CN106754567A - A kind of biological and ecological methods to prevent plant disease, pests, and erosion composite bacteria agent LAS for efficiently preventing and treating various crop droop - Google Patents

Abstract

The invention discloses a kind of biological and ecological methods to prevent plant disease, pests, and erosion composite bacteria agent LAS for efficiently preventing and treating various crop droop, it is made up of bacillus JC65, Bacillus cercus CH01, Bacillus subtillis SM16.By 3 kinds of bacterium single doses according to each bacterium number of viable 1:1:1 is made into mix preparation, and viable bacteria total concentration is 1 × 10 in mixture⁸~10¹⁰CFU/ml.LAS mixture is to the prevention effect of crop droop up to more than 70%.To the growth-promoting effect of disease-free plant up to 20%, the composite bacteria agent needs to carry out sowing and transplant two links.

Description

A biocontrol compound bacterial agent LAS that is highly effective in preventing and controlling various crop wilts

technical field

The invention relates to a biocontrol composite microbial agent LAS for efficiently preventing and treating various crop wilts, and belongs to the technical field of agricultural biological control.

Background technique

Watermelons, cucumbers, melons and pumpkins play a pivotal role in the production of agricultural crops worldwide, and wilt disease is one of the main diseases that affect the yield and quality of the above four agricultural crops. Fusarium wilt is a devastating soil-borne disease caused by Fusarium oxysporum f.sp., commonly known as wilt disease, dead seedling disease, etc., and occurs worldwide. With the expansion of planting area and continuous cropping, the occurrence of Fusarium wilt has become more serious.

At present, the main measures of fusarium wilt disease prevention and control still use chemical agents. Although chemical agents can control Fusarium wilt to a certain extent, at the same time, many beneficial microorganisms in the soil will also be damaged to varying degrees. The use of some chemicals can cause serious problems such as soil pollution and chemical pesticide residues. In addition, breeding disease-resistant varieties and strengthening field management measures also play a certain role in disease prevention and control. However, the breeding cycle of disease-resistant varieties is long, and the pathogenicity of Fusarium wilt varies greatly, and the resistance is easily lost.

The more effective agricultural measures are crop rotation, especially paddy and dry crop rotation, which can effectively reduce the amount of bacteria in the soil and reduce the occurrence of diseases, but there are certain difficulties in practical application. First, watermelons, cucumbers, pumpkins and melons are crops of high economic value; second, because Fusarium wilt can survive in the soil for ten to decades, short-term crop rotation cannot achieve the expected purpose; third, most non-host plants cannot be planted. Reduce the number of microsclerotia in the soil, and non-host root exudates can still provide nutrients for the survival of microsclerotia.

In this development situation, many eyes began to shift to biological control means. Compared with other methods, biological control is safe, effective, and durable, especially avoiding a series of problems caused by chemical control. Biocontrol preparations have a good effect on disease control, are non-toxic to humans and animals, do not pollute the environment, and have no residue; they have strong specificity for killing diseases and insect pests, do not harm natural enemies, are beneficial to organisms, and can maintain ecological balance; raw materials and active ingredients are natural products and are easy to Degradation can return to nature to ensure sustainable development; microorganisms can be modified by biotechnology and genetic engineering methods; various factors and components play a role, and it is difficult for pathogenic bacteria to develop drug resistance. According to reports, the main biocontrol fungi for fusarium wilt are Trichoderma and Gliocladium, but the control effect of the above biocontrol fungi is not ideal, mainly because these biocontrol fungi have poor colonization ability in the host rhizosphere. The more promising species of Fusarium wilt biocontrol bacteria are Bacillus, Bacillus subtilis, Pseudomonas fluorescens, Bacillus and so on. Generally speaking, the mixed use of multiple bio-control bacteria is better than a single bio-control bacteria. In addition, the combination of biocontrol agents and agricultural cultivation measures can provide a good habitat for biocontrol bacteria so that they can maximize their control potential.

Contents of the invention

The purpose of the present invention is to provide a microbial composition for efficiently preventing and treating various crop wilts and a microbial composite bacterial agent prepared therefrom.

Another object of the present invention is to provide the application of the microbial composition and the microbial composite bacterial agent prepared therefrom.

The purpose of the present invention can be achieved through the following technical solutions:

A microbial composition for effectively preventing and treating various crop wilt diseases, which is composed of the following three kinds of bacteria, namely: Bacillus JC65 (Bacillus sp.), which was preserved in the China Microorganism Culture Collection Management Committee on December 15, 2010. Microbiology Center, the strain preservation number is CGMCC No.4475; Bacillus cereuse CH01 (Bacillus cereuse), was preserved in the General Microbiology Center of China Committee for the Collection of Microorganisms on November 17, 2015, and the strain preservation number is CGMCC NO .11677; Bacillus subtilis SM16 (Bacillus subtilis), was preserved in the General Microorganism Center of China Microbiological Culture Collection Management Committee on June 18, 2009, and the strain preservation number is CGMCC No.3127.

The application of the microbial composition of the invention in the prevention and treatment of fusarium wilt and the promotion of crop growth.

Preferably, the wet bacteria after the fermentation of the three bacterial strains JC65, CH01 and SM16 are mixed with the thalline preservation liquid of this laboratory (belonging to the authorized patent of this laboratory, patent No. ZL02112559.7, open patent No. CN1358838, open date: July 17, 2002) formulated as a bacterial agent at a ratio of 1:40 (g/ml), which can be stored at room temperature for 2 to 3 years. The composite bacterial agent can be diluted and applied by watering when watermelon seedlings are transplanted.

The application of the microbial composition of the present invention in the preparation of microbial composite bacterial agents for preventing and controlling fusarium wilt and promoting crop growth.

The microbial composite bacterial agent prepared by the microbial composition of the present invention.

The microbial composite bacterial agent described in the present invention is preferably prepared by the following method: inoculate the described bacterial strains JC65, CH01 and SM16 respectively on LB plates, and culture them in a biochemical incubator at 28°C for 14-16h, and wait until they grow out. After a single colony, pick a single colony and insert it into a test tube containing 5mL of LB culture solution, place it in a shaker at 28°C and 200r/min for cultivation, and make a seed solution; inoculate the seed solution with 1% inoculum Put 500mL of LB culture medium in a conical flask, place it in a shaker at 28°C and 200r/min for 48h, dilute the concentration of the three bacterial solutions to 5×10 ⁷ CFU/mL and then follow the ratio of 1:1:1 The ratio is mixed evenly, which is the described microbial compound bacterial agent.

The preparation method of the microbial compound bacterial agent of the present invention comprises: respectively inoculating the described bacterial strains JC65, CH01 and SM16 on the LB plate and marking them, cultivating them in a biochemical incubator at 28°C for 14-16 hours, and waiting for single bacteria to grow Later, pick a single colony and insert it into a test tube containing 5mL of LB culture solution, place it in a shaker at 28°C and 200r/min for cultivation, and make a seed solution; inoculate the seed solution with a 1% inoculation amount Place 500mL of LB culture medium in a conical flask, place it in a shaker at 28°C and 200r/min for 48h, dilute the concentration of the three bacterial solutions to 5×10 ⁷ CFU/mL and mix them in a ratio of 1:1:1 uniform.

The application of the microbial compound bacterial agent of the invention in preventing and treating fusarium wilt and promoting crop growth.

Said application is preferably used for dilution and root irrigation when crops are transplanted and sown.

Beneficial effect

The advantages and positive effects of the present invention are as follows: the present invention is a biological control preparation specially developed for the Fusarium wilt disease. Because it is a biological agent, there are no series of problems caused by the use of chemical pesticides, which is conducive to the pollution-free production of watermelons, cucumbers, pumpkins and melons. Farmers can avoid or reduce the use of other chemical pesticides. This will not only benefit farmers It saves expenses and is conducive to the export trade of these crops.

Greenhouse experiments show that LAS mixture can effectively control the infestation of Fusarium wilt to watermelon, and achieve the effect of controlling the occurrence of the disease. The effect of disease prevention is 86.7%, and the effect of promoting growth is 33.3%. The field experiment shows that LAS mixture can control the occurrence of the disease on the one hand. , the control effect was maintained at 75.0%, and at the same time, it could promote the growth of the above four crops, increase production and income, and the growth promoting effect reached 66.6%. The comprehensive greenhouse and field growth promotion experiments found that the LAS mixture could promote the growth of the above four crops and increase the yield. .

Description of drawings

Fig. 1 Antagonistic effects of different biocontrol bacteria and Fusarium wilt

Figure 2 Inhibition diagram of different biocontrol bacteria on the growth of Fusarium wilt

Fig. 3 The growth-promoting effect diagram of watermelon 30 days after transplanting

Biological Material Deposit Information

JC65, Bacillus sp., was preserved on December 15, 2010 in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures. The preservation address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, The strain preservation number is CGMCC No.4475.

CH01, Bacillus cereuse, was preserved on November 17, 2015 in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures, and the preservation address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences , the strain preservation number is CGMCC NO.11677.

SM16, Bacillus subtilis, was preserved on June 18, 2009 in the General Microbiology Center of China Committee for the Collection of Microbial Cultures, and the preservation address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences , the strain preservation number is CGMCC No.3127.

detailed description

Example 1

1. Source of strains: JC65 was isolated from cucumber rhizosphere soil; SM16 and CH01 were isolated from root soil of healthy tomato plants.

2. Strain isolation method: using plate dilution method (refer to Microbiology Laboratory of Nanjing Institute of Soil Science, Chinese Academy of Sciences. Soil Microbial Research Method [M]. Beijing: Science Publishing Du, 1985.).

3. Basis for strain screening: Determination of antagonism between watermelon fusarium wilt pathogen and biocontrol bacteria (confrontation culture method, Lin Fucheng, Li Debao. Bacillus subtilis (Bacillus subtilis) on phytopathogenic fungi Lysis effect[J].Plant Acta Pathologica Sinica, 2003, 33(2): 174~177. Greenhouse and field experiments on the effect of biocontrol bacteria on the control and growth promotion of watermelon Fusarium wilt.

4. Strain identification method: identification by sequence sequencing of prokaryotic 16S rRNA coding gene fragments. The identification results are shown in Table 1.

Table 1: Sequencing comparison results

5. Plate Antagonism Experiment Method

Method 1: Inoculate the Fusarium wilt of watermelon stored at 4°C on a PDA plate for activation. After the fungus has covered the plate, use a sterilized puncher to evenly punch out circular bacterial blocks with a diameter of 8mm from the outer edge of the colony. Inoculate the mycelium block in the center of a new PDA plate, and inoculate a strain of biocontrol bacteria at an equidistant distance from the center around it, as shown in Figure 1. Cultivate at 25°C, observe the growth of fungi every two days, and record the size of the inhibition zone. Each treatment was replicated three times. See Table 2 and Figure 1 for recorded data and treatment group information.

Table 2 The inhibition rate of different biocontrol bacteria to Fusarium wilt

Note: 1. Values are average values, and different letters indicate significant differences between treatments at the significant level of P=0.05.

2. Inhibition rate (%) = (R-r)/R*100%

Method 2: Take 5.0×10 ⁷ CFU/ml of biocontrol bacteria 1mL in 99mL of PDA, mix and pour it into the plate as treatment 1, take 5.0×10 ⁷ CFU/ml of biocontrol bacteria 5mL in 95mL of PDA, mix and pour Plate for treatment 2. Take the PDA plate without any biocontrol bacteria as the control. Place the beaten fungus dish in the center of the plate. Cultivate at 25°C, observe the growth of fungi every two days and record the growth of fungi. Each treatment was repeated three times. See Table 3 and Figure 2 for treatment group information.

Table 3 Inhibition of growth of Fusarium wilt by different biocontrol bacteria

Note: 1. Values are average values, and different letters indicate significant differences between treatments at the significant level of P=0.05.

2. Inhibition rate (%) = (R-r)/R*100%

Example 2

Inoculate the strains JC65, CH01 and SM16 on the LB plate and streak them respectively, and culture them in a biochemical incubator at 28°C for 14-16 hours. After a single colony grows, pick a single colony and insert it into a test tube containing 5 mL of LB culture medium. Cultivate in a shaker at 28°C and 200r/min to make a seed solution; inoculate the seed solution with 1% of the inoculum in a conical flask containing 500mL of LB culture solution, place at 28°C and 200r/min Cultivate in a shaker for 48 hours, dilute the concentrations of the three bacterial solutions to 5×10 ⁷ CFU/mL, and then mix them uniformly according to the ratio of 1:1:1, which is the microbial composite bacterial agent.

Embodiment 3 greenhouse growth-promoting experiment

When the watermelon seedlings grow to the 3-4 leaf stage, select watermelon seedlings with consistent growth and transplant them into pots containing nutrient soil, and transplant 10 watermelon seedlings for each treatment. On the day after transplanting, 20 mL of 5×10 ⁷ CFU/ml of corresponding bio-control bacteria was irrigated by the root irrigation method, and the bio-control bacteria were re-watered again 30 days after transplanting. Take clear water as a control. The growth promotion of each group was observed every day.

The results are shown in Table 4 and Figure 3.

Total (average) fresh weight = underground fresh weight + aboveground fresh weight

Growth-promoting effect=[(average fresh weight of treatment-average fresh weight of clear water control)/average fresh weight of clear water control]×100%

Table 4 The growth-promoting effect of compound bacterial agent LAS on watermelon (transplanting 30d)

Note: 1. Values are average values, and different letters indicate significant differences between treatments at the significant level of P=0.05.

2. Biomass increase=[(average fresh weight of treatment-average fresh weight of clear water control)/average fresh weight of clear water control]×100%

Embodiment 4 greenhouse disease prevention experiment

When the watermelon seedlings grow to the 3-4 leaf stage, select watermelon seedlings with consistent growth and transplant them into pots containing nutrient soil, and transplant 10 watermelon seedlings for each treatment. On the day after transplanting, the watermelon seedlings in the treatment group were irrigated with 5×10 ⁷ CFU/ml of corresponding biocontrol bacteria by the root irrigation method, and then the watermelon seedlings in the treatment group were irrigated with 5×10 ⁶ spores/mL by the root irrigation method 20 mL of spore suspension. Take clear water as a control. The disease prevention situation of each group was observed every day, and the observation was stopped after the incidence rate of the control group reached more than 95%. See Table 5 for disease prevention information.

Fusarium wilt disease grade standard:

Level 0: no symptoms;

Level 1: Leaf yellowing or wilting area <50%;

Grade 2: Yellowing or wilting area of leaves > 50%;

Grade 3: The leaves wilt or die, and only the growth point survives;

Level 4: The whole bead is dead.

Disease severity (%) = 100% × Σ (number of diseased plants × number of disease grades) / (total number of plants × highest number of disease grades)

Disease prevention effect (%)=100%×(control disease severity-treatment disease severity)/control disease severity

Table 5 The disease prevention effect of compound bacterial agent LAS on watermelon (transplanting 30d)

Note: 1. Values are average values, and different letters indicate significant differences between treatments at the significant level of P=0.05.

2. Control effect% = (control disease severity - treatment disease severity) / control disease severity × 100

Embodiment 5 field disease prevention test

The field experiment was carried out at the Nannong Pailou Experimental Base. The watermelon variety is Zaojia (84-24)

The experimental treatments are: biocontrol complex bacterial agent LAS, water control. Treatment method: treatment on the day of transplanting, 15d, 30d, and 75d respectively by root irrigation.

Each treatment was replicated 3 times, and the area of each plot was 24m ² (6m*4m,), the distance between rows was 100cm, and the distance between plants was 40cm. 60 watermelons in each plot. See Table 6 for details.

Table 6 The disease prevention effect of compound bacterial agent LAS on watermelon in the field (transplanting 105d)

Note: 1. Values are average values, and different letters indicate significant differences between treatments at the significant level of P=0.05.

2. Control effect=[(average fresh weight of treatment-average fresh weight of clear water control)/average fresh weight of clear water control]×100%

The field disease control experiment of cucumber, melon and pumpkin is the same as the watermelon field disease control experiment, and the specific information is shown in Table 6, 7, and 8.

Table 7 The disease prevention effect of compound bacterial agent LAS on cucumber in the field (transplanting 105d)

Note: 1. Values are average values, and different letters indicate significant differences between treatments at the significant level of P=0.05.

2. Control effect=[(average fresh weight of treatment-average fresh weight of clear water control)/average fresh weight of clear water control]×100%

Table 8 The effect of compound bacterial agent LA on the disease prevention of melon in the field (transplanting 105d)

Note: 1. Values are average values, and different letters indicate significant differences between treatments at the significant level of P=0.05.

2. Control effect=[(average fresh weight of treatment-average fresh weight of clear water control)/average fresh weight of clear water control]×100%

The disease prevention effect of compound bacterial agent LA on pumpkin in the table 9 field (transplanting 105d)

Note: 1. Values are average values, and different letters indicate significant differences between treatments at the significant level of P=0.05.

2. Control effect=[(average fresh weight of treatment-average fresh weight of clear water control)/average fresh weight of clear water control]×100%

Embodiment 7 field growth promoting effect

The field experiment was carried out at the Nannong Pailou Experimental Base. The watermelon variety is Zaojia (84-24)

The experimental treatments are: biocontrol complex bacterial agent LAS, water control. Treatment method: Treatment on the day of transplanting, 15d, 30d, and 75d respectively by root irrigation.

Each treatment was replicated 3 times, and the area of each plot was 24m ² (6m*4m,), the distance between rows was 100cm, and the distance between plants was 40cm. 60 watermelons in each plot. See Table 10 for details.

The field growth promotion experiment of cucumber, melon and pumpkin is the same as the field growth promotion experiment of watermelon. See Tables 11, 12 and 13 for specific information.

Table 10 The growth-promoting effect of compound bacterial agent LAS on watermelon in the field (transplanting 35d)

Note: 1. Values are average values, and different letters indicate significant differences between treatments at the significant level of P=0.05.

2. Biomass increase=[(average fresh weight of treatment-average fresh weight of clear water control)/average fresh weight of clear water control]×100%

Table 11 The growth-promoting effect of compound bacterial agent LAS on cucumber in the field (transplanting 35d)

Note: 1. Values are average values, and different letters indicate significant differences between treatments at the significant level of P=0.05.

2. Biomass increase=[(average fresh weight of treatment-average fresh weight of clear water control)/average fresh weight of clear water control]×100%

Table 12 The growth-promoting effect of compound bacterial agent LAS on melon in the field (transplanting 35d)

Note: 1. Values are average values, and different letters indicate significant differences between treatments at the significant level of P=0.05.

2. Biomass increase=[(average fresh weight of treatment-average fresh weight of clear water control)/average fresh weight of clear water control]×100%

Table 13 The growth-promoting effect of compound bacterial agent LAS on pumpkin in the field (transplanting 35d)

Note: 1. Values are average values, and different letters indicate significant differences between treatments at the significant level of P=0.05.

2. Biomass increase=[(average fresh weight of treatment-average fresh weight of clear water control)/average fresh weight of clear water control]×100%.

Claims (8)

1. it is a kind of efficiently to prevent and treat the microbial composite of various crop droop, it is characterised in that to be made up of following 3 kinds of bacteriums, point It is not：Bacillus JC65 (Bacillus sp.), Chinese microorganism strain preservation management is preserved on December 15th, 2010 Committee's common micro-organisms center, culture presevation number is CGMCC No.4475；Bacillus cercus CH01 (Bacillus Cereuse), it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, bacterium on November 17th, 2015 It is CGMCC NO.11677 to plant preserving number；Bacillus subtillis SM16 was (Bacillus subtilis), in 06 month 2009 18 It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center day, culture presevation number is CGMCC No.3127.

2. application of the microbial composite described in claim 1 in terms of preventing and treating droop and promoting plant growth.

3. the microbial composite described in claim 1 prevents and treats droop and promotes the microbial composite bacteria of plant growth in preparation Application in agent.

4. the complex microbial inoculum that prepared by the microbial composite described in claim 1.

5. the complex microbial inoculum described in claim 4, it is characterised in that be prepared by the following method and obtain：By claim Bacterial strain JC65, CH01 and SM16 described in 1 are inoculated into the flat lining outs of LB respectively, and 14-16h is cultivated in 28 DEG C of biochemical cultivation cases, After single bacterium colony is grown, picking single bacterium colony is accessed in the test tube of the LB nutrient solutions containing 5mL, is placed in 28 DEG C, and 200r/min's shakes Cultivated in bed, be made seed liquor；Seed liquor is inoculated in the triangular flask of the LB nutrient solutions equipped with 500mL with 1% inoculum concentration, 28 DEG C are placed in, 48h is cultivated in the shaking table of 200r/min, 3 kinds of bacterial concentrations are diluted to 5 × 10⁷According to 1 after CFU/mL:1:1 Ratio is well mixed, as described complex microbial inoculum.

6. the preparation method of the complex microbial inoculum described in claim 4, it is characterised in that including：By described in claim 1 Bacterial strain JC65, CH01 and SM16 be inoculated into the flat lining outs of LB respectively, cultivate 14-16h in 28 DEG C of biochemical cultivation cases, wait to grow After single bacterium colony, picking single bacterium colony is accessed in the test tube of the LB nutrient solutions containing 5mL, is placed in 28 DEG C, is trained in the shaking table of 200r/min Support, be made seed liquor；Seed liquor is inoculated in the triangular flask of the LB nutrient solutions equipped with 500mL with 1% inoculum concentration, is placed in 28 DEG C, 48h is cultivated in the shaking table of 200r/min, 3 kinds of bacterial concentrations are diluted to 5 × 10⁷According to 1 after CFU/mL:1:1 ratio is mixed Close uniform.

7. application of the complex microbial inoculum described in claim 4 in terms of preventing and treating droop and promoting plant growth.

8. application according to claim 7, it is characterised in that being diluted pouring root when crop is transplanted and sows makes With.