CN112646734B - Orchid mycorrhizal fungus PF06 and application thereof - Google Patents
Orchid mycorrhizal fungus PF06 and application thereof Download PDFInfo
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Images
Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
Abstract
The invention discloses an orchid mycorrhizal fungus PF06 which is characterized in that the orchid mycorrhizal fungus PF06 is classified and named as Phialophora sp PF06, is preserved in China general microbiological culture Collection center (CGMCC) in 11 and 13 days of 2020, and has the address of No. 3 Siro No.1 Beijing Kogyang area, and the preservation number is CGMCC NO:21052. the invention separates and screens a strain of Phialophora sp.PF06 from wild orchid for the first time, the strain can promote the growth of orchid, and can promote the rapid breeding of orchid seedlings and shorten the seedling period when being applied to orchid mycorrhizal seedling raising practice, thereby providing a theoretical basis for the conservation of rare or endangered plants and providing technical support for the promotion of 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 PF06 and application thereof.
Background
Almost all orchids have symbiotic relationship with mycorrhizal fungi, and the existing research shows that the mycorrhizal fungi have unique ecological functions on the orchids, such as promoting seed germination and morphogenesis of seedlings, helping ecological invasion of the orchids, influencing the composition of biological communities, preparing biological elicitors with specific effects, and being beneficial to the aspects of protection, recovery or reconstruction of an ecological system and the like. Based on the mechanism, the recent research shows that mycorrhizal fungi provide essential nutrition for embryo cells through digestion 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, application and efficacy in horticulture and plant protection cause high attention of some students, researches on improving the transplanting survival rate of rare tissue culture seedlings, promoting plant growth and conservation and the like by utilizing the orchid mycorrhizal fungi have been widely reported, for example, dendrobium (Dendrobium sp.), anoectochilus (Anoectochilus sp.), paphiopedilum, cymbidium (Cymbium sp.), and Doriti sp) and the like are introduced into artificial cultivation by a mycorrhizal seedling raising technology, so that the seedling transplanting survival rate, the fresh mass growth rate, the dry matter accumulation and the mineral element absorption are remarkably improved, and metabolites of part of the mycorrhizal fungi can secrete gibberellin, IAA and other plant growth regulators.
Disclosure of Invention
The invention aims to provide an orchid mycorrhizal fungus PF06 and application thereof, wherein the orchid mycorrhizal fungus is a strain capable of promoting the growth of orchid plants.
In order to realize the purpose, the technical scheme provided by the invention is as follows:
an orchid mycorrhizal fungus PF06 which is classified and named as Phialophora sp PF06 and is preserved in China general microbiological culture Collection center (CGMCC) at 11 and 13 days 2020, with the address of No. 3 Homex 1 of Beijing Chaoyang district, and the preservation number of CGMCC NO:21052.
the application of the orchid mycorrhizal fungus PF06 in promoting the growth of orchid is described.
Preferably, the orchid mycorrhizal fungi PF06 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 fungi PF06 into the liquid fungicide comprises the following steps: inoculating orchid mycorrhizal fungi PF06 on a PDA culture medium flat plate, placing in an illumination incubator for 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 make fungus cakes, transferring the fungus cakes made by the strains into a bottle containing 150mL of liquid PDA culture medium, inoculating 2 fungus cakes (2 fungus cakes represent the inoculation amount), placing in a shaking table at 28 ℃ and 140r/min for shaking culture for 10d, crushing for 5min, diluting with sterile water to 40 times of the visual field of a microscope, observing average 20 fungi, and preparing into the liquid microbial inoculum.
Preferably, the orchid mycorrhizal fungus PF06 is prepared into a fungal elicitor with the concentration of 50-100 mL/L to promote the growth of orchid.
Preferably, the formulation of the fungal elicitor: beating the activated orchid mycorrhizal fungi PF06 into fungus cakes, inoculating the fungus cakes into a PDA liquid culture medium, inoculating 1 fungus cake per bottle, shaking and culturing for 7d in a shaking table at 120r/min, harvesting after the mycelia fully grow, smashing the mycelia, mixing with a bacterial liquid, and sterilizing at 121 ℃ for 20min to obtain the PF06 fungus elicitor.
Compared with the prior art, the invention has the following beneficial technical effects:
the invention separates and screens a strain of Phialophora sp.PF06 from wild orchid for the first time, the strain can promote the growth of orchid, and can promote the rapid breeding of orchid seedlings and shorten the seedling period when being applied to orchid mycorrhizal seedling raising practice, thereby providing a theoretical basis for the conservation of rare or endangered plants and providing technical support for the promotion of the large-scale development of the paphiopedilum industry.
Description of preservation information
The bottle mold Phialophora sp.PF06 is preserved in China general microbiological culture Collection center (CGMCC for short) in 11 month and 13 days in 2020, and the preservation number is CGMCC NO:21052.
drawings
FIG. 1 is a map of the topography of orchid mycorrhizal fungi PF06, wherein (a) is a PF06 hypha map at 40-fold magnification, and (b) is a PF06 colony map.
FIG. 2 Effect of orchid mycorrhizal fungus PF06 on the growth of biomass of paphiopedilum hirsutissimum test-tube plantlets.
FIG. 3 influence of the mycorrhizal fungi PF06 of orchid on the biomass growth of transplanted cup seedlings of paphiopedilum hirsutissimum.
FIG. 4 shows the effect of different concentrations of the inducer of the orchid mycorrhizal fungus PF06 on the growth of paphiopedilum hirsutissimum test-tube plantlets.
FIG. 5 Effect of orchid mycorrhizal fungus PF06 fungal elicitor on growth of paphiopedilum hirsutissimum test-tube plantlet.
FIG. 6 shows the effect of the mycorrhizal fungi PF06 on the growth of plants transplanted with leaf pockets.
Detailed Description
The following detailed description is to be read with reference to 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 identification of orchid mycorrhizal fungi PF06
1.1 isolation of orchid mycorrhizal fungi PF06
The isolation of the orchid mycorrhizal fungi PF06 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 dead (primary screening) or beneficial strains (secondary screening), and beating a bacterial cake with the diameter of 0.5cm from the edge of a bacterial colony to be used as an inoculation material;
(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 in each bottle, selecting 1 strain cake, inoculating into the center of the culture bottle, and comparing without inoculating strains; repeating the treatment for 3 times, placing the mixture into a culture room for symbiotic culture at the temperature of 23 +/-1 ℃, illuminating for 12-14 h every day at the illumination intensity of 1-2 000Lux, and observing the growth conditions of the bacteria and the seedlings every two days;
(3) Determination of orchid growth indexes: determining 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 (%) = (end weight-initial weight)/initial weight of the seedling multiplied by 100;
(4) Mycorrhization detection: randomly extracting 2-3 seedlings per treatment, shearing 3-4 fresh nutritive roots per seedling, soaking root segments in 75% ethanol in a super clean bench for 30s, washing with sterile water for 2 times, disinfecting with 0.1% mercury bichloride 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 the separation condition 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 carrying out full mycorrhization on 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 PF06.
1.2 identification of orchid mycorrhizal fungi PF06
(1) DNA extraction mycelium acquisition: inoculating the PF06 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 in a PDA liquid culture medium, shake culture is carried out for 3-5 days by a shaking table, mycelium is obtained by filtration and is dried for standby;
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);
(3) and (3) PCR amplification: the amplification primers are general ITS1 and ITS4, and the forward primer ITS1: TCCGTAGGTGAACCTGCGG; reverse primer ITS4: TCCTCCGCTTATTGATATGC; the amplification sequence is internal transcription interval regions 1 and 2, the PCR length is about 600 bp;
(4) and (3) detecting the DNA concentration and purity: 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;
(5) ITS sequencing and alignment: 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; the 16SrDNA sequences were aligned on the ribosomal database http:// rdp.cme.msu.edu/index.jsp; performing molecular level identification on mycorrhizal fungi through homology analysis; according to the similarity, the species of the test strains are identified by combining the morphological observation results;
PCR reaction system
| Reagent | Volume (mu L) |
| Template (genomic DNA 20-50 ng/. Mu.l) | 0.5 |
| 10×Buffer(with Mg 2+ ) | 2.5 |
| dNTPs (2.5 mM each) | 1 |
| Enzyme | 0.2 |
| F(10uM) | 0.5 |
| R(10uM) | 0.5 |
| Double steam adding H 2 O to | 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 PF06 and the Phialophora sp is 100 percent, so that the PF06 is identified as the Phialophora sp, is named as Phialophora sp 06, is preserved in China general microbiological culture Collection center (CGMCC for short), and has the preservation number of CGMCC NO:21052 and the sequence is shown in SEQ ID NO. 1.
Example 2
Growth and physiological action of orchid mycorrhizal fungi PF06 on paphiopedilum hirsutum
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 the strong seedling stage of paphiopedilum hirsutum and have 3-4 roots and 3-4 leaves; the transplanting seedling in the nutrition cup adopts a tissue culture seedling in a rooting stage, and has 5-8 roots and 4-5 leaves;
2.1.2 test strains: selecting orchid mycorrhizal fungi PF06 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 = 4.
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 circular plates with the same size, namely bacterial cakes, transferring the bacterial cakes prepared by the strains into 300mL triangular bottles containing 150mL of liquid PDA culture medium, inoculating 2 bacterial cakes (2 bacterial cakes represent the inoculation amount) to each triangular bottle, placing the triangular bottles in a shaking table at 28 ℃ and 140r/min for culture for 10d, crushing the triangular bottles by using a tissue triturator for 5min, diluting the triangular bottles by using sterile water 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 the strain: activating the screened orchid mycorrhizal fungi PF06 (activating to inoculate the tested strains on a PDA culture medium plate, placing the activated strains in a light incubator at a constant temperature of 28 ℃ for dark culture for 5-7 days), selecting strains with good growth, punching holes on the edges of bacterial colonies by using a puncher with the diameter of 5mm to make bacterial cakes, inoculating the bacterial cakes into 2.1.1 sterile tissue culture seedlings under the sterile condition, inoculating 2 seedlings on the periphery of a bottle, and repeating for 10 times; after inoculation, the mixture is placed in a culture room at 25 +/-2 ℃ for symbiotic culture, the illumination is 12-14 h every day, and the illumination intensity is 2000Lux; counting growth indexes such as fresh weight, leaf length, leaf width, root length, growth potential and the like by taking a bottle as a unit after symbiotic culture for 120 days;
(2) Transplanting seedling inoculation strain: pouring 10ml of 2.2.1 obtained liquid microbial inoculum for each cluster of seedlings on the same transplanting day, taking a poured equivalent liquid PDA culture medium as a reference (CK), pouring 1 time of microbial inoculum every 20 days, continuously pouring for 3-4 times, pouring 1 time of sterile water every 5-7 days after transplanting, properly spraying leaf surfaces according to the air humidity condition in the period, keeping the temperature (26 +/-2) DEG C, the humidity of 70-85 percent and the shading rate of 70-80 percent, and not applying or using the pesticide in the period; and after 120 days of symbiotic culture, 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 120 days of transplantation culture in 2.3 were measured according to the method given in the kit description of Peroxidase (POD) of Suzhou Ke Ming Biotechnology Co., ltd, the method given in the kit description of Catalase (CAT), and the method given in the kit description of Superoxide Dismutase (SOD).
2.5 statistical analysis of data
Experimental data statistics were performed using the EXCEL table and Duncan's new range test using DPS7.05 software. Fresh weight growth rate (%) = (final weight-initial weight)/initial weight × 100; leaf area = (leaf length x leaf width) × leaf shape index (0.8317).
Results and analysis
1. Inoculating orchid mycorrhizal fungi PF06 on test-tube plantlet
Symbiotic culture is carried out on the orchid mycorrhizal fungi PF06 and the paphiopedilum hirsutissimum tissue culture seedlings, the fact that the average fresh weight of biomass of mycorrhizal seedlings is obviously higher than that of a Control (CK) is confirmed, and the result shows that the inoculated orchid mycorrhizal fungi PF06 has a relatively obvious growth promoting effect. The influence of the orchid mycorrhizal fungi PF06 on the fresh weight growth and leaf area index of the tissue culture seedling is not obvious, but the fresh weight growth amount is obviously improved compared with the contrast, and the addition of the orchid mycorrhizal fungi PF06 enables the treatment to be 398.9% higher than the contrast, which shows that the inoculation treatment has a very obvious effect of promoting the growth of the orchid biomass. The leaf area and new root length of the tissue culture seedling treated by the orchid mycorrhizal fungi PF06 strain are not obviously different from those of the control, and are 1.2% and 33.3% higher than those of the control (figure 2).
2. Inoculating mycorrhizal fungi for transplanting cup seedlings
In a matrix (pine bark: volcanic rock: charcoal =4: 1 volume ratio), the biomass change of plants was significantly different after 3 inoculations of paphiopedilum folicum with the liquid inoculum obtained in 2.2.1 (10 ml per inoculation). The leaf width of PF06 is the largest, and compared with the control, the difference is obvious, and is improved by 44.9%. (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, after live bacteria are inoculated, the moisture content and the chlorophyll content of a plant are obviously improved, the activity of 3 kinds of enzymes of the plant treated by the Phialophora sp.PF06 is stronger, especially the activity of POD and CAT enzymes is obviously or extremely obvious compared with that of a control, and the activity of SOD enzyme is not obviously different from that of the Control (CK) plant (Table 1);
TABLE 1 influence of the inoculated orchid mycorrhizal fungi PF06 on the physiological indexes of paphiopedilum hirsutissimum test-tube plantlet
| Strain numbering | POD(U/g) | CAT(nmol/g) | SOD(U/g) | Chlorophyll Total amount (mg/g) |
| PF06 | 117.76 | 267.94 | 1434.67 | 0.505 |
| CK | 36.72 | 120.65 | 1666.67 | 0.435 |
(2) Transplanting cup seedlings physiological indexes: after transplanting, POD enzyme activity of the plants inoculated with the Phialophora sp.PF06 is enhanced, and SOD and CAT enzyme activities are weakened, but 3 enzyme activities are strongest in Control (CK) plants, and the difference of the total chlorophyll amount of each treatment is not obvious (Table 2).
TABLE 2 influence of the inoculated orchid mycorrhizal fungi PF06 on the physiological indexes of paphiopedilum hirsutissimum test-tube plantlet
| Strain numbering | POD(U/g) | CAT(nmol/g) | SOD(U/g) | Chlorophyll Total amount (mg/g) |
| PF06 | 288.33 | 65.32 | 91.30 | 0.376 |
| 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 PF06 obtained after screening in the example 1, wherein the PF06 is separated from fresh roots of wild paphiopedilum hirsutissimum in Tian-Anthri county of Guangxi and is an immortal strain of paphiopedilum hirsutissimum seedlings;
3.1.3 culture substrate: the symbiotic culture medium of the test-tube plantlets is a DE culture medium; the symbiotic culture medium of the nutrition cup seedlings is pine bark: volcanic rock: charcoal = 4.
3.2 test methods
3.2.1 Strain activation: inoculating the test strains on a PDA culture medium flat plate, and placing the flat plate into an illumination incubator to perform dark culture at the constant temperature of 28 ℃ for 5-7 days to activate the strains;
3.2.2 preparation of fungal elicitor: perforating the strains obtained by 3.2.1 culture and 7d activation by using a perforator with the diameter of 0.5cm, perforating a small disc with the same size as the discs to obtain a fungus cake, inoculating the fungus cake into a PDA liquid culture medium which is subpackaged in advance and sterilized, inoculating 1 fungus cake per bottle, performing shaking culture on a 120r/min shaking table for 7d, harvesting after the mycelia fully grow, smashing the mycelia by using a tissue triturator, mixing the smashed mycelia with a bacterial liquid, and sterilizing at 121 ℃ for 20min to obtain a PF06 fungus elicitor for later use;
3.2.3 tissue culture seedling treatment: placing the test-tube plantlet in the rooting stage of 3.1.1 in a greenhouse for hardening 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 water, placing in a shade place, spreading and airing the water on the surface of the plant, and moving into a transparent nutrition bowl with the caliber of 5cm for 2 plants/bundle, wherein the treatment is repeated for 3 times every 30 bundles;
3.2.4 preparation and inoculation of test-tube plantlet fungal elicitor culture Medium
Taking DE as a basal culture medium, adding the PF06 fungal elicitor for standby into the basal culture medium according to 3 gradients of 50, 100 and 150mL/L, and taking the DE culture medium without the fungal elicitor as a Control (CK); before inoculation, weighing the fresh weight (accurate to 0.000 1g) of a 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 fungal elicitors on 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 with leaves of about 5-6 leaves and 3-4 roots in a culture medium containing a fungal elicitor for a period of time, placing the paphiopedilum with leaves under a 85% shading net for hardening seedlings for 1 week, taking out, cleaning, sterilizing and airing the water on the surface of the plants, transplanting 2 plants/cluster into a seedling cup with the diameter of 4.5cm, wherein the matrix adopts 3.1.3 nutrient cup seedling symbiotic culture matrix, and the roots are required to be stretched and not to be 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 to 14 hours every day, the illumination intensity is 2500 to 3000Lux, the temperature is 28 +/-1 ℃, and the humidity is 70 to 75 percent; 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 the test-tube plantlet after 120d of culture, washing the culture medium at the root with clear water, naturally drying, and then weighing the fresh weight (accurate to 0.000 1g) of the tissue culture plantlet by using an electronic balance, wherein the method for measuring the physiological index is the same as the method in 2.4;
(2) Transplanting cup seedlings: counting the growth index of the 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 (50 mL/L, 100mL/L and 150 mL/L) of orchid mycorrhizal fungi PF06 in a test-tube plantlet 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 strains have different degrees of improvement on the fresh weight growth rate of plants under the influence of the concentrations; especially at low concentration (50 mL/L), the growth rate of the fresh weight of the cultivated plants by the Phialophora sp.pf06 fungal elicitor is significantly higher than that of the control (fig. 4);
(2) Fungal elicitor effects: based on the strain of the fungal elicitor with the optimal concentration, the growth promoting effect of the fungal elicitor added is better than that of a Control (CK); wherein, the fresh weight growth rate of the paphiopedilum hirsutissimum plants treated by adding the Phialophora sp.PF06 fungal elicitor is remarkably higher than that of a control (figure 5), and the paphiopedilum hirsutissimum fungal elicitor is a better paphiopedilum hirsutum seedling-raising fungal elicitor, and the concentration is properly controlled at 50-100 mL/L.
3.4.2 Effect of orchid mycorrhizal fungi PF06 fungal elicitor on the growth of transplanted cup seedlings
In a transplanting cup seedling test, the growth promoting effect of the Phialophora sp.PF06 fungal elicitor on the paphiopedilum hirsutum is optimal, the growth rate of the fresh weight of the plant is very obvious compared with that of a Control (CK) (figure 6), 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, in the test-tube plantlet, the nutrient consumption of the culture medium is reduced along with the prolonging of the 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 remarkable positive effect in the overall growth stage of the test-tube plantlet of the plant compared with a control group.
(1) 3.5 adding a fungal elicitor to affect physiological indexes of paphiopedilum hirsutissimum to transplant cup seedlings: after the orchid mycorrhizal fungi Phialophora sp.PF06 fungal elicitor is applied to the transplanted seedling, the enzyme activities of 3 kinds of Phialophora sp.PF06 fungal elicitors with the best plant growth are obviously lower than those of a contrast (table 3), and further the influence of the elicitor on the enzyme activity in the plant body is dynamically changed, so that the optimal application time of the fungal elicitor needs to be further discussed.
Table 3 Effect of addition of cymbidium mycorrhizal fungi PF06 fungal elicitor on physiological index of paphiopedilum hirsutissimum transplanted cup seedling
| Fungal elicitor treatment | POD(U/g) | CAT(nmol/g) | SOD(U/g) | Chlorophyll Total amount (mg/g) |
| PF06 | 293.33 | 54.01 | 59.29 | 0.358 |
| CK | 346.67 | 126.41 | 240.86 | 0.446 |
3.6 conclusion
The strain PF06 with the best growth promoting effect on paphiopedilum hirsutissimum at different growth stages of the plant:
the fungal elicitor has an extremely important promoting effect on the growth of paphiopedilum hirsutissimum test-tube plantlets, 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 added fungal elicitor of the genus Calycota is a better fungal elicitor for the seedling culture of paphiopedilum hirsutissimum, and the concentration is properly controlled to be 50-100 mL/L.
The foregoing description of specific exemplary embodiments of the invention has been presented for the 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 PF06 and application thereof
<130> JC
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 587
<212> DNA
<213> Phialophora sp.
<400> 1
ctgcggaagg atcattaccg agttagggtg cctcgtcgcg cccgacctcc aaccctttgc 60
ttacttgacc tattttgttg cttcggcagg cccgccgccc ggaaacgggt ggccgccggg 120
ggcgtttcac cgccccgggc ccgcgcctgt cgatggccct attaaaactc ttgtcaaaac 180
gtgtcgtctg agtttatcta aacaaataaa aaccaaaact ttcaacaacg gatctcttgg 240
ttctggcatc gatgaagaac gcagcgaaat gcgataagta atgcgaattg cagaatccgt 300
gagtcatcga atctttgaac gcacattgcg ccctctggta ttccggaggg catgcctgtt 360
cgagcgtcat tatcacccct caagcccggc ttgttgttgg atgcagcgct tatcccgctc 420
ctcccaaaga taatgacggc gtctgcgacg actcctgtac actgagcttt cgggcacgta 480
cacggctaga agtccagacc cggtcgccgt cccccccgcg gggacaccca ttaccacaag 540
gttgacctcg gatcaggtag gaatacccgc tgaacttaag catatca 587
Claims (6)
1. An orchid mycorrhizal fungus PF06 which is classified and named as Phialophora sp PF06 and is preserved in China general microbiological culture Collection center (CGMCC) at 11-13.2020, with the address of Beijing Chaoyang district Beichen Xilu No.1 Hospital No. 3 and the preservation number of CGMCC NO:21052.
2. use of the orchid mycorrhizal fungus PF06 according to claim 1 for promoting the growth of paphiopedilum hirsutissimum.
3. Use according to claim 2, characterized in that: the orchid mycorrhizal fungi PF06 is prepared into a liquid microbial inoculum or a fungal elicitor to promote the growth of paphiopedilum hirsutum.
4. The use according to claim 2, characterized in that the orchid mycorrhizal fungus PF06 is prepared into a liquid microbial inoculum by the following steps: inoculating orchid mycorrhizal fungi PF06 on a PDA culture medium flat plate, placing the flat plate in an illumination incubator for dark culture of an activated strain at a constant temperature of 28 ℃ for 5-7 d, then perforating at the edge of a bacterial colony to prepare a bacterial cake, transferring the bacterial cake prepared by the strain into a bottle containing 150mL of liquid PDA culture medium, inoculating 2 bacterial cakes, placing the bottle in a shaking table at a temperature of 28 ℃ and 140r/min for shake culture for 10d, crushing for 5min, diluting with sterile water to 40 times of a microscope visual field, observing average 20 bacteria, and preparing into a liquid microbial inoculum.
5. Use according to claim 2, characterized in that: the orchid mycorrhizal fungus PF06 is prepared into a fungal elicitor with the concentration of 50-100 mL/L to promote the growth of paphiopedilum hirsutissimum.
6. Use according to claim 3, characterized in that: preparing the fungal elicitor: beating the activated orchid mycorrhizal fungi PF06 into fungus cakes, inoculating the fungus cakes into a PDA liquid culture medium, inoculating 1 fungus cake per bottle, shaking and culturing for 7d in a shaking table at 120r/min, harvesting after the mycelia fully grow, smashing the mycelia, mixing with a bacterial liquid, and sterilizing at 121 ℃ for 20min to obtain the PF06 fungus elicitor.
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