CN102994419B - Bacillus pumilus LNXM12 and application thereof - Google Patents

Abstract

The invention relates to a bacillus pumilus LNXM12 China general microbiological culture collection center (CGMCC) No. 3380 and an application of the bacillus pumilus LNXM12 in preventing and treating damages of wheat scab, wherein the bacillus pumilus LNXM12 can grow easily at low temperature, has growth-promoting effect and disease prevention effect and is scattered in Tibet. The bacillus pumilus is biocontrol bacillus obtained by separating wheat rhizosphere soil in Nyemo County, Lhasa, Tibet; the bacillus pumilus can grow well under the low temperature condition, thereby being beneficial to the growth-promoting function and the biocontrol function; the biocontrol effect on the wheat scab can reach 68%, thereby increasing the yield of the wheat by 12%; and therefore, the bacillus pumilus has good application prospect.

Description

A kind of Bacillus pumilus LNXM12 and its application

technical field

The invention belongs to the technical field of microbes and agricultural disease prevention and control, and relates to Bacillus pumilus, in particular to a low-temperature-suitable Bacillus pumilus LNXM12 and its application in preventing and treating wheat scab disease.

Background technique

Wheat head blight, aliased with dead ears, rotten wheat heads, and red wheat heads, is an important disease of wheat and barley. It spreads all over the world and is mainly distributed in humid and semi-humid areas, especially in temperate areas with humid and rainy climates. It can be damaged from seedlings to heading, mainly causing seedling rot, stem base rot, stalk rot and ear rot, among which ear rot is the most harmful. Wheat scab has long been one of the most serious diseases in the wheat regions south of the Huaihe River and the middle and lower reaches of the Yangtze River in my country. Yield losses in pandemic years can range from 10% to 40%. Fusarium scab not only reduces the yield of wheat, but also makes the diseased grain lose its seed use and industrial value. At the same time, because the low-metabolism product of Gibberella contains toxin, it will cause poisoning to humans and animals after eating.

At present, the main means of preventing and controlling wheat head blight is spraying chemical pesticides. The extensive application of the latter not only causes air, water, soil and food pollution, but more importantly, through the directional selection of chemical pesticides, many pathogenic bacteria are resistant to pesticides. It has brought more difficulties for the prevention and treatment of the disease and seriously threatened the safety of wheat production in my country. In recent years, more and more areas have adopted integrated control methods to control wheat scab, among which biopesticides play an important role. There are many kinds of rhizosphere microorganisms, many of which can promote plant growth, prevent plant diseases, and increase crop yield. These microorganisms are generally called Plant Growth-Promoting Rhizobacteria (PGPR for short). There is a good mutualistic symbiotic relationship between PGPR and plants, which can not only make plants produce induced disease resistance, but also improve the yield and quality of crops. Therefore, it is of great theoretical significance and application value to isolate and utilize probiotics with growth-promoting and disease-preventing effects from PGPR and apply them to the prevention and control of plant diseases and increase yield.

CN 101836638A discloses a fungicide for preventing and treating wheat scab, the active ingredients of which are triadimefon and tebuconazole. The fungicide has stable chemical properties. When mixed, the two components of triadimefon and tebuconazole are mutually compatible, and no chemical reaction occurs. It is widely used, has significant synergistic effects, and is safe to use. It has a good effect on different types of wheat gibberella populations in the field. The antibacterial activity is significantly higher than that of triadimefon or tebuconazole alone, that is, the combination of the two has a synergistic effect. The compound agent also has a good control effect on other important diseases in wheat production such as wheat powdery mildew, has a wide bactericidal spectrum, and can delay the resistance of pathogenic bacteria to the single agent in the formula, which is of great significance for resistance management.

CN 101617698A discloses a preparation method of plant medicine for preventing and treating wheat scab, comprising 5,000 grams of pine needles, 2,000 grams of tobacco leaves, 3,000 grams of nuxychonzi, 8,000 grams of tea seed, 3,000 grams of tripterygium wilfordii powder, and 5,000 grams of lime sulfur mixture. Take pine needles, tobacco leaves, nuxya chinensis, tea dead, and tripterygium twig powder in proportion, grind them together and put them together in a large iron pot, add 200 catties of warm water to soak for 5 minutes, then boil on high heat for 30 minutes, cease fire and filter to get the medicine solution and The lime sulfur mixture is fully mixed and stirred for 10 minutes to complete the finished product.

CN 102405908A discloses a chitosan oligosaccharide composition for resisting wheat scab and its use and method, specifically, relates to a pesticide composition for resisting wheat scab, which contains chitosan Oligosaccharides, chitosan oligosaccharides account for 0.003%-0.028% by weight of the pesticide composition. The invention also provides the above-mentioned pesticide composition containing chitosan oligosaccharide for preventing and treating wheat scab, characterized in that the weight percentage of chitosan oligosaccharide in the pesticide composition containing chitosan oligosaccharide is 0.003%-0.028%. The composition of the invention solves the important problem of resistance to wheat scab, especially after treating the seeds and plants of wheat with the pesticide composition containing chitosan oligosaccharide, it not only resists wheat scab, but also does not produce pesticides on crops. Harmful, no pesticide residues.

CN 1305721A discloses a plant active oligosaccharide pesticide for preventing and treating crop diseases such as wheat head blight. The plant active oligosaccharide pesticide uses plant slag as raw material and obtains it by the following process: in the plant slag raw material Add pectinase with 100,000-150,000 pectinase units per kilogram of raw material slag, stir and heat to 30-48°C for hydrolysis and inactivation, the inactivated solution is filtered and sieved, and then concentrated under vacuum and reduced pressure. The product is obtained by sterilizing and sealing in jars.

There are no detailed reports on the use of PGPR to control wheat scab.

Contents of the invention

One of the objectives of the present invention is to provide a Bacillus pumilus which has a good antibacterial effect on pathogenic bacteria such as wheat scab and has potential biocontrol value in view of the problems existing in the prevention and treatment of wheat scab. . The second object of the present invention is to provide the application of the bacillus pumilus in preventing and treating wheat head blight.

In order to achieve one of the above objects, the present invention adopts the following technical solutions:

A Bacillus pumilus, classified as Bacillus pumilus (Bacillus pumilus) LNXM12, was preserved on November 2, 2009 in the General Microbiology Center of China Committee for the Collection of Microorganisms, and the preservation number is CGMCC No.3380.

The Bacillus pumilus LNXM12 CGMCC No.3380 was isolated from wheat rhizosphere soil in Nim County, Lhasa, Tibet.

The morphological characteristics and physiological and biochemical characteristics of the Bacillus pumilus LNXM12 CGMCC No.3380: Gram stain positive, rod-shaped, capable of producing spores, able to grow at 10°C, but not at 50°C, resistant to Under the culture of pH 5.7 and 7% NaCl, the contact enzyme test, V-P test, citrate utilization test, glucose fermentation test and casein test were all positive, starch hydrolysis test, nitrate reduction test, anaerobic growth test and C Salt utilization tests were negative.

The molecular identification characteristics of the Bacillus pumilus LNXM12 CGMCC No.3380: using PCR technology to amplify part of the 16S rRNA sequence of the Bacillus pumilus LNXM12 CGMCC No.3380 to obtain a fragment with a length of 485bp, which was sequenced The results are shown in the sequence table. After homology comparison with the known 16S rRNA sequence in GenBank, it was found that the similarity with Bacillus pumilus GQ911554.1 was 99%.

According to the results of Gram staining, morphological characteristics, physiological and biochemical characteristics and 16S rRNA sequence analysis of the strains, refer to "Bacillus" (translated by Cai Miaoying, Agricultural Press, 1988) and "Microbial Taxonomy" (Zhang Jizhong, Shanghai) Fudan University Press, 1995) retrieval table, the bacterial strain of the present invention is identified as Bacillus pumilus (Bacillus pumilus) through multiphase classification, named as LNXM12.

The microbial agent containing Bacillus pumilus (Bacillus pumilus) LNXM12 CGMCC No.3380 also belongs to the protection scope of the present invention.

In order to achieve the above-mentioned object two, the present invention adopts the following technical solutions:

Application of the Bacillus pumilus LNXM12 CGMCC No.3380 in the prevention and treatment of wheat head blight. The specific technical scheme is as follows:

The Bacillus pumilus LNXM12 CGMCC No.3380 has significant antibacterial effect on Gibberella.

The Bacillus pumilus LNXM12C GMCC No.3380 has significant antibacterial effect on rice bacterial blight.

The Bacillus pumilus LNXM12 CGMCC No.3380 has a significant antibacterial effect on yeast.

The Bacillus pumilus LNXM12 CGMCC No.3380 has a significant growth-promoting effect on wheat.

The Bacillus pumilus LNXM12 CGMCC No.3380 has a significant growth-promoting effect on Arabidopsis.

The application method of the Bacillus pumilus LNXM12 CGMCC No.3380 strain is as follows: fermentation culture→dilution→spraying→effect measurement.

The fermentation culture of the Bacillus pumilus (Bacillus pumilus) LNXM12 CGMCC No.3380 bacterial strain, its culture medium is composed of: glucose 20g/L, L-glutamic acid 5g/L, MgSO ₄ 0.5g/L, KCl0.5g/L L, KH ₂ PO ₄ 1g/L, Fe ₂ SO ₄ 6H ₂ O 0.15mg/L, MnSO ₄ H ₂ O 5.0mg/L, CuSO ₄ 5H ₂ O 0.16mg/L, the rest is filtered water, Adjust to pH 7.0.

Bacillus pumilus (Bacillus pumilus) LNXM12 CGMCC No.3380 provided by the present invention is a bacillus that has a good control effect on wheat head blight, and is a low-temperature suitable bacterium that can grow at a temperature of 10°C. It is non-polluting to the environment, can significantly inhibit Gibberella, rice bacterial blight and yeast, and can also significantly promote the growth of wheat and Arabidopsis, and has potential biocontrol value.

Description of drawings

Fig. 1 is the spore photograph of the Bacillus pumilus LNXM12 CGMCCNo.3380 of embodiment 2.

Fig. 2 is the bacteriostasis photograph of Bacillus pumilus (Bacillus pumilus) LNXM12 CGMCCNo.3380 of embodiment 6 to Gibberella.

3 is a photo of the antibacterial effect of Bacillus pumilus LNXM12 CGMCCNo.3380 of Example 7 on rice bacterial blight.

4 is a photo of the bacteriostatic effect of Bacillus pumilus LNXM12 CGMCCNo.3380 of Example 8 on yeast.

The technical scheme of the present invention will be further described below in conjunction with the accompanying drawings and through specific implementation methods, but the following examples are only simple examples of the present invention, and do not represent or limit the scope of protection of the present invention. The scope of rights of the present invention is based on rights Requirements shall prevail.

Detailed ways

For better illustrating the present invention, facilitate understanding technical scheme of the present invention, typical but non-limiting embodiment of the present invention is as follows:

Example 1:

Using the five-point sampling method, soil samples were collected at a depth of 5-25 mm in the wheat rhizosphere soil in Nim County, Lhasa, Tibet, and put into sampling bags, and the sampling time, location, and vegetation were marked. Weigh 5g of the soil sample, dissolve it in 45ml of normal saline, shake and mix for 30min on a shaker, and let it stand for clarification. Aspirate the supernatant and dilute with sterile water respectively in five concentration gradients of 10 _-1 , 10 _-2 , 10 _-3 , 10 _-4 , and 10 _-5 , and bathe in 80°C water bath for 10 minutes. Aspirate 200μl of coated LB plates and culture overnight at 37°C. After the colonies grew on the plate, single colonies were picked, streaked and purified, and stored in numbers, and screened to obtain Bacillus pumilus LNXM12 CGMCC No.3380.

Example 2:

With reference to the test methods of "Bacillus" (translations such as Cai Miaoying, Agricultural Press, 1988) and "Microbiological Taxonomy" (Zhang Jizhong, Shanghai Fudan University Press, 1995), the Bacillus pumilus (Bacillus pumilus) obtained in Example 1 pumilus) LNXM12 CGMCC No.3380 morphological characteristics and physiological and biochemical characteristics were detected.

The results show that the bacterium is Gram-positive, rod-shaped, and capable of producing spores, as shown in Figure 1. It can grow at 10°C, but cannot grow at 50°C. It can tolerate the cultivation of pH 5.7 and 7% NaCl. Enzyme test, V-P test, citrate utilization test, glucose fermentation test and casein test were all positive, and starch hydrolysis test, nitrate reduction test, anaerobic growth test and propionate utilization test were all negative.

Example 3:

The Bacillus pumilus LNXM12 CGMCCNo.3380 obtained in Example 1 was inoculated in liquid LB medium, 180rpm, 37°C shaking culture for 10h, the bacteria were collected by centrifugation, after suspension, lysozyme and SDS were added to break the wall, and phenol- Genomic DNA was extracted by chloroform method.

Using genomic DNA as a template, use forward primer PRB1601 (5'-GGATCCTAATACATGCAAGTCGAGCGG-3') (SEQ ID NO:1) and reverse primer PRB1602 (5'-GGATCCACGTATTACCGCGGCTGCTGGC-3') (SEQ ID NO:2) to 16S rRNA was amplified by PCR. The PCR conditions are: 95°C, 4min; 94°C, 1min; 50°C, 1min; 72°C, 2min; 34cycle; 72°C, 10min, stored at 4°C. The PCR product was recovered and sent for sequencing. It was confirmed that the length of the 16S rRNA partial gene sequence was 485 bp. The result was shown in the sequence table, and the similarity with Bacillus spumilus GQ911554.1 in GenBank was 99%.

Example 4:

Prepare 1C medium according to the formula in Table 1:

Table 1

Composition concentration 1×1C 5×1C Peptone 0.7 4.2g 21g Sodium chloride 0.5 3g 15g soy flour 0.3 1.8g 9g final volume 600ml 600ml

Prepare the soil leach solution as follows:

Add 500g of soil to 1L of distilled water, heat at 98°C for 45min, filter through filter paper, and store at 4°C after autoclaving.

Prepare the main medium according to the formula in Table 2:

Table 2

Composition Final concentration Volume (ml) 5×1C 1× 50 20% glucose 0.1% 1.25

Soil Leachate 10% 25 water 173.75

Inoculate the Bacillus pumilus LNXM12 CGMCCNo.3380 obtained in Example 1 into the above-mentioned main medium, cultivate overnight at 37°C, take 5 μl of the bacterial liquid and spot it at the cross point 2.5cm away from the center of the solid medium plate, every Five replicates of each strain were cultured in incubators at 4°C, 10°C, 14°C, 18°C, and 30°C, respectively, and the growth of the strains was recorded every day for 15 days.

The results show that the Bacillus pumilus LNXM12 CGMCCNo.3380 of the present invention can grow in an environment with a temperature of ≤10°C. The strain that can develop colonies within a week at a temperature of 10°C is recorded as Bacillus pumilus LNXM12 CGMCCNo.3380, and further tests show that it can grow at 4°C.

Example 5:

The Bacillus pumilus (Bacillus pumilus) LNXM12 CGMCC No.3380 obtained in Example 1 was inoculated on the LB medium, cultivated overnight, and then the activated Bacillus pumilus (Bacillus pumilus) LNXM12 CGMCC No.3380 was transferred to Landy culture Shake flask fermentation was carried out in the medium at a temperature of 30° C., a shaking table rotation speed of 200 rpm, and a fermentation time of 38 hours to obtain a fermentation broth of Bacillus pumilus LNXM12 CGMCC No.3380.

The composition of the fermentation medium is: glucose 20g/L, L-glutamic acid 5g/L, MgSO ₄ 0.5g/L, KCl 0.5g/L, KH ₂ PO ₄ 1g/L, Fe ₂ SO ₄ 6H ₂ O 0.15mg/L, MnSO ₄ ·H ₂ O 5.0mg/L, CuSO ₄ ·5H ₂ O 0.16mg/L, and the rest is filtered water, adjusted to pH 7.0.

Embodiment 6:

Using the plate confrontation method, pour a PDA plate, place a gibberella block with a diameter of 1 cm in the center of the plate, place four filter paper sheets symmetrically at a distance of 2 cm around it, and beat 5 μl of Bacillus pumilus (Bacillus pumilus) in Example 5 on the filter paper sheet The fermentation broth of LNXM12CGMCC No.3380 was repeated three times and cultured in a light incubator at 25°C for 3 days to observe the antibacterial effect of Bacillus pumilus LNXM12CGMCC No.3380 on Gibberella.

The results are shown in accompanying drawing 2, it can be seen that the average diameter of the inhibition zone is 10 mm, that is, Bacillus pumilus LNXM12 CGMCC No.3380 provided by the present invention has significant antibacterial effect on Gibberella.

Embodiment 7:

Adopt plate face-off method, in the NA culture medium of 150ml, add cultured rice bacterial blight 5ml, pour NA flat plate, put four sterilized filter paper pieces on each flat plate, draw the bacillus pumilus of embodiment 5 of 5 μ l ( The fermentation broth of Bacillus pumilus) LNXM12CGMCC No.3380 was poured on the filter paper and cultured at 28°C for 3 days to observe the antibacterial effect of Bacillus pumilus LNXM12 CGMCC No.3380 on rice bacterial blight.

The result is as shown in accompanying drawing 3, and it can be seen that the average diameter of the inhibition zone of left side and following bacterial colony is 16.5mm, namely Bacillus pumilus (Bacillus pumilus) LNXM12 CGMCC No.3380 provided by the present invention has significant inhibition to bacterial blight of rice. Bacteria.

Embodiment 8:

Inoculate the Bacillus pumilus LNXM12 CGMCCNo.3380 of Example 4 into 1C medium for 8 hours, then transfer to 10ml of Landy medium at a ratio of 1:100, and ferment for about 40 hours. The supernatant was collected by centrifugation and stored at 4°C for later use.

Inoculate the fungal strain yeast to be tested into a suitable medium and cultivate overnight. After measuring the OD value, dilute the bacterial solution to 10 ⁷ cfu/ml, and mix the medium to make a bacteria-containing plate. After the cooling is complete, punch a cross hole (2.5 cm from the center of the plate) and inoculate the fermentation supernatant of the Landy medium with Bacillus pumilus LNXM12CGMCC No.3380. Place it at room temperature and cultivate it for 2 days to observe the bacteriostatic effect on yeast.

The results are shown in Figure 4. It can be seen that the average diameter of the inhibition zone is 28mm, that is, the Bacillus pumilus LNXM12 CGMCC No.3380 provided by the present invention has a significant antibacterial effect on yeast.

Embodiment 9:

Soak wheat seeds in clear water for 2 hours, accelerate germination under 28°C humid conditions, sow them in a turnover box equipped with sterile soil, irrigate the roots when the wheat seedlings grow to 5 true leaves, and pour 20ml of each wheat seedling on average. Example 5 The fermentation broth of Bacillus pumilus LNXM12 CGMCC No.3380 (concentration: 10 ⁸ cfu/ml) was used as control with clean water. After two weeks, 10 seedlings were taken to measure plant height, root length and plant fresh weight.

The results showed that the growth of wheat seedlings treated with bacteria solution was obviously better than that of the control group. After measurement, the increase rate of plant height reached 61.5%, the increase rate of root length reached 3.4%, and the increase rate of fresh weight reached 124.5%. That is, the Bacillus pumilus LNXM12 CGMCC No.3380 provided by the present invention can significantly promote the growth of wheat.

Example 10:

Arabidopsis thaliana seeds were surface-sterilized by sodium hypochlorite (effective Cl content: 5%), then sowed on MS medium plates (9 cm), and cultivated in the greenhouse for one week for later use.

The low temperature suitable Bacillus pumilus (Bacillus pumilus) LNXM12 CGMCCNo.3380 of Example 4 was inoculated into LB liquid medium and cultivated overnight. After measuring the OD value, the concentration of the bacterial solution was quantified as 10 ⁵ cfu/ml.

Arabidopsis seedlings were soaked in the above bacterial solution for 5 minutes, and planted on MS plates (20×20 cm), with 4 seedlings per plate, and 3 replicates for each strain. After 21 days of cultivation in the greenhouse, the plate was taken out and the fresh weight of each Arabidopsis was weighed. The negative control was root soaked in fresh liquid medium.

The results showed that the average fresh weight of Arabidopsis thaliana treated with the bacterial solution was 0.04935g, and the negative control was 0.039444g, that is, Bacillus pumilus LNXM12 CGMCC No.3380 provided by the present invention can significantly promote the growth of Arabidopsis thaliana.

Example 11:

The fermented liquid of Bacillus pumilus (Bacillus pumilus) LNXM12 CGMCC No.3380 of embodiment 5 is diluted 100 times, before earing 15 days before beginning to spray fermented liquid to wheat, spray 1 time every 7 days afterwards, spray 3 times altogether Second-rate.

The results show that the fermentation broth of Bacillus pumilus LNXM12 CGMCCNo.3380 of the present invention has significant effects of preventing and controlling wheat scab and improving wheat yield, and its biocontrol effect can reach 68%, and the yield can be increased by 12%.

It should be noted and understood that various modifications and improvements can be made to the invention described in detail above without departing from the spirit and scope of the invention as claimed in the appended claims. Accordingly, the scope of the claimed technical solution is not limited by any particular exemplary teaching given.

The applicant declares that the present invention illustrates the ratio of raw materials and preparation steps of the present invention through the above-mentioned examples, but the present invention is not limited to the above-mentioned ratio and preparation steps, that is, it does not mean that the present invention must rely on the above-mentioned preparation steps to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent replacement of the selected raw materials in the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the present invention.

Sequence Listing: (SEQ ID NO:3)

TGAGCGTAGTTGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTGCGAGCAGTTACTCTCGCACTTGTTCTTCCCTAACAACAGTAGCTTTACGATCCGAAGACCTTCATCACTCACGCGGCGTTGCTCCGTCAGGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTCGCCTTGGTGAGCCATTACCCCACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGACAGCCGAAACCGTCTTTCATCCTTGAACCATGCGGTTCAAGGAACTATCCGGTATTAGCTCCGGTTTCCCGGAGTTATCCCAGTCTTACAGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTAACATCCGGGAGCAAGCTCCCTTCTGTCCGCTCGACTTGCATGTATTAGGATCCA

Claims (1)

1. Application of Bacillus pumilus LNXM12CGMCC No.3380 in inhibiting yeast; The Bacillus pumilus (Bacillus pumilus) LNXM12 was preserved on November 2, 2009 in the General Microorganism Center of the China Microbiological Culture Collection Management Committee, and the preservation number is CGMCCNo.3380, which is isolated from the wheat rhizosphere soil in Nimu County, Lhasa, Tibet. Obtained; its morphological and physiological and biochemical characteristics are: Gram staining positive, rod-shaped, able to produce spores, able to grow at 10°C, but not at 50°C, and able to tolerate the cultivation of pH 5.7 and 7% NaCl, The contact enzyme test, V-P test, citrate utilization test, glucose fermentation test and casein test were all positive, and the starch hydrolysis test, nitrate reduction test, anaerobic growth test and propionate utilization test were all negative.