CN111117910B - Enterobacter ludwigii PN6 and application thereof - Google Patents
Enterobacter ludwigii PN6 and application thereof Download PDFInfo
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- C05—FERTILISERS; MANUFACTURE THEREOF
- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
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Abstract
The invention discloses enterobacter ludwigii PN6 and application thereof. The classification name of the strain is Enterobacter ludwigii, the strain number is PN6, the strain is preserved in China center for type culture Collection, and the preservation number is: CCTCC NO: M2019881. The strain PN6 screened from the root system of the moso bamboo has obvious bacteriostatic action on the pathogenic bacteria of the blight of the moso bamboo, the bacteriostatic rate of the fermentation liquor can reach 49.28 percent, and the strain has high-efficiency capability of dissolving phosphorus and secreting IAA; the strain PN6 has a promoting effect on the growth of moso bamboos, and test data show that the liquid microbial inoculum prepared from PN6 has a remarkable growth promoting effect on growth indexes such as the ground diameter, the seedling height and chlorophyll of the moso bamboos, and can increase the stress resistance of the moso bamboos and reduce the probability of occurrence of the blight of the moso bamboos. Therefore, the invention provides excellent strain resources for developing environment-friendly fertilizer special for moso bamboo organisms.
Description
Technical Field
The invention relates to the technical field of microorganisms and biofertilizers, and particularly relates to enterobacter ludwigii PN6 and application thereof.
Background
Phyllostachys edulis is widely distributed in bamboo plants in China, has the most accumulation amount and the most application, and is a bamboo species with excellent economic and ecological benefits. In recent years, with the increase of demand for moso bamboos, nutrient exhaustion of the moso bamboo artificial forest land has increased dramatically. In addition, the moso bamboos are attacked by various diseases in the growth process, such as the sooty mould of the moso bamboos, the rust disease of the moso bamboos, the blight disease of the moso bamboos and the like, wherein the blight disease of the moso bamboos is more harmful. The disease mainly damages lateral branches and young shoots of new bamboos growing in the current year, pathogenic bacteria can invade through natural orifices or wounds of the new bamboos, so that the young shoots wither, the lateral branches wither or the whole body withers, and the cultivation and the productivity of moso bamboos are seriously damaged. Since 1959, the moso bamboo blight was first discovered in Huang Jiang, it has now spread to Jiang, Shanghai, Su, Min, etc. provinces and cities. The risk evaluation and analysis result shows that the deinococcus bambusicola is a relatively high-risk forest pest, has potential threat to the cultivation of bamboo forest resources and the development of bamboo industry in China, and is expected to strengthen the prevention and treatment of the phyllostachys pubescens blight. Moso bamboo is mainly distributed in hilly areas with deficient nutrients in south China, at present, weak soil fertility and insufficient nutrient supply are one of the main problems in moso bamboo resource cultivation, and the problems of increased disease conditions, reduced survival rate and the like are often caused by various adversity stresses on the growth of moso bamboo. Therefore, it is urgent to promote the growth of moso bamboo and improve the stress tolerance of moso bamboo. However, the means for promoting the growth of moso bamboos mainly focuses on the aspects of enhancing the cultivation measures and the like for a long time, and research is limited, and the effect of promoting the growth and resisting the diseases of the moso bamboos by using a microbial preparation means is rarely reported.
In the past decades, the research on endophytes of plants has been a hot research spot at home and abroad. Endophytes are defined as all microorganisms that are able to colonize the plant cell space or inside the cell and have a beneficial effect on host plants with a complex symbiotic relationship. Holman et al later proposed a more realistic definition: microorganisms isolated from plant tissue that have undergone stringent surface sterilization can be considered endophytes if they are not pathogenic to the plant host at all. Endophytic bacteria have many advantages over microorganisms screened from soil or plant rhizosphere soil: they are distributed in different tissues of plants, have sufficient nutrients, are protected by the plant tissues, are not interfered by indigenous microorganisms in the soil, are not influenced by external severe environments such as strong sunlight, ultraviolet rays, wind and rain and the like, and have stable ecological environment. So far, the research on endophytic bacteria is mainly focused on crops, while the research on endophytic bacteria in woody plants is few, and the research on the endophytic bacteria of moso bamboos is more rarely reported. Therefore, the method has great potential and value for improving the stress resistance of the artificial bamboo forest of the moso bamboos and enhancing the productivity of the bamboo forest by digging the functions and practical characteristics of the endophytes of the moso bamboos.
Disclosure of Invention
The first object of the present invention is to provide an Enterobacter ledebei (Enterobacter ludwigii) PN6 for promoting the growth of moso bamboos.
The method selects a root system of 3-4 years old moso bamboo with good growth vigor from a mao bamboo artificial forest in a Dagang forest farm, a Guangshan forest farm and a Dajing mountain forest farm in Yichun city in Jiangxi province, and obtains a strain of Enterobacter ludwigii (Enterobacter ludwigii) for promoting the growth of the moso bamboo by screening, wherein the strain number is PN 6. Has been preserved in China Center for Type Culture Collection (CCTCC) with the address: wuhan university in Wuhan City, Hubei province, China, zip code: 430072, with the preservation number: CCTCC NO of M2019881, and the preservation date is 10 months and 31 days in 2019.
The main biological characteristics of the Enterobacter ludwigii PN6 strain: the bacillus-free gram-negative (G-) Brevibacterium has round colonies, neat edges and wet and viscous surface. The strain is light milky white, catalase-positive, oxidase-negative, 3% potassium hydroxide-positive, starch hydrolysis-positive, gelatin hydrolysis-positive, nitric acid reduction-positive, methyl red-positive, VP-negative, and indole-negative. The 16S rDNA sequence of the Enterobacter ludwigii PN6 strain is shown in SEQ ID NO. 1. The 16S rDNA sequence was compared with the sequence in GeneBank database. The result shows that the strain PN6 has high homology with Enterobacter ludwigii, and the degree of acquaintance reaches 100 percent. The binding morphology, physiological and biochemical characteristics (as shown in Table 1) and 16S rDNA sequence analysis were identified as Enterobacter ludwigii.
The second purpose of the invention is to provide the application of the Enterobacter ludwigii PN6 in promoting the growth of moso bamboos, in particular to the application in promoting the growth of moso bamboo seedlings.
The third purpose of the invention is to provide the application of the Enterobacter ludwigii PN6 in inhibiting the pathogenic bacteria of the bamboo blight and the pecurococcus bambusicola.
The fourth purpose of the invention is to provide the application of the Enterobacter ludwigii PN6 in dissolving the phosphate which is difficult to dissolve.
Preferably, the sparingly soluble phosphate comprises tricalcium phosphate, iron phosphate, calcium hydrogen phosphate, aluminium phosphate or calcium phytate.
The fifth purpose of the invention is to provide the application of the Enterobacter ludwigii PN6 in preparing the auxin IAA.
It is a sixth object of the present invention to provide a biofertilizer containing the above Enterobacter ludwigii (Enterobacter ludwigii) PN 6. Preferably, the biological fertilizer is a bacterial fertilizer.
The seventh object of the present invention is to provide a preparation for inhibiting trichoderma bambusicola pathogenic bacteria deinococcus bambusicola, which contains the above Enterobacter ledoides (Enterobacter ludwigii) PN 6.
Compared with the prior art, the invention has the beneficial effects that:
the Enterobacter ludwigii PN6 has obvious bacteriostatic activity on the bamboo blight pathogenic bacteria deinococcus bambusicola. Under the condition of liquid shaking culture, the phosphate solubilizing and IAA secreting ability is strong. The Enterobacter ludwigii (Enterobacter ludwigii) PN6 is prepared into a microbial inoculum to be inoculated to the moso bamboo seedlings, and the result shows that the microbial inoculum can remarkably promote the growth of the moso bamboo seedlings. Therefore, the invention provides excellent strain resources for developing bacterial fertilizers for promoting the growth of moso bamboos.
The Enterobacter ludwigii PN6 is preserved in China Center for Type Culture Collection (CCTCC) in 2019 at 31.10.9, and the address is Wuhan university in Wuhan City, Hubei province, China, zip code: 430072, preservation number of CCTCC NO: M2019881.
Drawings
FIG. 1 is a phylogenetic tree of the 16S rDNA gene sequence of Enterobacter ludwigii PN6, in which PN6 represents Enterobacter ludwigii PN6 of the present invention.
FIG. 2 is a colony morphology of Enterobacter ludwigii PN 6.
FIG. 3 shows the resistance of Enterobacter ludwigii PN6 against a plate of pathogenic bacteria (Rhynchosia bambusicola).
FIG. 4 shows the phosphate solubilizing loop produced by Enterobacter ludwigii PN6 in NBRIP medium.
FIG. 5 shows the results of determination of calcium phosphate-solubilizing ability of Enterobacter ludwigii PN6 in NBRIP medium at 3d and 6 d.
FIG. 6 shows the results of the detection of IAA secretion ability of Enterobacter ludwigii PN 6.
FIG. 7 is a graph showing the effect of Enterobacter ludwigii PN6 inoculation for 180d on the growth of annual Phyllostachys pubescens seedlings.
Detailed Description
The present invention will be better understood by those skilled in the art from the following examples. The examples are described only to illustrate the invention and should not be construed as limiting the invention as detailed in the claims.
Example 1: separation and identification of Phyllostachys pubescens endophytic bacterium PN6
Selecting good-growing-condition moso bamboo of 3-4 years from the artificial moso bamboo trees in Hongkong forest farm, Guanshan forest farm and Jinggang mountain Dajing forest farm in Yichun city in Jiangxi province, shoveling off the surface soil, digging out the root system of the moso bamboo by a shovel, and collecting the fine root of the moso bamboo. Washing the root system of the moso bamboo with distilled water for 3 times, and carrying out surface disinfection according to the following steps: soaking with 75% v/v alcohol for 1min, 3.25% v/v sodium hypochlorite for 3min, 75% v/v alcohol for 30s, and washing with sterile water for 3 times. Grinding the treated root tissue, and collecting grinding fluid. And (3) coating an NA plate (obtained by uniformly mixing and sterilizing a formula of an NA culture medium, namely 3g of beef extract, 10g of peptone, 5g of NaCl, 16g of agar and 1000mL of distilled water with the pH value of 7.2) with the sterile water after the last washing, and checking whether the surface is sterilized completely. The plate was incubated at 30 ℃ by the dilution plate method.
After 36h of culture, bacterial colonies appeared on the NA plates, and most of the colonies became milky white and very similar in appearance. The colonies were further purified by the present inventors and found by morphological and molecular characterization to be round, slightly milky in color and opaque (FIG. 2). Microscopic observation of the strain showed that the strain was bacilli, and analysis of physiological and biochemical characteristics showed that the strain was aerobic gram-negative bacilli (Table 1). The 16S rDNA sequence of the strain is shown in SEQ ID NO. 1, a phylogenetic tree is constructed by utilizing the 16S rDNA sequence, and the result shows that the strain is Enterobacter ludwigii which is closest to the bamboo endophytic bacteria, has the strain number of PN6 and is named as Enterobacter ludwigii (Enterobacter ludwigii) PN6 (figure 1). The physiological and biochemical characteristics of PN6 were also consistent with the standard strain descriptions of e.ludwigii. PN6 is therefore an isolate of e.ludwigii.
TABLE 1 physiological and biochemical characteristics of Enterobacter ledwigii PN6
Remarking: "-" indicates negative reaction; "+" indicates positive reaction
Enterobacter ledebensis (Enterobacter ludwigii) PN6 has been deposited with the China Center for Type Culture Collection (CCTCC) at the address: wuhan university in Wuhan City, Hubei province, China, zip code: 430072, with the preservation number: CCTCC NO of M2019881, and the preservation date is 10 months and 31 days in 2019.
Example 2: strain PN6 for endogenous antagonism of pathogenic bacteria of phyllostachys pubescens dieback
Using a puncher (the diameter is 5mm) to punch bacterial cakes at the colony edge of trichoderma harzianum blight pathogenic bacteria trichoderma reesei (c. phyllostachydis) cultured for 5d, inoculating the bacterial cakes to the center of a PDA plate, streaking bacterial strains to be detected activated by an NA inclined plane to two sides of the PDA plate, and using pathogenic bacteria only as a Control (CK), and repeating the treatment for 3 times. Culturing at 28 deg.C for 4 days to observe the existence of bacteriostatic zone, and measuring the width of bacteriostatic zone.
The Enterobacter ledebi PN6 with good bacteriostasis in the plate confronting test is inoculated in an NB culture medium (the formula of the NB culture medium comprises 3g of beef extract, 10g of peptone, 5g of NaCl, 1000mL of distilled water and pH 7.2, and the Enterobacter ledebi PN6 is obtained by uniformly mixing and then sterilizing the mixture). The fermentation was carried out in a 150mL shake flask containing 50mL of liquid per flask and incubated at 30 ℃ for 2 days (200 r/min). Centrifuging the fermentation liquid at 10000r/m for 20min, collecting supernatant, and filtering with 0.22 μm microporous membrane to obtain sterile filtrate. Mixing sterile filtrate 5mL and 20mL PDA culture medium (formula is potato 200g, glucose 20g, agar 15g, distilled water 1000mL, natural pH) cooled to 50 deg.C, and pouring into flat plate by cleaning potato, peeling, weighing 200g potato, cutting into small pieces, adding water, decocting, filtering with gauze, collecting filtrate, mixing with glucose and agar, dissolving in water, stirring for dissolving, sterilizing at 121 deg.C for 20min, and cooling; sterile water was used as a control instead of sterile filtrate. And then the pathogenic bacteria block is spotted in the middle of the flat plate. After 3 replicates of each treatment and 4 days of incubation at 28 ℃, the rate of inhibition of the filtrate on the growth of rhynchophthora bambusi was determined. The calculation formula is as follows: the growth inhibition ratio (%) × (control plate colony diameter-diameter of plate colony with filtrate)/(control plate colony diameter-diameter of mass) × 100%. The result shows that the Enterobacter ledebye PN6 has better inhibiting effect on the deinococcus bambusicola (figure 3), and the inhibiting rate can reach 49.28 percent (table 2).
TABLE 2 antibacterial Activity of Enterobacter ledwigii PN6 against Pediococcus bambusicola
Note: the lower case letters in the same column represent significant differences at the 0.05 level.
Example 3: strain PN6 phosphorus-dissolving capability determination test
Enterobacter ledebensis PN6 was inoculated in NBRIP solid medium (glucose 10g, Ca)3(PO4)2 5g，MgCl25g，KCl 0.2g，MgSO4.7H2O 0.25g，(NH4)2SO40.1g, 1000mL of distilled water and 15g of agar, and uniformly mixing and sterilizing to obtain the product), culturing for 4 days, and observing whether a phosphorus dissolving ring is generated. The phosphorus solubilizing ability of Enterobacter ledwigii PN6 was quantified using the method of Pikovskaya et al. After the Enterobacter ledeb PN6 was shake-cultured in NB medium for 24h, it was inoculated into a 100mL Erlenmeyer flask containing 50mL NBRIP culture medium at an inoculum size of 1% v/v, and NBRIP culture medium inoculated with the same volume of blank seed solution (no inoculated strain) was used as a control. The inoculation treatment is repeated five times, the shaking culture is carried out for 6d at the temperature of 30 ℃ and at the speed of 180r/min, the fermentation liquor is centrifuged for 10min (4 ℃ and at the speed of 10000r/min) respectively at the 3 rd culture time and the 6 th culture time, and the content of soluble phosphorus in the supernatant is measured by adopting a molybdenum-antimony colorimetric method under different culture time periods. The results show that Enterobacter ledebarkii PN6 has phosphate solubilizing rings in NBRIP solid culture medium, and shows that the culture medium has better phosphate solubilizing capability (figure 4). Enterobacter ledebensis PN6 has strong dissolving capacity to calcium phosphate in NBRIP culture solution in 3d and 6d, and the phosphate dissolving amount is 203.67mg/L and 278.21mg/L respectively (figure 5).
Example 4: strain PN6 IAA production capacity test
The Salkowski colorimetric method is adopted to measure the IAA producing capability of the Enterobacter ledebensis PN 6. Taking IAA standard, configuring into concentration gradient of 0, 0.5, 2.5, 5.0, 7.5, 10, 12.5, 15.0, 17.5, 25.0 and 50.0mg/L, taking 2mL of IAA with each concentration, adding equal amount of ferric chloride colorimetric solution (PC colorimetric solution), keeping the temperature in the dark at 30 ℃ for 30min, measuring absorbance at the wavelength of 530nm by using a spectrophotometer, drawing a standard curve, and obtaining the equation y which is 31.868x (R)20.9947). Inoculating the activated Enterobacter ludwigii PN6 strain to King culture medium (the formula of the King culture medium is peptone 2g, glycerol 1g and K)2SO4 0.15g、MgSO4 4.7g、H20.15g of O, 1.5g of agar and H2O1000 mL, pH 7.2, uniformly mixing and then sterilizing) shake culture for 15d, and measuring the IAA content in the fermentation broth according to the standard curve preparation method. As shown in FIG. 6, the test results show that Enterobacter ludwigii PN6 has strong capability of secreting IAA, and the IAA secretion amount is 12.36 mg/L.
Example 5: greenhouse potting test of strain PN6
Inoculating 2-3 times of activated Enterobacter ludwigii PN6 to a culture medium containing50mL of NB medium (NB medium formula beef extract 3g, peptone 10g, NaCl 5g, distilled water 1000mL, pH 7.2, mixed uniformly and sterilized) in a 100mL triangular flask, and carrying out shaking culture at 30 ℃ for 48h at 180r/min to obtain fermentation liquor. Centrifuging the fermentation liquid at 4 deg.C and 6000r/min for 5min, collecting thallus, washing thallus precipitate with sterile normal saline for 3 times to obtain bacterial suspension, and adjusting bacterial suspension to 10 with sterile normal saline8cfu/mL is prepared into a microbial inoculum. The microbial inoculum is inoculated to the rhizosphere of a 1-year-old moso bamboo seedling, and the inoculation amount is 5 mL/plant by taking equivalent sterile normal saline as a Control (CK). And (5) repeating the treatment every 10 times, uniformly managing in a greenhouse with the illumination of 12h/d, and watering at proper time. The effect of inoculation of Enterobacter ledebye PN6 for 180 days on the growth of annual Phyllostachys pubescens seedlings is shown in FIG. 7. As can be seen from fig. 7, the application of the enterobacter ledebei PN6 microbial inoculum significantly promoted the growth of the bamboo seedlings. The application of bacteria treatment obviously improves the seedling height and the ground diameter of the moso bamboo seedlings, and the seedling height and the ground diameter are respectively increased by 62.51 percent and 42.59 percent compared with a Control (CK) (see table 3).
TABLE 3 Effect on the growth of Phyllostachys Pubescens seedlings after inoculation with Enterobacter ledebye PN 6180 d
Note: the lower case letters in the same column represent significant differences at the 0.05 level.
The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.
Sequence listing
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<120> Enterobacter ludwigii PN6 and application thereof
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atgtctggga aactgcccga tggaggggga taactactgg aaacggtagc taataccgca 120
taacgtcgca agaccaaagt gggggacctt cgggcctcac accatcggat gtgcccagat 180
gggattagct agtaggtggg gtaatggctc acctaggcga cgatccctag ctggtctgag 240
aggatgacca gccacactgg aactgagaca cggtccagac tcctacggga ggcagcagtg 300
gggaatattg cacaatgggc gcaagcctga tgcagccatg ccgcgtgtat gaagaaggcc 360
ttcgggttgt aaagtacttt cagcggggag gaaggcggtg gagttaatag cttcaccgat 420
tgacgttacc cgcagaagaa gcaccggcta actccgtgcc agcagccgcg gtaatacgga 480
gggtgcaagc gttaatcgga attactgggc gtaaagcgca cgcaggcggt ctgtcaagtc 540
ggatgtgaaa tccccgggct taacctggga actgcattcg aaactggcag gctagagtct 600
tgtagagggg ggtagaattc caggtgtagc ggtgaaatgc gtagagatct ggaggaatac 660
cggtggcgaa ggcggccccc tggacaaaga ctgacgctca ggtgcgaaag cgtggggagc 720
aaacaggatt agataccctg gtagtccacg ccgtaaacga tgtcgacttg gaggttgttc 780
ccttgaggag tggcttccgg agctaacgcg ttaagtcgac cgcctgggga gtacggccgc 840
aaggttaaaa ctcaaatgaa ttgacggggg cccgcacaag cggtggagca tgtggtttaa 900
ttcgatgcaa cgcgaagaac cttacctact cttgacatcc agagaactta gcagagatgc 960
tttggtgcct tcgggaactc tgagacaggt gctgcatggc tgtcgtcagc tcgtgttgtg 1020
aaatgttggg ttaagtcccg caacgagcgc aacccttatc ctttgttgcc agcggttcgg 1080
ccgggaactc aaaggagact gccagtgata aactggagga aggtggggat gacgtcaagt 1140
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gacctcgcga gagcaagcgg acctcataaa gtgcgtcgta gtccggatcg gagtctgcaa 1260
ctcgactccg tgaagtcgga atcgctagta atcgtagatc agaatgctac ggtgaatacg 1320
ttcccgggcc ttgtacacac cgccc 1345
Claims (9)
1. Enterobacter ludwigii (Enterobacter ludwigii) PN6, deposited in the chinese culture collection center (CCTCC) at 31/10/2019, and addressed to wuhan university, wuhan city, north huichi, china, zip code: 430072, preservation number of CCTCC NO: M2019881.
2. Use of Enterobacter ludwigii (Enterobacter ludwigii) PN6 according to claim 1 for promoting the growth of moso bamboo.
3. The use of claim 2, wherein the phyllostachys pubescens is a phyllostachys pubescens seedling.
4. Use of Enterobacter ludwigii (Enterobacter ludwigii) PN6 according to claim 1 for inhibiting Phyllostachys pubescens Kupffer pathogenic bacteria Rhamnoideus.
5. Use of Enterobacter ludwigii (Enterobacter ludwigii) PN6 according to claim 1 for dissolving poorly soluble phosphates.
6. The use according to claim 5, wherein the sparingly soluble phosphate comprises tricalcium phosphate, iron phosphate, calcium hydrogen phosphate, aluminum phosphate or calcium phytate.
7. Use of Enterobacter ludwigii (Enterobacter ludwigii) PN6 of claim 1 for the preparation of auxin IAA.
8. A biofertilizer characterized by containing Enterobacter ludwigii (Enterobacter ludwigii) PN6 according to claim 1.
9. A preparation for inhibiting Phyllostachys bambusicola Kuntze disease pathogenic bacteria Rhynchosia bambusicola Kuntze, comprising Enterobacter ludwigii PN6 of claim 1.
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| CN107629980A (en) * | 2017-09-30 | 2018-01-26 | 中南民族大学 | The Ludwig enterobacteria SXZ N5 bacterial strains and purposes of one plant of production huperzine and Huperzine B |
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| CN109280628A (en) * | 2018-08-08 | 2019-01-29 | 江西农业大学 | A kind of enterobacteria strain and its application in promotion Bamboo Growth |
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| CN103525721A (en) * | 2013-09-06 | 2014-01-22 | 中国科学院烟台海岸带研究所 | Probiotic with bacteriostatic ability and application thereof |
| CN105586300A (en) * | 2016-03-18 | 2016-05-18 | 中国农业科学院油料作物研究所 | Enterobacter ludwigii BG10-1 and application thereof in biological prevention and control over aspergillus flavus |
| CN107629980A (en) * | 2017-09-30 | 2018-01-26 | 中南民族大学 | The Ludwig enterobacteria SXZ N5 bacterial strains and purposes of one plant of production huperzine and Huperzine B |
| CN108085275A (en) * | 2017-12-19 | 2018-05-29 | 山东省科学院生态研究所 | A kind of Ludwig enterobacteria for inhibiting family's muscardine disease fungus and its application |
| CN109280628A (en) * | 2018-08-08 | 2019-01-29 | 江西农业大学 | A kind of enterobacteria strain and its application in promotion Bamboo Growth |
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