CN116836814B - Endophytic fungus and application thereof in improving aluminum tolerance of alfalfa - Google Patents
Endophytic fungus and application thereof in improving aluminum tolerance of alfalfa Download PDFInfo
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- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 title claims abstract description 72
- 229910052782 aluminium Inorganic materials 0.000 title claims abstract description 72
- 235000017587 Medicago sativa ssp. sativa Nutrition 0.000 title claims abstract description 60
- 241000219823 Medicago Species 0.000 title claims abstract description 59
- 241000233866 Fungi Species 0.000 title abstract description 20
- 241000203233 Aspergillus versicolor Species 0.000 claims abstract description 17
- 241000228212 Aspergillus Species 0.000 claims abstract description 8
- 238000009304 pastoral farming Methods 0.000 claims abstract description 3
- 241000894006 Bacteria Species 0.000 claims description 7
- 239000002068 microbial inoculum Substances 0.000 claims description 4
- 230000002906 microbiologic effect Effects 0.000 claims description 4
- 239000003795 chemical substances by application Substances 0.000 claims description 2
- 240000006439 Aspergillus oryzae Species 0.000 claims 1
- 235000002247 Aspergillus oryzae Nutrition 0.000 claims 1
- 239000004411 aluminium Substances 0.000 claims 1
- 239000002028 Biomass Substances 0.000 abstract description 17
- 230000012010 growth Effects 0.000 abstract description 13
- 238000000926 separation method Methods 0.000 abstract description 5
- 238000004321 preservation Methods 0.000 abstract description 4
- 231100000419 toxicity Toxicity 0.000 description 27
- 230000001988 toxicity Effects 0.000 description 27
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- 238000011081 inoculation Methods 0.000 description 15
- 241000196324 Embryophyta Species 0.000 description 8
- 238000011160 research Methods 0.000 description 6
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- 244000005700 microbiome Species 0.000 description 5
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- 239000002253 acid Substances 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 239000011777 magnesium Substances 0.000 description 3
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- 108090000623 proteins and genes Proteins 0.000 description 3
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- 241000219195 Arabidopsis thaliana Species 0.000 description 2
- ZOXJGFHDIHLPTG-UHFFFAOYSA-N Boron Chemical compound [B] ZOXJGFHDIHLPTG-UHFFFAOYSA-N 0.000 description 2
- 235000008733 Citrus aurantifolia Nutrition 0.000 description 2
- 108091023242 Internal transcribed spacer Proteins 0.000 description 2
- 240000004658 Medicago sativa Species 0.000 description 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 2
- 235000011941 Tilia x europaea Nutrition 0.000 description 2
- 238000007792 addition Methods 0.000 description 2
- 229910052796 boron Inorganic materials 0.000 description 2
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- 230000000116 mitigating effect Effects 0.000 description 2
- 150000007524 organic acids Chemical class 0.000 description 2
- 229910052698 phosphorus Inorganic materials 0.000 description 2
- 239000011574 phosphorus Substances 0.000 description 2
- YGSDEFSMJLZEOE-UHFFFAOYSA-N salicylic acid Chemical compound OC(=O)C1=CC=CC=C1O YGSDEFSMJLZEOE-UHFFFAOYSA-N 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- PORMUFZNYQJOEI-UHFFFAOYSA-N sumatriptan succinate Chemical compound OC(=O)CCC(O)=O.CNS(=O)(=O)CC1=CC=C2NC=C(CCN(C)C)C2=C1 PORMUFZNYQJOEI-UHFFFAOYSA-N 0.000 description 2
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 101100438273 Arabidopsis thaliana CAN1 gene Proteins 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 244000025254 Cannabis sativa Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 235000005979 Citrus limon Nutrition 0.000 description 1
- 244000131522 Citrus pyriformis Species 0.000 description 1
- 241000222290 Cladosporium Species 0.000 description 1
- GUTLYIVDDKVIGB-OUBTZVSYSA-N Cobalt-60 Chemical compound [60Co] GUTLYIVDDKVIGB-OUBTZVSYSA-N 0.000 description 1
- RWSOTUBLDIXVET-UHFFFAOYSA-N Dihydrogen sulfide Chemical compound S RWSOTUBLDIXVET-UHFFFAOYSA-N 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 235000010624 Medicago sativa Nutrition 0.000 description 1
- 240000007817 Olea europaea Species 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 241000220317 Rosa Species 0.000 description 1
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000009858 acid secretion Effects 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- JGDITNMASUZKPW-UHFFFAOYSA-K aluminium trichloride hexahydrate Chemical compound O.O.O.O.O.O.Cl[Al](Cl)Cl JGDITNMASUZKPW-UHFFFAOYSA-K 0.000 description 1
- 229940009861 aluminum chloride hexahydrate Drugs 0.000 description 1
- XKMRRTOUMJRJIA-UHFFFAOYSA-N ammonia nh3 Chemical compound N.N XKMRRTOUMJRJIA-UHFFFAOYSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
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- 239000011575 calcium Substances 0.000 description 1
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- 230000003203 everyday effect Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 229910000037 hydrogen sulfide Inorganic materials 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- FJKROLUGYXJWQN-UHFFFAOYSA-N papa-hydroxy-benzoic acid Natural products OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 description 1
- 230000002688 persistence Effects 0.000 description 1
- 230000008121 plant development Effects 0.000 description 1
- 229920000768 polyamine Polymers 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
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- 230000002441 reversible effect Effects 0.000 description 1
- 230000002786 root growth Effects 0.000 description 1
- 229960004889 salicylic acid Drugs 0.000 description 1
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- 239000010703 silicon Substances 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
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- 231100000331 toxic Toxicity 0.000 description 1
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Classifications
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- 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
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
-
- 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
-
- 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
- C12N1/14—Fungi; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/66—Aspergillus
Abstract
The invention discloses endophytic fungi aspergillus omutatus and application thereof in improving aluminum resistance of alfalfa, wherein the endophytic fungi are aspergillus omutatus (Aspergillus versicolor) F6, and the preservation number is GDMCC No:63473 the endophytic fungus aspergillus omutatus (Aspergillus versicolor) F6 obtained by separation can increase the aluminum stress tolerance of alfalfa long grazing No. 801, increase the growth of alfalfa and obviously increase the aboveground biomass and underground biomass of the alfalfa.
Description
Technical Field
The invention belongs to the field of microorganisms, and particularly relates to endophytic fungi aspergillus omutatus and application thereof in improving aluminum alfalfa tolerance.
Background
Although alfalfa (Medicago Sativa l.) is a leguminous grass of important socioeconomic value (Tian et al 2023), alfalfa is more sensitive to soil aluminum toxicity (Wang et al 2016, liu et al 2017, sun et al 2020), and acidic soil potentiates the effects of aluminum toxicity in the southwest china (Jiang et al 2022) and throughout the tropical and subtropical zones (Zhou et al 2015). Aluminum toxicity affects approximately 50% of cultivated land worldwide (Awasthi et al 2017, shi et al 2022), aluminum toxicity inhibits root growth, plant development (Rahman et al 2018), is the primary growth constraint on global acid soil that limits alfalfa yield and persistence (Campbell et al 1989, cui et al 2013, zhou et al 2014, zhou et al 2015, wang et al 2016, awasthi et al 2017, liu et al 2017, buhaiov et al 2018, charles g.willis et al 2020, sun et al 2022, yu 2022). Soil aluminum toxicity is a major threat to alfalfa yield in south China. Although the effect of aluminum toxicity on alfalfa seedlings has been studied, it is still not completely clear whether aluminum alters germination of alfalfa seeds. Aluminum toxicity is a major limiting factor in alfalfa planting in Guizhou and even southern acid soil areas (Xu and Ji 1998). Aluminum toxicity obviously reduces the reproduction rate and survival rate of rhizobia, and seriously inhibits the growth of alfalfa (Li et al 2005) so that large-area planting is difficult to carry out, thereby severely restricting the development of south grassland animal husbandry. The research carried out by us shows that the variety and the concentration of the aluminum in the soil have obvious influence on the germination vigor, germination rate, index and relative aluminum damage of the alfalfa, and the influence of the toxicity of the aluminum in the soil on the germination is regulated and controlled by the variety of the alfalfa (Tian et al 2023).
At present, the alfalfa aluminum resistance is studied, and extensive research is conducted for reducing the toxicity of soil aluminum and improving the aluminum resistance of plants. Lime has traditionally been used to alleviate soil aluminum toxicity (Liu et al 2017, zhang et al 2019) and to raise soil pH (Haque et al 2020). However, this method is costly (Liu et al 2017) and is considered to be environmentally (Liu et al 2017) and climatic disadvantageous because lime for acid soils produces inorganic carbon emissions (zamannian et al 2018, zamanian and Kuzyakov 2019). Because aluminum toxicity can cause boron deficiency, the addition of boron also slows down the aluminum toxicity hazard (Lenable et al 1996, zhou et al 2015, yan et al 2021). Since aluminum toxicity affects the absorption of moisture and nutrients by plants (Rahman et al 2018), the application of substances such as phosphorus, calcium, magnesium, sulfur, silicon, hormones, salicylic acid, polyamines, biofertilizers and biochar plays an important role in alleviating the toxicity of aluminum to plants (Pandey et al 2013, rahman et al 2018, rahman and Upadhyaya 2020). Since aluminum toxicity also inhibits growth of rhizobia, inoculation of rhizobia can promote growth of alfalfa under aluminum toxicity (Shi et al 2022). In addition, ammonium nitrogen (Shi et al 2022) and hydrogen sulfide (Zhu et al 2018) reduce the toxic effect of aluminum on alfalfa, and adjusting the ratio of potassium to magnesium to an appropriate ratio enhances the aluminum resistance of alfalfa (Huang and Grunes 1992). However, long-term, large-scale, extensive additions are not practical, increasing costs on the one hand, and creating new environmental and ecological problems on the other hand, and perhaps reducing aluminum toxicity by soil improvement is not sustainable. Cultivation of aluminum tolerant alfalfa varieties is one way to overcome this limitation. Over 200 alfalfa varieties exist in the world, but few varieties are available that can accommodate aluminum toxicity (Jiang et al 2022).
However, current research into aluminum toxicity mitigation has focused mainly on the selection of alfalfa varieties, seed germination and seedling growth (Qiu et al 2010, sun et al 2018), and the mitigation mechanisms of exogenous organic acids (Qiu 2010,Liu et al.2019). Among many mechanisms against aluminum stress, organic acid secretion is the most important mechanism of plant against aluminum stress. Researches prove that zinc, magnesium and phosphorus can relieve the toxicity of aluminum to alfalfa seedlings. Some researchers (Sun et al 2018) have also found that an increase in aluminum concentration can reduce alfalfa root tip oxidase activity. In addition, from the research of gene angle, it is found that the overexpression of alfalfa MsLEA2 gene in Arabidopsis thaliana can promote the growth of transgenic lines, protect the antioxidant enzyme system of plants and improve the capability of plants to resist aluminum toxicity under the action of aluminum toxicity stress (Liu et al 2019). The gene obviously shows the function of promoting the aluminum toxicity resistance of the arabidopsis thaliana (Liu et al 2019) in the arabidopsis thaliana. However, researches on solving the problem of aluminum toxicity resistance of alfalfa by utilizing microorganisms are rarely reported at present.
Disclosure of Invention
The technical problems to be solved by the invention are as follows: provides an endophytic fungus capable of improving the aluminum tolerance of alfalfa and application thereof.
The technical scheme of the invention is as follows: aspergillus versicolor (Aspergillus versicolor) F6 was deposited at 5.17.2023 with the Guangdong province microbiological strain collection center, no. 100, mitrex, guangzhou City, accession number GDMCC No:63473.
a microbial inoculum containing aspergillus omutatus (Aspergillus versicolor) F6.
The application of the aspergillus versicolor (Aspergillus versicolor) F6 or the microbial inoculum containing the aspergillus versicolor (Aspergillus versicolor) F6 in improving the aluminum alfalfa tolerance of Dragon pasture 801 is provided.
Compared with the prior art, the invention has the following beneficial effects:
the endophytic fungus aspergillus omutatus (Aspergillus versicolor) F6 obtained by separation can increase the aluminum stress tolerance of alfalfa dragon grazing No. 801, increase the growth of alfalfa and obviously increase the aboveground biomass and underground biomass of the alfalfa.
Preservation information:
aspergillus versicolor (Aspergillus versicolor) F6 was deposited at 5 months and 17 days 2023 with the Guangdong province microbiological strain collection center, no. 100, mitrex, guangzhou, city, accession number GDMCC No:63473.
drawings
FIG. 1 isolated endophyte plate colony morphology;
FIG. 2 effect of inoculation of endophytes on alfalfa mortality under aluminum stress treatment;
FIG. 3 effect of inoculation of endophytes on alfalfa survival under aluminum stress treatment;
FIG. 4 effect of inoculation of endophytes on alfalfa on-ground biomass under aluminum stress treatment;
FIG. 5 effect of inoculation of endophytes on alfalfa height under aluminum stress treatment;
FIG. 6 effect of inoculation of endophytes on alfalfa root weight under aluminum stress treatment;
FIG. 7 effect of inoculation of endophytes on alfalfa root volume under aluminum stress treatment;
FIG. 8 effect of inoculation of endophytes on alfalfa length under aluminum stress treatment;
FIG. 9 Effect of endophyte inoculation on alfalfa root surface area under aluminum stress treatment
FIG. 10 effect of inoculation of endophytes on root length per unit root volume of alfalfa under aluminum stress treatment;
FIG. 11 effect of inoculation of endophytes on alfalfa root tip number under aluminum stress treatment;
FIG. 12 effect of endophyte inoculation under aluminum stress treatment on alfalfa root bifurcation number.
Detailed Description
The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from commercial sources.
Example 1 Albumin resistant alfalfa seed endophyte screening
(1) Isolation and purification of endophytic bacteria of alfalfa seeds with aluminum toxicity resistance
The alfalfa seeds with the surfaces disinfected are placed on a culture dish paved with water-absorbing filter paper, after natural air drying, the alfalfa seeds are transferred into a sterile grinding pot for grinding, 1g of seed powder is weighed from the alfalfa seeds, 9ml of sterile water is added into each g of seed powder, and the alfalfa seeds are fully and uniformly mixed. Then diluting the homogenate to prepare seed suspension, taking 10 0 、10 -1 、10 -2 、10 -3 And 10 -4 Five dilution gradients. 10ul of the culture medium is inoculated on LB culture medium, cultured in an incubator at 28 ℃ and observed every 6 hours for 2 to 4 days. As endophyte in the seeds is unevenly distributed, the inspection results of different times are greatly changed, the operation is repeated for 15 times, and 15g is weighed.
The seed homogenate after surface disinfection is cultivated in the dark for 2 to 5 days in an LB plate constant temperature incubator at 28 ℃, and colony growth conditions are observed every 24 hours. And purifying the bacteria obtained by culture, picking out representative single colonies with different forms and colors from an LB plate, streaking and purifying, repeating for 6 times, and screening out the bacteria capable of culturing.
(2) Separation and purification of endophytic fungi of alfalfa seeds resistant to aluminum toxicity
1g alfalfa seeds were weighed and surface sterilized. Cutting herba Medicaginis seeds with surface sterilized by tissue separation method into two sections, half burying in PDA culture medium, culturing at 25deg.C for 5-7 days until mycelia grow out at the edge of tissue block, and inoculating needle to obtain mycelia tip, and transferring onto new PDA plate for purification culture.
(3) By morphological observation, representative 5 strains of bacteria and 2 strains of fungi, designated B1, B7, B9, B10, B14 and F1, F6, respectively, were selected (fig. 1).
TABLE 1 colony morphology characterization of alfalfa seed-culturable endophytic bacteria
F1: colony powder or velvet, olive green, black green on the back, conidium acrogenesis or lateral growth, forming branched spore chain, oval, cylindrical, lemon, near spherical, light brown, smooth, 0-1 septum, most no septum. The preliminary morphology was identified as Cladosporium.
F6: the velvet shape, the flocculence shape or both have quite wide color change range, and on the colony of different strains, light green, light yellow or pink and other colors can sometimes appear locally, while the reverse side of the colony is nearly colorless, yellow orange or rose; colorless to purplish red droplets form on some colonies. The conidiophore head is loose and radial, colorless or slightly yellow. The preliminary morphology was identified as aspergillus.
Example 2 Effect of isolated seed Source culturable microorganisms on alfalfa growth
1. Preparation of microbial inoculum
The selected endophytes are cultivated and expanded, the bacteria grow on LB medium, and the fungi grow on PDA medium.
(1) Preparation of bacterial suspensions: the single bacterial colony obtained by purification and screening is picked up by 10ul and inoculated into sterilized 50ml of LB culture solution, and placed in a shaking incubator at 37 ℃ for 180r/min, and shake culture is carried out until turbidity appears.
(2) Preparation of fungal spore suspension: inoculating 4 strains of fungi on a PDA (personal digital assistant) plate by the method of the reference prescription, respectively inoculating the fungi on a PDA solid culture medium, and culturing for 7 days at 25 ℃ in a dark place; after the whole flat plate is fully grown, the spores in the flat plate are gently scraped by a sterile shovel, transferred into a 50mL conical flask containing 5mL of sterile water and glass beads, placed in a shaking table at 37 ℃ and 180rpm for shaking for 1h to break up the mycelia, and then the liquid in the conical flask is filtered into the conical flask by sterile filter paper to obtain the spore suspension of the fungi.
TABLE 2 determination of the concentration of the seed Source culturable microbial agent strain (CFU/ml, measured by a cytometer)
2. Experimental materials
Alfalfa Yu alfalfa No.1 (abbreviated as MS_YM1) and Dragon herding No. 801 (abbreviated as MS_LM801) were selected as experimental materials.
3. Experimental method
(1) The flowerpot and cover of the required material and acid soil are sterilized with cobalt-60 gamma rays for 2 days.
(2) And (5) placing the sterilized seeds in a culture dish for germination, and uniformly transplanting after 9 days.
(3) Roots of seedlings were immersed in the cultured bacterial suspension or fungal spore suspension and dark treated for 6 hours before transplanting.
(4) Transplanting the treated seedlings into a small flowerpot, wherein 250g of soil and 120ml of sterile water are filled in the flowerpot, and the seedlings soaked by the same strain are singly covered by a cover after being transplanted. Each variety was replicated 6 times, and they were incubated in a sterile room temperature environment for 16 hours with light and 8 hours with darkness.
(5) After 5 days, the transplanted seedlings are treated by adding aluminum stress (specific method of aluminum stress comprises simulating soil aluminum concentration to 100ppm by using an aluminum chloride hexahydrate solution, and uniformly injecting 20ml by using a 20ml sterile injector in a cross reciprocating way for each pot), and the growth state of the seedlings is observed and recorded every day.
4. Sampling
1) Uniformly sampling after 14 days of aluminum stress, taking the root and overground part of the whole plant, and rapidly rinsing soil on the root by using 1 XPBS;
2) Sucking excessive water with water absorbing paper, and placing the root into a centrifuge tube;
3) Re-storing in refrigerator at 4 deg.c;
4) Measuring and recording plant height, root weight and stem weight in the sampling process;
5) The cleaned roots were scanned under a Epson Perfection V800 Photo scanner, and then the root morphology indexes were analyzed using the WinRHIZO Pro2015 software.
5. Results and analysis
Inoculation under aluminum stress has been found to be potentially detrimental to inoculated endophytes, as they have significantly increased mortality and significantly decreased survival compared to control CK (with aluminum stress); while the inoculation of the culturable endophytic fungus F1 showed no significant difference in the number of deaths and survival of ms_ym1 compared to the control (p > 0.05), the inoculation of the culturable endophytic fungi F1 and F6 significantly reduced the number of deaths of ms_lm801 compared to the control CK (p < 0.05), significantly lower than the control CK0 (no aluminum stress), significantly increased the number of survival (p < 0.05), significantly higher than the control CK0, see fig. 2 and 3.
Seed source culturable endophytes have a significant effect on aboveground biomass under aluminum stress (p < 0.05). Under aluminum stress, the inoculated endophytes significantly reduced the aerial biomass and height (p < 0.05) compared to the control CK, while the inoculated fungi F1 significantly reduced the aerial biomass of ms_ym1 (p < 0.05), the inoculated fungi F6 significantly increased the height of ms_ym1 (p < 0.05), but the inoculated fungi F1 and F6 significantly increased the aerial biomass and height of ms_lm801 (p < 0.05), and the inoculated fungi F6 significantly increased the aerial biomass and height of ms_lm801 over the control CK0 (p < 0.05), see fig. 4 and 5.
Seed source culturable microorganisms have a significant effect on root biomass under aluminum stress (p < 0.05). Under aluminum stress, root biomass of two varieties inoculated with endophytes was significantly reduced (p < 0.05) compared to control CK, but inoculated endophytes F1 and F6 significantly increased root biomass of ms_lm801 (p < 0.05), inoculated endophytes F6 significantly exceeded the control CK0, but inoculated endophytes F1 significantly reduced root biomass of ms_ym1 (p < 0.05), while inoculated endophytes F6 had no significant effect on root biomass of ms_ym1 (p < 0.05) (p > 0.05), as shown in fig. 6.
The seed source can culture microorganisms with obvious difference (p < 0.05) on root morphology under aluminum stress. Under aluminum stress, the inoculated endophyte treatment significantly reduced the root volume, length, surface area, root length per root volume, root tip number, and root bifurcation number (p < 0.05) of ms_lm801 and ms_ym1 compared to control CK, while inoculated endophytes F1 and F6 significantly increased the root volume, length, surface area, root length per root volume, root tip number, and root bifurcation number (p < 0.05) of ms_lm801 and inoculated endophyte F6 significantly increased the root volume, length, surface area, root length per root volume, root tip number, and root bifurcation number (p < 0.05) of ms_ym1, as shown in fig. 7, 8, 9, 10, 11, and 12.
Therefore, the endophytic fungus strains F1 and F6, particularly the strain F6, obtained by separation can obviously increase the aluminum stress tolerance of alfalfa nomadic 801 (MS_LM801), increase the growth of alfalfa and obviously increase the aboveground biomass and the underground biomass of the alfalfa.
EXAMPLE 3 identification and preservation of endophytic fungus Strain F6
(1) Molecular characterization of endophytic fungus Strain F6
ITS1 of strain F6 was sequenced, with a sequenced front primer sequence CTTGGTCATTTAGAGGAAGTAA and a sequenced rear primer sequence GCTGCGTTCTTCATCGATGC. The ITS1 sequence of the strain F6 is shown as SEQ ID No. 1.
The comparison of the ITS sequences of strain F6 according to the BLAST software and known strain sequences in the GenBank database shows that the consistency of strain F6 and multiple strains Aspergillus versicolor reaches 99.81%, and the strain F6 is determined to be Aspergillus versicolor by combining morphology.
(2) Preservation of endophytic fungus strain F6:
aspergillus versicolor (Aspergillus versicolor) F6 was deposited at 5.17 of 2023 with the Guangdong province microbiological strain collection center, accession number GDMCC No:63473.
Claims (3)
1. aspergillus versicolor strainAspergillus versicolor) F6, deposited 5.17 days 2023 with the cantonese microbiological strain collection center, 100 th of the middle road of pioneer, guangzhou, accession No. GDMCC No:63473.
2. comprising the following claimsThe aspergillus omutatus strain isAspergillus versicolor) F6 bacteria agent.
3. The koji mold according to claim 1Aspergillus versicolor) Use of F6 or the microbial inoculum of claim 2 for increasing aluminium tolerance of alfalfa No. 801 in nomadic grazing.
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