CN1314325C - Biological preparation and its application for controlling soft rot of konjac - Google Patents

Abstract

The present invention relates to a biological agent for controlling konjak bacterial soft rot and an application thereof, which belongs to the technical field of the biological pesticide. The production strain of the present invention is bacillus amyloliquefaciens C3 of which the preservation number is CGMCC No. 1553. The strain of the present invention has the following distinctive characteristics that 1, colony presents an irregular round shape, and the edge of the colony presents a cracking shape; the colony is viscous in the initial stage, and wrinkles and is milky and nontransparent in the late stage; the mycelium is difficult to disperse in liquid, generates spore, and presents a rod shape; 2, the strain is colonized so as to have strong bacteriostatic capacity, promote the konjak to grow and has the functions of disease prevention and yield increase. The present invention obtains liquor or wettable powder which are prepared by adding wetting agent, spreader, auxiliary materials, etc. to bacteria mycelium culture by the liquid fermentation production of the strain, and has the advantages of obvious stable control efficiency for konjak bacterial soft rot, good integral character and easy commercial process.

Description

Biological preparation and its application for controlling soft rot of konjac

Technical field:

The invention relates to a biological preparation for preventing and treating bacterial soft rot of konjac and its application, belonging to the technical field of biological pesticides.

Background technique:

Konjac is rich in glucomannan. It is one of the few plants containing natural glucomannan in nature, and it is also the only plant with wide distribution, strong adaptability, and large-scale extraction of glucomannan. It is a medical health food and industrial raw material with great development potential. With the continuous development of konjac products and the adjustment of rural industrial structure, konjac has become a major economic crop in the mountainous areas of central and western my country, and it is one of the effective ways for farmers to get rid of poverty and revitalize the local economy. According to incomplete statistics, in 2005, the planting area in Yunnan Province reached 150,000 mu, and the national planting area was about 800,000 mu. Whether it is abroad or at home, the soft rot caused by bacteria is the first limiting factor for the development of konjac industry. Konjac often has a good development momentum in the past few years, but with the extension of the planting period and the expansion of the cultivation area, the epidemic cycle of soft rot is getting shorter and shorter, and the disease is getting worse year by year. The increase in the yield and quality of konjac eventually led to a sharp decrease in the planting area. Seriously dampened the enthusiasm of farmers to plant konjac and hindered the development of local economy.

Konjac bacterial soft rot is a worldwide plant disease that is devastating to konjac production. Our research has confirmed that it is mainly caused by Erwinia carotovora subsp.carotovora, followed by Erwinia chrysanthemum E. chrysanthermi. According to incomplete statistics, only in Yunnan Province, the annual comprehensive incidence rate of konjac soft rot is more than 35%, the loss rate of konjac is 21.5%, and the economic loss per mu is 774 yuan. The loss amounted to more than 12 million yuan. For konjac bacterial soft rot, at present, agricultural measures such as crop rotation and large doses of chemical pesticides are mainly used to prevent and control konjac bacterial soft rot. It is getting lower and lower; chemical control not only has high cost and poor control effect, but also pollutes the environment and affects the quality, and some pathogenic bacteria show resistance to the commonly used agricultural streptomycin in production, making the control effect even worse. In addition, due to the use of chemical pesticides and pesticide residues exceeding international standards, the growth of its exports has been affected.

With the development of science and technology and the improvement of people's living standards, people are paying more and more attention to health and environmental issues. The development of high-yield, high-quality, and efficient konjac production has become a key issue in improving the economic income of farmers in taro areas, especially in recent years. Organic konjac Or pollution-free konjac production has become the leading direction of the development of konjac industry. In terms of disease prevention and control, agricultural and biological control and other disease prevention measures that do not cause pesticide residues and environmental pollution are more needed. Therefore, screening and developing a highly efficient, non-toxic, safe, non-residue, and easy-to-use biological agent for the prevention and treatment of bacterial soft rot in konjac will help reduce the damage of the disease, improve the quality, enhance the stability of the konjac farmland ecosystem, and ensure the protection of the konjac industry. Healthy development has important practical significance and broad application prospects to increase farmers' production and income.

Invention content:

The purpose of the present invention is to overcome the shortcomings of the existing methods and provide a highly efficient, non-toxic, safe, residue-free and easy-to-use biological agent for preventing and treating bacterial soft rot of konjac.

The bacterium used in the present invention is Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) C ₃ , and the depository unit is: General Microbiology Center of China Microorganism Culture Preservation Management Committee; Address: China. Beijing. Zhongguancun; Preservation Date: December 05, 2005; Registered number CGMCC No.1553.

The production strain Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) C ₃ of the present invention is isolated from the rhizosphere soil of Amorphophallus konjac in Fuyuan, Yunnan. It is determined by morphology, culture properties, conventional physiology and biochemistry and Biolog automatic identification system that the biocontrol bacteria is amyloliquefaciens A new strain of Bacillus amyloliquefaciens C ₃ . The bacterial strain has the following characteristics: (1) the colony is irregularly round, the edge of the colony is cracked, the colony is sticky at the beginning, wrinkled in the later stage, milky white, opaque, the thalline is not easy to disperse in the liquid, does not produce pigment, and produces spores; Observation under a microscope: the bacterium is straight rod-shaped, with a size of 0.7-0.8×2.0-2.4 μm; (2) the strain has strong colonization ability, strong antibacterial ability, can promote the growth of konjac, and has the functions of sterilization, disease prevention and production increase. The strain can colonize the rhizosphere and body of Amorphophallus konjac. The main pathogen Erwinia carotovora on this crop has a significant inhibitory effect, and the average diameter of the inhibition zone is 14.0-20.0mm. (3) Possess the following physiological and biochemical characteristics: positive Gram staining, glycogen utilization, Tween 40, N-acetyl-D-glucosamine, ribitol, L-arabinose, D-arabinitol, L- Fucose, D-galactose, gentiobiose, meso-inositol, lactulose, D-mannitol, D-psicose, D-raffinose, L-rhamnose, D-sorbose Alcohol, methylpyruvate, acetic acid, citric acid, formic acid, D-galactonic acid, D-gluconic acid, A-glycolic acid, Y-glycolic acid, itaconic acid, a-butyronic acid, a-ketone valeric acid, propionic acid, succinic acid, D-alanine, L-alanine, L-alanyl-glycine, L-aspartic acid, L-glutamic acid, L-leucine, L -Serine, L-Threonine, DL-Carnitine, Y-Aminobutyric Acid, Uridine, Inosine, Uridine, Thymidine, Phenylethylamine, Butylenediamine, 2-aminoethanol, Glucose- 6-phosphate; cannot utilize cyclodextrin, dextrin, N-acetyl-D-galactose, cellobiose, D-fructose, aD-glucose, aD-lactose, maltose, sucrose, D-trehalose, xylose Alcohol, Malonate, L-Proline, D-Serine, Glycerin.

The present invention is achieved in this way: the test tube culture of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) C3 bacterial strain, the expansion culture of shaking table, the fermentation culture is prepared as biological preparation, it is applied on the konjac, measures its to konjac bacterial soft rot Control effectiveness and yield.

The cultivation method of biocontrol bacterial strain Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) _C3 of the present invention (below is weight percent):

1. Test tube culture

The formula of the test tube slant medium is: beef extract 0.3%, yeast extract 0.1%, peptone 0.5%, glucose 1.0%, agar 1.7-2.0%, pH 6.8-7.0.

Move the Bacillus amyloliquefaciens C ₃ to the slant medium of the test tube, and culture it at a constant temperature of 28° C. for 1-2 days to obtain the test tube species.

2. Liquid fermentation culture

The liquid fermentation medium formula is: soybean powder 2.0%, starch 1.0%, glucose 0.30%, KH ₂ PO ₄ 0.1%, peptone 0.2% (NH ₄ ) ₂ SO ₄ 0.05%, MgSO ₄ 7H ₂ O 0.03%, CaCO ₃ 0.1%, peanut oil 0.1%, defoamer 0.01%, pH 7.5-8.0.

Inoculate the test tube seed into 500ml Erlenmeyer flask liquid culture medium, fill each bottle with 200ml, shake and cultivate at 28°C for 14 hours at a constant temperature of 150-200rpm, the obtained culture solution can be used as a preparation, or transferred to a fermenter for fermentation Manufactured to obtain a liquid formulation.

Liquid fermentation conditions

Tank temperature: The tank temperature of the fermenter is controlled at 25-32°C, measured by a thermometer inserted into the medium, and adjusted by passing cooling water or hot water into the interlayer.

Tank pressure: The tank pressure of the fermenter is controlled at 0.5/square centimeter, which is adjusted through the sterile air inlet and exhaust gas outlet.

Stirring: the stirring speed of the fermenter is 200 rpm.

Sampling inspection and culture condition adjustment: take a sample from the sampling tube once every 2 hours, measure the pH, and smear the smear with crystal violet staining to check the shape of the bacteria, whether there is any contamination by bacteria, observe whether the bacteria produce spores, etc. When the number of bacterial spores in the sample accounts for more than 5% of the total bacterial cells, reduce the ventilation rate and lower the culture temperature to about 22°C, and keep the other conditions unchanged. When it is above 85%, stop cultivating and putting the tank in. Microscopic examination counts when the fermenter is put in the tank, and counts viable bacteria with the culture medium plate method.

Culture cycle: The culture cycle of the fermenter is about 36-48 hours.

Liquid fermentation preparation: the bacteria content was determined by plate counting, and the bacteria content reached 18 billion CFU/ml, which is used for field disease prevention and control.

3. Solid fermentation culture

First carry out the liquid fermentation culture of bacteria (same as above). Then, it is made into wettable powder by adding filling agent, filtering with plate and frame, beating, drying and pulverizing. The specific process is as follows:

Specifically add filler: Put the fermentation hydraulic pressure into the storage tank, add diatomaceous earth (light calcium carbonate) as filler according to the following calculation formula, and stir for 30 minutes after adding.

The amount of filler diatomite (light calcium carbonate) added (kg) = 〖number of fermentation broth bacteria (100 million/ml) x tank volume (liter) x yield〗/number of finished product bacteria-residue in fermentation broth (Kilogram).

Plate and frame filtration: Press the material from the storage tank into the plate and frame with a pressure of 2 kg/cm2, and filter through the No. 7 filter cloth. The pressure should be adjusted at any time so that the bacteria content in the filtrate does not exceed 0.2 billion/ml.

Beating: unload the filter cake into the beating tank, add thick milk No. 100 at 8% of the amount of calcium carbonate added, or add SDS at 3%, add an appropriate amount of filtrate and stir evenly for 30 minutes.

Mixing: crush the bacterium-containing diatomaceous earth after beating, dry in the shade, then dilute by 5 times the volume, stir and mix evenly.

Drying: Ventilate and dry in a drying room with a temperature below 60°C to a moisture content of 5%-8%.

Pulverization: The discharge temperature should not exceed 60°C during pulverization to prevent the inactivation of bacteria.

Measurement of finished product quality indicators: Bacteria content, water content, suspension rate, and fineness are all measured according to the national enterprise standard (Q/KWL02-2003). The bacteria content is 1 billion/g, and other indicators meet the standards.

Field efficacy test and yield increase test of biological agent of the present invention for preventing and treating soft rot

1. Preparation of biological preparations: prepare test preparations according to the aforementioned Bacillus amyloliquefaciens C ₃ culture method.

2. Field efficacy and yield increase test of biocontrol bacteria C ₃ liquid preparation on konjac soft rot

2.1 Test agent and treatment:

A. 250-fold dilution of the liquid preparation of biocontrol bacteria _C3 : adopt the root-watering method, pour 50 ml of liquid medicine on the root of each konjac plant, and apply the medicine for the first time at the early stage of the disease, and apply medicine twice with an interval of 7 days.

B. 500-fold dilution of the liquid preparation of biocontrol bacteria _C3 : adopt the root-watering method, pour 50 ml of liquid medicine on the root of each konjac plant, and apply the medicine for the first time at the early stage of the disease, and apply medicine twice with an interval of 7 days.

C. 1000-fold dilution of the liquid preparation of biocontrol bacteria _C3 : adopt the root-watering method, pour 50 milliliters of liquid medicine on the root of each konjac plant, and apply the medicine for the first time at the early stage of the disease, and apply medicine twice with an interval of 7 days.

D, 500-fold dilution of the contrast fungicide chlorothalonil: adopt the root-watering method, pour 50 milliliters of liquid medicine on the root of each konjac plant, apply the first medicine at the initial stage of the disease, and apply medicine twice with an interval of 7 days.

E, blank control: apply clear water, adopt the method of watering the roots, pour 50 milliliters of medicinal liquid on the root of each konjac plant, apply for the first time at the initial stage of the disease, and apply 2 times altogether, with an interval of 7 days.

2.2. Test method: the test crop is Amorphophallus konjac (a local variety in Fuyuan County), the control object is bacterial soft rot of konjac, and the test site is Zhuyuan Town, Fuyuan County, Yunnan Province.

The experiment consisted of 5 treatments, 3 repetitions, and a total of 15 plots. The area of the residential area is not less than 20 square meters, arranged randomly.

Set up protective rows around, and measure the yield of each plot when harvesting.

2.3. Investigation method: In the late stage of konjac onset, disease investigation was carried out in the experimental plot. According to the symptoms and characteristics of the aboveground part of konjac soft rot and the development trend of the disease, we divided the lesions into the surrounding type and the longitudinal type. The former lesions surround the petiole, and the disease Fast development. It will quickly cause lodging of compound leaves, which is very harmful; the latter lesion is only on one side of the petiole, and mostly extends longitudinally along the vascular bundle, and later expands laterally, causing the entire petiole to rot.

The grading standard of konjac soft rot (the condition is divided into 5 grades):

Grade 0: no symptoms; grade 1: small water-soaked lesions less than 2 cm on one side of the petiole, or less than 1/10 of the total length of the petiole, and mild yellowing of the leaves on the same side; grade 2: one side of the petiole Lesions of more than 2 cm, or 1/10--5/10 of the total length of the petiole, and yellowing of the leaves on the same side; Grade 3: circular lesions, or longitudinal lesions accounting for 5/10 of the total length of the petiole /10 or more, most of the leaves are yellowed or partially dead; Grade 4: the whole leaf is yellow or lodging and rotten.

If the entire compound leaf starts to yellow from the top, and there is soft rot at the junction of the petiole and the tuber, it is a tuber disease, and the disease is classified as Grade 3.

3. Field efficacy and yield increase test of biocontrol bacteria C ₃ wettable powder against konjac soft rot

1. Test agent and treatment:

A. 250-fold dilution of biocontrol bacteria C ₃ wettable powder: use the root watering method, pour 50 ml of liquid medicine on the roots of each konjac plant, apply the first medicine at the early stage of the disease, and apply medicine twice with an interval of 7 days.

B. 500-fold dilution of biocontrol bacteria C ₃ wettable powder: use the root-watering method, pour 50 ml of liquid medicine on the roots of each konjac plant, apply the first medicine at the early stage of the disease, and apply medicine twice with an interval of 7 days.

C. 1000-fold dilution of biocontrol bacteria _C3 wettable powder: use the root-watering method, pour 50 ml of liquid medicine on the roots of each konjac plant, apply the first medicine at the early stage of the disease, and apply medicine twice with an interval of 7 days.

D, 500-fold dilution of the contrast fungicide chlorothalonil: adopt the root-watering method, pour 50 milliliters of liquid medicine on the root of each konjac plant, apply the first medicine at the initial stage of the disease, and apply medicine twice with an interval of 7 days.

E, blank control: apply clear water, adopt the method of watering the roots, pour 50 milliliters of medicinal liquid on the root of each konjac plant, apply for the first time at the initial stage of the disease, and apply 2 times altogether, with an interval of 7 days.

2. Test method: the test crop is Amorphophallus konjac (a local variety in Fuyuan County), the control object is bacterial soft rot of konjac, and the test site is Zhuyuan Town, Fuyuan County, Yunnan Province.

The experiment consisted of 5 treatments, 3 repetitions, and a total of 15 plots. The area of the residential area is not less than 20 square meters, arranged randomly.

Set up protective rows around, and measure the yield of each plot when harvesting.

3. Investigation method: In the late stage of konjac onset, disease investigation was carried out in the experimental plot. According to the symptoms and characteristics of the aboveground part of konjac soft rot and the development trend of the disease, we divided the lesions into the surrounding type and the longitudinal type. The former lesions surround the petiole, and the disease Rapid development will quickly cause compound leaf lodging, which is very harmful; the latter lesion is only on one side of the petiole, and mostly expands longitudinally along the vascular bundle, and later expands laterally, causing the entire petiole to rot.

The grading standard of konjac soft rot (the condition is divided into 5 grades):

Grade 0: no symptoms; grade 1: small water-soaked lesions less than 2 cm on one side of the petiole, or less than 1/10 of the total length of the petiole, and mild yellowing of the leaves on the same side; grade 2: one side of the petiole Lesions of more than 2 cm, or 1/10--5/10 of the total length of the petiole, and yellowing of the leaves on the same side; Grade 3: circular lesions, or longitudinal lesions accounting for 5/10 of the total length of the petiole /10 or more, most of the leaves are yellowed or partially dead; Grade 4: the whole leaf is yellow or lodging and rotten. If the entire compound leaf starts to yellow from the top, and there is soft rot at the junction of the petiole and the tuber, it is a tuber disease, and the disease is classified as Grade 3.

4. Test results

4.1 Field efficacy and yield increase test of biocontrol bacteria C ₃ liquid preparation on konjac soft rot

Table 1 Field control effect of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) C ₃ liquid preparation on konjac soft rot deal with Disease index Total number of plants (plants) Average rate of diseased plants (%) average disease index Control effect (%) Repeat I Repeat II Repeat III CK diluted 250 times and diluted 500 times 19.173.334.67 21.675.929.33 17.175.8313.08 900941953 123.593.22 19.344.269.03 -77.953.3 Diluted 1000 times 6.33 11.75 12.83 952 3.68 10.30 46.7 Chlorothalonil 500 times 10.17 15.08 13.67 927 5.23 12.97 32.9

Table 2 Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) C ₃ liquid preparations to the yield-increasing effect of konjac deal with Yield per mu (kg) Incremental output (kg) Yield increase rate (%) CK diluted 250 times diluted 500 times diluted 1000 times 1600.82267.82134.42134.4 -667533.6533.6 -41.6733.3333.33 Chlorothalonil 500 times 1734.2 133.4 8.33

From Table 1 and Table 2, it can be seen that the biocontrol bacterium C ₃ liquid preparation has obvious effects on the control and production increase of konjac soft rot, among which the 250-fold dilution treatment has the best control and production effect, and the control effect reaches 77.9%. Root irrigation 500 times and 1000 times The control effects on konjac soft rot were 53.3% and 46.7% respectively. Compared with diluting 500 times of chlorothalonil, the disease strain rate of each treatment (250 times, 500 times, 1000 times,) of applying biocontrol bacteria _C3 liquid preparation is lower than it by 1.64%, 2.01%, 1.55%, respectively. The index is 8.71, 3.94, and 2.67 lower than chlorothalonil, and the control effect is 45.00%, 20.40%, and 13.80% higher than it, showing that the biocontrol agent has better control effect; Application of each treatment of biocontrol bacteria _C3 (250 times, 500 times, 1000 times,) the yield increase rate is higher than it by 33.34%, 25.00%, 25.00%, showing that the product has a certain effect of promoting growth and increasing production, so the The product has the basic conditions for mass promotion in production.

The spraying method used the root watering method. In the study, it was found that the probability of pathogenic bacteria invading from the base of the stem was significantly reduced in the konjac plants treated with root irrigation, but some rotten plants in the aboveground part of the field were still found, mainly invading from the aboveground parts such as leaves. Therefore, in the future, the combination of root watering method and foliar spraying method will be used in the method of pesticide application to achieve the purpose of effectively controlling the soft rot of konjac.

4.2 Field efficacy and yield increase test of biocontrol bacteria C ₃ wettable powder on Amorphophallus soft rot

Table 3 field control effect of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) C ₃ wettable powder on konjac soft rot deal with Disease index Total number of plants (plants) Average rate of diseased plants (%) average disease index Control effect (%) Repeat I Repeat II Repeat III CK diluted 250 times and diluted 500 times 19.174.007.05 21.674.509.30 17.173.5310.14 900950959 123.203.32 19.344.018.83 -79.354.3 Diluted 1000 times 8.23 10.11 9.05 960 3.57 9.13 52.8 Chlorothalonil 500 times 10.17 15.08 13.67 927 5.23 12.97 32.9

Table 4 Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) C ₃ wettable powder is to the production-increasing effect of konjac deal with Yield per mu (kg) Incremental output (kg) Yield increase rate (%) CK diluted 250 times diluted 500 times diluted 1000 times 1600.82301.32190.22103.4 -700.5589.4502.6 -43.7636.8231.40 Chlorothalonil 500 times 1734.2 133.4 8.33

From Table 3 and Table 4, it can be seen that the biocontrol bacterium _C3 wettable powder has obvious effects on the control and production increase of konjac soft rot, and wherein the 250-fold dilution treatment is the best in control and production increase, and the control effect reaches 79.3%. 500 times of root irrigation and With 1000 times of treatment, the control effects on konjac soft rot were 54.3% and 52.8%, respectively. Compared with diluting 500 times of chlorothalonil, the sick strain rate of each treatment (250 times, 500 times, 1000 times) of applying biocontrol bacteria _C3 wettable powder is lower than it by 2.03%, 1.91%, 1.66%, respectively. The disease index is 8.96, 4.14, and 3.84 lower than that of chlorothalonil, and the control effect is 46.40%, 21.40%, and 19.90% higher than it, showing that the biocontrol agent has better control effect; , the yield increase rate of each treatment (250 times, 500 times, 1000 times) of applying biocontrol bacteria _C3 was 35.43%, 28.49%, and 23.07% higher than it respectively, showing that the product has a certain growth-promoting and production-increasing effect, so The product has the basic conditions for mass promotion in production.

The overall results show that the biological preparation (no matter liquid preparation or wettable powder) of the present invention has significant effect on preventing and treating bacterial soft rot of konjac, and has the effects of promoting growth and increasing production.

The main control object of the preparation of the invention is konjac soft rot. This preparation can be applied by soaking seeds, root irrigation, spraying and other methods. The preparation of the present invention has been carried out in the field plot control effect test in Fuyuan County, Qujing City, Yunnan Province, and it is proved that the control effect of the biocontrol preparation is remarkable and stable, and can effectively solve the problem of prevention and control of konjac bacterial soft rot that has plagued taro farmers for a long time. The control effect on Konjac bacterial soft rot is 46.7-79.30%.

Detailed ways:

The biocontrol agent can be mixed with other bactericidal pesticides and plant growth regulators, but it must be ensured that the mixed agents have no side effects on the Bacillus amyloliquefaciens _C3 used in the present invention.

The bacterial biocontrol agent of the present invention is generally applied at the stage of crop sowing or disease initiation. The method is to soak (mix) the seeds with the fermented preparation and the bulbs before sowing, then dry and apply it to the soil, or apply the preparation to the base of the konjac stem through root irrigation or spray at the initial stage of the disease, and dilute it 300-500 times use. For areas with severe bacterial soft rot of konjac, topdressing can be applied 2-3 times during the growth of konjac.

The invention has obvious prevention and treatment effects on konjac bacterial soft rot, is suitable for the whole growth period of konjac, and has the characteristics of low cost, convenient use, long validity period and the like.

The present invention is further described in detail below with examples, but the content of the present invention is not limited thereto.

Embodiment one:

Transfer Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) C ₃ to the test tube slant medium, the formula of the test tube slant medium is: beef extract 0.3%, yeast extract 0.1%, peptone 0.5%, glucose 1.0%, agar 1.7%, pH : 7.0.

Incubate at 28°C for 2 days to obtain test tube species.

The liquid fermentation medium formula is: soybean powder 2.0%, starch 1.0%, glucose 0.30%, KH ₂ PO ₄ 0.1%, peptone 0.2% (NH ₄ ) ₂ SO ₄ 0.05%, MgSO ₄ 7H ₂ O 0.03%, CaCO ₃ 0.1%, peanut oil 0.1%, defoamer 0.01%, pH 7.5;

The test tube seed is inoculated in 500ml triangular flask (200ml of every bottle) liquid medium, and medium formula is: beef extract 0.3%, yeast extract 0.1%, peptone 0.5%, glucose 1.0%, agar 1.7. Shake culture at a constant temperature of 28° C. for 14 hours at a rotation speed of 150 rpm, and the obtained culture solution is the biological preparation of the present invention.

Embodiment two:

At first obtain test tube species (same as embodiment one), test tube species is inoculated in 500ml triangular flask (200ml of every bottle) liquid medium, medium formula is: beef soak 0.3%, yeast extract 0.1%, peptone 0.5%, glucose 1.0%, agar 2.0%. Shake culture at a constant temperature of 28° C. for 14 hours at a rotational speed of 200 rpm, transfer the obtained culture solution to a fermenter for fermentation production, and obtain a liquid preparation.

The liquid fermentation medium formula is: soybean powder 2.0%, starch 1.0%, glucose 0.30%, KH ₂ PO ₄ 0.1%, peptone 0.2% (NH ₄ ) ₂ SO ₄ 0.05%, MgSO ₄ 7H ₂ O 0.03%, CaCO ₃ 0.1%, peanut oil 0.1%, defoamer 0.01%, pH 8.0.

Liquid fermentation conditions

Tank temperature: the tank temperature of the fermenter is controlled at 25°C, measured by a thermometer inserted into the medium, and adjusted by passing cooling water or hot water into the interlayer.

Tank pressure: The tank pressure of the fermenter is controlled at 0.5/square centimeter, which is adjusted through the sterile air inlet and exhaust gas outlet.

Stirring: the stirring speed of the fermenter is 200 rpm.

Sampling inspection and culture condition adjustment: take a sample from the sampling tube once every 2 hours, measure the pH, and smear the smear with crystal violet staining to check the shape of the bacteria, whether there is any contamination by bacteria, observe whether the bacteria produce spores, etc. When the number of bacterial spores in the sample accounts for more than 5% of the total bacterial cells, reduce the ventilation rate and lower the culture temperature to about 22°C, and keep the other conditions unchanged. When it is above 85%, stop cultivating and putting the tank in. Microscopic examination counts when the fermenter is put in the tank, and counts viable bacteria with the culture medium plate method.

Culture cycle: The culture cycle of the fermenter is about 36 hours.

Liquid fermentation preparation: the bacteria content was determined by plate counting, and the bacteria content reached 18 billion CFU/ml, which is used for field disease prevention and control.

The biological preparation of the present invention is obtained.

Embodiment three:

At first carry out the liquid fermentation culture of bacterium (same as embodiment two). Then, it is made into wettable powder by adding filling agent, filtering with plate and frame, beating, drying and pulverizing. The specific process is as follows:

Specifically add the filler: put the fermentation hydraulic pressure into the storage tank, add the filler diatomite (light calcium carbonate) according to 10% of the volume of the tank, and stir for 30 minutes after adding.

Plate and frame filtration: Press the material from the storage tank into the plate and frame with a pressure of 2 kg/cm2, and filter through the No. 7 filter cloth. The pressure should be adjusted at any time so that the bacteria content in the filtrate does not exceed 0.2 billion/ml.

Beating: unload the filter cake into the beating tank, add thick milk No. 100 at 8% of the amount of calcium carbonate added, or add SDS at 3%, add 2% of the filtrate and stir evenly for 30 minutes.

Mixing: crush the bacterium-containing diatomaceous earth after beating, dry in the shade, then dilute by 5 times the volume, stir and mix evenly.

Drying: Ventilate and dry in a drying room with a temperature below 60°C to a moisture content of 8%.

Pulverization: The discharge temperature should not exceed 60°C during pulverization to prevent the inactivation of bacteria.

Measurement of finished product quality indicators: Bacteria content, water content, suspension rate, and fineness are all measured according to the national enterprise standard (Q/KWL02-2003). The bacteria content is 1 billion/g, and other indicators meet the standards.

The biological preparation of the present invention is obtained.

Claims (3)

1, a kind of biologic product of preventing and treating konjaku bacterial slimy soft rot disease is characterized in that: the production bacterial strain of said preparation is bacillus amyloliquefaciens (Bacillus amyloliquefaciens) C ₃, preserving number CGMCC No.1553.

2, the described bacillus amyloliquefaciens of claim 1 (Bacillus amyloliquefaciens) C ₃The cultural method of bacterial strain is characterized in that, described bacterial strain is to cultivate acquisition through conventional liquid and solid fermentation, and its liquid and solid fermentation culture medium prescription are as follows:

(1) liquid fermentation medium prescription: analysis for soybean powder 2.0%, starch 1.0%, glucose 0.30%, KH ₂PO ₄0.1%, peptone 0.2% (NH ₄) ₂SO ₄0.05%, MgSO ₄7H ₂O 0.03%, CaCO ₃0.1%, peanut oil 0.1%, defoamer 0.01%, pH 7.5-8.0;

(2) solid fermentation culture medium prescription: beef soaks 0.3%, yeast extract 0.1%, peptone 0.5%, glucose 1.0%, agar 1.7-2.0%, pH6.8-7.0.

3, the described bacillus amyloliquefaciens of claim 1 (Bacillus amyloliquefaciens) C ₃The application of bacterial strain in control konjaku bacterial slimy soft rot disease.