CN106701618B - Bacillus amyloliquefaciens capable of resisting powdery mildew of melon crops and producing spores at high yield - Google Patents
Bacillus amyloliquefaciens capable of resisting powdery mildew of melon crops and producing spores at high yield Download PDFInfo
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Abstract
The invention discloses a Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) KN-527 with high spore yield through normal-pressure room-temperature plasma mutagenesis, wherein the preservation number is CCTCC NO: M2015709. The strain has the advantages that the fermentation spore number is increased by 161% compared with that before mutagenesis, the spore formation rate is high, the heat resistance is good, the strain has a remarkable disease resistance effect on melon powdery mildew, the prevention effect reaches more than 95%, and the strain can be applied to cucumber, pumpkin, bitter gourd, watermelon and other melon crops. According to melon field test verification, after the microbial agent produced by the strain is applied with conventional fertilization, the plant grows vigorously, the capability of growing hair and side branches is strong, and the leaf color is bright green.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a bacillus amyloliquefaciens (Bacillus amyloliquefaciens) strain, and a microbial agent for resisting powdery mildew of melon crops.
Background
Powdery mildew is a common disease of melon crops, 3 powdery mildews belong to 6 species, the monascus cucurbitae and the erysiphe bisporus are main pathogens, and the monascus cucurbitae is reported to discover a plurality of physiological races. Powdery mildew mainly harms leaves and also infects leafstalks, melon vines and melons. The leaf blade is firstly attacked to generate small nearly circular white mildew spots (namely pathogenic bacteria hypha and conidium) on the leaf surface, then the small nearly circular white mildew spots are expanded to form mildew spots, the mildew spots are quickly expanded under proper conditions and are connected with each other, white powder can be fully distributed on the whole leaf blade to cause the leaf blade to become brittle, the mildew spots on the later period become grey, and black small granules, namely the cyst closure shell of the pathogenic bacteria, grow on the white powder. The diseased leaves are irregular yellowish-brown spots, and the spots are connected to cause the brown of the whole leaves to be dry but not fall off. When the petiole, the melon vine and the melon are infected with diseases, white powdery spots are also generated, and the diseased parts are covered with white powder after development. Powdery mildew can reduce plant canopies, reduce the single plant fruit setting rate and reduce fruits, thereby causing yield reduction and loss of market value and causing serious economic loss for cultivators.
China is a big agricultural country, and pesticides are used in a large variety and large amount, wherein 70-80% of the pesticides are directly scattered in the environment. After the chemical pesticide is applied, the plant can adsorb a large amount of pesticide, and the pesticide enters the interior of the plant through the osmosis effect, so that serious residue is caused, and the pollution is caused to agricultural products. The organisms continuously absorb low-dose toxic substances from environmental media or food and gradually accumulate and concentrate in the bodies, in the whole ecological system, pesticides are continuously concentrated step by step and amplified step by step through biological enrichment and transmission of a food chain, and human beings are positioned at the top of the food chain, so that the damage is the most serious. The continuous use of chemical pesticides causes the decrease of soil fertility, hardening and the like, which can cause the drastic change of a soil microbial system, further influence the activity of enzymes in soil and the transformation of nutrient substances, change the efficiency and speed of nutrient cycle of an agronomy ecosystem, and reduce the continuous productivity of land. Aiming at the physiological characteristics of diseases, timely and symptomatic administration of the pesticides and selection of the high-efficiency, safe and high-bioactivity biopesticides are particularly important.
At present, the powdery mildew is mostly prevented and controlled by a chemical method, but the powdery mildew pathogenic bacteria are fast in differentiation and many in subspecies, and chemical reagents are difficult to effectively guarantee the pollution-free production of crops for a long time, so that a new safe, efficient and economic prevention and control means becomes a hot door. A number of antagonistic microorganisms have been reported in recent years including bacteria, yeast, actinomycetes, and the like. However, the stress resistance of common bacteria, yeasts and actinomycetes is poor, the requirement on the growth environment is high, and the method cannot be popularized and applied in batches. The spore is used as the organism with the strongest stress resistance in the whole biological world and is very prominent in the aspects of heat resistance, chemical medicine resistance, radiation resistance and the like. The bacillus amyloliquefaciens is used as an aerobic spore-producing gram-positive rod-shaped bacterium, has no report on causing diseases to human and livestock at present, has simple nutritional requirement, easy survival, field planting and propagation and good stability, can improve the yield of crops, increase the stress resistance, greatly increase the soil fertility, can achieve the effect of inhibiting pathogenic bacteria after propagating into dominant flora in soil, and is very suitable for mass production and application as a biological control bacterium and a biological fertilizer.
Disclosure of Invention
The invention aims to make up the blank of bacillus amyloliquefaciens in the field of preventing and controlling powdery mildew of melon crops, and provides a bacillus amyloliquefaciens with high spore yield and a microbial agent thereof.
The invention takes Bacillus amyloliquefaciens as an original strain, obtains a Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) KN-527 with high yield of spores through normal-pressure room-temperature plasma mutagenesis, is preserved in China Center for Type Culture Collection (CCTCC) in Wuhan university in Wuhan, Han dynasty in 2015 at 11-26 days, and has a preservation number of M2015709.
The invention provides a microbial agent for resisting powdery mildew of melon crops, which comprises the bacillus amyloliquefaciens KN-527.
The microbial agent can be used as a biological fertilizer for promoting the growth of melon crops.
The invention has the beneficial effects that:
after the bacillus amyloliquefaciens is subjected to plasma mutagenesis and screening, the spore production number can reach 1.19E +10 CFU/g, and is improved by 161 percent compared with the original strain 7.37E +09 CFU/g before mutagenesis. The field test result shows that after 14 days of pesticide application, the control effect of KN-527 on melon powdery mildew reaches more than 95%, and is obviously superior to the chemical pesticide carbendazim. The strain can effectively prevent the powdery mildew of melons, the disease indexes of cucumbers, pumpkins and watermelons in experimental groups are far lower than those of control groups, 3000 times of the strain solution is diluted by 0.1 hundred million cfu/g of bacillus amyloliquefaciens microbial inoculum (applied for 14 days), the powdery mildew of melons can be effectively prevented and controlled, the disease index is controlled to be below 6%, and the prevention effect reaches more than 90%; after 14 days of application, the disease index of the control group reaches more than 97 percent, and the control effect on powdery mildew of non-melon crops such as strawberries, wheat, hot peppers and the like is low.
Detailed Description
The present invention will be described in detail below with reference to examples.
Example 1
(1) Purification culture of bacillus amyloliquefaciens original strain
Selecting a single colony of a starting strain bacillus amyloliquefaciens KN-52 (obtained by natural screening from soil of Wuhan vegetable planting bases), inoculating the single colony into a simple culture medium consisting of peptone 1%, yeast extract powder 0.5%, sodium chloride 1% and pH7.2-7.5, culturing on a constant temperature shaking table at the temperature of 32 ℃ and 180rpm for 16-20 hours, taking 10ml of fermentation liquor, centrifuging at the temperature of 4 ℃ and 6000r/min, and collecting bacterial pulp for later use;
(2) primary screening: normal temperature and pressure plasma induced change method
And (2) dissolving the bacteria prepared in the step (1) with 10ml of sterile physiological saline, uniformly shaking, coating 10 mu l of the bacteria on a sterilized metal slide, placing the slide on an objective table, adjusting the height of the objective table, and irradiating the bacillus amyloliquefaciens after setting the treatment time. Helium is used as a carrier, the irradiation power is 150W, the working air flow is 10SLPM, the irradiation distance is 2mm, and the irradiation time is 60-240 s. And transferring the slide to an EP tube filled with 1ml of sterile normal saline after irradiation treatment, and oscillating for 1-2 min to enable thalli attached to the slide to be eluted into the normal saline to form a new sensitive bacteria suspension. Diluting the sensitive bacteria suspension to 10-4、10-5、10-6There are 3 gradients.
(3) Collecting muskmelon powdery mildew pathogenic bacteria, preparing pressure screening plate
Collecting muskmelon leaves seriously infected with powdery mildew in a field, spraying sterile water on the surfaces of the leaves, and standing overnight in a refrigerator at 4 ℃. In a clean bench, the water agar plate is blown in by an ear washing ball, and the water agar plate is put into an incubator and cultured for 7d at 20 ℃.
Preparing a pressure screening flat solid culture medium, wherein the culture medium consists of 5-10% of sucrose, 0.1-0.5% of yeast extract, 0.02-0.05% of sodium chloride, 0.02-0.05% of magnesium sulfate, 0.05-0.2% of calcium carbonate, 2% of agar powder and pH7.2-7.5, and adding 2% of powdery mildew pathogenic bacteria at about 45 ℃ after sterilization;
each gradient of diluted susceptible bacterial suspension was coated onto 3 pressure screening plates. The coated plate was placed in an incubator at 30 ℃ for 16 h. Picking single bacterial colony with large zone of inhibition zone and transparent band, inoculating the single bacterial colony into a slant culture medium, culturing the single bacterial colony in a constant temperature incubator at 30 ℃ to form spores, and storing the spores in a refrigerator at 4 ℃ for later use;
the slant culture medium consists of peptone 1%, yeast extract powder 0.5%, sodium chloride 1%, agar powder 1.5-2% and pH7.2-7.4;
(4) selection of highly productive strains of resistant strains
Taking the slant strain in the step (3), washing with 25ml of sterile water, loading into a sterile 100ml triangular flask with glass beads, shaking and mixing uniformly to prepare a bacterial suspension, putting into a 70 ℃ water bath kettle for water bath for 30min, taking out, cooling to room temperature immediately, inoculating into a fermentation seed shake flask, culturing at 32 ℃ and 180rpm for 16-24h, transferring into a fermentation shake flask, and culturing at 32 ℃ and 180rpm for 40-48 h; taking shake flask fermentation liquor, performing conventional smear, performing red-recovery dyeing by using alkaline carbolic acid, and observing spore formation rate after fermentation by using an Olinbas microscope oil scope (16 × 100 times).
Selecting the strain with typical bacillus amyloliquefaciens spore shape and high spore forming rate. And comprehensively judging the disease resistance and the spore formation rate of the mutagenic strain according to the results to obtain the strain KN-527 with high spore formation rate.
Compared with CFU (carbon fiber Unit) after fermentation, the number of spores of KN-527 can reach 1.19E +10, which is increased by 161% compared with 7.37E +09 of the original strain CFU before mutagenesis, and is shown in Table 1.
TABLE 1
| Bacterial strains | Numbering | Sample volume (ml) | Test tube | 1 | 2 | 3 | Blank space | CFU |
| KN-52 (original strain) | 1-1 | 0.5 | 7 | 67 | 82 | 71 | 3 | 7.43E+09 |
| KN-52 (original strain) | 1-2 | 0.5 | 7 | 73 | 72 | 68 | 6 | 7.30E+09 |
| KN-527 | 2-1 | 0.5 | 7 | 110 | 117 | 119 | 5 | 1.18E+10 |
| KN-527 | 2-2 | 0.5 | 7 | 120 | 118 | 119 | 2 | 1.20E+10 |
Example 2 field inhibition effect of bacillus amyloliquefaciens KN-527 on melon powdery mildew was verified.
1. Material
Reagent to be tested: the bacillus amyloliquefaciens KN-527 and the chemical pesticide are traditional powdery mildew control agents.
Test work: melon (sweet melon)
The control object is: powdery mildew
The experiment was set up with 3 treatments:
(1) control group: treating with clear water;
(2) treatment group: 0.1 hundred million CFU/g of the Bacillus amyloliquefaciens diluted 1000, 2000 and 3000 times
(3) 50% carbendazim wettable powder;
the processing is carried out in a melon greenhouse in a scientific research base. Before the tested melons are applied, the lateral vines of the melons are removed, the application is carried out for the first time at the early stage of disease occurrence, the application is carried out for 1 time after 7 days, and the liquid medicine is uniformly sprayed on plant leaves.
2. Method and results
And (3) performing fixed-point plant-positioning investigation, namely 10 plants are selected in each section, 5 leaves with the length of more than 5cm are investigated according to growth conditions of each plant, disease progression is investigated before medicine, 7d and 14d after medicine are investigated once respectively, and the disease progression is counted in a grading manner according to the disease degree of the leaves.
Grading standard: according to the grade index standard of 0-9 disease conditions
Level 0: healthy leaves without spots
Level 1: the lesion spots account for less than 10% of the area of the whole leaf;
and 3, level: the disease spots account for 11 to 25 percent of the area of the whole leaf;
and 5, stage: the disease spots account for 16 to 40 percent of the area of the whole leaf;
and 7, stage: the lesion spots account for 41 to 65 percent of the area of the whole leaf;
and 9, stage: the lesion spots account for more than 65% of the area of the whole leaf;
the drug effect calculation method comprises the following steps:
disease index (%) ═ sigma (number of diseased leaves at each level x number of corresponding disease stages) × 100%/survey total number of leaves x number of highest disease stages;
the disease index increase rate is (after-medicine disease index-before-medicine disease index)/before-medicine disease index is multiplied by 100 percent;
the control effect is [1- (treatment area drug-back disease means x control area drug-front disease means)/(treatment area drug-front disease means x control area drug-back disease means) ] x 100%.
TABLE 2 field inhibition effect of Bacillus amyloliquefaciens on melon powdery mildew
As can be seen from the data in Table 2, 0.1 hundred million cfu/g of bacillus amyloliquefaciens microbial inoculum diluted 1000, 2000 and 3000 times of the microbial inoculum (applied for 14 days) can effectively prevent and control powdery mildew of melons, the disease index is controlled to be below 5 percent, and the prevention effect reaches more than 95 percent; after 14 days of pesticide application, the control group disease means 98.2%, the control effect of 50% carbendazim wettable powder on the melon powdery mildew is 65.4%, and the control effect of 3000 times diluted bacterial liquid on the melon powdery mildew can reach 95.9%.
Example 3 field effect verification of Bacillus amyloliquefaciens KN-527 as a biofertilizer on melon.
Reagent to be tested: bacillus amyloliquefaciens KN-527
Test work: melon (sweet melon)
Nutrient solution: israel Polyfood all-element nutrient solution
The test method comprises the following steps:
the muskmelons planted for one week are divided into two groups, wherein the group A is applied with nutrient solution and clear water, and the group B is applied with nutrient solution and bacillus amyloliquefaciens wettable powder.
And (4) investigating plant growth condition, fruit growth condition, yield and quality in the vine-pulling stage, the melon-setting stage and the melon-expanding stage.
TABLE 3 field Effect verification of Bacillus amyloliquefaciens as a biofertilizer on melon
Test results show that the bacillus amyloliquefaciens KN-527 can be used as a biological fertilizer, has an obvious effect of promoting the growth of melon plants, enhances the capability of growing hair and lateral branches, and makes the plants robust.
Example 4 field inhibition of bacillus amyloliquefaciens KN-527 against powdery mildew of melons and other crops.
1. Material
Reagent to be tested: bacillus amyloliquefaciens CCTCC NO of M2015709
Test work: cucumber, pumpkin, watermelon, strawberry, wheat and hot pepper
The control object is: powdery mildew
The experiment was set up with 2 treatments:
(1) treatment group: clear water treatment (CK);
(2)0.1 hundred million CFU/g 3000-fold liquid of bacillus amyloliquefaciens
The above treatment is carried out in each greenhouse of the scientific research base. The test article is applied for the first time at the early stage of disease onset, and is sprayed with the medicine for 1 time after 7 days, and the medicine liquid is uniformly sprayed on plant leaves.
2. Method and results
And (3) performing fixed-point plant-positioning investigation, wherein 20 plants are selected in each area, 5 leaves with the length of more than 5cm are investigated for each plant according to growth conditions, disease progression is investigated before medicine, 7d and 14d after medicine are investigated once respectively, and the disease progression is counted in a grading manner according to the disease degree of the leaves.
Grading standard: according to the grade index standard of 0-9 disease conditions
Level 0: healthy leaves without spots
Level 1: the lesion spots account for less than 10% of the area of the whole leaf;
and 3, level: the disease spots account for 11 to 25 percent of the area of the whole leaf;
and 5, stage: the disease spots account for 16 to 40 percent of the area of the whole leaf;
and 7, stage: the lesion spots account for 41 to 65 percent of the area of the whole leaf;
and 9, stage: the lesion spots account for more than 65% of the area of the whole leaf;
the drug effect calculation method comprises the following steps:
disease index (%) ═ sigma (number of diseased leaves at each level x number of corresponding disease stages) × 100%/survey total number of leaves x number of highest disease stages;
the disease index increase rate is (after-medicine disease index-before-medicine disease index)/before-medicine disease index is multiplied by 100 percent;
the control effect is [1- (treatment area drug-back disease means x control area drug-front disease means)/(treatment area drug-front disease means x control area drug-back disease means) ] x 100%.
TABLE 4 field inhibition effect verification of Bacillus amyloliquefaciens on powdery mildew of melons and other crops
The powdery mildew has 3 species belonging to 6 species, and the monascus cucurbit and the powdery mildew are main pathogens. The pathogenic bacteria of powdery mildew of melons such as cucumber, pumpkin, watermelon and the like are mainly lecanomyces cucumerinum, while the pathogenic bacteria of powdery mildew of strawberry, wheat and pepper are mainly lecanomyces lupulifera, poaceae powdery mildew and leveillula taurica. As can be seen from the data in Table 4, 3000 times of bacteria solution (applied for 14 days) diluted by 0.1 hundred million cfu/g of Bacillus amyloliquefaciens can effectively prevent and treat the powdery mildew of melons, the disease index is controlled to be below 6 percent, and the prevention effect reaches more than 90 percent; after 14 days of application, the disease index of the control group reaches more than 97 percent, the control effect on powdery mildew of strawberries, wheat and hot peppers is 81.3 percent, 50.2 percent and 63.8 percent respectively, and the control effect is not ideal.
Claims (2)
1. A strain of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) KN-527 capable of resisting powdery mildew of melon crops and producing spores at high yield is characterized in that: is preserved in China center for type culture Collection with the preservation number of CCTCC NO: M2015709.
2. A microbial agent for resisting powdery mildew of melon crops is characterized in that: the active ingredient of the microbial agent is the bacillus amyloliquefaciens of claim 1.
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| CN105503391A (en) * | 2015-12-21 | 2016-04-20 | 云南农业大学 | Functional foliage fertilizer for preventing and controlling powdery mildew of tomatoes and cucumbers and application |
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