CN112592838A - Orchid mycorrhizal fungus PF07 and application thereof - Google Patents
Orchid mycorrhizal fungus PF07 and application thereof Download PDFInfo
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- 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
- A01G22/60—Flowers; Ornamental plants
- A01G22/63—Orchids
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- 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
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- A—HUMAN NECESSITIES
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- 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
- A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
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- 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
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Abstract
The invention discloses an orchid mycorrhizal fungus PF07, wherein the orchid mycorrhizal fungus PF07 is septoria cupulosa (Cypherophora sp.) PF07, is stored in the common microorganism center of the China Committee for culture Collection of microorganisms in 11 months and 13 days in 2020, has the address of No. 3 Siro-1 Bichen of Beijing Chaoyang district, and has the preservation number of CGMCC NO: 21053. the invention separates and screens a strain of septoria cupulophora sp.PF07 from wild orchid for the first time, the strain can promote the growth of orchid, can promote the rapid breeding of orchid seedlings and shorten the seedling period when being applied to orchid mycorrhizal seedling raising practice, provides a theoretical basis for the conservation of rare or endangered plants and provides a technical support for promoting the large-scale development of the paphiopedilum industry.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to orchid mycorrhizal fungi PF07 and application thereof.
Background
Almost all orchid plants have symbiotic relationship with mycorrhizal fungi, and the existing research shows that the mycorrhizal fungi have unique ecological functions on the orchid plants, such as promoting seed germination and seedling morphogenesis, helping ecological invasion of the orchid plants, influencing the composition of biological communities and preparing biological elicitors with specific efficacies, and being beneficial to the aspects of protection, recovery or reconstruction of an ecological system and the like. By studying the mechanism, the recent research shows that mycorrhizal fungi provide essential nutrition for embryo cells through digesting hypha, stimulate plants to produce hormones such as gibberellin and IAA, and vitamins such as nicotinic acid and nicotinamide. In addition, the mycorrhizal fungi can synthesize the phytoalexin in the plant body, and further activate the enzymatic activities of SOD, POD, CAT, PAL and the like to enhance the resistance of the host plant. Meanwhile, when the mycorrhizal fungi are successfully colonized and become beneficial dominant flora, the released antagonistic substances can effectively enhance the disease resistance of the orchids, and have important significance for improving the survival rate of seedlings and promoting the growth of plants. In recent years, orchid mycorrhizal fungi become a new research hotspot, and particularly, research on the aspects of improving the transplanting survival rate of certain rare species of tissue culture seedlings, promoting the growth and the conservation of plants and the like by utilizing the orchid mycorrhizal fungi has been widely reported, for example, dendrobe (Dendrobium sp), Anoectochilus (Anoectochilus sp), paphiopedilum, Cymbidium (Cymbium sp), Pediobolus (Doriti sp) and the like, after the mycorrhizal seedling raising technology is introduced into artificial cultivation, the seedling transplanting survival rate, the fresh quality growth rate, the dry matter accumulation and the mineral element absorption are obviously improved, and metabolites of part of the mycorrhizal fungi can secrete plant growth regulators such as gibberellin, IAA and the like.
Disclosure of Invention
The invention aims to provide an orchid mycorrhizal fungus PF07 and application thereof, wherein the orchid mycorrhizal fungus PF07 is a strain capable of promoting the growth of orchid plants.
In order to achieve the purpose, the technical scheme provided by the invention is as follows:
an orchid mycorrhizal fungus PF07 is classified and named as septoria californica (Cypherophora sp.) PF07, is preserved in China general microbiological culture Collection center on 11-13.2020, and is No. 3 Hospital No.1 of Beijing Chaoyang district, Beicheng, with the preservation number of CGMCC NO: 21053.
the application of the orchid mycorrhizal fungus PF07 in promoting the growth of orchid is disclosed.
Preferably, the orchid mycorrhizal fungus PF07 is prepared into a liquid microbial inoculum or a fungal elicitor to promote the growth of orchid.
Preferably, the method for preparing the orchid mycorrhizal fungus PF07 into the liquid microbial inoculum comprises the following steps: inoculating orchid mycorrhizal fungi PF07 to a PDA culture medium flat plate, placing the flat plate in an illumination incubator, carrying out dark culture at a constant temperature of 28 ℃ for 5-7 d to activate strains, then punching holes at the edges of the colonies to prepare fungus cakes, respectively transferring the fungus cakes prepared by the strains into bottles containing 150mL of liquid PDA culture medium, inoculating 2 fungus cakes (the 2 fungus cakes represent the inoculation amount), placing the inoculated 2 fungus cakes in a shaking table at 28 ℃ and 140r/min for shake culture for 10d, crushing for 5min, diluting with sterile water to 40 times of the visual field of a microscope, observing average 20 bacteria, and preparing into a liquid microbial inoculum.
Preferably, the orchid mycorrhizal fungus PF07 is prepared into a fungal elicitor with the concentration of 100-150mL/L to promote the growth of orchid.
Preferably, the formulation of the fungal elicitor: beating the activated orchid mycorrhizal fungi PF07 into fungus cakes, inoculating the fungus cakes into a PDA liquid culture medium, inoculating the fungus cakes into a bottle of 250mL, inoculating 1 fungus cake, shaking and culturing for 7d in a shaking table of 120r/min, harvesting after the mycelia fully grow, smashing the mycelia, mixing the mycelia with a bacterial liquid, and sterilizing at 121 ℃ for 20min to obtain the PF07 fungus elicitor.
Compared with the prior art, the invention has the following beneficial technical effects:
the invention separates and screens a strain of septoria cupulophora sp.PF07 from wild orchid for the first time, the strain can promote the growth of orchid, can promote the rapid breeding of orchid seedlings and shorten the seedling period when being applied to orchid mycorrhizal seedling raising practice, provides a theoretical basis for the conservation of rare or endangered plants and provides a technical support for promoting the large-scale development of the paphiopedilum industry.
Description of preservation information
The septoria cupulophora sp.PF07 is preserved in China general microbiological culture Collection center (CGMCC for short) in 11-13.2020, and the preservation number is CGMCC NO: 21053.
drawings
FIG. 1 is a topographical map of mycorrhizal fungi PF07, wherein (a) is a PF07 hypha map at 40-fold magnification, and (b) is a PF07 colony map.
FIG. 2 Effect of the orchid mycorrhizal fungus PF07 on the biomass growth of paphiopedilum hirsutissimum test-tube plantlets.
FIG. 3 effect of orchid mycorrhizal fungus PF07 on biomass growth of transplanted paphiopedilum hirsutissimum seedlings.
FIG. 4 shows the effect of different concentrations of the inducer of the orchid mycorrhizal fungus PF07 on the growth of paphiopedilum hirsutissimum test-tube plantlets.
FIG. 5 Effect of orchid mycorrhizal fungus PF07 fungal elicitor on growth of paphiopedilum hirsutissimum test-tube plantlets.
FIG. 6 Effect of orchid mycorrhizal fungus PF07 on the growth of plants with leaf pocket transplanted cup seedlings.
Detailed Description
The following detailed description is to be read in connection with the accompanying drawings, but it is to be understood that the scope of the invention is not limited to the specific embodiments. The raw materials and reagents used in the examples were all commercially available unless otherwise specified. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. In the quantitative experiments in the following examples, three replicates were set up and the results averaged.
Example 1
Isolation and characterization of orchid mycorrhizal fungus PF07
1.1 isolation of the orchid mycorrhizal fungus PF07
The isolation of orchid mycorrhizal fungi PF07 comprises sampling and screening, and specifically comprises the following steps:
separating strains from fresh wild orchid nutrition roots of 7 batches by adopting a tissue block separation method or a tissue liquid smearing method, wherein the separation culture medium is a potato glucose culture medium (PDA), 154 strains are separated, the mycorrhizal fungi are purified by adopting a tip picking method, and 60 strains are obtained after the mycorrhizal fungi are combined;
primary screening and secondary screening of excellent character fungal strains:
(1) solid strain: selecting activated strains which are not lethal (primary screening) or beneficial strains (secondary screening), and respectively beating bacterial cakes with the diameter of 0.5cm from the edges of bacterial colonies as inoculation materials;
(2) the inoculation method comprises the following steps: cleaning the culture medium adhered to the tissue culture seedling roots with sterile water on a superclean bench, sucking water with sterile absorbent paper, weighing fresh weight by taking a bottle as a unit, transferring into a symbiotic culture medium, taking 2 strains per bottle, selecting 1 strain cake, inoculating into the center of the culture bottle, and comparing with no strain; repeating the treatment for 3 times, putting the mixture into a culture room for symbiotic culture at the temperature of 23 +/-1 ℃ for 12-14 h of illumination every day with the illumination intensity of 1600-2000 Lux, and observing the growth conditions of the bacteria and the seedlings every two days;
(3) and (3) measuring the orchid growth index: respectively measuring the fresh weight of the tissue culture seedlings on the day of inoculation and at 90 days after inoculation, and counting the survival condition and growth vigor of the plants after inoculation; calculating the fresh weight growth rate (%) of the seedling as (end weight-initial weight)/initial weight x 100;
(4) mycorrhization detection: randomly drawing 2-3 seedlings every time, shearing 3-4 fresh nutrient roots for each seedling, soaking root segments in 75% ethanol in an ultra-clean workbench for 30s, washing with sterile water for 2 times, then disinfecting with 0.1% mercuric chloride solution for 1-3 min for surface sterilization, carrying out the following operation and culture conditions in the same fungus separation method of 1.1, counting the separation condition and calculating the separation rate after 7d, comparing with the colony morphology and growth characteristics of the original strain, and if the separated strain is the target strain, indicating that the strain can form mycorrhiza, and fully mycorrhizing the plant;
inoculating orchid tissue culture seedlings and strains by adopting a symbiotic (DE) culture medium, primarily screening and culturing for 60 days, selecting strains which do not cause death to the seedlings, then carrying out secondary screening, measuring and recording to obtain a beneficial symbiotic strain which has an obvious promotion effect on the growth of the seedlings and is named as PF 07.
1.2 identification of orchid mycorrhizal fungi PF07
Obtaining mycelium by extracting DNA: inoculating the PF07 strain which grows vigorously on a solid culture medium on a solid PDA culture medium for culture, and carefully scraping off the mycelia for later use after the mycelia grow out; some strains which grow slowly and have no hypha developed on a solid culture medium are inoculated into a PDA liquid culture medium, shake culture is carried out for 3-5 days by a shaking table, mycelium is obtained by filtration, and the mycelium is dried for later use;
observation of culture characteristics of beneficial strains: the characteristics of the colonies of each culture were observed and recorded: shape, size, color and variation, texture, as shown in FIG. 1;
② extracting genome DNA: the kit is operated according to SK8255 (bacteria), SK8259 (fungi) and SK8257 (yeast);
PCR amplification: the amplification primers are the universal ITS1 and ITS4, and the forward primer ITS 1: TCCGTAGGTGAACCTGCGG, respectively; reverse primer ITS 4: TCCTCCGCTTATTGATATGC, respectively; the amplified sequences are internal transcription spacers 1 and 2, and the PCR length is about 600 bp;
detecting the concentration and purity of the DNA: the size of the obtained genome DNA fragment is related to factors such as sample storage time, shearing force in operation and the like, and the concentration and purity of the recovered DNA fragment are detected by agarose gel electrophoresis and an ultraviolet spectrophotometer;
ITS sequencing and comparison: recovering and purifying the PCR reaction result, and determining ITS fragment sequences by adopting a DNA sequencer to obtain a forward ITS sequence and a reverse ITS sequence; 16SrDNA sequences are aligned on a ribosome database http:// rdp.cme.msu.edu/index.jsp; performing molecular level identification on mycorrhizal fungi through homology analysis; identifying the species of the test strains according to the similarity and the morphological observation result;
PCR reaction system
| Reagent | Volume (μ L) |
| Template (genomic DNA 20-50 ng/. mu.l) | 0.5 |
| 10×Buffer(with Mg2+) | 2.5 |
| dNTPs (2.5 mM each) | 1 |
| Enzyme | 0.2 |
| F(10uM) | 0.5 |
| R(10uM) | 0.5 |
| Double steam adding H2O | 25 |
PCR cycling conditions:
16SrDNA sequences are aligned on a ribosome database http:// rdp.cme.msu.edu/index.jsp;
the alignment results are shown in the following table:
the matching degree of the orchid mycorrhizal fungi PF07 and Cypheophora sp is 100%, so that the orchid mycorrhizal fungi PF07 is identified as Cypheophora sp, named as Cypheophora sp PF07, and is preserved in the common microorganism center of China Committee for culture Collection of microorganisms (CGMCC for short), and the preservation number is CGMCC NO: 21053, the sequence is shown in SEQ ID NO. 1.
Example 2
Orchid mycorrhizal fungus PF07 having growth and physiological effects on paphiopedilum hirsutissimum
2.1 test materials
2.1.1 test plants: the symbiotic culture of test-tube plantlets adopts sterile tissue culture plantlets which grow vigorously at a strong seedling stage of paphiopedilum hirsutum and have 3-4 roots and 3-4 leaves; the transplanting seedlings in the nutrition cups adopt tissue culture seedlings in a rooting stage, and the tissue culture seedlings have 5-8 roots and 4-5 leaves;
2.1.2 test strains: selecting orchid mycorrhizal fungi PF07 obtained after screening in the embodiment 1;
2.1.3 culture Medium: the symbiotic culture medium in the bottle is a DE culture medium; the culture medium of the transplanted seedling in the nutrition cup is pine bark: volcanic rock: charcoal was mixed at a volume ratio of 4:2: 1.
2.2 test methods
2.2.1 preparation of liquid microbial inoculum: inoculating the test strains to a PDA culture medium flat plate, placing the flat plate in an illumination incubator for dark culture at a constant temperature of 28 ℃ for 5-7 d to activate the strains, then punching holes on the edges of bacterial colonies by using a puncher with the diameter of 5mm to prepare small round plates with the same size, namely bacterial cakes, respectively transferring the bacterial cakes prepared by the strains into 300mL triangular flasks containing 150mL of liquid PDA culture medium, inoculating 2 bacterial cakes (2 bacterial cakes represent the inoculation amount) into the triangular flasks, placing the triangular flasks in a shaking table with the inoculation amount of 28 ℃ and 140r/min for shake culture for 10d, then crushing the bacterial cakes for 5min by using a tissue triturator, diluting the bacterial cakes to 40 times of the visual field of a microscope, observing average 20 bacteria, and preparing a liquid microbial inoculum for later use;
2.2.2 tissue culture seedling treatment: placing the tissue culture seedlings in the rooting stage in 2.1.1 in a greenhouse for hardening for about 7-10 days, washing off a culture medium, soaking the tissue culture seedlings in 1000 times of aqueous solution of 50% carbendazim wettable powder for 10min, placing the tissue culture seedlings in a shade, airing, and transplanting the tissue culture seedlings into 50-hole seedling culture hole trays, wherein 2 plants/clump are planted for 3 times after 30 clumps are treated;
2.3 inoculation method
(1) Inoculating the test-tube plantlet with a strain: activating the screened orchid mycorrhizal fungi PF07 (activating to inoculate the tested strains on a PDA culture medium plate, placing the PDA culture medium plate in an illumination incubator for dark culture at the constant temperature of 28 ℃ for 5-7 d to activate the strains), selecting the strains with good growth, punching the edges of the colonies by using a puncher with the diameter of 5mm to prepare a bacterial cake, inoculating the bacterial cake into 2.1.1 aseptic tissue culture seedlings under aseptic conditions, inoculating 2 seedlings at the periphery of a bottle, and repeating for 10 times; after inoculation, putting the inoculated seeds into a culture room at 25 +/-2 ℃ for symbiotic culture, and illuminating for 12-14 h every day at the illumination intensity of 2000 Lux; counting the fresh weight, the leaf length, the leaf width, the root length and the growth potential growth indexes by taking a bottle as a unit after symbiotic culture for 120 days;
(2) transplanting seedling inoculation strain: pouring 10ml of liquid inoculum obtained by 2.2.1 bacteria per seedling on the same day of transplanting, taking a poured equivalent liquid PDA culture medium as a Control (CK), pouring 1 time of bacteria liquid every 20 days, continuously pouring 3-4 times, pouring 1 time of sterile water every 5-7 days after transplanting, properly spraying the leaves according to the air humidity, keeping the temperature (26 +/-2) DEG C, the humidity at 70-85%, the shading rate at 70-80%, and not applying or using the fertilizer in the period; and after symbiotic culture for 120 days, counting growth indexes of leaf width, leaf length, leaf width, root length and the like of the tissue culture seedlings.
2.4 measurement of physiological indices
(1) POD, CAT and SOD enzyme activity is measured by a micro method: the activities of POD, CAT and SOD enzymes in the leaves of the seedlings obtained after the seedlings were transplanted and cultured for 120 days in 2.3 were measured according to the method given in the kit specification of Peroxidase (POD) of Suzhou Ke Ming Biotechnology, Inc., the method given in the kit specification of Catalase (CAT), and the method given in the kit specification of Superoxide Dismutase (SOD).
2.5 statistical analysis of data
Test data statistics were performed using EXCEL tables and Duncan's new pole error test using DPS7.05 software. Fresh weight increase (%) (end weight-initial weight)/initial weight × 100; leaf area (leaf length x leaf width) x leaf shape index (0.8317).
Results and analysis
1. Inoculating orchid mycorrhizal fungi PF07 to test-tube plantlet
The cymbidium mycorrhizal fungi PF07 and paphiopedilum hirsutissimum tissue culture seedlings are subjected to symbiotic culture, the average fresh weight of the biomass of the mycorrhizal seedlings is proved to be obviously higher than that of a Control (CK), and the result shows that the inoculated cymbidium mycorrhizal fungi PF07 has a relatively obvious growth promoting effect. The influence of the orchid mycorrhizal fungus PF07 on the fresh weight growth and leaf area index of the cultured seedlings is not obvious, but the fresh weight growth amount is obviously improved compared with that of a control, the addition of the orchid mycorrhizal fungus PF07 strain treatment is 395% higher than that of the control respectively, and the inoculation treatment has a very obvious effect of promoting the growth of orchid biomass. The leaf area and the new root length of the tissue culture seedlings treated by the orchid mycorrhizal fungi PF07 strain are not obviously different from those of a control, and are respectively 22.2% and 25% higher than that of the control (figure 2).
2. Inoculating mycorrhizal fungi to transplanted cup seedlings
In a matrix (pine bark: volcanic rock: charcoal: 4:2:1 volume ratio), after paphiopedilum hirsutum is inoculated with the liquid microbial inoculum obtained in 2.2.1 for 3 times (10 ml for each inoculation), the plant biomass change is remarkably different, and the overall growth promotion effect of the orchid mycorrhizal fungus PF07 is better. The orchid mycorrhizal fungus PF07 and the control were not significantly different in terms of leaf length, leaf width and root length (FIG. 3).
3. Influence of inoculated mycorrhizal fungi on physiological indexes of paphiopedilum hirsutissimum
(1) The physiological indexes of the test-tube plantlet are as follows: because the nutrition of the culture medium of the test-tube plantlet is limited, the moisture content and the chlorophyll content of the plant are obviously improved after the live bacteria are inoculated, the biomass of the plant inoculated with the orchid mycorrhizal fungi PF07 is better increased, but the POD, CAT and SOD enzyme activities of the plant treated by the inoculated bacteria are weaker after the plant is cultured for 120 days (Table 1);
TABLE 1 Effect of orchid mycorrhizal fungi inoculated PF07 on physiological indices of paphiopedilum hirsutissimum test-tube plantlets
| Strain numbering | POD(U/g) | CAT(nmol/g) | SOD(U/g) | Total amount of chlorophyll (mg/g) |
| PF07 | 42.84 | 46.15 | 490.33 | 0.717 |
| CK | 36.72 | 120.65 | 1666.67 | 0.435 |
(2) Transplanting cup seedlings physiological indexes: after transplanting, the biomass of a plant inoculated with the septoria cupulophora sp (Cypherophora sp) PF07 is better increased, but after culturing for 120d, the POD enzyme activity of the inoculated plant is weakened, and the SOD enzyme activity is enhanced; the CAT enzyme activity of the inoculated P.californica (Cypherophora sp.) PF07 was not significantly different, but 3 enzyme activities were all the strongest in the Control (CK) plant, and the difference in the total chlorophyll amount was not significant (Table 2).
TABLE 2 Effect of orchid mycorrhizal fungi inoculated PF07 on physiological indexes of paphiopedilum hirsutissimum test-tube plantlets
| Strain numbering | POD(U/g) | CAT(nmol/g) | SOD(U/g) | Total amount of chlorophyll (mg/g) |
| PF07 | 333.33 | 36.72 | 94.94 | 0.473 |
| CK | 290.33 | 131.58 | 244.76 | 0.445 |
Example 3
Action of fungal elicitor on growth and physiology of paphiopedilum hirsutissimum
3.1 materials and methods
3.1.1 test plants: the symbiotic culture of test-tube plantlets adopts aseptic plantlets which grow robustly in the strong seedling stage of paphiopedilum hirsutum and have 3-4 roots and 3-4 leaves; the symbiotic culture of the nutrition cup seedlings adopts test tube seedlings in a rooting stage, and the test tube seedlings have 5-8 roots and 4-5 leaves;
3.1.2 test strains: selecting the strain PF07 obtained after screening in the example 1, wherein PF07 is separated from fresh roots of wild paphiopedilum hirsutissimum in Guangxi Tian county and is an immortal strain of paphiopedilum hirsutissimum seedlings;
3.1.3 culture Medium: the test-tube plantlet symbiotic culture medium is a DE culture medium; the nutrient cup seedling symbiotic culture medium is pine bark: volcanic rock: charcoal was mixed at a volume ratio of 4:2: 1.
3.2 test methods
3.2.1 Strain activation: inoculating the test strains to a PDA culture medium plate, and placing the plate into an illumination incubator to perform dark culture at the constant temperature of 28 ℃ for 5-7 d of activated strains;
3.2.2 preparation of fungal elicitor: perforating the strain obtained by culturing for 7d and activating for 3.2.1, perforating by using a perforator with the diameter of 0.5cm, perforating a small disc with the same size, namely a fungus cake, inoculating the fungus cake into a PDA liquid culture medium which is subpackaged in advance and sterilized, inoculating 1 block/bottle of the fungus cake, performing shake culture on a 120r/min shaking table for 7d, harvesting after the sufficient growth of the mycelium, smashing the mycelium by using a tissue triturator, mixing the smashed mycelium with a bacterial liquid, and sterilizing at 121 ℃ for 20min to obtain an orchid mycorrhizal fungi PF07 fungal elicitor for later use;
3.2.3 tissue culture seedling treatment: placing the test-tube plantlet at the rooting stage of 3.1.1 in a greenhouse for hardening for about 7-10 days, washing off culture medium attached to the root, soaking for 10min by 1000 times of aqueous solution of 50% carbendazim wettable powder, draining, placing in a shade, spreading and drying the water on the surface of the plant, and moving the plant into a transparent nutrition pot with the caliber of 5cm for 2 plants/clump, wherein the treatment is repeated for 3 times at each clump;
3.2.4 preparation and inoculation of test-tube plantlet fungal elicitor culture Medium
Taking DE as a basic culture medium, adding the reserved cymbidium mycorrhizal fungi PF07 fungal elicitors into the basic culture medium according to 3 gradients of 50, 100 and 150mL/L, and taking the DE culture medium without the fungal elicitors as a Control (CK); before inoculation, weighing the fresh weight (accurate to 0.0001 g) of the tissue culture seedling obtained after 3.2.3 treatments on an ultra-clean workbench under aseptic condition, then inoculating 6 seedlings to a culture medium containing a fungal elicitor at the periphery of a tissue culture bottle, repeating for 10 times, namely 6 seedlings in each bottle, and 5 bottles in total, and inoculating 30 seedlings in each treatment, namely test-tube seedlings; culturing at 25 deg.C under the condition of 2000Lux and 12h/d at relative humidity of 70-75%, and counting the growth indexes such as fresh weight, leaf length, leaf width, new root number, root length and growth potential by taking bottle as unit after culturing for 120 d;
3.2.5 transplanting cup seedling watering fungus elicitor
Selecting 3.2.4 paphiopedilum hirsutissimum tissue culture seedlings with leaves of 5-6 leaves and roots of 3-4 paphiopedilum hirsutissimum, culturing in a culture medium containing a fungal elicitor for a period of time, putting the paphiopedilum hirsutissimum tissue culture seedlings under a 85% shading net, hardening the seedlings for 1 week, taking out, cleaning, sterilizing, airing the water on the surfaces of the plants, transplanting 2 plants/cluster into a seedling culture cup with the caliber of 4.5cm, wherein the substrate adopts 3.1.3 nutrient cup seedling symbiotic culture substrate, and the roots are required to be stretched and not damaged;
before pouring the fungal elicitor, poking the matrix around the root system of the paphiopedilum hirsutissimum tissue culture seedling, pouring 10mL of the fungal elicitor diluted by 30 times by using a 10mL injector at a position about 1cm away from the root system, then covering the matrix, and pouring 10mL of sterile water in a contrast way; the illumination is carried out for 12-14 h every day, the illumination intensity is 2500-3000 Lux, the temperature is 28 +/-1 ℃, and the humidity is 70-75%; spraying with appropriate amount every day to keep plant and matrix wet, keeping ventilation, supplementing and applying fungal elicitor for 1 time every 10 days, continuously applying for 3 months, continuously culturing for 120 days, and counting growth indexes such as transplanting survival rate, fresh and heavy leaf length, leaf width, new root number and growth potential.
3.3 items and methods of measurement
(1) Test-tube plantlet: harvesting test-tube plantlets after 120 days of culture, washing culture medium at the root with clear water, naturally drying, and weighing fresh weight (accurate to 0.0001 g) of the tissue culture plantlets by using an electronic balance, wherein the method for measuring the physiological indexes is the same as the method in 2.4;
(2) transplanting cup seedlings: counting the growth index of fresh weight growth of the cup seedlings after 120 days of culture; the method for measuring physiological index is the same as that described in 2.4 above.
3.4 results and analysis
3.4.1 growth Effect of fungal elicitors on test-tube plantlets
(1) Concentration of fungal elicitor: inoculating 3 fungal elicitors with different concentrations (50mL/L, 100mL/L and 150mL/L) of orchid mycorrhizal fungi PF07 in test-tube plantlets of paphiopedilum hirsutissimum, and finding that the fungal elicitors have an extremely important promoting effect on the growth of paphiopedilum hirsutissimum, but the fungal elicitors with different concentrations added with the strains have different degrees of improvement on the fresh weight growth rate of plants under the influence of the concentrations; the fresh weight growth rate of a plant cultured by the calix brevicola (Cyphellophora sp.) PF07 in the fungal elicitors with 3 concentrations is obviously or extremely higher than that of a Control (CK), particularly 150mL/L of the calix brevicola (Cyphellophora sp.) PF07, and the fresh weight growth rate is improved by 262.9 percent compared with that of the control (figure 4);
(2) fungal elicitor effects: based on the strain of the fungal elicitor with the optimal concentration, the growth promotion effect of the added fungal elicitor is better than that of a Control (CK); wherein, the growth rate of the fresh weight of the paphiopedilum hirsutissimum plant treated by the fungus elicitor added with the stemona (Cypherophora sp.) PF07 is remarkably higher than that of a control, is improved by 262.9 percent (figure 5) compared with the control, is a better paphiopedilum hirsutissimum seedling culture fungus elicitor, and the concentration is properly controlled at 100-150 mL/L.
3.4.2 orchid mycorrhizal fungi PF07 fungal elicitor has effect on the growth of transplanted cup seedlings
In a cup seedling transplanting test, a Pediobolus sp (Cypherophora sp.) PF07 fungal elicitor has the best growth promoting effect on paphiopedilum hirsutum, the difference of the fresh weight growth rate of a plant is very obvious compared with that of a Control (CK), 377.8 percent (figure 6) is improved, and the effect of the fungal elicitor on the growth of the plant is very obvious. After transplanting, the growth environment of the plant depending on survival is changed, the growth space of the plant is enlarged, the nutrient, moisture and illumination in the environment can be actively received, the plant grows vigorously, the number of new buds and tillers is large, and the biomass is rapidly increased. In contrast, the test-tube plantlet has reduced nutrient consumption of the culture medium along with the prolonged culture time, and the environment in the bottle is not favorable for the growth of the plant, so that the action strength of the fungal elicitor in the growth of the plant is reduced to a certain extent, but the fungal elicitor still generates a significant positive effect in the overall growth stage of the test-tube plantlet of the plant compared with the control.
3.5 Effect of addition of fungal elicitor on physiological indices of paphiopedilum hirsutum
(1) Test-tube plantlet: with the increase of the concentration of the added fungal elicitor, the POD enzyme activity of the plants treated by the P.californica (Cypherophora sp.) PF07 fungal elicitor is enhanced, the total chlorophyll content is remarkably increased, and the 2 enzyme activities of CAT enzyme and SOD enzyme are weakened, particularly the CAT enzyme activity is remarkably weakened (Table 3).
TABLE 3 influence of addition of inducers of orchid mycorrhizal fungi PF07 fungi on physiological indexes of paphiopedilum hirsutissimum test-tube plantlet
(2) Transplanting cup seedlings: after the orchid mycorrhizal fungi PF07 fungal elicitor is applied to the transplanted seedlings, the difference between the total quantity of POD and chlorophyll of the plants treated by the fungal elicitor and a control is not obvious, and the activities of CAT and SOD enzymes are lower than that of the control (table 4); the enzyme activity in the plant is dynamically changed, and the optimal watering time of the fungal elicitor needs to be further discussed.
Table 4 Effect of addition of inducers of orchid mycorrhizal fungi PF07 fungi on physiological indexes of paphiopedilum hirsutissimum transplanted cup seedlings
| Fungal elicitor treatment | POD(U/g) | CAT(nmol/g) | SOD(U/g) | Total amount of chlorophyll (mg/g) |
| PF07 | 329.67 | 65.13 | 47.00 | 0.392 |
| CK | 346.67 | 126.41 | 240.86 | 0.446 |
3.6 conclusion
The strain PF07 which has the best effect on growth promotion of paphiopedilum hirsutissimum at different growth stages of the plant:
(1) the fungal elicitor has an extremely important promoting effect on the growth of paphiopedilum hirsutissimum test-tube plantlets, but the activity of different fungal elicitors is different, and the addition of the fungal elicitor with the same strain and different concentrations can improve the fresh weight growth rate of plants to different degrees. The Pachysolen cupulophora sp PF07 fungal elicitor has the best growth promoting effect on paphiopedilum hirsutissimum test-tube plantlets, the fresh weight growth rate of treated plants is remarkably higher than that of a Control (CK), the Pachysolen cupulophora sp fungal elicitor is a better paphiopedilum hirsutissimum seedling-raising fungal elicitor, and the concentration is properly controlled to be 100-150 mL/L;
(2) the Pediobolus sp (Cypherophora sp.) PF07 fungal elicitor has good growth promoting effect on the paphiopedilum hirsutum transplanted cup seedlings; after transplanting, the action strength of the fungal elicitor in the plant growth is reduced to a certain extent, but still generates a significant positive effect compared with a control;
(3) with the increase of the concentration of the added fungal elicitor, the POD enzyme activity of plants treated by the Penicillium chrysosporium (Cypherophora sp.) PF07 fungal elicitor is enhanced, the total chlorophyll amount is remarkably increased, and the enzyme activities of 2 CAT enzyme and SOD enzyme are weakened.
The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.
SEQUENCE LISTING
<110> Guangxi Zhuang nationality autonomous region forestry science research institute
<120> orchid mycorrhizal fungus PF07 and application thereof
<130> JC
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 590
<212> DNA
<213> Cyphellophora sp.
<400> 1
cctgcggaag gatcattacc gagttagggt gcctcgtcgc gcccgacctc caaccctttg 60
cttacttgac ctattttgtt gcttcggcag gcccgccgcc cggaaacggg tggccgccgg 120
gggcgtttca ccgccccggg cccgcgcctg tcgatggccc tattaaaact cttgtcaaaa 180
cgtgtcgtct gagtttatct aaacaaataa aaaccaaaac tttcaacaac ggatctcttg 240
gttctggcat cgatgaagaa cgcagcgaaa tgcgataagt aatgcgaatt gcagaatccg 300
tgagtcatcg aatctttgaa cgcacattgc gccctctggt attccggagg gcatgcctgt 360
tcgagcgtca ttatcacccc tcaagcccgg cttgttgttg gatgcagcgc ttatcccgct 420
cctcccaaag ataatgacgg cgtctgcgac gactcctgta cactgagctt tcgggcacgt 480
acacggctag aagtccagac ccggtcgccg tcccccccgc ggggacaccc attaccacaa 540
ggttgacctc ggatcaggta ggaatacccg ctgaacttaa gcatatcaaa 590
Claims (6)
1. An orchid mycorrhizal fungus PF07 is characterized in that the orchid mycorrhizal fungus PF07 is a septoria cupulosa (Cypherophora sp.) PF07, is stored in the common microorganism center of China general microbiological culture Collection center on 11-13 months in 2020, has the address of No. 3 Siro-1 Bichen of Beijing Chaoyang district, and has the preservation number of CGMCC NO: 21053.
2. use of the orchid mycorrhizal fungus PF07 according to claim 1 for promoting the growth of orchid.
3. Use according to claim 2, characterized in that: the orchid mycorrhizal fungi PF07 is prepared into liquid microbial inoculum or fungal elicitor to promote the growth of orchid.
4. The use of claim 3, wherein the orchid mycorrhizal fungus PF07 is prepared into a liquid microbial inoculum by the following steps: inoculating orchid mycorrhizal fungi PF07 to a PDA culture medium flat plate, placing the flat plate in an illumination incubator, performing dark culture at a constant temperature of 28 ℃ for 5-7 d to activate strains, then punching holes at the edges of the colonies to prepare fungus cakes, respectively transferring the fungus cakes prepared by the strains into bottles containing 150mL of liquid PDA culture medium, inoculating 2 fungus cakes, placing the bottles in a shaking table at a temperature of 28 ℃, performing shake culture at a speed of 140r/min for 10d, crushing for 5min, diluting with sterile water to a visual field of 40 times of a microscope, observing average 20 fungi, and preparing into a liquid microbial inoculum.
5. Use according to claim 3 or 4, characterized in that: the orchid mycorrhizal fungi PF07 is prepared into a fungal elicitor with the concentration of 100-150mL/L to promote the growth of orchid.
6. The use according to claim 3, wherein the fungal elicitor is formulated: beating the activated orchid mycorrhizal fungi PF07 into fungus cakes, inoculating the fungus cakes into a PDA liquid culture medium, inoculating the fungus cakes into a bottle of 250mL, inoculating 1 fungus cake, shaking and culturing for 7d in a shaking table of 120r/min, harvesting after the mycelia fully grow, smashing the mycelia, mixing the mycelia with a bacterial liquid, and sterilizing at 121 ℃ for 20min to obtain the PF07 fungus elicitor.
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| 陈宝玲 等: "有益菌根真菌及其互作对带叶兜兰试管苗生理生长的影响", 《西南林业大学学报》 * |
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Application publication date: 20210402 Assignee: GUANGXI YUKOU INNOVATION AGRICULTURE CO.,LTD. Assignor: GUANGXI ZHUANG AUTONOMOUS REGION FORESTRY Research Institute Contract record no.: X2023980045104 Denomination of invention: An orchid mycorrhizal fungus PF07 and its application Granted publication date: 20221129 License type: Common License Record date: 20231102 |