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

A kind of pseudomonas, bacterial agent and application thereof

technical field

The present invention relates to a microorganism, a microbial inoculum and its application.

Background technique

Root rot is caused by Fusarium spp., a soil-borne disease that seriously hinders soybean production in soybean production. It is seriously harmful and affects the yield and quality of soybeans in my country. Chemicals are mostly used in agriculture, which has unsatisfactory effects and pollutes soil ecology. surroundings. Soybean root rot usually occurs frequently in continuous cropping plots, which can generally cause yield losses of 15 to 30%, and yield losses as high as 80% in severe areas. Rice root-knot nematodes can parasitize various crops such as rice and onion, and weeds such as barnyardgrass and parasite. The host range is very wide, and it is easy to spread with water flow, which brings great difficulties to control.

At present, soil sterilization is mainly carried out by chemical agents for the prevention and control of root rot, but the cost of chemical agents is high, and the negative impact on the ecological environment and human health is relatively large. Using disease-resistant varieties to control root rot is one of the most economical and effective measures, but it is difficult to ensure that a resistant variety can be effectively promoted for a long time. Long-term use of a single-resistant variety will easily lead to soybean loss of resistance to root rot. sex. Therefore, the development and utilization of environment-friendly green prevention and control technologies, and the development of biological control preparations with low cost, good effect, and harmless to the environment, humans and animals, can effectively control the damage of root rot and protect soybeans and other root rot caused by Fusarium spp. It is of great significance to the production safety of food crops affected by rot disease.

SUMMARY OF THE INVENTION

In order to solve the problems of high cost and environmental pollution of the existing methods for preventing and treating root rot, the present invention provides a pseudomonas, a fungicide and an application thereof.

The Pseudomonas of the present invention is Pseudomonas chlororaphis P1, which is preserved in the General Microbiology Center of the China Microorganism Culture Collection and Administration Commission, and the preservation number is CGMCC No.19577.

The Pseudomonas inoculum of the present invention is prepared by using the above-mentioned Pseudomonas genus Pseudomonas chlororaphis P1 as a strain, through activation, fermentation and filtration.

The preparation method of described Pseudomonas microbial inoculum is carried out according to the following steps:

1. Activation: coating the Pseudomonas chlororaphis P1 described in claim 1 on the potato solid medium PDA, and culturing it in the dark at 26-30°C for 4-6 days to obtain mycelium;

2. Fermentation: pick the mycelium of step 1 and inoculate it into the potato liquid medium PDB, and shake and culture for 100-130h under the conditions of 26-30°C and 140-160rpm to obtain a fermentation broth;

3. Filtration of the fermentation broth in step 3 to obtain a bacterial agent.

The application of a fungicide using the above-mentioned Pseudomonas as an active ingredient in preventing and treating crop root rot.

The Pseudomonas of the present invention is safe and non-toxic, the preparation process of the agent containing the Pseudomonas of the present invention is simple, the production cycle is short, the cost of the raw materials used is low, the use is convenient, and it is not used for preventing and treating root rot of crops. It will pollute the environment, can also improve the ecological diversity of microorganisms, and inhibit the growth of harmful bacteria. In the application of preventing and controlling root rot of crops, the control effect is good. In the greenhouse experiment, the root rot index control effect reached more than 70%. The rot index control effect has reached more than 55%, and the output of crops has increased by more than 8%. In preventing and treating root rot, the Pseudomonas fungus agent of the present invention is safe, non-toxic, does not endanger human health, has remarkable effect on preventing and controlling root rot of crops, and is favorable for popularization and use.

The preservation date of Pseudomonas chlororaphis P1 of the present invention is April 15, 2020, and the preservation address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No. .19577.

Description of drawings

Fig. 1 is the colony morphology diagram of Pseudomonas of the present invention;

Fig. 2 is the bacteriostatic effect diagram of Pseudomonas of the present invention.

Detailed ways

Embodiment 1: The Pseudomonas in this embodiment is Pseudomonas chlororaphis P1, which is deposited in the General Microbiology Center of the China Microorganism Culture Collection and Management Committee, and the deposit number is CGMCC No.19577.

The screening and identification method of Pseudomonas chlororaphis (Pseudomonas chlororaphis) P1 of the present embodiment is as follows:

1. In the autumn of 2018, soil samples were collected from the soybean continuous cropping field in Qiqihar, Heilongjiang, and PDA medium was used to separate and purify bacteria in the soil, and Fusarium was used as the pathogen to screen biocontrol bacteria by the plate confrontation method;

2. Coat the suspension with bacteria isolated from the soil on the potato solid medium (PDA), cultivate in the dark for 5 days at 28°C, and then pick a single colony for purification to obtain biocontrol bacteria; Antibacterial colonies were streak-inoculated on PDA medium, and after culturing in the dark at 28°C for 5 days, single colonies were obtained for identification.

3. The single colony obtained in the second step is identified by morphological and 16sDNA and other molecular biological methods. According to the identification results, it can be determined that the strain obtained in the second step is Pseudomonas chlororaphis, named P1.

The colony of Pseudomonas of the present invention is smooth, uplifted, mucus-like, embedded in the culture medium, and is not easy to be provoked and is milky white. Observed under a microscope, the thalline of Pseudomonas of the present invention is rod-shaped, slightly curved, has flagella, has capsule, and has no spores.

FIG. 1 is a colony diagram of Pseudomonas in the present embodiment. From FIG. 1 , it can be seen that the appearance of the biocontrol bacteria P1 and the ability of the biocontrol bacteria P1 to secrete pigments. Fig. 2 is a graph showing the bacteriostatic effect of Pseudomonas in this embodiment, LD is a control graph of Fusarium oxysporum, A1, A2, and A3 are graphs of the confrontation experiment between Fusarium and Pseudomonas in this embodiment. The cultivation time is 72h, the cultivation time of A2 is 120hh, and the cultivation time of A3 is 168h. It can be seen from FIG. 2 that the Pseudomonas of this embodiment has a significant bacteriostatic effect on the root rot pathogen Fusarium oxysporum. , and with the increase of culture time (72h, 120h and 168h), under the action of Pseudomonas in this embodiment, the growth of Fusarium oxysporum hyphae is continuously inhibited, and the colony diameter is continuously reduced.

Embodiment 2: The difference between this embodiment and Embodiment 1 is that the culture conditions of Pseudomonas genus Pseudomonas chlororaphisP1 are: culturing in the dark at a temperature of 26-30°C for 4- 6 days, the pH is 6-7.5. Others are the same as the first embodiment.

Embodiment 3: The Pseudomonas bacterial agent of this embodiment is prepared by activation, fermentation and filtration by using the Pseudomonas chlororaphis P1 described in Embodiment 1 as the strain.

Embodiment 4: The difference between this embodiment and Embodiment 3 is that the preparation method of the Pseudomonas bacterial agent is carried out according to the following steps:

1. Activation: The Pseudomonas chlororaphis P1 described in the specific embodiment 1 is coated on the potato solid medium PDA, and cultured in the dark at 26-30°C for 4-6 days to obtain mycelium;

2. Fermentation: pick the mycelium of step 1 and inoculate it into the potato liquid medium PDB, and shake and culture for 100-130h under the conditions of 26-30°C and 140-160rpm to obtain a fermentation broth;

3. Filtration of the fermentation broth in step 3 to obtain a bacterial agent. Others are the same as the third embodiment.

The formula of the potato solid culture medium PDA in step 1 of this embodiment: 200 g of potato, 20 g of sucrose, 20 g of agar, heated to dissolve and filtered, and distilled water to 1 L.

The formula of the potato liquid culture medium PDB in step 2 of this embodiment: 200 g of potato, 20 g of sucrose, dissolved by heating and filtered, and distilled water to 1 L.

In the third step of this embodiment, a bacterial filter with a pore size of 0.22 μm is used.

Specific embodiment 5: the application of the pseudomonas bacterial agent described in the specific embodiment 2 in the prevention and treatment of crop root rot.

The spore concentration of Pseudomonas chlororaphis P1 in the Pseudomonas inoculum is 1×10 ⁷ to 1×10 ⁸ spores/ml when the Pseudomonas inoculum of the present embodiment is used for preventing and treating root rot of crops. The amount of crops per mu is 10-12L.

The crops described in this embodiment are soybeans, corn, and the like.

Embodiment 6: The preparation method of the microbial inoculum with Pseudomonas as an active ingredient described in Embodiment 3 is characterized in that the preparation method of the microbial inoculum is carried out according to the following steps:

1. Activation: Spread Pseudomonas chlororaphis P1 on the potato solid medium PDA, and cultivate at 28°C for 5 days in the dark to obtain mycelium;

2. Fermentation: pick the mycelium of step 1 and inoculate it into the potato liquid medium PDB, and shake and culture for 120h at 28°C and 150rpm to obtain a fermentation broth;

3. Filtration of the fermentation broth in step 3 to obtain a bacterial agent.

The potato solid culture medium PDA formula in step 1 of the present embodiment: 200g of potato, 20g of sucrose, 20g of agar, heated to dissolve and filtered, and distilled water to dilute to 1L.

The potato liquid culture medium PDB formula in step 2 of this embodiment: 200g of potato, 20g of sucrose, heated to dissolve and filtered, and distilled water is settled to 1L.

In the third step of this embodiment, a bacterial filter with a pore size of 0.22 μm is used.

Test 1: Influence of the Pseudomonas agent P1 of the present invention on soybeans suffering from root rot

1. The Pseudomonas inoculum prepared in Embodiment 6 is diluted with distilled water to a spore concentration of 1×10 ⁷ to 1×10 ⁸ /ml to obtain an aqueous spore solution;

2. After germinating soybeans for 3 days, transplant them into PVC flowerpots, one plant per pot, set up three test groups, each group has 20 pots, test group 1 (LD) is the root rot soybean group, test group 2 (LD) +P1) is to use the Pseudomonas inoculum of the present invention on soybeans suffering from root rot, and test group 3 (P1) is to directly use the Pseudomonas inoculum of the present invention on soybeans;

3. Step 1 The spore aqueous solution and water were mixed according to the volume ratio of 1:9 to obtain the irrigation liquid, and the experiment group 2 and the experiment group 3 were irrigated, and each pot was irrigated with 20ml, and the irrigation was repeated 4 times; the soybeans in the experiment group 1 were growing 15 days later, Fusarium was inoculated (inoculation of the root rot pathogen Fusarium oxysporum LD, which caused the onset of soybean root rot).

The results regarding the effects of soybean root rot LD and Pseudomonas P1 of the present invention on soybean growth are shown in Table 1.

Table 1 Effects of P1 on soybean growth in greenhouse experiments

As can be seen from Table 1, after the Pseudomonas P1 of the present invention is used for soybean root rot, the plant height and fresh weight of soybeans tend to increase, and the Pseudomonas of the present invention can promote the growth of soybean, thereby It can be seen that the P1 inoculum of the present invention has a promoting effect on soybean growth.

Experiment 2: Use the Pseudomonas microbial inoculum of the present invention to carry out potted experiments for crops in greenhouse

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

1. The Pseudomonas inoculum prepared in Embodiment 6 is diluted with distilled water to a spore concentration of 1×10 ⁷ to 1×10 ⁸ /ml to obtain an aqueous spore solution;

2. After germinating soybeans for 3 days, transplant them into PVC flowerpots, one plant per pot, and set up four experimental groups, namely control group, repetition I, repetition II, and repetition III groups, with 20 pots in each group;

3. The ratio of spore aqueous solution and water is 1:9 mixing to obtain irrigation liquid, repeating I, repeating II, repeating III groups in each pot is irrigated 20ml, then use equal amount of PDB medium as a control group to irrigate, Each group was irrigated 4 times; 15 days later, each pot was inoculated with Fusarium (the inoculation of root rot pathogenic bacteria Fusarium oxysporum LD, which caused soybean root rot to develop);

4. Investigate the influence of the Pseudomonas inoculum of the present invention on soybean growth 15 days after inoculation, and then randomly select soybean roots to investigate the incidence of soybean root rot, and calculate the quantitative control effect of root rot; greenhouse experiment soybean root rot The formula for calculating the number of diseases is as follows:

According to Burpee et al. 1980, soybean root rot disease is divided into 5 disease grades:

Level 0: No disease on soybean roots;

Grade 1: The main root of soybean has no obvious change, and the tip of the fibrous root is slightly browned, but it does not affect the growth of soybean;

Level 3: The color of the soybean taproot has turned slightly black, and the root tip of the soybean fibrous root has become severely black, but it does not affect the growth of soybean;

Level 5: The color of the main root of soybean becomes black and heavy, and the fibrous root of soybean is sharply reduced, which seriously affects the growth of soybean;

Level 7: Soybean roots are rotted and soybean plants cannot grow.

Soybean root rot disease index = ∑ (number of diseased plants at all levels of soybean root rot × corresponding disease grade value)/(total number of soybean plants under investigation × 7) × 100. Among them, 0<disease index≤30 is weak pathogenicity; 30<disease index≤60 is moderate pathogenicity; 60<disease index≤100 is weak pathogenicity.

The number of root rot control effect (%) = (the number of root knots in the control group - the number of root knots in the treatment group) x 100%/the number of root knots in the control group.

Among them, the number of cases investigated and the number of root rot calculated control effects are shown in Table 2.

Table 2 Calculation of the control effect of P1 on soybean root rot in greenhouse experiments using the number of root rots

It can be seen from Table 2 that the disease index of using the P1 bacterial agent of the present invention is significantly lower than that of the control group, and the control effect on the number of root knots of rice root knot in the greenhouse is all greater than 70%, and the P1 bacterial agent of the present invention has a good control effect. .

Test 3 uses the Pseudomonas bacterial agent of the present invention to carry out the field experiment detection of crops

The specific method is:

Select soybean fields with severe disease for 30 consecutive years, and set up four experimental groups, namely control group, repeat I, repeat II, and repeat III groups, each of 667 m ² ; Soybean seeds are sown in the field that is not submerged in water, and the P1 bacterial agent prepared by the specific embodiment six is used to spray the repetition I, repetition II, and repetition III groups, and the dosage per mu is about 10-12L, and the control group uses clear water. treatment as a control.

After 30 days, the root rot index was investigated to calculate the control effect of root rot index; the plot yield of soybean was measured during the harvest period, and the effect of P1 biocontrol agent on soybean yield was calculated.

The grading standard of field symptoms of root knot nematode is based on Burpee et al. 1980, which divided soybean root rot disease into 5 incidence grades:

Level 0: No disease on soybean roots;

Grade 1: The main root of soybean has no obvious change, and the tip of the fibrous root is slightly browned, but it does not affect the growth of soybean;

Level 3: The color of the soybean taproot has turned slightly black, and the root tip of the soybean fibrous root has become severely black, but it does not affect the growth of soybean;

Level 5: The color of the main root of soybean becomes black and heavy, and the fibrous root of soybean is sharply reduced, which seriously affects the growth of soybean;

Level 7: Soybean roots are rotted and soybean plants cannot grow.

Soybean root rot disease index = ∑ (number of diseased plants at all levels of soybean root rot × corresponding disease grade value)/(total number of soybean plants under investigation × 7) × 100. Among them, 0<disease index≤30 means weak pathogenicity; 30<disease index≤60 means moderate pathogenicity; 60<disease index≤100 means strong pathogenicity.

The calculation formula of soybean root rot control effect in field experiments is as follows: root knot index control effect (%) = (root rot index of control group - root rot index of treatment group) x 100% / root rot index of control group

The root rot index control effect results are shown in Table 3, and the effects of the results of the harvest period investigation on soybean yield are shown in Table 4.

Table 3 Control effect of P1 on soybean root rot in field experiment

deal with root rot index Root rot index control effect (%) control 54.6 ± 3.4 Repeat I 22.5±4.8 58.79 Repeat II 21.5±3.2 60.6 Repeat III 20.9±4.6 61.72

As shown in Table 3, the root rot index control effect of P1 inoculum on soybean root rot in the field is 53.2% to 62.3%.

Table 4 Effects of field experiment P1 on soybean yield

deal with Plot yield (kg/66.7m²) Yield increase rate (%) control 6.4 Repeat I 4.8 8.69 Repeat II 6.2 13.04 Repeat III ±8.6 13.04

The experimental results were all analyzed by Duncan's new multiple range method of SAS software for significant difference analysis, the same letter in the same column indicates that the difference is not significant, P≤0.05). As can be seen from Table 4, the results of the survey during the harvest period showed that P1 had an effect on increasing the yield of soybean in the field, increasing the yield by 8.69% to 13.04%. The P1 inoculum of the invention can not only prevent and treat root rot, but also improve the yield of crops, which is beneficial to popularization and use.

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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