CN107325980B - Radiation-resistant paenibacillus KH9 and application thereof in biological antitranspirant - Google Patents
Radiation-resistant paenibacillus KH9 and application thereof in biological antitranspirant Download PDFInfo
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Abstract
The invention discloses a radiation-resistant paenibacillus KH9 and application thereof in a biological antitranspirant, wherein the radiation-resistant paenibacillus (KH 9) with higher polysaccharide production capability and plant transpiration reduction function is obtained by separating, screening and culturing in soil samples collected in arid desert regions around Xinjiang apocynum venetumPaenibacillus sp.) KH9CGMCC number 12799, which has obvious physiological and biochemical characteristic difference and molecular level difference with common paenibacillus strains, when the screened radiation-resistant paenibacillus strain with the number of KH9 is compounded with an antitranspirant to be applied in drought resistance, a biofilm is formed on the leaves of crops, the transpiration of the crops can be effectively reduced, the photosynthesis is enhanced, the chlorophyll content is increased by 4.50%, the crop has stronger growth capability, osmotic adjustment capability and active oxygen defense capability, and the damage degree of the crops under drought stress can be reduced.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and relates to the technical field of a novel radiation-resistant paenibacillus strain and application thereof in a biological antitranspirant.
Technical Field
The drought trend will continue further as the greenhouse effect increases. At present, 1/3 lands in the world are in arid and semiarid states, and 48.9 percent of cultivated lands in China are also in arid and semiarid regions. Drought stress is a main abiotic stress, seriously restricts the sustainable development of agriculture, and causes the phenomena of slow growth of crops, reduced yield, aggravation of plant diseases and insect pests and the like, so that efficient drought-resisting and water-saving measures are necessary to be developed.
In recent years, chemical water-saving technology is increasingly applied to drought resistance of agriculture, and the use of an antitranspirant is one of the effective measures. After the transpiration resistant agent is sprayed on the leaf surfaces of plants, the transpiration rate of the plants can be reduced, the water loss is reduced, and the drought resistance of the plants is improved by influencing the activity of a protective enzyme system. However, research shows that the application of the existing antitranspirant can cause closed leaf stomata and further reduce photosynthesis, net photosynthetic products are reduced, and further, the growth of crops is slowed down. Therefore, a new transpiration resistant agent needs to be developed, the defects are overcome, and the transpiration resistant agent can be more effectively utilized in the agricultural drought-resistant production.
The drought resistance of microorganisms can induce plants to generate resistance by regulating the metabolic activity of the microorganisms, and certain microorganisms can form a biological film taking an extracellular complex (EPS) as a main component on the surface of leaves to induce a stomatal reaction due to the special high polymer structure of the polysaccharide substances, so that the plant transpiration is reduced, the water utilization efficiency is improved, the water deficiency is resisted, and the functions of preserving water and fertility, improving the soil structure and the like are achieved. The microorganisms and the antitranspirant are compounded to develop a novel biological antitranspirant, on the basis of keeping respective advantages of the microorganisms and the antitranspirant, a more efficient and more reliable effect can be exerted, but at present, reports about synergistic action of the microorganisms and the antitranspirant on plant drought resistance are few, so that the drought resistance of the microorganisms needs to be further researched, a resistant strain with the plant transpiration reducing effect and stable performance is provided for the drought resistance of the microorganisms, and the novel biological antitranspirant has a potential application and development prospect in the field of plant drought resistance.
Disclosure of Invention
Aiming at the current situation that the related reports about the novel radiation-resistant paenibacillus strain and the application of the novel radiation-resistant paenibacillus strain in the biological antitranspirant are not found in the prior art, and the current antitranspirant can cause the closing of leaf surface stomata to weaken photosynthesis so as to slow down the growth of crops, the invention aims to provide a resistant strain with plant transpiration reduction and stable performance in the biological antitranspirant. According to the invention, a new radiation-resistant paenibacillus strain is separated and screened from a soil sample, and the new radiation-resistant paenibacillus strain has higher polysaccharide production capability, so that the plant transpiration is obviously reduced. By providing a radiation-resistant Paenibacillus sp KH9CGMCC No.12799, a method for compounding the bacillus with an antitranspirant and an application technical scheme in drought resistance.
The invention adopts the main technical scheme that:
the soil sample is collected from arid desert areas around Xinjiang Apocynum by Xinjiang agricultural academy of sciences microbial application research, the collected soil sample is irradiated by a 5000KGy cobalt source, a large number of radiation-resistant bacterial strains which grow well, are sticky and are not easy to pick up are selected and optimized by taking a PDA culture medium as a separation culture medium, a 16S rRNA gene sequence is determined, and a new radiation-resistant bacillus strain (Paenibacillus sp) with higher polysaccharide production capacity is selected from the radiation-resistant bacterial strains.
The invention specifically provides a radiation-resistant Paenibacillus (Paenibacillus sp.) KH9CGMCC No.12799, which is obtained by separating, screening and culturing collected soil samples by providing a determined screening method to obtain a batch of Paenibacillus microbial strains, screening a Paenibacillus strain KH9 with higher polysaccharide production capacity from the Paenibacillus microbial strains, and temporarily naming the Paenibacillus (Paenibacillus sp. KH9) as the Paenibacillus strain through microbiological classification and identification.
Specifically, the collected soil sample is irradiated by a 5000KGy cobalt source, and then is separated, screened and cultured, a strain with the number of KH9 is screened out, and the strain belongs to a Paenibacillus sp strain through microbiological classification and identification. The strain has been deposited in the international collection of microorganisms under the Budapest treaty before the filing date: china general microbiological culture Collection center (CGMCC). Address: west road No.1, north west of the republic of kyo, yang, institute of microbiology, academy of sciences of china, zip code: 100101. the preservation date is 2016, 7, month and 20 days, and the preservation number of the strain is CGMCC No. 12799. Is identified as bacillus (Paenibacillus sp.) KH9 by microbiology. The optimal growth conditions of the strain are as follows: the temperature is 30 ℃, the culture medium adopts PDA culture medium (potato 200g/L, glucose 20g/L, agar 15g/L, distilled water 1L, pH is natural), and the culture time is 3 d; after 3d culture, the colony is round, neat in edge, convex, smooth, pink, opaque, sticky and not easy to lift on the surface of the PDA culture medium; the bacteria are gram-positive bacteria, aerobic, motile, rod-shaped, peribiotic flagellum and endospore. According to the morphological characteristics, the KH9 strain is subjected to morphological, physiological and biochemical identification and molecular biological sequencing by referring to Bergey's Manual of systematic bacteriology identification, eighth edition and Manual of systematic identification of common bacteria, the strain is preliminarily identified as a member of Paenibacillus sp, but the strain has distinctive characteristics of a new strain, and is tentatively named as radiation-resistant monospora fungus Paenibacillus sp.KH9 from the taxonomic point of view.
Meanwhile, the invention extracts the total DNA of the strain KH9, adopts the universal amplification primer of the bacteria 16S rDNAPCR to carry out PCR amplification, and the PCR product is sequenced after being cut and purified. The tested 16S rRNA gene sequence is compared with the sequence in the GenBank database, and the result shows that: the strain KH9 has the maximum homology of 97.3% with a model strain Paenibacillus xylaexedens NRRL B-1090T (CLG48537), has the homology of less than 97.0% with other strains in the same genus, cannot be determined to be exactly classified, is determined to be a new strain, and is tentatively named as Paenibacillus sp.KH9 from the taxonomic point of view.
Further, the invention provides a radiation-resistant Paenibacillus (Paenibacillus sp.) KH9CGMCC No.12799 and an antitranspirant compound and application thereof in drought resistance. The anti-transpirant used in the invention is produced by taking camel thorn alkaline essential oil and fulvic acid as main raw materials through chemical washing, purification and activation processes, and is rich in amino acid, proline, abscisic acid, major, middle and trace elements and the like, and KH9 thalli are compounded when the anti-transpirant is used. The strain has the obvious effects of reducing transpiration of crops, enhancing photosynthesis, obviously increasing chlorophyll content, enhancing physiological activity of plant enzyme systems and stimulating the growth of plant root systems.
By implementing the specific technical indexes of the invention, the content of the invention is realized, and the following beneficial effects can be achieved:
(1) the radiation-resistant Paenibacillus sp KH9CGMCC No.12799 provided by the invention has the advantages of simple culture conditions, fast propagation, stable genetic characteristics and higher polysaccharide production capacity.
(2) When the radiation-resistant Paenibacillus sp KH9CGMCC No.12799 and the antitranspirant are compounded and applied in drought resistance, a biological film is formed on the leaves of crops, the transpiration of the crops can be effectively reduced, the photosynthesis is enhanced, the chlorophyll content is increased by 4.50%, the crop has stronger growth capacity, permeation regulation capacity and active oxygen defense capacity, and the damage degree of the crops under drought stress can be reduced.
Drawings
FIG. 1 shows the colony and the cell photograph of radiation-resistant Paenibacillus sp KH9CGMCC No. 12799.
FIG. 2 shows a phylogenetic dendrogram of radiation-resistant Paenibacillus sp KH9CGMCC No. 12799.
FIG. 3 shows the effect of a biological antitranspirant based on radiation-resistant Paenibacillus sp KH9CGMCC No.12799 on the relative water content of leaves of wheat seedlings under drought stress.
FIG. 4 shows the effect of biological antitranspirant based on radiation-resistant Paenibacillus sp KH9CGMCC No.12799 on proline content in leaves of wheat seedlings under drought stress.
FIG. 5 shows the effect of biological antitranspirant based on radiation-resistant Paenibacillus sp KH9CGMCC No.12799 on malondialdehyde content in leaves of wheat seedlings under drought stress.
FIG. 6 shows the effect of a biological antitranspirant based on radiation-resistant Paenibacillus sp KH9CGMCC No.12799 on the activity of superoxide dismutase (SOD) in wheat seedling leaves under drought stress.
Detailed Description
The present invention will be described below by way of examples, but the present invention is not limited to the following examples. All raw and auxiliary materials selected for use in the present invention, as well as methods for culturing the selected bacterial species, are well known and used in the art, and all percentages referred to herein are by weight unless otherwise indicated.
The first embodiment is as follows: separation, screening and identification of radiation-resistant Paenibacillus sp KH9CGMCC No.12799
1. Isolation and selection of bacterial species
The bacillus (Paenibacillus sp.) KH9 used in the invention is obtained by sampling soil collected from a large amount in arid desert areas around Apocynum venetum by Xinjiang academy of agricultural sciences microbial application research institute, irradiating by 5000KGy cobalt source, separating polysaccharide-producing bacteria in a soil layer by using a traditional plate culture method, purifying the strains by using a plate scribing method, optimizing and screening a batch of good-growing bacterial strains with strong polysaccharide-producing capability, and preferably selecting a bacterial strain with the number of KH9 from the bacterial strains.
A separation step:
(1) preparing a soil sample diluent: dissolving the soil sample irradiated by 5000KGy cobalt source in a triangular flask containing sterile water according to a solid-to-liquid ratio of 1: 10, stirring with a glass rod, culturing at 30 deg.C under constant temperature oscillation (160r/min) for 30min, taking out the triangular flask, and standing on a horizontal desktop for 30 min. Taking 1mL of supernatant fluid, placing in a test tube containing 9mL of sterilized water, mixing well, and gradually diluting to 10 degrees by gradient dilution method-4。
(2) Separation and purification: get 10-2、10-3、10-41mL of each dilution was applied to PDA solid medium and immediately spread with a glass spatula. The plate was placed upside down in a 30 ℃ incubator for 5 days. After the bacterial colony grows out, selecting a single bacterial colony which is smooth and sticky in surface and not easy to lift, and continuously separating and purifying by adopting a scribing method until pure culture is obtained.
2. Culture conditions of the strains
(1) The growth medium of the strain No. KH9 was PDA medium: 200g/L of potato, 20g/L of glucose, 15g/L of agar and 1L of distilled water, wherein the pH is natural, and the potato is cultured for 96 hours at 30 ℃.
(2) The strain with the number of KH9 can grow under the condition of 20-40 ℃, the optimal growth temperature is 30 ℃, and the culture time is 3-5 days.
(3) The strain No. KH9 had the optimum growth pH of 7.
Specifically, the collected soil sample is irradiated by a 5000KGy cobalt source, and then is separated, screened and cultured, a strain with the number of KH9 is screened out, and the strain belongs to a Paenibacillus sp strain through microbiological classification and identification. The strain has been deposited in the international collection of microorganisms under the Budapest treaty before the filing date: china general microbiological culture Collection center (CGMCC). Address: west road No.1, north west of the republic of kyo, yang, institute of microbiology, academy of sciences of china, zip code: 100101. the preservation date is 2016, 7, month and 20 days, and the preservation number of the strain is CGMCC No. 12799. The new strain is identified as Paenibacillus microbiologically, and is tentatively classified as Paenibacillus (Paenibacillus sp.) KH9 from the taxonomic point of view. The optimal growth conditions of the strain are as follows: the temperature is 30 ℃, the culture medium adopts PDA culture medium (potato 200g/L, glucose 20g/L, agar 15g/L, distilled water 1L, pH is natural), and the culture time is 3 d.
3. Physiological and biochemical identification of strain KH9
Morphological characteristics: the KH9 strain is streaked and inoculated on a PDA plate for culture for 3d, and the bacterial colony is circular, has neat edges, is convex, smooth, pink, opaque, sticky and is not easy to pick up; the bacteria are gram-positive bacteria, aerobic bacteria, motility, thallus rod shape, periphytic flagellum, and endospore formation, and their colony and thallus morphology are shown in figure 1.
Physiological and biochemical characteristics of the strain, detected by a Biolog GN2 plate, the available carbon source of the strain is cyclodextrin, dextrin, mannan, Tween 40, Tween 80, acetic acid, gamma-hydroxybutyric acid, α -ketoglutaric acid, pyruvic acid, succinamic acid, succinic acid, 2-deoxyadenosine, inosine and D-ketorolose.
Through the thallus morphology, culture characteristic observation and physiological and biochemical index measurement of the radiation-resistant Paenibacillus sp KH9CGMCC No.12799, namely through tests such as thallus morphology observation, strain culture characteristic observation, growth temperature measurement and the like, by referring to methods of Bergey's systematic bacteriology identification handbook eighth edition and common bacteria system identification handbook, a strain with the number of KH9 has obvious physiological and biochemical characteristic difference compared with common Paenibacillus strains, and the capability of producing antibiotics and polysaccharides is stronger, so that the KH9 strain is a typical new strain, and from the taxonomic angle, the strain belongs to a member of the Paenibacillus sp, and is temporarily named as the radiation-resistant monospora fungus Paenibacillus sp.KH9.
Example two: molecular level identification of radiation-resistant Paenibacillus sp KH9CGMCC No.12799
Extracting total DNA of the strain KH9, performing PCR amplification by using a bacterial 16S rDNA PCR amplification universal primer, and sequencing after the PCR product is subjected to gel cutting and purification. BLAST comparison of the obtained sequence of the experimental strain with known sequences in GenBank database was performed to determine the species relationship closest to the genetic relationship with the experimental strain. And combining sequence alignment in EzTaxon (http:// www.ezbiocloud.net/EzTaxon) and calling related mode strain sequences to perform phylogenetic tree analysis on standard mode strain sequences, and performing cluster analysis and phylogenetic tree construction by using a MEGA 5.0 software package and adopting an adjacency method (Neighbor-Joining method). As shown by a phylogenetic tree, the maximum homology of a bacterial strain KH9 and a model bacterial strain Paenibacillus xylosexexendens NRRL B-1090T (CLG48537) is 97.3 percent, the homology of the bacterial strain KH9 and other bacterial strains in the same genus is less than 97.0 percent, the phylogenetic tree refers to an attached figure 2, a bacterial strain with the number of KH9 is clearly different from a common bacillus-like (Paenibacillus sp.) member bacterium, has obvious molecular level difference, is determined to be a new bacterial strain of the bacillus-like, and from the taxonomic point of view, the bacterial strain belongs to the member of the bacillus-like (Paenibacillus sp.) and is temporarily named as Paenibacillus sp.KH9.
Example three: preparation of radiation-resistant Paenibacillus sp KH9CGMCC No.12799 thallus
The activated strain of the invention, namely, the radiation-resistant Paenibacillus (Paenibacillus sp.) KH9CGMCC No.12799 is inoculated into a seed test tube filled with 5ml of PDA liquid culture medium, the seed test tube is cultured at 30 ℃, after shaking culture is carried out at 200rpm for 36h, the seed test tube is inoculated into the PDA liquid culture medium according to the inoculation amount of 2% (100 ml of PDA liquid culture medium filled into a 500ml triangular bottle), the seed test tube is cultured at 30 ℃, shaking fermentation is carried out at 200rpm for 3-4 days, and the effective viable count is more than or equal to 2 hundred million/ml.
Example four: application of biological antitranspirant based on radiation-resistant Paenibacillus sp KH9CGMCC No.12799 in drought resistance
1. The using method comprises the following steps:
the antitranspirant comprises the following components: the liquid fulvic acid is more than or equal to 80g/L, the nitrogen phosphorus and potassium are more than or equal to 40g/L, the alhagi sparsifolia essential oil is more than or equal to 80g/L, and the amino acid is more than or equal to 5 g/L.
Compounding: before use, the antitranspirant is diluted by 500 times, and the spraying amount of the bacterial liquid is 20% of the diluted antitranspirant.
2. Pot experiment
The invention adopts a potting method, and the soil to be tested is desert soil. Removing stones and grass roots, sieving, adding urea, triple superphosphate and potassium sulfate as base fertilizers (calculated according to the nitrogen application amount of 200mg/kg, N: P2O5: K2O: 18:20: 8), and stirring thoroughly. 3 irrigation quantity levels are set in the test, and are respectively as follows: fully supplying water, and maintaining the water content of the soil in the growth period to be 75% of the field water capacity; slight water stress, and the water content of the soil in the growth period is maintained to be 65% of the field water capacity; severe water stress, and the water content of the soil in the growth period is maintained to be 55 percent of the field water capacity. 3 treatments are respectively set under each irrigation quantity level as follows: CK: no antitranspirant and microbial inoculum, T1: 50-fold dilution of antitranspirant, T2: 50 times of the dilution of the antitranspirant and 20 percent of microbial bacteria.
CK. The treatment of T1 and T2 respectively adopts water, 50 times of diluted antitranspirant and 20% microbial inoculum to mix with wheat seeds, then the mixture is tightly sealed for 30min, and then the mixture is aired and sowed, and water is fully supplied. And controlling the water content of the soil in the leaf 3 period, adopting different drought resisting treatments, and uniformly spraying the soil on the wheat leaves. The consumed water was replenished every two days after spraying.
3. Influence of radiation-resistant Paenibacillus sp KH9CGMCC No.12799 biological antitranspirant on relative water content of wheat leaves under different water stresses
The relative water content of the wheat leaves is measured after 5 days of spraying, and the result is shown in figure 3, the relative water content of the leaves of the biological antitranspirant containing the new paenibacillus strain is higher than that of the leaves treated by the single-application antitranspirant by 15.23 percent under the condition of 55 percent of water. Therefore, the biological antitranspirant containing the new paenibacillus strain can enhance the relative water content of plant leaves and reduce the transpiration capability of the leaves.
4. Influence of radiation-resistant Paenibacillus sp KH9CGMCC No.12799 biological antitranspirant on growth of wheat seedlings under different water stresses
Under mild water stress (65%) and severe water stress (55%), the spraying of the bacillus-containing biological antitranspirant has a certain promotion effect on the growth and chlorophyll content of wheat seedlings, and the results are shown in table 1:
table 1: effect of differently treated biological antitranspirants on wheat seedling growth
As can be seen from Table 1, compared with the treatment of single application of the antitranspirant, under the condition of 65% of water, the biomass of wheat seedlings compounded with KH9 thalli is increased by 11.86%, the plant height is increased by 2.35%, and the chlorophyll content is increased by 4.50%; the differences between the T1 and T2 treated biomass were not significant at 55% moisture.
5. Influence of radiation-resistant Paenibacillus sp KH9CGMCC No.12799 biological antitranspirant on Proline (PRO) and Malondialdehyde (MDA) of wheat seedling leaves under different water stresses
As can be seen from the attached figure 4, the content of Proline (PRO) in the leaves of the wheat seedlings treated by the antitranspirant compounded with the new bacillus species under the conditions of 65% moisture and 55% moisture is respectively reduced by 11.08% and 16.93% compared with the treatment of the antitranspirant applied singly.
As can be seen from FIG. 5, the content of Malondialdehyde (MDA) in leaves of wheat seedlings is reduced by 10.48% under 55% water condition, but is increased under 65% water condition compared with the single application of the antitranspirant.
6. Influence of radiation-resistant Paenibacillus sp KH9CGMCC No.12799 biological antitranspirant on superoxide dismutase (SOD) of wheat seedling leaves under different water stresses
As can be seen from FIG. 6, the activity of SOD enzyme was 5.00% in 55% water but increased in 65% water in the antitranspirant treatment containing the new species of Paenibacillus, as compared to the treatment with the antitranspirant alone.
The experiments show that the radiation-resistant Paenibacillus sp KH9CGMCC No.12799 is developed into the biological antitranspirant, the transpiration of crops can be effectively reduced and the photosynthesis is enhanced under the low-moisture condition compared with the single application of the antitranspirant, the chlorophyll content is increased by 4.50%, and meanwhile, the biological antitranspirant has stronger growth capacity, permeation regulation capacity and active oxygen defense capacity on the crops and can improve the damage degree of the crops under drought stress.
The above examples are merely illustrative for clearly illustrating the present invention and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications can be made while remaining within the scope of the present invention.
SEQUENCE LISTING
<110> institute of microorganism application of Sinkiang academy of agricultural sciences (Xinjiang-Yameiya bioengineering research and development center, China)
<120> radiation-resistant paenibacillus KH9 and application thereof in biological antitranspirant
<130>KH9
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Claims (2)
1. Radiation-resistant paenibacillus (B)Paenibacillus sp.) KH9, characterized in that said radiation-resistant Paenibacillus (B.) (Paenibacillus sp.) KH9 has CGMCC accession No. 12799.
2. The radiation-resistant Paenibacillus (B.radiodurans) of claim 1Paenibacillus sp.) KH9 is used as biological antitranspirant.
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| CN111727847B (en) * | 2020-07-08 | 2022-07-15 | 浙江省农业科学院 | Straw fiber seedling culture substrate for colonization biocontrol bacteria, production method thereof and two-step colonization biocontrol bacteria method |
| CN114181850B (en) * | 2021-11-18 | 2023-01-31 | 新疆农业科学院微生物应用研究所(中国新疆—亚美尼亚生物工程研究开发中心) | UV radiation resistant bacillus and application thereof |
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| CN105733966A (en) * | 2016-04-29 | 2016-07-06 | 新疆农业科学院微生物应用研究所 | Radiation-resistant filamentous fungi M30 and application thereof in biological treatment of adsorbing cadmium |
| CN105886407A (en) * | 2016-01-28 | 2016-08-24 | 新疆农业科学院微生物应用研究所 | Radiation-resistant filamentous fungi F35 and application thereof in lead-adsorption biological treatment |
| WO2016179046A1 (en) * | 2015-05-01 | 2016-11-10 | Indigo Agriculture, Inc. | Isolated complex endophyte compositions and methods for improved plant traits |
| CN106631412A (en) * | 2016-12-11 | 2017-05-10 | 孙祎 | Preparation method of non-toxic degradable transpiration inhibiter |
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| WO2016179046A1 (en) * | 2015-05-01 | 2016-11-10 | Indigo Agriculture, Inc. | Isolated complex endophyte compositions and methods for improved plant traits |
| CN105886407A (en) * | 2016-01-28 | 2016-08-24 | 新疆农业科学院微生物应用研究所 | Radiation-resistant filamentous fungi F35 and application thereof in lead-adsorption biological treatment |
| CN105733966A (en) * | 2016-04-29 | 2016-07-06 | 新疆农业科学院微生物应用研究所 | Radiation-resistant filamentous fungi M30 and application thereof in biological treatment of adsorbing cadmium |
| CN106631412A (en) * | 2016-12-11 | 2017-05-10 | 孙祎 | Preparation method of non-toxic degradable transpiration inhibiter |
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