CN112592838B - 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
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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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Abstract
The invention discloses an orchid mycorrhizal fungus PF07, wherein the orchid mycorrhizal fungus PF07 is a septoria cupulosa (Cypherophora sp.) PF07, is preserved 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 Hospital No.1 North Chen of Beijing Chaoyang district and the preservation number of CGMCC NO:21053. the cypherophora sp.PF07 can promote the growth of orchid, can promote the rapid propagation of orchid seedlings and shorten the seedling period when being applied to the mycorrhizal seedling cultivation practice of orchid, provides a theoretical basis for the conservation of rare or endangered plants, and provides 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 PF07 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. 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, application and efficacy in horticulture and plant protection are highly regarded by some students, researches on improving the transplanting survival rate of certain rare species of tissue culture seedlings, promoting plant growth and conservation and the like by utilizing the orchid mycorrhizal fungi have been widely reported, such as dendrobe (Dendrobium sp.), anoectochilus (Anoectochilus sp.), paphiopedia, cymbidium (Cymbium sp.), dolichia (Doriti sp.) and the like, after the mycorrhizal seedling raising technology is introduced into artificial cultivation, the seedling transplanting survival rate, the fresh mass growth rate, dry matter accumulation and mineral element absorption are remarkably 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 Cypherophora sp PF07, is preserved in China general microbiological culture Collection center in 11 months and 13 days in 2020, is No. 3 of Beijing Chaoyang district Beichen Xilu No.1, and has a 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 fungi PF07 into the liquid microbial inoculum comprises the following steps: inoculating orchid mycorrhizal fungi PF07 on a PDA culture medium plate, placing the plate in an illumination incubator for dark culture of activated strains at a constant temperature of 28 ℃ for 5-7 d, then perforating at the edges of bacterial colonies to prepare bacterial cakes, respectively transferring the bacterial cakes prepared by the strains into bottles containing 150mL of liquid PDA culture medium, inoculating 2 bacterial cakes (the inoculum size is shown by the 2 bacterial cakes), placing the bottles 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 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 1 fungus cake per bottle, shaking and culturing for 7d in a shaking table at the speed of 120r/min, harvesting after mycelia fully grow, smashing the mycelia, mixing with a bacterial liquid, and sterilizing at the temperature of 121 ℃ for 20min to obtain the PF07 fungal 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 map of the topography of orchid mycorrhizal fungus PF07, wherein (a) is PF07 hypha map at 40 magnification, and (b) is PF07 colony map.
FIG. 2 Effect of orchid mycorrhizal fungus PF07 on the growth of biomass 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 PF07 fungal elicitor of the orchid mycorrhizal fungus on the growth of paphiopedilum hirsutissimum.
FIG. 5 Effect of orchid mycorrhizal fungus PF07 fungal elicitor on growth of paphiopedilum hirsutissimum test-tube plantlet.
FIG. 6 shows the effect of the mycorrhizal fungi PF07 on the growth of plants with leaf-pocket transplanted cup seedlings.
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 characterization of orchid mycorrhizal fungus PF07
1.1 isolation of orchid mycorrhizal fungus PF07
The separation 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 combination;
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 respectively beating fungus cakes with the diameter of 0.5cm from the edges of the 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 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) And (3) measuring the orchid growth index: respectively measuring the fresh weight of the tissue culture seedlings on the day of inoculation and at 90d 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 PF07.
1.2 identification of orchid mycorrhizal fungi PF07
(1) DNA extraction mycelium acquisition: 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; inoculating some strains which grow slowly and have no hypha on a solid culture medium into a PDA liquid culture medium, performing shake culture on a table concentrator for 3-5 days, filtering to obtain mycelium, and drying 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) kits;
(3) and (3) PCR amplification: the amplification primers are universal 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; 16SrDNA sequences are aligned on a ribosome database https:// 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 | 25 |
PCR cycling conditions:
16SrDNA sequences are aligned on a ribosome database https:// 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 Cyphellophora sp is 100 percent, so the orchid mycorrhizal fungi PF07 is identified as Cyphellophora sp, named as goby-stemona (Cyphellophora sp) PF07, and is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, (CGMCC for short) with the preservation number of CGMCC NO:21053, the sequence of which is shown in SEQ ID NO. 1.
Example 2
Orchid mycorrhizal fungus PF07 on growth and physiology of 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 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 the orchid mycorrhizal fungi PF07 obtained by screening in the example 1;
2.1.3 culture Medium: the symbiotic culture medium in the bottle is a DE culture medium; the culture medium of the transplanted seedlings in the nutrition cups 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, 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 temperature of 28 ℃ and the speed of 140r/min for shake culture for 10d, crushing the bacterial cakes by using a tissue triturator for 5min, diluting the bacterial cakes to the visual field of a microscope by 40 times, 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 strain on a PDA culture medium plate, placing the activated strain in an illumination incubator at the constant temperature of 28 ℃ for dark culture for 5-7 d), selecting the well-grown strain, punching the edge of a bacterial colony by using a puncher with the diameter of 5mm to prepare a bacterial cake, inoculating the bacterial cake into 2.1.1 sterile tissue culture seedlings under the sterile condition, inoculating 2 seedlings at the periphery of a bottle, and repeating for 10 times; after inoculation, placing the mixture in a culture room at 25 +/-2 ℃ for symbiotic culture, and illuminating for 12-14 h every day at the illumination intensity of 2000Lux; 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 times of bacterial liquid 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 bacterial liquid every 20 days later, 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 condition during the period, keeping the temperature (26 +/-2) DEG C, the humidity at 70-85 percent and the shading rate at 70-80 percent, and not applying or using the pesticide during 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 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 growth rate (%) = (end 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 PF07 to test-tube plantlet
Symbiotic culture is carried out on the orchid mycorrhizal fungi PF07 and the paphiopedilum hirsutissimum tissue culture seedlings, 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 orchid mycorrhizal fungi PF07 inoculated treatment 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 the control, the addition of the orchid mycorrhizal fungus PF07 strain treatment is 395% higher than the control, and the inoculation treatment has a very obvious effect of promoting the orchid biomass growth. The leaf area and the new root length of the tissue culture seedling treated by the orchid mycorrhizal fungus PF07 strain are not obviously different from those of a control, and are respectively 22.2% and 25% 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), after paphiopedilum hirsutissimum is inoculated for 3 times with the liquid fungicide obtained in 2.2.1 (10 ml for each inoculation), the plant biomass change is remarkably different, and the overall growth promotion effect of orchid mycorrhizal fungus PF07 is better. The differences between the orchid mycorrhizal fungi PF07 and the control are not obvious in the aspects of leaf length, leaf width and root length (figure 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 water content and the chlorophyll content of the plant are obviously improved after the inoculation of live bacteria, 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 inoculation are weaker after the plant is cultured for 120 days (Table 1);
TABLE 1 Effect of orchid mycorrhizal fungi PF07 inoculated 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 | 42.84 | 46.15 | 490.33 | 0.717 |
| CK | 36.72 | 120.65 | 1666.67 | 0.435 |
(2) Transplanting cup seedlings according to physiological indexes: after transplanting, the biomass of a plant inoculated with the Calycophora (Cypherophora sp.) PF07 is increased well, but after culturing for 120d, POD enzyme activity of the inoculated plant is weakened, and SOD enzyme activity of the inoculated plant is enhanced; the CAT enzyme activity of the plant of the inoculated cup-shaped spore (Cypherophora sp.) PF07 is not obviously different, but 3 enzyme activities are the strongest in the Control (CK) plant, and the difference of the total chlorophyll amount of each treatment is not obvious (Table 2).
TABLE 2 influence of orchid mycorrhizal fungi PF07 on physiological indexes of paphiopedilum hirsutissimum test-tube plantlet
| 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 the PF07 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 plate, and placing the PDA culture medium 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 a strain 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 to obtain a fungus cake, inoculating the fungus cake into a PDA liquid culture medium which is subpackaged and sterilized in advance, inoculating 1 block/bottle of the fungus cake, shaking and culturing for 7d by using a 120r/min shaking table, 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 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 elicitor into the basic 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 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 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 moist, 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 roots with clear water, naturally drying, and weighing fresh weight (accurate to 0.000 1g) of the tissue culture plantlets by using an electronic balance, wherein the method for measuring 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 (50 mL/L, 100mL/L and 150 mL/L) of orchid mycorrhizal fungi PF07 into 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 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 plants cultured by the calix brella (Cypherphora sp.) PF07 in the fungal elicitors with 3 concentrations is obviously or extremely obviously higher than that of a Control (CK), particularly 150mL/L of the calix brella (Cypherphora sp.) PF07, and the fresh weight growth rate is improved by 262.9% 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 fresh weight growth rate of the paphiopedilum hirsutissimum plants 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 hirsutum seedling culture fungus elicitor, and the concentration is properly controlled at 100-150mL/L.
3.4.2 Effect of orchid mycorrhizal fungi PF07 fungal elicitor on the growth of transplanted cup seedlings
In a cup seedling transplanting test, a Pediobolus cupulophora sp PF07 fungal elicitor has the best growth promotion 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 is improved (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 changes, the growth space of the plant is enlarged, the plant can actively accept nutrients, moisture and illumination in the environment, the plant grows vigorously, more sprouts are produced, 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 activity of POD (POD enzyme) of plants treated by the P.californica (Cypherophora sp.) PF07 fungal elicitor is enhanced, the total amount of chlorophyll is remarkably increased, and the activities of 2 enzyme activities of CAT enzyme and SOD enzyme are weakened, particularly CAT enzyme, and the activity is remarkably weakened (Table 3).
TABLE 3 influence of addition of cymbidium mycorrhizal fungus PF07 fungal elicitor on physiological indices 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 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 timing of applying the fungal elicitor needs to be further discussed.
Table 4 influence of addition of orchid mycorrhizal fungus PF07 fungal elicitor 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 with the best growth promoting effect on 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 Pediobolus cupulophora sp PF07 fungal elicitor has the best growth promotion 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 treated plants are better paphiopedilum hirsutum seedling fungal elicitors, and the concentration is properly controlled to be 100-150 mL/L;
(2) PF07 fungal elicitor (Cypherophora sp.) has good growth promoting effect on transplanted paphiopedilum hirsutissimum 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 calix (Cypheromona sp.) PF07 fungal elicitor is enhanced, the total chlorophyll amount is remarkably increased, and the 2 enzyme activities of 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. 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 scientific 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 a septoria cupulosa (Cypherophora sp.) PF07 which is preserved in China general microbiological culture Collection center on 11-13 months in 2020, and the address is No. 3 Homex 1 of Beijing Korean district Beichen Xilu, the preservation number is CGMCC NO:21053.
2. use of the orchid mycorrhizal fungus PF07 of claim 1 for promoting the growth of paphiopedilum hirsutissimum.
3. Use according to claim 2, characterized in that: the orchid mycorrhizal fungi PF07 is prepared into a liquid microbial inoculum or a fungal elicitor to promote the growth of paphiopedilum hirsutum.
4. The use according to claim 3, characterized in that the orchid mycorrhizal fungus PF07 is prepared into a liquid microbial inoculum by the following steps: inoculating orchid mycorrhizal fungi PF07 on a PDA culture medium flat plate, placing the flat plate in a light incubator for dark culture at a constant temperature of 28 ℃ for 5-7 d to activate strains, then perforating at the edges of 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 fungus cakes in a shaking table at a temperature of 28 ℃ and a speed of 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 fungi, and preparing into a liquid microbial inoculum.
5. Use according to claim 3 or 4, characterized in that: the orchid mycorrhizal fungus PF07 is prepared into a fungal elicitor with the concentration of 100-150mL/L to promote the growth of paphiopedilum hirsutissimum.
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 1 fungus cake per bottle, shaking and culturing for 7d in a shaking table at the speed of 120r/min, harvesting after mycelia fully grow, smashing the mycelia, mixing with a bacterial liquid, and sterilizing at the temperature of 121 ℃ for 20min to obtain the PF07 fungal elicitor.
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