CN114456981B - Salt-alkali-tolerant land nitrogen-fixing soybean rhizobium and application thereof - Google Patents
Salt-alkali-tolerant land nitrogen-fixing soybean rhizobium and application thereof Download PDFInfo
- Publication number
- CN114456981B CN114456981B CN202210193179.1A CN202210193179A CN114456981B CN 114456981 B CN114456981 B CN 114456981B CN 202210193179 A CN202210193179 A CN 202210193179A CN 114456981 B CN114456981 B CN 114456981B
- Authority
- CN
- China
- Prior art keywords
- strain
- nitrogen
- dian
- bradyrhizobium
- soybean
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
- A01G22/40—Fabaceae, e.g. beans or peas
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G7/00—Botany in general
- A01G7/06—Treatment of growing trees or plants, e.g. for preventing decay of wood, for tingeing flowers or wood, for prolonging the life of plants
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Environmental Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Microbiology (AREA)
- Ecology (AREA)
- Virology (AREA)
- Genetics & Genomics (AREA)
- Biodiversity & Conservation Biology (AREA)
- Forests & Forestry (AREA)
- Botany (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Pest Control & Pesticides (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Dentistry (AREA)
- Plant Pathology (AREA)
- Agronomy & Crop Science (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a nitrogen-fixing soybean bradyrhizobium japonicum strain for saline-alkali tolerant land and application thereof. The Dian strain of the Wttawa bradyrhizobium obtained by separation is preserved in the Guangdong province microbial strain preservation center in 2022 at 1 month and 11 days, and the preservation number is GDMCC No:62202. the research of the invention shows that the strain has the characteristics of high nodulation rate and strong nitrogen fixation capacity, and can optimize the micro-ecological environment for the root growth of crops in alkaline soil; the plant is treated by the Dian strain, so that the nodulation and nitrogen fixation of the soybean can be effectively promoted, the nitrogen nutrition of the plant can be increased, the growth of the plant can be promoted, and the biomass, the number of nodules and the nitrogen content of the soybean plant can be obviously improved. The strain is particularly suitable for the cultivation and production of soybeans in stony desertification areas, expands a strain resource library of nitrogen-fixing rhizobia, is favorable for promoting plant nodulation and nitrogen fixation, and increases the nitrogen nutrition of plants.
Description
Technical Field
The invention belongs to the technical field of agricultural microorganisms. More particularly, relates to a nitrogen-fixing soybean rhizobium in a saline-alkali tolerant land and application thereof.
Background
Rhizobia (Rhizobium) is a group of gram-negative bacteria widely distributed in soil. The symbiotic nitrogen fixation system between leguminous plants and rhizobia has been a research hotspot. The physicochemical characteristics of the soil are important factors influencing the symbiotic nitrogen fixation of the rhizobia and leguminous plants. In the karst ecosystem, the symbiotic rhizobia with leguminous plants predominate in the genus bradyrhizobium (liuluo et al, 2018). Researches show that the leguminous plants and rhizobia form a symbiotic nitrogen fixation system, so that the problems of soil structure deterioration, insufficient soil nutrients and the like caused by pollution and degeneration can be solved. For example, plum blossom (2010) was inoculated with rhizobia while planting white clover in reclaimed soil in the mountainous and western mines. The result shows that the average plant height and the average crown width of the white clover after inoculation of the rhizobium are respectively 53.87 percent and 32.33 percent higher than those of the control group, and are obviously higher than those of the planting mode without inoculation of the rhizobium. In addition, liu Xiaoguang et al (2006) also found in field experiments of Wuchuan drought farmers in inner Mongolia province that direct inoculation of rhizobia in wind-eroded degraded soil can significantly promote the formation of soil aggregates, improve the soil structure, increase the soil fertility, improve the water storage condition of the soil, and improve the wind erosion resistance of the soil. In addition, rhizobia can also improve the physicochemical property of soil in the stony desertification region and promote the growth of vegetation. Luchengchong et al (2008) find that rhizobia of Acacia confusa Merr in Guangxi karst region has strong adaptability to high-calcium and alkaline earth, and the rhizobia can secrete acid, thereby improving karst alkaline soil and accelerating the renewal of vegetation in the forest.
The soybean is a typical nitrogen fixation crop and is also a main grain and oil crop in China. Rhizobia forms a symbiotic nitrogen fixation system with soybeans and provides nitrogen for soybean growth. Inoculating high-efficiency rhizobia is an important measure for promoting symbiotic nitrogen fixation of soybeans and improving nitrogen nutrition of the soybeans. At present, most rhizobia are separated from fresh rhizobia and belong to Bradyrhizobium (Bradyrhizobium), for example, chinese patent CN101182478A discloses a rhizobium nitrogen-fixing strain BDYD1 and application thereof, wherein the strain BDYD1 is of Bradyrhizobium and has the characteristics of acid-aluminum resistance and high-efficiency nodulation, and the rhizobia is suitable for acid soil areas in south China. Most of the slow rhizobia identified at present are only suitable for acid soil areas in south China, but not suitable for alkaline soil, such as saline-alkali soil in stony desertification areas. Therefore, in order to expand the microbial strain resource library of nitrogen-fixing rhizobium, more new microbial strains with efficient nitrogen-fixing capacity need to be screened out to fill the microbial strain resource library.
Disclosure of Invention
The invention aims to overcome the defects of the problems and provide a nitrogen-fixing soybean rhizobium strain capable of resisting saline-alkali soil and an application thereof.
The first purpose of the invention is to provide a saline-alkali soil resistant nitrogen-fixing soybean rhizobium.
The second purpose of the invention is to provide the application of the strain.
The third purpose of the invention is to provide a nitrogen-fixing microbial preparation.
The fourth purpose of the invention is to provide the application of the nitrogen-fixing microbial preparation.
The fifth object of the invention is to provide a method for promoting nodulation and nitrogen fixation of soybean.
The above purpose of the invention is realized by the following technical scheme:
the invention separates and screens a rhizobium Dian from soybean rhizobium planted in the Guizhou Xingyi stony desertification area. The strain has the morphological characteristics as follows: slow growing type, the optimum growth temperature is 28 ℃, and on YMA plate culture medium, a colony which is approximately round, viscous, smooth, protruded and milky white is formed. The rod shape is short under the optical microscope, most of the movement is carried out, and spores are not generated. Gram staining was red, gram negative bacteria. The sequence of the 16S rDNA gene of the strain is sequenced and analyzed, the nucleotide sequence of a Dian strain is shown as SEQ ID NO 1, similarity search is carried out on a sequencing result by utilizing a BLAST program in a GenBank database, multiple sequence comparison and construction of a phylogenetic tree are carried out by MEGA4.1 software, and the sequence similarity of the 16S rDNA gene of the rhizobium strain provided by the invention and the sequence similarity of a Bradyrhizobium otawaense strain OO99 of a strain of Bradyrhizobium (Bradyrhizobium sp.) is up to 99.34 percent, so that the strain is identified as the Didyrhizobium strain of Bradyrhizobium (Bradyrhizobium otawaense) from Bradyrhizobium sp).
The invention provides a salt and alkali tolerant nitrogen fixing soybean Bradyrhizobium sp Dian strain, which is preserved in Guangdong province microbial culture collection center (GDMCC) in 1-11 days 2022, wherein the strain is deposited as GDMCC No:62202.
the research of the invention shows that the Dian strain has the characteristics of acid production and starch hydrolysis, while the general rhizobia has no starch hydrolysis capacity, so that the Dian strain can be used in the food or chemical industry to replace amylase to hydrolyze starch; because the stony desertification area is alkaline soil, the acid production characteristic of the Dian strain can help to optimize the root growth micro-ecological environment of alkaline soil crops and promote the growth of plants. The strain is inoculated to a plant, can effectively promote the nodulation and nitrogen fixation of the soybean, increase the nitrogen nutrition of the plant, promote the growth of the plant and obviously improve the biomass, the root nodule number and the nitrogen content of the soybean plant. The strain is particularly suitable for the cultivation and production of soybeans in stony desertification areas, and is favorable for promoting plant nodulation and nitrogen fixation and increasing the nitrogen nutrition of plants.
Therefore, the application of the Dian rhizobium strain provided by the invention in enhancing the adaptability of soybeans to stony desertification soil environment, promoting vegetation recovery, promoting nodulation and nitrogen fixation of soybeans, promoting plant growth and improving soybean yield is in the protection range of the invention.
The invention provides an application of the rhizobium Dian strain in preparation of a nitrogen-fixing microbial preparation suitable for rocky desertification areas.
The invention provides a nitrogen-fixing microbial preparation which contains a rhizobium Dian strain and/or a bacterial liquid thereof.
Preferably, the bacterial liquid is bacterial suspension.
Preferably, the OD value of the bacterial suspension is 0.6 ± 0.05.
Preferably, the fermentation conditions for preparing the bacterial suspension are 24-32 ℃ and the pH is 6.8-7.0.
More preferably, the fermentation conditions are 28 ℃ and pH 7.0.
The invention provides application of the nitrogen-fixing microbial preparation in promoting nodulation and nitrogen fixation of soybeans and promoting plant growth, in particular application in promoting nodulation and nitrogen fixation of soybeans and plant growth in stony desertification areas.
The invention also provides a method for promoting the nodulation and nitrogen fixation of the soybeans, which adopts the Dian strain, the bacterial liquid or the microbial preparation to treat plants.
The invention has the following beneficial effects:
the slow rooting rhizobium Dian strain is separated and screened from the soybean root nodule, and has the characteristics of high nodulation rate and strong nitrogen fixation capacity; the inoculation of the bradyrhizobium Dian has obvious influence on the biomass of the soybean, increases the fresh weight of soybean plants by 21.69 percent, also obviously improves the SPAD value and the nitrogen content of soybean leaves, and increases the SPAD value and the nitrogen content by 12.26 percent and 40.02 percent respectively, thereby promoting the growth of the plants. The tieback test shows that the inoculated Dian strain can obviously improve the total nitrogen content of soybean plants, so that the microbial preparation prepared by the Dian strain can be widely popularized and applied in a large area, the adaptability of soybeans to the soil environment of the stony desertification saline-alkali soil is enhanced, the yield of the soybeans is improved, the local vegetation recovery is promoted, and the strain provides an important microbial resource for producing soybean microbial fertilizers suitable for the stony desertification soil.
Drawings
FIG. 1 is a morphological feature under a microscope of bradyrhizobium Dian after gram staining;
FIG. 2 is a dendrogram of the 16S rDNA sequence phylogenetic of Dian of bradyrhizobium rhizogenes (note: circles are soybean rhizobia isolated and purified in experiments, others are reference rhizobia);
FIG. 3 shows the reaction of bradyrhizobium Dian citrate, the liquefaction of gelatin, the detection of phosphorus dissolving capacity of 3-ketolactose reaction, starch hydrolysis and litmus milk reaction;
FIG. 4 shows the effect of slow rhizobium Dian sand inoculation of soybeans;
FIG. 5 shows the effects of sand-cultured Dian strains of bradyrhizobium on soybean biomass, nitrogen content and nitrogenase activity (Note: 0.01. Rho.: 0.05. Rho.: 0.001. Rho.: 0.01. Rho.: 0.001. Rho.: P < 0.001).
Detailed Description
The invention is further described with reference to the drawings and specific examples, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
The following examples employ the following media and nutrient formulations:
solid YMA medium: 10g of mannitol, 3g of yeast extract powder 2 HPO 4 0.25g，KH 2 PO 4 0.25g, anhydrous MgSO 4 0.2g, naCl 0.1g and agar 15g, adding water to a constant volume of 1000mL, adjusting the pH value to 7.0-7.2, and sterilizing at 121 ℃ for 30min.
Liquid YMA medium: 10g of mannitol, 3g of yeast extract powder 2 HPO 4 0.25g，KH 2 PO 4 0.25g, anhydrous MgSO 4 0.2g and 0.1g of NaCl, adding water to a constant volume of 1000mL, adjusting the pH value to 7.0-7.2, and sterilizing at 121 ℃ for 30min.
Starch solid medium: 20g of soluble starch, 10g of peptone, 5g of beef extract, 5g of sodium chloride and 15g of agar, diluting with water to 1000mL, and sterilizing at 121 ℃ for 15min.
The low-nitrogen nutrient solution comprises the following components: (1) macroelements: 56.676g/L Ca (NO) 3 ) 2 ·4H 2 O，13.24g/L(NH 4 ) 2 SO 4 ，38.418g/L KNO 3 ，147.89g/L MgSO 4 ·7H 2 O，34.025g/L KH 2 PO 4 ，106.54g/L CaCl 2 ·2H 2 O，264.88g/L K 2 SO 4 ；
(2) Trace elements: 0.254g/L MnSO 4 ·7H 2 O，0.431g/L ZnSO 4 ·7H 2 O，0.125g/L CuSO 4 ·5H 2 O，0.2g/L(NH 4 ) 6 MO 7 O 24 ·4H 2 O，14.68g/L Na 2 -EDTA，0.95g/L NaB 4 O 7 ·4H 2 O。
EXAMPLE 1 isolation and purification of the Strain
Collecting the root nodules on the soybean roots cultivated in the Guizhou Xingyi stony desertification area, selecting fresh and plump root nodules, soaking the root nodules in sterile water for 5-6 min in an ultra-clean bench, removing impurities on the surface, disinfecting the surface with 75% absolute ethyl alcohol for 3-5 min, then disinfecting the surface with 3.5% NaClO for 8min, and then washing the surface with sterile water for 9-10 times; cutting a single nodule with a sterilized knife, clamping the nodule with a forceps, streaking on a solid YMA culture medium, carrying out inverted culture in a constant-temperature incubator at 28 ℃ for 5-7 days until a clear colony grows out, carrying out streaking purification on the isolated nodule bacterium monoclonal for 3-4 times according to characteristics of thalli such as morphology, size, transparency, viscosity, color, luster and the like until the clear monoclonal grows out, picking the single colony, transferring the single colony to an inclined plane, storing at the low temperature of 4 ℃, and naming the obtained strain as a Dian strain.
Whether rhizobia is considered can be preliminarily judged according to the following method:
colony morphology: the rhizobium colonies are round, milky white, translucent, neat in edge and much in mucilage. The diameter of the bacterial colony reaches 2-4 mm after 3-5 days of culture, and the diameter of the bacterial colony reaches 1mm after 7-10 days of culture, the bacterial colony is fast-growing rhizobia.
EXAMPLE 2 identification of the strains
1. Morphological characterization of strains
The Dian strain isolated in example 1 was inoculated on a YMA solid medium for culture and observed for recording. After culturing for 5-7 days under the optimal growth condition of pH 7.0 and at the temperature of 28 ℃, observing the state of a single colony of the separated and purified strain Dian, wherein the state mainly comprises the size, the color, the transparency, the wettability, the surface state and the edge state of the colony. Meanwhile, the Dian strain in the logarithmic growth phase is stained by a smear and then the form of the strain is observed by adopting an optical microscope.
The results are shown in FIG. 1, which indicates that the Dian strain is gram-negative, short and rod-shaped, has no spores, and most of them are motile. Growing on a solid YMA culture medium plate, wherein the colony is approximately round, milky, semitransparent, regular in edge, more in mucilage, and 0.8-1 mm in diameter.
2. Physiological and biochemical characteristic identification of strain
The physiological and biochemical characteristics of the Dian strain were determined by reference to Bergey's Manual of bacteria identification (eighth edition of the Berkan Manual of bacteria identification, beijing, scientific Press, 2011). The results are shown in FIG. 2, which indicates that the Dian strain is chemoheterotrophic, grows aerobically and can grow by using glucose and lactose; citrate cannot be utilized, gelatin liquefaction is negative, 3-ketolactose is not utilized, and the capacity of activating an insoluble phosphorus source is not realized; the litmus milk reacts as acid coagulation.
3. Sequence sequencing and analysis of 16S rDNA gene of strain
Extracting the total DNA of the thallus obtained in the step, and amplifying a 16S rDNA sequence by using a bacterial universal primer pair 16S rDNA-F (5.
PCR reaction (50. Mu.L): 2 XPCR Buffer 25 uL, primer 16S rDNA-F/16S rDNA-R each 2.0 uL, dNTP 1.0 uL, taqDNA polymerase 1 uL, template 2 uL, sterilized ddH2O 17 uL.
And (3) PCR reaction conditions: pre-denaturation at 94 deg.C for 5min; denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 45s, extension at 72 ℃ for 2min, and amplification for 35 cycles; extension for 10min at 72 ℃.
After the amplification product is detected by 10g/L agarose gel electrophoresis, the PCR product is subjected to clone sequencing by Guangzhou Ongke biotechnology limited company, and the nucleotide sequence of the strain obtained by sequencing is shown as SEQ ID NO. 1.
The obtained sequence results were subjected to BLAST search at the National Center for Biotechnology Information (NCBI) of the United states, 10 model strains having high similarity were selected as reference strains, and the construction of a phylogenetic tree of the 16S rDNA gene was carried out by using the Neighbor-joining method (Neighbor-joining) in MEGA4.1 software. From the results of the alignment and phylogenetic tree as shown in FIG. 3, the sequence of the Dian 16S rDNA gene of Rhizobium was 99.34% similar to that of Bradyrhizobium otawaense strain OO99, which is a model strain of Bradyrhizobium (Bradyrhizobium sp.), and was located in the same evolutionary branch, thereby confirming that the Dian is a novel strain of Bradyrhizobium (Bradyrhizobium sp.) Wootaihua Bradyrhizobium (Bradyrhizobium otawaense).
In view of the above morphological, physiological and biochemical characteristic analysis and 16S rDNA sequence homology analysis results, it was determined that the isolated and purified strain Dian was identified as a salt and alkali resistant Bradyrhizobium (Bradyrhizobium sp.) named Bradyrhizobium worthwhile (Bradyrhizobium otawaense) Dian strain, which was deposited at the Guangdong province collection of microorganisms (GDMCC) 1/11 days 2022 with the deposit number being GDMCC No:62202, storage unit address: building No. 59, building No. 5 of the prefecture midroad No. 100 yard in Guangzhou city.
Example 3 Dian Strain characterization of Rhizobium japonicum
Preparing 1L YMA liquid culture medium, subpackaging in 3 triangular flasks, and sterilizing at 121 deg.C for 20min; sucking 100 μ L of purified Rhizobium bacterial liquid reaching logarithmic phase into a triangular flask containing YMA culture medium, and oscillating overnight at 28 deg.C with a shaker at 180r/min until bacterial liquid OD 600 And (3) after the value reaches 0.6 +/-0.05, subpackaging the mixture into sterilized 50mL centrifuge tubes, centrifuging the mixture in a centrifuge at 5000rpm for 10min, taking out the mixture, pouring out the supernatant, and carrying out heavy suspension by using a low-nitrogen nutrient solution to obtain a bacterial suspension for later use.
Taking 5 mu L of prepared rhizobium suspension, placing the rhizobium suspension on a soluble starch solid culture medium for spotting inoculation, culturing the rhizobium suspension in a constant-temperature incubator at 28 ℃ for 3-5 days until obvious monoclonals appear, uniformly dripping iodine solution on a flat plate, and showing that the strain has the ability of hydrolyzing starch by generating a transparent ring around a bacterial colony.
The experimental results are shown in the starch hydrolysis reaction results in fig. 2, and show that a relatively obvious transparent ring is generated around the bacterial colony, which indicates that the bradyrhizobium japonicum Dian has relatively strong starch hydrolysis capability. In the identification of the reaction of litmus milk, compared with CK, the Dian strain turns litmus red, which indicates that the strain is acidogenic, and the acidogenic property of the Dian strain can help to optimize the root system growth micro-ecological environment of alkaline soil crops and promote the growth of plants because the stony desertification area is alkaline soil.
Example 4 Backfield test of bradyrhizobium Dian
Soybean seed sterilization: selecting soybean seeds with undamaged, plump and consistent size seed coats, sterilizing the seed coats of the soybean seeds for 12 hours by adopting a chlorine dry disinfection method (100 mL of sodium hypochlorite and 4.8mL of hydrochloric acid), taking the soybean seeds out of a super-clean workbench, blowing for 10min, and placing at room temperature for later use.
And (3) culturing thalli: preparing 1L YMA liquid culture medium, subpackaging in 3 triangular flasks, and sterilizing at 121 deg.C for 20min; sucking 100 μ L of purified Rhizobium bacterial liquid reaching logarithmic phase into a triangular flask containing YMA culture medium, and oscillating overnight at 28 deg.C with a shaker at 180r/min until bacterial liquid OD 600 And (3) after the value reaches 0.6 +/-0.05, subpackaging the mixture into sterilized 50mL centrifuge tubes, centrifuging the mixture in a centrifuge at 5000rpm for 10min, taking out the mixture, pouring out the supernatant, and carrying out heavy suspension by using a low-nitrogen nutrient solution to obtain a bacterial suspension for later use.
Sand culture and inoculation: mixing the fine sand, the medium sand and the coarse sand according to a ratio of 1; when the seeds germinate and cotyledons turn green and are not opened, 100mL of the bacterial suspension obtained in the previous step is inoculated into the pot subjected to inoculation treatment.
And (3) taking non-inoculation as a control, repeating each treatment for 5 times, harvesting soybean plants 35 days after inoculation, and taking the fresh weight of the plants, the nodulation number, the azotase activity, the nitrogen content of the plants and the SPAD value as judgment indexes for measuring the associativity and the nitrogen fixation capacity of rhizobia.
In a blank control (-N), the phenomenon of nodulation does not occur, and the inoculation of the Dian rhizobium can promote the nodulation of the soybeans, namely, the backcross treatment can be carried out on the average of 16 large nodulation and 130 small nodulation per soybean, the section of the nodulation is shown to be dark red, the activity of the nitrogen-fixing enzyme of the nodulation is measured to be 78.48 mu mol/h/g, and the Dian strain to be tested is preliminarily judged to be the nodulation with the nitrogen-fixing efficiency.
As shown in FIG. 4, the leaves of soybean inoculated with Rhizobium were significantly turned green from nitrogen deficient yellow leaves after 35 days, as compared with the low-nitrogen non-inoculated treatment, indicating that the test strain Dian had a higher nitrogen fixation efficiency.
In FIG. 5, it is shown that the inoculation of Dian with rhizobia has a significant effect on the biomass of soybeans, i.e., the fresh weight of the inoculated soybean plants is increased by 21.69% compared to the plants without rhizobia. Meanwhile, the inoculation of the rhizobium Dian obviously improves the SPAD value and the nitrogen content of the soybean leaves, and the SPAD value and the nitrogen content are respectively increased by 12.26 percent and 40.02 percent compared with those of an uninoculated control. Further, the test strain Dian is the rhizobium with higher nitrogen fixation efficiency.
In conclusion, the invention separates and screens the Dian rhizobium strain from the soybean rhizobia planted in the Xingyi stony desertification areas of Guizhou. The research of the invention shows that the Dian strain has the capability of hydrolyzing starch, and has the characteristics of high nodulation rate and strong nitrogen fixation capability; the tieback test shows that the inoculation of the Dian strain can obviously improve the total nitrogen content of soybean plants, and can optimize the micro-ecological environment for root growth of alkaline soil crops, thereby promoting the growth of plants and having wide practical value in the aspect of improving the soil ecosystem of stony desertification saline-alkali soil.
The microbial preparation prepared by using the Dian strain can be popularized and applied in a large area, the adaptability of soybeans to the soil environment of stony desertification saline-alkali soil is enhanced, the yield of the soybeans is improved, and the local vegetation recovery is promoted.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
<110> south China university of agriculture
<120> saline-alkali soil resistant nitrogen fixation soybean rhizobium and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1384
<212> DNA
<213> Dian strain (SIPOSequenceListing 1.0) of Chroothecium worthiatum
<400> 1
gcgctgctta cctgcgagtc gagcgggcgt agcaatacgt cagcggcaga cgggtgagta 60
acgcgtggga acgtaccttt tggttcggaa caacacaggg aaacttgtgc taataccgga 120
taagccctta cggggaaaga tttatcgccg aaagatcggc ccgcgtctga ttagctagtt 180
ggtgaggtaa tggctcacca aggcgacgat cagtagctgg tctgagagga tgatcagcca 240
cattgggact gagacacggc ccaaactcct acgggaggca gcagtgggga atattggaca 300
atgggggcaa ccctgatcca gccatgccgc gtgagtgatg aaggccctag ggttgtaaag 360
ctcttttgtg cgggaagata atgacggtac cgcaagaata agccccggct aacttcgtgc 420
cagcagccgc ggtaatacga agggggctag cgttgctcgg aatcactggg cgtaaagggt 480
gcgtaggcgg gtctttaagt caggggtgaa atcctggagc tcaactccag aactgccttt 540
gatactgaag atcttgagtc cgggagaggt gagtggaact gcgagtggta gaggtgaaat 600
tcgtagatat tcgcaagaac acccagtggc gaaggcggct cactggcccg gtactgacgc 660
tgaggcacga aagcgtgggg agcaaacagg attagatacc ctggtagtcc acgccgtaaa 720
cgatgaatgc cagccgttag tgggtttact cactagtggc gcagctaacg ctttaagcat 780
tccgcctggg gagtacggtc gcaagattaa aactcaaagg aattgacggg ggcccgcaca 840
agcggtggag catgtggttt aattcgacgc aacgcgcaga accttaccag cccttgacat 900
gtccaggacc ggtcgcagag atgtgacctt ctcttcggag cctggagcac aggtgctgca 960
tggctgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga gcgcaacccc 1020
cgtccttagt tgctaccatt tagttgagca ctctaaggag actgccggtg ataagccgcg 1080
aggaaggtgg ggatgacgtc aagtcctcat ggcccttacg ggctgggcta cacacgtgct 1140
acaatggcgg tgacaatggg atgctaaggg gcgacccttc gcaaatctca aaaagccgtc 1200
tcagttcgga ttgggctctg caactcgagc ccatgaagtt ggaatcgcta gtaatcgtgg 1260
atcagcacgc cacggtgaat acgttcccgg gccttgtaca caccgcccgt cacaccatgg 1320
gagttggttt tacctgaaga cggtgcgcta acccgcaagg gaggcagccg gccacggtag 1380
ctca 1384
Claims (10)
1. A nitrogen-fixing bradyrhizobium strain (for saline-alkali tolerant land) (Bradyrhizobium sp.) Dian strain, wherein said strain is deposited in Guangdong province collection of microorganisms (GDMCC) at 11/1/2022, and the strain number is GDMCC No:62202.
2. use of a Dian strain of bradyrhizobium as defined in claim 1 to enhance the adaptability of soybean plants to stony desertification soil environments and/or to promote the recovery of soybean plants in stony desertification soil environments.
3. Use of the Dian strain of bradyrhizobium as defined in claim 1 for promoting nodulation and nitrogen fixation in soybean plants, promoting growth of soybean plants, and/or preparing nitrogen-fixing microbial agents.
4. A nitrogen-fixing microbial preparation comprising the Dian strain of bradyrhizobium according to claim 1 and/or a bacterial solution thereof.
5. The microbial preparation according to claim 4, wherein the bacterial liquid is a bacterial suspension.
6. The microbial preparation of claim 5, wherein the bacterial suspension has an OD of 0.6 ± 0.05.
7. The microbial preparation of claim 6, wherein the bacterial suspension is prepared under fermentation conditions of 24-32 ℃ and pH 6.8-7.0.
8. Use of the nitrogen-fixing microbial preparation of any one of claims 4-7 for promoting nodulation and nitrogen fixation of soybean plants and/or promoting growth of soybean plants.
9. Use of the Dian strain of bradyrhizobium of claim 1 to increase yield in soybean plants.
10. A method for promoting the nodulation and nitrogen fixation of soybean, characterized in that a soybean plant is treated with the Dian strain of bradyrhizobium of claim 1 or a bacterial solution thereof, or with the microbial preparation of any one of claims 4 to 7.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210193179.1A CN114456981B (en) | 2022-02-28 | 2022-02-28 | Salt-alkali-tolerant land nitrogen-fixing soybean rhizobium and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210193179.1A CN114456981B (en) | 2022-02-28 | 2022-02-28 | Salt-alkali-tolerant land nitrogen-fixing soybean rhizobium and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114456981A CN114456981A (en) | 2022-05-10 |
| CN114456981B true CN114456981B (en) | 2023-04-14 |
Family
ID=81415588
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210193179.1A Active CN114456981B (en) | 2022-02-28 | 2022-02-28 | Salt-alkali-tolerant land nitrogen-fixing soybean rhizobium and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114456981B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115287229B (en) * | 2022-08-04 | 2023-05-12 | 兰州大学 | Rhizobium capable of promoting growth of alfalfa beans and nodulation and nitrogen fixation and application thereof |
| CN116376755A (en) * | 2023-03-08 | 2023-07-04 | 华南农业大学 | Agate slow rhizobium HP25 strain and application thereof |
| CN117210366B (en) * | 2023-09-18 | 2024-06-04 | 中国科学院华南植物园 | Rhizobium japonicum strain and application thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113215037B (en) * | 2021-05-08 | 2022-09-13 | 华南农业大学 | High-efficiency nitrogen-fixing bradyrhizobium strain and application thereof |
-
2022
- 2022-02-28 CN CN202210193179.1A patent/CN114456981B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN114456981A (en) | 2022-05-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN114456981B (en) | Salt-alkali-tolerant land nitrogen-fixing soybean rhizobium and application thereof | |
| CN106987541B (en) | Efficient alfalfa rhizobium with stress resistance and growth promotion performance and application thereof | |
| CN113215037B (en) | High-efficiency nitrogen-fixing bradyrhizobium strain and application thereof | |
| CN110591957B (en) | Salt-tolerant alfalfa rhizobia and application thereof | |
| CN113388526B (en) | Endophytic fungus FO-R20 and application thereof | |
| CN111117924B (en) | Compound microbial inoculum and preparation method thereof, fertilizer and method for preventing and treating root rot | |
| CN115505536B (en) | Aspergillus niger CA-5 and application thereof | |
| CN113621532A (en) | Compound microbial agent containing saline-alkali-resistant bacillus paraclicheniformis and preparation method thereof | |
| CN114908025A (en) | Paenibacillus mucilaginosus HB-02 strain and application thereof in promoting crop growth | |
| CN115029278B (en) | Bacillus megaterium and application thereof in promoting crop growth | |
| CN112342173A (en) | Bacillus belgii and application thereof | |
| CN111073827A (en) | Bacillus beilesensis JTB8-2 and application thereof in biological control of orobanche meloidogyne | |
| CN112111432B (en) | Double-source humic acid biofertilizer and preparation method and application thereof | |
| CN106929433B (en) | Phosphate solubilizing penicillium and application thereof | |
| CN111334458B (en) | Biocontrol actinomycetes and application thereof in prevention and control of ginger stem basal rot or soybean epidemic disease | |
| CN114874953B (en) | Peanut rhizosphere biocontrol bacterium-Bacillus beleisi SW-1 and application thereof | |
| CN115287194B (en) | Medicinal wild rice endophytic fungi YYA21 and application thereof | |
| CN107099486B (en) | Bacillus amyloliquefaciens GN03 and application thereof | |
| CN114350559A (en) | Salt-tolerant growth-promoting slow-growing rhizobium Liaoning RY6 strain and application thereof | |
| CN109355235B (en) | Rhizobium FAMB126 and application thereof | |
| CN109370953B (en) | Juemina rhizobium JYN6 and application thereof | |
| CN117229985B (en) | Biocontrol strain bacillus atrophaeus BGB-98R and application thereof | |
| CN113444672B (en) | Lactobacillus buchneri and application thereof | |
| CN109439590B (en) | Cassia rhizobium WYS3R1 and application thereof | |
| CN116064288B (en) | Streptomyces roseoformis HC7-22 for plant iron-removing and growth-promoting and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB03 | Change of inventor or designer information |
Inventor after: Liang Cuiyue Inventor after: Chen Qianqian Inventor after: Lu Xing Inventor after: Xie Baoxing Inventor after: Liu Guoxuan Inventor after: Guan Zidi Inventor after: Tian Jiang Inventor after: Tian Jihui Inventor before: Liang Cuiyue Inventor before: Chen Qianqian Inventor before: Lu Xing Inventor before: Xie Baoxing Inventor before: Liu Guoxuan Inventor before: Guan Zidi Inventor before: Tian Jiang Inventor before: Tian Jihui |
|
| CB03 | Change of inventor or designer information | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |