium inclined plane;

and (3) preparing a casein peptone agar culture medium and a liquid culture medium according to the formula respectively, and preparing the bacterial slant.

2.2 plate antagonism test

2.2.1 Strain activation

The seed activation of the watermelon muskmelon fruit blotch and bacteria is carried out: the melon fruit blotch germs and the separated bacteria are respectively transferred to the inclined plane of a casein peptone agar culture medium and cultured for 16-28 hours at the temperature of 28-30 ℃ for later use.

2.2.2 preparation of West melon fruit blotch germ plate

Inoculating the activated melon fruit blotch germ into a liquid culture medium for shake flask culture at 25-28 ℃ and 180 rpm. Pouring the culture medium of the muskmelon fruit spot disease bacteria into plates every other day, and adding 0.2ml (diluting the concentration of the muskmelon fruit spot disease bacteria to be 10) into each plate after the culture medium is solidified³～10⁴cells/ml) muskmelon fruit blotch fermentation broth (about 36 hours of culture), uniformly coating, and culturing overnight at 25-28 ℃ for later use.

2.2.3 preparation of bacterial liquid fermentation broths

After the melon fruit blotch germs are cultured for 2 days in a liquid culture medium in a shake flask, the activated and separated bacteria are transferred into the liquid culture medium for shake flask culture at the temperature of 25-28 ℃ and 180 r/min for later use every other day (about 36 hours of culture).

2.2.4 plate antagonism assay

A sterilized filter paper (diameter of 9mm) is dipped in the separated bacteria fermentation liquid and put into the watermelon and muskmelon fruit blotch germ plates, 3 different antagonistic bacteria are put on each plate, and the steps are repeated for 3 times. The experimental results are observed under the culture condition of 25-28 ℃ for about 20 hours, and the existence and the size of the inhibition zone are mainly considered.

3. Results

The 3 bacterial strains with better antagonistic effect are obtained through a plate antagonistic test, and the bacterial strain N01 is one of the bacterial strains. The experiment is carried out by coating 0.5ml of watermelon melon fruit blotch original bacteria solution on a plate, dipping the bacterial strain N01 fermentation liquid on a filter paper sheet, and simultaneously culturing.

By observing the antagonistic effect of the strain N01 on the protoplasm of the fruit blotch, the inhibition zone is obvious, which shows that the strain N01 has high growth speed, strong diffusion capacity and obvious antagonistic effect on the protoplasm of the fruit blotch (see figure 1).

The bacterium was identified as Burkholderia sp. The new strain is preserved in China Center for Type Culture Collection (CCTCC) at 6-month and 30-month in 2020, is named Burkholderia N01(Burkholderia sp.N01), and has the preservation number of CCTCC NO: m2020248.

Example 3

Biocontrol microbial inoculum prepared from bacterium N01

(1) Mixing 1% of Burkholderia N01, liquid culture medium, inorganic salt calcium, magnesium and phosphorus by mass volume ratio, performing liquid shake flask fermentation, culturing at 25 ℃ and rotating at 120rpm to obtain seed liquid, wherein the bacterial count of the Burkholderia N01 reaches 10⁸cfu/ml；

(2) And (3) amplification culture: the raw materials are bran, inorganic salt calcium, magnesium and phosphorus with the weight percentage of 1%, the water adding amount is 99-89% (5L liquid fermentation tank), and the raw materials are stirred and mixed to obtain a mixed solution; after the autoclave sterilization at 115 ℃, when the temperature of the mixed liquid is cooled to below 35 ℃, inoculating the seed liquid in an aseptic operation mode according to the inoculation amount of 1 percent of the mass-volume ratio, always keeping the positive pressure environment of more than 0.01MPa, and carrying out aeration culture at 35 ℃ for 96 hours until the number of the fermentation bacteria reaches 10⁸More than cfu/ml, wherein in the steps (1) and (2), the final concentration of calcium is 0.9g/L, the final concentration of magnesium is 1.3g/L, and the final concentration of phosphorus is 0.8 g/L; and finally, aseptically packaging to obtain the microbial inoculum.

Example 4

(1) B, the burkholderia N01 with the mass volume ratio of 5 percent,Mixing liquid culture medium, inorganic salt calcium, magnesium and phosphorus, performing liquid shake flask fermentation, culturing at 30 deg.C and rotation speed of 200rpm to obtain seed solution, wherein the bacterial count of Burkholderia N01 is 10⁸cfu/ml；

(2) And (3) amplification culture: the raw materials comprise 3 weight percent of bran, 2 weight percent of vegetable cake, 2 weight percent of bean cake powder, 99-89 weight percent of inorganic salt calcium, magnesium and phosphorus, and the water is added, and the raw materials are stirred and mixed to obtain a mixed solution; sterilizing at 116 deg.C under high pressure, cooling to below 35 deg.C, inoculating seed liquid at 5% mass/volume in sterile operation mode, maintaining positive pressure environment of above 0.01MPa, and culturing at 30 deg.C for 60 hr until the number of fermentation bacteria reaches 10⁸More than cfu/ml, wherein in the steps (1) and (2), the final concentration of calcium is 1.2g/L, the final concentration of magnesium is 0.8g/L, and the final concentration of phosphorus is 15 g/L; and finally, aseptically packaging to obtain the microbial inoculum.

Example 5

(1) Mixing Burkholderia N01 with mass volume ratio of 10%, liquid culture medium, inorganic salt calcium, magnesium and phosphorus, performing liquid shake flask fermentation, culturing at 35 ℃ and rotating speed of 220rpm to obtain seed liquid, wherein the number of Burkholderia N01 reaches 10⁸cfu/ml；

(2) And (3) amplification culture: the raw materials comprise 5 weight percent of bran, 3 weight percent of vegetable cake, 3 weight percent of bean cake powder, 99-89 weight percent of inorganic salt calcium, magnesium and phosphorus, and the water is added, and the raw materials are stirred and mixed to obtain a mixed solution; after 120 ℃ high-pressure sterilization, when the temperature of the mixed solution is cooled to below 35 ℃, inoculating the seed solution in an aseptic operation mode according to the inoculation amount of 10 percent of the mass-volume ratio, always keeping a positive pressure environment of more than 0.01MPa, and carrying out aeration culture at 25 ℃ for 24 hours until the number of fermentation bacteria reaches 10⁸More than cfu/ml, wherein in the steps (1) and (2), the final concentration of calcium is 1.5g/L, the final concentration of magnesium is 0.5g/L, and the final concentration of phosphorus is 2.5 g/L; and finally, aseptically packaging to obtain the microbial inoculum.

Example 6

Preparing antagonistic (watermelon and muskmelon fruit blotch seedling raising) matrix

The mushroom residue rotten clinker, the grass peat, the vermiculite and the perlite are respectively mixed according to the volume percentage45: 35: 13: 7, mixing uniformly according to a ratio of 3kg/m³Adding a biocontrol microbial inoculum containing the Burkholderia strain N01, uniformly mixing, and adjusting the water content of the substrate to 30-40% by using tap water to ensure that the number of Burkholderia contained in the substrate reaches 10⁷The seedling substrate can be obtained by cfu/g or more (in the actual production, a biocontrol microbial inoculum or a bacterial liquid can be further added to adjust the bacterial content).

The statistical basic physicochemical properties of the seedling substrate prepared in example 6 are shown in table 1:

table 1:

total porosity	60.8～80.5
		Porosity of ventilation	14.0～18.5
Pore space for holding water	50～60
		Volume weight (g/cm3)	0.19～0.25
Conductivity (ms/cm)	0.16～0.20
		Total nitrogen (%)	0.26～0.34
Quick-acting nitrogen (g/kg)	2.0～2.2
		Quick-acting phosphorus (g/kg)	5.5～5.6
Quick-acting potassium (g/kg)	13.4～14.3
		Organic carbon (%)	20.2～30.1
pH value	6.5～7.2

Comparative experiment 1

Sowing, seedling raising, substrate management, water and fertilizer management and temperature and humidity control are the same.

Seeding

The two prepared substrates with the height of about three-quarters are respectively filled into each hole of a seedling raising hole tray (50 holes), the two substrates are lightly pressed by hands, 1-2 watermelon seeds are sowed into each hole, the hole tray is covered by the substrates, and a plastic film is used for covering the hole tray to prevent water from evaporating for later use.

Emergence of seedlings

And putting the prepared seeding hole tray into a greenhouse, and keeping the temperature difference between day and night to be more than 10 ℃ at the temperature of 25 ℃. And (4) sprouting in about 4-5 days, removing the plastic film after sprouting, and culturing in a plug until 3-4 true leaves (about 3-4 weeks) are obtained for later use.

And culturing to 100 watermelon seedlings in each group, and performing a disease resistance experiment. The grouping situation is as follows:

control group: inoculating no bacteria;

group of pathogenic bacteria: inoculating the cucumis melo fruit blotch without using the control microbial inoculum prepared in example 4;

antagonistic bacteria N01 group: the preventing and controlling agent prepared in example 4 is used for infecting the melon fruit blotch germs.

Daily incidence of the pathogenic strain was continuously recorded (see fig. 2), and the results indicated that: the prepared seedling substrate is superior to the seedling substrate with a conventional formula, and the disease of fruit blotch is reduced by about 45-60% (see figure 3).

Comparative experiment 2

Test for biocontrol effect on melon sigatoka in pots containing the substrate for antagonizing melon sigatoka prepared in example 6

The specific experimental steps are as follows:

treatment group:

1 sowing of seeds

Antagonistic substrates about three-quarters high are respectively put into each hole of seedling raising hole trays (32 holes, 50 holes, 60 holes and 70 holes) (the mass ratio of the mixture of nitrogen, phosphorus and potassium in the 32 holes to the antagonistic substrates is 5g/kg, wherein the mass ratio of nitrogen to phosphorus to potassium is 2-2.2: 3-3.2: 4-4.2, the mass ratio of the mixture of nitrogen, phosphorus and potassium in the 50 holes to the antagonistic substrates is 6g/kg, wherein the mass ratio of nitrogen to phosphorus to potassium is 3.2: 2.2: 4.3, the mass ratio of the mixture of nitrogen, phosphorus and potassium in the 72 holes to the antagonistic substrates is 8g/kg, wherein the mass ratio of nitrogen to phosphorus to potassium is 3.1: 3.6: 4.2, the mass ratio of nitrogen to phosphorus to potassium in the 72 holes to the antagonistic substrates is 8g/kg, the mass ratio of nitrogen to phosphorus to potassium is 3.1: 3.6: 4.2), melon seeds are lightly sowed in each hole by hand, and the west melon seeds are sowed in each hole, covering with matrix, and covering the tray with plastic film to prevent water evaporation.

2 emergence of seedlings

3 potted plant

Adopting soil of a plot with serious incidence of watermelon fruit blotch, weighing 3.0 kg of soil in each pot (the specification is 320 multiplied by 260mm), uniformly mixing 2% of organic fertilizer, and filling the organic fertilizer into the pots.

Transplanting the watermelon and melon seedlings in all the pots with root substrates, transplanting 5 seedlings in each pot, and supplementing water appropriately according to needs.

Control group: (the mixture of nitrogen, phosphorus and potassium is not contained in the nutrient supplement, the other components are the same, and the preparation process is the same) when transplanting, only 20ml of the diluted fermentation liquor (10) of the fruit blotch pathogen is poured into each plant⁵cfu/ml). The number of diseased plants was recorded daily. See Table 2 for statistics of disease incidence

TABLE 2

The results show that: the invention uses the characteristic of nutrient requirement of watermelon and melon seedling to prepare antibacterial and nutrient components which can meet the requirement of watermelon and melon seedling in different types of plug (32 holes, 50 holes, 60 holes and 72 holes). Not only can prevent and control the occurrence of fruit blotch, but also can reduce the using amount of pesticides and can promote the nutrient absorption of plants. Therefore, the application value of cultivating strong seedlings by reducing phytotoxicity is realized.

The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Burkholderia N01(Burkholderia sp.N01) with the collection number CCTCC NO: m2020248.

2. A preparation method of a biocontrol microbial inoculum is characterized by comprising the following steps:

(1) mixing 1-10% by mass volume of Burkholderia N01, a liquid culture medium, inorganic salt calcium, magnesium and phosphorus, performing liquid shake flask fermentation, culturing at 25-35 ℃ and rotating at 120-220 rpm to obtain a seed solution, wherein the number of Burkholderia N01 reaches 10⁸cfu/g；

(2) And (3) amplification culture: the raw materials comprise 1-5 wt% of bran, 0-3 wt% of vegetable cake, 0-3 wt% of bean cake powder, inorganic salt calcium, magnesium and phosphorus, the water addition amount is 99-89%, and the raw materials are stirred and mixed to obtain a mixed solution; after the mixture is sterilized under high pressure at 115-120 ℃, when the temperature of the mixture is cooled to below 35 ℃, the seed solution is inoculated in an aseptic operation mode according to the inoculum size of 1-10% by mass volume, the positive pressure environment of more than 0.01MPa is always kept, and the aerated culture is carried out for 24-96 hours at 25-35 ℃ until the number of fermentation bacteria reaches 10⁸More than cfu/ml, wherein in the steps (1) and (2), the final concentration of calcium is 0.9-1.5 g/L, the final concentration of magnesium is 0.5-1.3 g/L, and the final concentration of phosphorus is 0.8-2.5 g/L; and finally, aseptically packaging to obtain the microbial inoculum.

3. Use of a combination of a growth substrate and a nutritional supplement for controlling watermelon and melon fruit blotch, wherein the growth substrate comprises the burkholderia strain N01 of claim 1 or the biocontrol agent prepared by claim 2.

4. The use according to claim 3, characterized in that the method for preparing the seedling substrate comprises: respectively mixing mushroom residue clinker, turf, vermiculite and perlite according to the volume percentage of 45: 35: 13: 7, mixing uniformly according to a ratio of 3kg/m³Adding a biocontrol microbial inoculum containing the Burkholderia strain N01, uniformly mixing, and adjusting the water content of the substrate to 30-40% by using tap water to ensure that the number of Burkholderia contained in the substrate reaches 10⁷The seedling culture substrate is obtained above cfu/g.

5. The use according to claim 4, wherein said combination with a nutritional supplement: according to the number of holes of a hole tray selected by watermelon and muskmelon planting, a mixture of nitrogen, phosphorus and potassium is supplemented, and any one of the following schemes is adopted in sequence: and if the number of the holes is 32, the mass ratio of the nitrogen, phosphorus and potassium mixture to the seedling substrate is 4.8-5.2 g/kg, wherein the mass ratio of nitrogen: phosphorus: the mass ratio of potassium is 2.1-2.3: 3-3.2: 4.1 to 4.3;

and if the number of the holes is 50, the mass ratio of the nitrogen, phosphorus and potassium mixture to the seedling substrate is 5.8-6.2 g/kg, wherein the mass ratio of nitrogen: phosphorus: the mass ratio of potassium is 3.1-3.3: 2.1-2.3: 4.2 to 4.4;

and if the number of the holes is 60, the mass ratio of the nitrogen, phosphorus and potassium mixture to the seedling substrate is 6.8-7.2 g/kg, wherein the mass ratio of nitrogen: phosphorus: the mass ratio of potassium is 2.4-2.6: 3.2-3.4: 4-4.2;

and if the seedling raising substrate is a 72-hole plug tray, the mass ratio of the nitrogen, phosphorus and potassium mixture to the seedling raising substrate is 7.8-8.2 g/kg, wherein the mass ratio of nitrogen: phosphorus: the mass ratio of potassium is 3-3.2: 3.5-3.7: 4.1 to 4.3;

alternatively, the first and second electrodes may be,

6. The use according to claim 5, wherein said use is combined with a nutritional supplement: according to the hole number of the hole tray used for planting the watermelon and muskmelon, a mixture of nitrogen, phosphorus and potassium is supplemented, and any one of the following schemes is adopted in sequence: if the number of the holes is 32, the mass ratio of the nitrogen, phosphorus and potassium mixture to the seedling substrate is 5g/kg, wherein the mass ratio of nitrogen: phosphorus: the mass ratio of potassium is 2.2: 3.1: 4.2;

and if the number of the holes is 50, the mass ratio of the nitrogen, phosphorus and potassium mixture to the seedling substrate is 6g/kg, wherein the mass ratio of nitrogen: phosphorus: the mass ratio of potassium is 3.2: 2.2: 4.3;

and if the number of the holes is 60, the mass ratio of the nitrogen, phosphorus and potassium mixture to the seedling substrate is 7g/kg, wherein the mass ratio of nitrogen: phosphorus: the mass ratio of potassium is 2.5: 3.3: 4.1;

and if the seedling raising substrate is a 72-hole plug tray, the mass ratio of the nitrogen, phosphorus and potassium mixture to the seedling raising substrate is 8g/kg, wherein the mass ratio of nitrogen: phosphorus: the mass ratio of potassium is 3.1: 3.6: 4.2;

alternatively, the first and second electrodes may be,

7. the use as claimed in claim 6, wherein the phosphorus and potassium supplementation is with potassium dihydrogen phosphate, wherein the P content is greater than or equal to 52.0%, and the K content is greater than or equal to 34.0%; potassium sulfate is used for supplementing potassium, wherein the content of K is more than or equal to 51.0 percent; the nitrogen is supplemented by urea, wherein the content of N is more than or equal to 46.0 percent.