CN111690555B - Pseudomonas, microbial inoculum and application thereof - Google Patents

Abstract

The invention discloses pseudomonas, a microbial inoculum and application thereof, and relates to a microorganism, a microbial inoculum and application thereof. The invention solves the problems of high cost and environmental pollution of the existing method for preventing and treating the root rot. The Pseudomonas is Pseudomonas chlororaphis (Pseudomonas chlororaphis) P1; the pseudomonas agent is prepared by taking P1 as a strain; the application of pseudomonas inoculum in preventing and treating root rot of crops. The pseudomonas is low in cost, safe and non-toxic, does not harm human health, has a remarkable effect of preventing and treating the root rot of crops, and is beneficial to popularization and use.

Description

Pseudomonas, microbial inoculum and application thereof

Technical Field

The invention relates to a microorganism, a microbial agent and application thereof.

Background

The root rot is caused by fusarium, which seriously hinders the soil-borne diseases of soybean production in soybean production, has serious harm, influences the yield and quality of soybeans in China, adopts chemical agents in agriculture, has unsatisfactory continuous effect and pollutes the soil ecological environment. The soybean root rot generally occurs in continuous cropping plots, the yield loss is 15-30%, and the yield loss in severe regions is as high as 80%. The rice root-knot nematode can parasitize various crops such as rice, shallot and the like and weeds such as barnyard grass, nomadic weeds and the like, has a very wide host range and is easy to spread along with water flow, thereby bringing great difficulty to prevention and control.

At present, soil sterilization is mainly carried out for preventing and treating root rot through chemical agents, but the chemical agents have higher cost and have larger negative effects on the ecological environment and the human health. The use of disease-resistant varieties for preventing and treating root rot is one of the most economic and effective measures, but it is difficult to ensure that one resistant variety can be effectively popularized for a long time, and the loss of the resistance of soybeans to the root rot is easily caused by using single resistant varieties for a long time. Therefore, the development and utilization of environment-friendly green prevention and control technology, the development cost is low, the effect is good, and the biological prevention and control preparation is harmless to the environment, people and livestock, and has important significance for effectively controlling the harm of root rot and ensuring the production safety of other grain crops such as soybean and the like which are harmed by the root rot caused by fusarium.

Disclosure of Invention

The invention provides pseudomonas, a microbial inoculum and application thereof, aiming at solving the problems of high cost and environmental pollution of the existing method for preventing and treating root rot.

The Pseudomonas is Pseudomonas chlororaphis (P1) and is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No.19577.

The Pseudomonas agent is prepared by taking Pseudomonas chlororaphis (Pseudomonas chlororaphis) P1 of Pseudomonas as a strain and performing activation, fermentation and filtration.

The preparation method of the pseudomonas microbial inoculum comprises the following steps:

1. and (3) activation: coating the Pseudomonas chlororaphis (PDA) P1 of claim 1 on potato solid medium, and culturing at 26-30 deg.C in dark for 4-6 days to obtain mycelium;

2. fermentation: inoculating the hypha obtained in the step one into a potato liquid culture medium PDB, and performing shake culture for 100-130 h at 26-30 ℃ and 140-160 rpm to obtain a fermentation liquid;

3. and C, filtering the fermentation liquor obtained in the step three to obtain the microbial inoculum.

The application of the microbial inoculum with the pseudomonas as an active component in preventing and treating crop root rot.

The pseudomonas is safe and nontoxic, the pseudomonas containing fungicide of the invention has simple preparation process, short preparation period, low cost of used raw materials and convenient use, does not pollute the environment when being used for preventing and treating the root rot of crops, can improve the ecological diversity of microorganisms and inhibit the growth of harmful bacteria, and has good prevention effect when being applied to preventing and treating the root rot of crops, the root rot index prevention effect in greenhouse experiments reaches more than 70 percent, the root rot index prevention effect in field experiments reaches more than 55 percent, and the yield of crops is increased by more than 8 percent. The pseudomonas bacterial agent is safe and nontoxic when preventing and treating root rot, does not harm human health, has obvious effect of preventing and treating root rot of crops, and is beneficial to popularization and use.

The preservation date of the Pseudomonas chlororaphis P1 is 4 months and 15 days in 2020, the preservation address is the institute of microbiology of China academy of sciences No. 3 of West Lu No.1 Hopkins of Naja district, chaozhou, and the preservation number is CGMCC No.19577.

Drawings

FIG. 1 is a colony morphology of Pseudomonas bacteria of the present invention;

FIG. 2 is a diagram showing the bacteriostatic effect of Pseudomonas bacteria according to the present invention.

Detailed Description

The first embodiment is as follows: the Pseudomonas is Pseudomonas chlororaphis (Pseudomonas chlororaphis) P1, is preserved in the China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.19577.

The method for screening and identifying P1 of Pseudomonas chlororaphis (Pseudomonas chlororaphis) of the Pseudomonas genus according to the present embodiment is as follows:

1. collecting soil samples in the continuous cropping land of the black dragon river ziqihaer soybeans in 2018 autumn, separating and purifying bacteria in the soil by adopting a PDA (potato dextrose agar) culture medium, and screening biocontrol bacteria by adopting a plate confronting method by taking fusarium as a pathogenic bacterium;

2. coating the suspension with bacteria separated from soil on a potato solid culture medium (PDA), culturing in the dark at 28 deg.C for 5 days, and selecting single colony for purification to obtain biocontrol bacteria; and streaking and inoculating the biocontrol bacterial colony on a PDA culture medium, and performing dark culture at 28 ℃ for 5 days to obtain a single bacterial colony for identification.

3. The single colony obtained in the second step is identified by the methods of morphology, molecular biology such as 16sDNA and the like, and according to the identification result, the strain obtained in the second step can be determined to be Pseudomonas chlororaphis of Pseudomonas and named as P1.

The pseudomonad of the invention has smooth and raised bacterial colony in a mucus shape, is embedded into a culture medium, is not easy to pick up and is milk white, and the bacterial colony cultured for 5 days generates red water soluble pigment, and the back surface is dark red. The pseudomonas thallus is rod-shaped, slightly bent, flagellated, capsulated and spore-free when observed under a microscope.

FIG. 1 is a colony diagram of Pseudomonas bacteria according to the present embodiment, and it can be seen from FIG. 1 that the appearance of biocontrol bacteria P1 and the secretion of pigment by biocontrol bacteria P1 are apparent. FIG. 2 is a diagram showing the bacteriostatic effect of Pseudomonas in this embodiment, LD shows a diagram of Fusarium oxysporum, A1, A2, and A3 show the experimental effect of Fusarium and Pseudomonas in this embodiment, A1 has a culture time of 72h, A2 has a culture time of 120hh, and A3 has a culture time of 168h, and it can be seen from FIG. 2 that Pseudomonas in this embodiment has a significant bacteriostatic effect on Fusarium oxysporum, which is the root rot pathogen, and with the increase of the culture time (72 h, 120h, and 168 h), fusarium oxysporum hyphae are inhibited from growing continuously and the colony diameter is reduced continuously under the effect of Pseudomonas in this embodiment.

The second embodiment is as follows: the first difference between the present embodiment and the specific embodiment is: the culture conditions of Pseudomonas chlororaphis (P.chlororaphispP 1) were: culturing in dark at 26-30 deg.c for 4-6 days and pH 6-7.5. The rest is the same as the first embodiment.

The third concrete implementation mode: the Pseudomonas chlororaphis P1 is a strain of Pseudomonas aeruginosa (Pseudomonas chlororaphis) described in the first embodiment, and is prepared by activation, fermentation and filtration.

The fourth concrete implementation mode: the third difference between the embodiment and the specific embodiment is that the preparation method of the pseudomonas agent is carried out according to the following steps:

1. activation: coating Pseudomonas chlororaphis P1 of the first embodiment on potato solid culture medium PDA, and culturing in the dark at 26-30 deg.C for 4-6 days to obtain mycelium;

2. fermentation: inoculating the hypha obtained in the step one into a potato liquid culture medium PDB, and performing shake culture for 100-130 h at 26-30 ℃ and 140-160 rpm to obtain a fermentation liquid;

3. and C, filtering the fermentation liquor obtained in the step three to obtain the microbial inoculum. The rest is the same as the third embodiment.

The formula of the potato solid culture medium PDA in the first step of the embodiment is as follows: 200g of potato, 20g of cane sugar and 20g of agar, heating to dissolve, filtering and then fixing the volume of distilled water to IL.

The formula of the potato liquid culture medium PDB in step two of this embodiment: 200g of potato and 20g of cane sugar, heating to dissolve, filtering, and then adding distilled water to a constant volume until IL is obtained.

In the third step of the present embodiment, a 0.22 μm pore size bacterial filter is used for the filtration.

The fifth concrete implementation mode: the application of the pseudomonas microbial inoculum according to the second embodiment in preventing and treating the root rot of crops.

When the pseudomonas preparation is applied to preventing and treating root rot of crops, the spore concentration of pseudomonas chlororaphis (P.chlororaphisp1) in the pseudomonas preparation is 1x10 ⁷ ～1x10 ⁸ The dosage of each mu of crops is 10-12L.

The crops in the embodiment are soybeans, corns and the like.

The sixth specific implementation mode is as follows: the preparation method of the microbial inoculum taking the pseudomonas as the active ingredient is characterized by comprising the following steps of:

1. and (3) activation: coating Pseudomonas chlororaphis P1 on potato solid culture medium PDA, and culturing in dark at 28 deg.C for 5 days to obtain mycelium;

2. fermentation: inoculating the hypha obtained in the step one into a potato liquid culture medium PDB, and performing shake culture at 28 ℃ and 150rpm for 120 hours to obtain a fermentation liquid;

3. and C, filtering the fermentation liquor obtained in the step three to obtain the microbial inoculum.

The formula of the potato solid culture medium PDA in the first step of the embodiment comprises 200g of potatoes, 20g of cane sugar and 20g of agar, and the ingredients are heated, dissolved and filtered, and then distilled water is added to the solution to reach the constant volume IL.

In the second step of the embodiment, the formula of the liquid culture medium PDB for potatoes is 200g and 20g of sucrose, and the volume of the liquid culture medium PDB is fixed to IL by distilled water after heating, dissolving and filtering.

In the third step of the present embodiment, a 0.22 μm pore size bacterial filter is used for filtration.

Test 1: the influence of the pseudomonas agent P1 on the soybeans with root rot

1. Diluting the pseudomonas bacterial agent prepared by the sixth specific embodiment with distilled water until the spore concentration is 1x10 ⁷ ～1x10 ⁸ Obtaining spore water solution per ml;

2. after accelerating germination of soybeans for 3 days, transplanting the soybeans into a PVC flowerpot, wherein each pot is provided with one strain, three test groups are arranged, each group comprises 20 pots, a test 1 group (LD) is a soybean group with root rot, a test 2 group (LD + P1) is that the pseudomonas microbial inoculum provided by the invention is used for soybeans with the root rot, and a test 3 group (P1) is that the pseudomonas microbial inoculum provided by the invention is directly used for the soybeans;

3. step one, spore water solution and water are mixed according to the volume ratio of 1:9, obtaining irrigation liquid, irrigating the test 2 group and the test 3 group, irrigating 20ml of irrigation liquid in each pot, and repeatedly irrigating for 4 times; the soybeans of the test 1 group were inoculated with fusarium after 15 days of growth (inoculated with fusarium oxysporum LD, a root rot pathogen, causing the onset of soybean root rot).

The results regarding the effect of soybean root rot LD and pseudomonas P1 of the present invention on soybean growth are shown in table 1.

TABLE 1 Effect of P1 on Soybean growth in greenhouse experiments

As can be seen from the table 1, after the pseudomonas P1 is used for soybean root rot, the plant height and fresh weight of soybean tend to increase, and the pseudomonas can promote the growth of soybean, so that the P1 microbial inoculum has a promoting effect on the growth of soybean.

Test 2: using the pseudomonas bacterial agent to perform crop greenhouse potting experiment

The specific test method is carried out according to the following steps:

1. diluting the pseudomonas bacterial agent prepared by the sixth specific embodiment with distilled water until the spore concentration is 1x10 ⁷ ～1x10 ⁸ Obtaining spore water solution per ml;

2. after accelerating germination of soybeans for 3 days, transplanting the soybeans into a PVC flowerpot, wherein one plant in each pot is provided with four experimental groups, namely a control group, a repeat I group, a repeat II group and a repeat III group, and each group comprises 20 pots;

3. proportionally mixing spore water solution and water according to the volume ratio of 1:9, obtaining irrigation liquid, irrigating 20ml of each pot in the repeat I, repeat II and repeat III groups, then irrigating by using an equivalent PDB culture medium as a control group, and repeatedly irrigating each group for 4 times; inoculating fusarium in each pot after 15 days (inoculating fusarium oxysporum LD which is a root rot pathogen to cause the soybean root rot to be attacked);

4. investigating the influence of the pseudomonas microbial inoculum on the growth of the soybean 15 days after inoculation, randomly selecting the roots of the soybean, investigating the incidence quantity of the soybean root rot, and calculating the quantity control effect of the root rot; the greenhouse experiment soybean root rot quantity control effect calculation formula is as follows:

soybean root rot disease was classified into 5 disease grades according to Burbee et al 1980:

level 0: no disease occurs to the soybean roots;

level 1: the main root of the soybean is not obviously changed, the tip of the fibrous root is slightly browned, but the growth of the soybean is not influenced;

and 3, stage: the color of the main root of the soybean is slightly blackened, the tip of the fibrous root of the soybean is seriously blackened, but the growth of the soybean is not influenced;

and 5, stage: the color of the main root of the soybean is changed to be black and heavy, and the fibrous root of the soybean is reduced rapidly, so that the growth of the soybean is seriously influenced;

and 7, stage: the soybean roots are rotten, and the soybean plants cannot grow.

Disease index of soybean root rot = ∑ (number of diseased plants at each level of soybean root rot x corresponding number of diseased plants)/(total number of investigated soybean plants x 7) × 100. Wherein the disease index is more than 0 and less than or equal to 30, and the pathogenicity is weak; the disease index is more than 30 and less than or equal to 60, and the medium pathogenicity is obtained; the disease index is less than 60 and less than or equal to 100, and the pathogenicity is weak.

Root rot quantity control effect (%) = (control group root knot quantity-treatment group root knot quantity) x 100%/control group root knot quantity.

Wherein, the investigation of the number of attacks and the calculation of the number control of root rot are shown in table 2.

Table 2 calculation of the prevention Effect of P1 on Soybean root rot in greenhouse experiments by means of the amount of the root rot

From table 2, it can be seen that the disease index of the P1 fungicide is obviously lower than that of a control group, and the control effect on the root knot number of the rice root knot worms in a greenhouse is more than 70%, so that the P1 fungicide has a good control effect.

Experiment 3 Using the Pseudomonas bacteria preparation of the present invention to perform field experimental detection on crops

The specific method comprises the following steps:

selecting soybean field blocks with serious morbidity for 30 years continuously, and setting four experimental groups, namely a control group, a repeat I group, a repeat II group and a repeat III group, wherein each group is 667m ² (ii) a And then leveling the soil of each group by using a rotary cultivator, sowing soybean seeds in a field which is not immersed in water, spraying the P1 microbial inoculum prepared by the sixth embodiment by using the repeat groups I, II and III, wherein the dosage of each mu is about 10-12L, and the control group is treated by applying clear water as a control.

Investigating the root rot index after 30 days, and calculating the control effect of the root rot index; and measuring the yield of the soybean in the cell in the harvest period, and calculating the influence of the P1 biocontrol microbial inoculum on the yield of the soybean.

The grading standard of the field symptoms of the root knot nematode disease is to divide the root rot disease of the soybean into 5 disease grades according to Burbee et al in 1980:

stage 0: no disease occurs to soybean roots;

level 1: the main roots of the soybeans are not obviously changed, the tips of the fibrous roots are slightly browned, but the growth of the soybeans is not influenced;

and 3, level: the color of the main root of the soybean is slightly blackened, the tip of the fibrous root of the soybean is seriously blackened, but the growth of the soybean is not influenced;

and 5, stage: the color of the main roots of the soybeans turns black and is heavier, and the fibrous roots of the soybeans are reduced suddenly, so that the growth of the soybeans is seriously influenced;

and 7, stage: the soybean roots are rotten, and the soybean plants cannot grow.

Disease index of soybean root rot = ∑ (number of diseased plants at each level of soybean root rot x corresponding number of diseased plants)/(total number of investigated soybean plants x 7) × 100. Wherein the disease index is more than 0 and less than or equal to 30, and the pathogenicity is weak; the disease index is more than 30 and less than or equal to 60, and the medium pathogenicity is obtained; the disease index is more than 60 and less than or equal to 100, and the pathogenicity is strong.

The formula for calculating the root rot control effect of the field experiment soybean comprises the following steps of root knot index control effect (%) = (the number of the control group root rot is one treatment group root rot index) x 100%/the control group root rot index

The root rot index control results are shown in table 3, and the effect of the harvest period survey results on soybean yield is shown in table 4.

TABLE 3 prevention of Soybean root rot by field experiment P1

Treatment of	Index of root rot	Root rot index control effect (%)
			Control	54.6 Sha 3.4
Repetition of I	22.5 Sha 4.8	58.79
			Repeat II	21.5 Sha 3.2	60.6
Repeat III	20.9 shi 4.6	61.72

As shown in Table 3, the P1 microbial inoculum has a root rot index prevention effect of 53.2-62.3% on soybean root rot in a field, and the P1 microbial inoculum has a remarkable effect on preventing and treating crop root rot.

TABLE 4 Effect of field experiment P1 on Soybean yield

Treatment of	Cell output (kg/66.7 m) 2 )	Yield increase (%)
			Control of	6.4 Share
Repetition of I	Shi 4.8	8.69
			Repeat II	6.2	13.04
Repeat III	8.6	13.04

And the experimental results are subjected to difference significance analysis by adopting a Duncan's new repolarization method of SAS software, the same letters in the same column indicate that the difference is not significant, and P is less than or equal to 0.05). As can be seen from Table 4, the results of the harvest period survey show that P1 has yield increase effect on field soybeans, and the yield increase effect is 8.69-13.04%. The P1 microbial inoculum can prevent and treat root rot, can also improve the yield of crops, and is beneficial to popularization and application.

Claims (4)

1. The Pseudomonas is Pseudomonas chlororaphis (P1) and is preserved in the China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No:19577.

2. a Pseudomonas chlororaphis P1 of Pseudomonas in claim 1, which is activated, fermented and filtered to obtain the Pseudomonas chlororaphis P1.

3. The pseudomonas agent according to claim 2, wherein the preparation method of the pseudomonas agent comprises the following steps:

1. and (3) activation: coating the Pseudomonas chlororaphis (PDA) P1 of claim 1 on potato solid medium, and culturing at 26-30 deg.C in dark for 4-6 days to obtain mycelium;

2. fermentation: inoculating the hypha obtained in the first step into a potato liquid culture medium PDB, and performing shake culture for 100-130 h at 26-30 ℃ and 140-160 rpm to obtain a fermentation liquid;

3. and C, filtering the fermentation liquor obtained in the step three to obtain the microbial inoculum.

4. The use of a pseudomonas agent as claimed in claim 2 for controlling root rot of crops caused by fusarium oxysporum.

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