CN111903412B - Method for producing citrus stock bitter orange mycorrhiza seedlings - Google Patents
Method for producing citrus stock bitter orange mycorrhiza seedlings 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
- A01G17/00—Cultivation of hops, vines, fruit trees, or like trees
- A01G17/005—Cultivation methods
-
- 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
- A01G18/00—Cultivation of mushrooms
- A01G18/10—Mycorrhiza; Mycorrhizal associations
-
- 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
- A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
- A01G24/10—Growth substrates; Culture media; Apparatus or methods therefor based on or containing inorganic material
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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
- A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
- A01G24/10—Growth substrates; Culture media; Apparatus or methods therefor based on or containing inorganic material
- A01G24/12—Growth substrates; Culture media; Apparatus or methods therefor based on or containing inorganic material containing soil minerals
- A01G24/13—Zeolites
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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
- A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
- A01G24/20—Growth substrates; Culture media; Apparatus or methods therefor based on or containing natural organic material
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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
- A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
- A01G24/20—Growth substrates; Culture media; Apparatus or methods therefor based on or containing natural organic material
- A01G24/22—Growth substrates; Culture media; Apparatus or methods therefor based on or containing natural organic material containing plant material
- A01G24/23—Wood, e.g. wood chips or sawdust
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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
- A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
- A01G24/20—Growth substrates; Culture media; Apparatus or methods therefor based on or containing natural organic material
- A01G24/22—Growth substrates; Culture media; Apparatus or methods therefor based on or containing natural organic material containing plant material
- A01G24/25—Dry fruit hulls or husks, e.g. chaff or coir
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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
- A01G31/00—Soilless cultivation, e.g. hydroponics
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Abstract
The invention discloses a method for producing mycorrhiza seedlings of citrus rootstock bitter orange, which comprises the following operation steps of (1) detoxification treatment of bitter orange seeds; (2) sowing seeds of fructus aurantii: according to the using amount of inoculating 100-300 spores to 10 bitter orange seeds, firstly spreading a burkesler flavedo FS-1-106 microbial inoculum on a sowing substrate, then covering the substrate, sowing the bitter orange seeds obtained after detoxification treatment in the step (1) in the substrate, then covering river sand, sowing and germinating, wherein the bitter orange with 8-10 leaves is obtained after sowing and germinating, and the bitter orange with the plant height of 10-15 cm is the mycorrhiza seedling. According to the method, the germination, mycorrhization seedling and transplanting technologies of citrus stock bitter orange seeds are combined, a set of citrus stock bitter orange mycorrhizal seedling breeding technical system is established, technical support is provided for large-scale production of citrus mycorrhizal seedlings, and related technical bases are provided for mycorrhization seedling and rapid propagation of other plants.
Description
Technical Field
The invention belongs to the technical field of plant mycorrhiza, and particularly relates to a production technology of citrus stock bitter orange mycorrhiza seedlings.
Background
Citrus is the fruit tree with the largest cultivation area in south China and an important agricultural supporting industry, the citrus yellow shoot is a destructive disease of citrus production in the world, and pathogenic bacteria of the citrus yellow shoot are gram-negative bacteria parasitized in phloem and are spread by psyllids. The plants infected with the yellow shoot have the symptoms of yellow leaves, withering, root rot and the like, and the fruits are malformed, so that the citrus yellow shoot is a worldwide problem and the development of the citrus industry is seriously restricted.
Arbuscular Mycorrhizal (AM) fungi are widely distributed in soil, and more than 90% of terrestrial plant root systems can establish a symbiotic relationship with the Arbuscular Mycorrhizal (AM) fungi. The AM fungi can promote the absorption and utilization of water and soil mineral elements, particularly phosphorus, by host plants, improve the soil ecology, increase the pH value of the soil and regulate the synthesis of plant secondary metabolites; improving the microenvironment of plant rhizosphere, promoting the growth of plant root systems, enhancing the resistance of host plants to adverse environments such as barren, drought, plant diseases and insect pests and the like, and improving the quality and the yield of crops.
Citrus is a mycorrhizal plant, and root hairs of citrus root systems are short and few, and the absorption function of citrus is enhanced mainly by AM fungi. The mycorrhiza technology is utilized to produce citrus rootstock and bitter orange mycorrhiza seedlings in a large scale, so that the growth of the bitter orange seedlings can be promoted, and the quality of the citrus seedlings taking bitter orange as rootstock can be improved. Meanwhile, the citrus mycorrhizal seedlings are cultured by means of mycorrhiza to promote the roots to absorb soil nutrients and water, stress resistance is enhanced, the important effect on improving the transplanting survival rate of the citrus seedlings is achieved, and the disease resistance of plants to soil-borne pathogenic bacteria is improved by the citrus mycorrhizal seedlings. Therefore, the cultivation of the detoxified citrus mycorrhizal seedlings is a development trend of citrus seedling cultivation.
The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.
Disclosure of Invention
The invention separates and obtains a dominant strain of citrus soil Arbuscular Mycorrhizal (AM) fungi-Clarkey yellow bursa fungus FS-1-106, the strain is used for preparing AM microbial inoculum, and the AM microbial inoculum has obvious growth promoting function on citrus stocks and bitter orange under the condition of a proper symbiotic culture system.
In order to achieve the purpose, the technical scheme provided by the invention is as follows:
a method for producing citrus rootstock bitter orange mycorrhiza seedlings comprises the following operation steps:
(1) detoxification treatment of the seeds of the bitter orange;
(2) sowing seeds of fructus aurantii: according to the using amount of inoculating 100-300 spores to 10 bitter orange seeds, firstly spreading a burkesler flavedo FS-1-106 microbial inoculum on a sowing substrate, then covering the substrate, sowing the bitter orange seeds obtained after detoxification treatment in the step (1) in the substrate, then covering river sand, sowing and germinating, wherein the bitter orange with 8-10 leaves is obtained after sowing and germinating, and the bitter orange with the plant height of 10-15 cm is the mycorrhiza seedling.
Preferably, the detoxification treatment of the seeds of fructus aurantii in the step (1) is to pretreat the seeds of fructus aurantii in a water bath at 40 ℃ for 10 minutes, and then soak the seeds of fructus aurantii in warm water at 53 ℃ for 50 minutes, while stirring continuously, so that the temperature is uniform.
Preferably, the substrate in the step (2) is obtained by sieving fresh soil through a 20mm × 20mm sieve, mixing the fresh soil with river sand according to the volume ratio of 1:1, scattering a burkesleya flaviviridis FS-1-106 microbial inoculum on a seeding substrate, covering the substrate with the thickness of 2-3 cm, seeding the fructus aurantii seeds obtained after the detoxification treatment in the step (1) in the substrate, and covering the river sand with the thickness of 1-2 cm.
Preferably, the preparation method of the agent of the Clarkey yellow fungus FS-1-106 in the step (2) comprises the following steps:
(a) taking clover as a host, inoculating spores of the Sorangium flavum FS-1-106 into root systems of the seedlings of the clover, wherein the inoculation amount is 1000-1500 spores per pot of the clover; planting the seeds in a culture medium, and performing illumination culture for 12-16 weeks to obtain the fulvia fulvidraco filaments; a potted plant (780mm multiplied by 350mm multiplied by 250mm) production mode is adopted, and the planting density of the clovers is 50-80 plants/pot;
(b) stopping watering the clovers cultured for 12-16 weeks in the step (a), removing stem and leaf parts after the plants are dried, reserving root systems, obtaining hypha in a culture medium, the culture medium and the reserved root systems of the clovers, namely the burkesleya flavedo FS-1-106 microbial inoculum, wherein the spore amount in the microbial inoculum is 80-120 spores/g, and the culture medium contains spores of the burkeslea flavedo besides the hypha.
Preferably, the Clarkey yellow fungus FS-1-106 in the step (a) is classified and named as Clarkey yellow fungus (Redeckara fulvum) FS-1-106, the nucleotide sequence table is shown as SEQ ID NO:1, the preservation unit is: china general microbiological culture Collection center, preservation Address: the microbiological research institute of western road No.1, 3, national academy of sciences, north-kyo, the rising area, the preservation date: year 2020, month 05, day 25, accession number: CGMCC No. 19902.
Preferably, the culture substrate in step (a) is prepared by mixing river sand and zeolite at a volume ratio of 3:1, and sterilizing the mixed culture substrate at 121 deg.C under high pressure steam for 1 h.
Preferably, 300-500 mL of Hoagland nutrient solution with a concentration of 50% is poured every 2 weeks during the culture in step (a).
The application of the obtained fructus aurantii mycorrhiza seedlings comprises the following steps: transplanting the trifoliate orange mycorrhiza seedlings obtained in the step (2) to a special citrus seedling cup containing a matrix, and carrying out cultivation management for 8-9 months, wherein the seedlings are used for scion grafting of citrus varieties taking trifoliate orange as stocks.
Preferably, the cultivation management is to shade the seedlings of the mycorrhiza of the bitter orange for 15 to 20 days after the transplantation, and manage the seedlings of the bitter orange according to normal seedlings after 30 days; then, spraying 50% Hoagland nutrient solution every 2 weeks; after 90 days, 1/1000 high-nitrogen low-phosphorus compound fertilizer (N: P: K: 21: 7: 12) is sprayed every 2-3 weeks.
Preferably, the matrix in the special citrus seedling raising cup containing the matrix is obtained by mixing 2 parts of fresh soil, 1 part of chaff, 1 part of sawdust and 0.5 part of cow dung according to the volume part ratio.
Compared with the prior art, the invention has the following beneficial effects:
the microbial inoculum obtained by the invention has good promotion effect on the growth of citrus taking fructus aurantii as a stock, has strong adaptability and colonization ability, can promote the absorption of plant root systems to nutrients in soil, promotes the growth of plants and improves the stress resistance; according to the method, the germination, mycorrhization seedling and transplanting technologies of citrus stock bitter orange seeds are combined, a set of citrus stock bitter orange mycorrhizal seedling breeding technical system is established, technical support is provided for large-scale production of citrus mycorrhizal seedlings, and related technical bases are provided for mycorrhization seedling and rapid propagation of other plants.
Drawings
FIG. 1 is a spore morphology characteristic of the strain of Clarkey yellow Chaetomium FS-1-106 of the present invention, wherein a is a dark field hypha and spore morphology graph (vehicle: sterile water), b is a plot of continuous points and spore wall structure morphology (vehicle: Melzer's reagent) in a bright field, c is a graph of hypha, continuous spore hypha and spore morphology (vehicle: sterile water) with legend of 100 μm in a bright field, and d is a graph of spore color reaction (gossypol blue lactate) with legend of 50 μm in a bright field.
FIG. 2 shows specific data of growth indexes of mycorrhiza seedlings of fructus Aurantii of the present invention, where AM is a seedling of mycorrhiza of Clarkey yellow fungus FS-1-106, and CK is a control group.
FIG. 3 shows the mycorrhiza seedlings of fructus Aurantii of the present invention, AM is the mycorrhiza seedlings of Clarkey yellow fungus FS-1-106, CK is the control group.
FIG. 4 is a view showing the root infection of the mycorrhiza of fructus Aurantii of the present invention with the control group, wherein a is the hypha in the root, b is the control group (CK), C is the arbuscular structure, and D is the vesicle structure.
FIG. 5 is a comparison of the root infection rate of the mycorrhiza of fructus Aurantii and the root infection rate of the control fructus Aurantii.
FIG. 6 is a diagram showing the growth of clover in the process of preparing the F.flavedo FS-1-106 microbial inoculum by the method of the invention.
FIG. 7 shows the navel orange mycorrhiza seedling obtained by grafting the navel orange mycorrhiza seedling to the navel orange scion, wherein AM is the mycorrhiza seedling obtained by the method of the present invention, and CK is the seedling obtained by the control group.
FIG. 8 shows a phylogenetic tree of the fungus Leidedeza flavedo FS-1-106.
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 used in the examples were all commercially available unless otherwise specified.
The units in the following examples: days are denoted d, daily is denoted d-1Hours are expressed as h, minutes are expressed as min, and seconds are expressed as s; volume liters is denoted as L and milliliters is denoted as mL; the units of measurement are m in meters, cm in centimeters and mm in millimeters.
Example 1
The screening process of the strain of the Clarkey yellow bursa of Fabricius FS-1-106 is as follows:
(1) taking 10-20 g of an original soil sample as a culture, putting the culture into a food mixer cup, adding 600ml of deionized water, centrifuging at high speed for 3-5 s (when the content of bacterial strains in the original soil sample is not large, in order to avoid the situation that the bacterial strains cannot be extracted, enriching firstly, collecting 500 g of citrus rhizosphere soil (the original soil sample) from Chongxisei county in Guangxi, mixing the orange rhizosphere soil with sterilized river sand (the diameter is 0.5-2 mm) 1:1, sowing 3-5 grains/pot of corn, culturing for 4 months in a greenhouse by illumination, obtaining a corn root system and a culture medium which are enriched soil samples, and putting the enriched soil samples into a video mixer cup to be extracted instead of the original soil samples);
(2) pouring out substances obtained by high-speed centrifugation in the step (1), sequentially passing through 3 soil standard sieves (the aperture is 0.8mm on the upper layer, 0.25mm in the middle and 0.0385mm on the lower layer), leaving most gravels in a cup of a food stirring machine, and flushing each layer of sieve by flowing water until the flowing water is clean water;
(3) transferring the residue in the lower layer sieve into a centrifuge tube containing 60% sucrose, centrifuging at 1500 rpm for 3min, and rapidly pouring the supernatant in the centrifuge tube into a sieve with the aperture of 0.0385 mm;
(4) the obtained supernatant fluid remnants contain AM fungal spores, the supernatant fluid remnants in the sieve are washed by water for 1-2 min and then transferred to a glass culture dish, and the spores are observed by a stereoscopic microscope;
(5) firstly observing and recording the color, the size, the characteristics of the hyphomycete, the shape of spore fruits and the like of spores under a stereoscopic microscope, on the basis, picking fresh AM fungal spores by using a capillary pipette, placing the spores on a glass slide, adding a floating carrier (such as water, lactic acid, glycerol lactate and PVLG), and observing under a Nikon E-600 microscope to record the characteristics of the spores such as shape, size, color, surface ornamentation, spore content, the number, width and shape of the hyphomycete, spore wall structure, auxiliary cells (soil-borne vesicles), exogenic hyphae and accessory structure spore-producing cysts; meanwhile, Melzer's reagent and cotton blue reagent are used for assisting in observing the specific reaction of the spores, and representative or specific characteristics are photographed. According to the morphological characteristics of the spores of the AM fungus, as shown in FIG. 1, the classification system of Schu β ler & Walker (2010) is used and is referred to the "VA mycorrhizal fungi identification manual and the related websites of Schenck & Perez (1988): http:// INVAM. caf. wvu. edu (INVAM, West Virginia University, USA); http:// www.zor.zut.edu.pl/Glomeromycota/Taxolomy. html (Department of Plant Pathology, University of Agriculture in Szczecin, Poland) and http:// www.lrz.de/. Schuessler/amylo/amylology. html and in recent years the original descriptions of new species were published for species search and identification. Further performing monospore culture on the species difficult to determine or possible new species and new record species to obtain a large number of homologous spores, determining the species, and performing auxiliary identification on part of the species by adopting a molecular biological method. AM fungal spores were prepared into slide specimens using Melzer's PVLG 1:1 or PVLG as the vehicle, sealed, numbered and stored, the invention relates to spores numbered FS-1-106 therein.
Results
(I) morphological characteristics of the Strain
Spore: loose clustering in soil, nearly spherical to spherical, yellow to orange yellow, size: 58.1-138.3 (87.2) mu m, the outer surface of the spore has scale-shaped separation, and the spore wall: 3 layers; l1 was colorless and transparent, and had a thickness of about 1 μm, and the wall was bright yellow in Melzer's reagent and light blue in gossypol lactate blue; l2 is 1-2.4 μm thick, orange yellow; l3 is 2.17-3.61 μm thick, and is layered, and is composed of 4-6 layers of layered walls with thickness of about 0.5-1 μm;
inclusion of: colorless to light yellow, flocculent, difficult to overflow when spores are broken by pressure in water, lake blue is dyed in a lactic acid phenol cotton blue reagent, the color is not changed in a Melzer's reagent,
hypha of Neurospora: 7 to 10 μm, colorless and transparent, and occasionally biased to one side at the connecting points, with the connecting points separated by a diaphragm formed of L3.
The molecular phylogenetic tree of FS-1-106 is shown in FIG. 8.
(II) identification results
The strain is identified as the Clarithromyces flavidus (Redeckara fulvum) by combining morphological and molecular biological characteristics, is classified and named as the Clarithromyces flavidus (Redeckara fulvum) FS-1-106, has a nucleotide sequence table shown as SEQ ID NO:1, and has a preservation unit: china general microbiological culture Collection center, preservation Address: the microbiological research institute of western road No.1, 3, national academy of sciences, north-kyo, the rising area, the preservation date: year 2020, month 05, day 25, accession number: CGMCC No. 19902.
Example 2
Growth promoting effect of bacterial strain microbial inoculum on potted citrus rootstock bitter orange
Preparation of bacterial strain agent
The preparation method of the Leideogrammitis flava FS-1-106 microbial inoculum comprises the following specific operation steps:
(a) taking clover as a host, inoculating spores of single leprosy FS-1-106 which is obtained by separation, purification, culture and propagation into a root system of the seedling of the clover, and planting the spores in a culture medium according to the inoculation amount that 1000-1500 spores of the leprosy FS-1-106 are used for each pot of clover, wherein the culture medium is river sand: mixing zeolite at a volume ratio of 3:1, sterilizing for 1h under high-pressure steam at 121 ℃, and culturing for 12-16 weeks under illumination to obtain the fulvia fulva hyphae; pouring 300-500 mL of Hoagland nutrient solution with the concentration of 50% every 2 weeks during the illumination culture period; wherein, the planting adopts a pot culture (780mm multiplied by 350mm multiplied by 250mm) production mode, and the host density is 50-80 strains/pot (as shown in figure 6).
(b) And (b) stopping watering the clovers cultured for 12-16 weeks in the step (a), removing stem and leaf parts after the plants are dried, reserving root systems, wherein hypha obtained in the culture medium, the culture medium and the reserved root systems of the clovers are the burkeslea flavedo FS-1-106 microbial inoculum, and the amount of spores in the obtained microbial inoculum is 80-120 spores/g.
Application of dachia flavonidus FS-1-106 microbial inoculum in production of citrus rootstock and bitter orange mycorrhiza seedlings
2.1 the application of the bursa flavipes FS-1-106 microbial inoculum prepared in the step (I) in the production of mycorrhiza seedlings of citrus rootstocks and bitter oranges, and the specific operation steps are as follows:
(1) detoxification treatment and sowing of bitter orange seeds: pretreating fructus Aurantii seed in 40 deg.C water bath for 10 min, soaking in 53 deg.C warm water for 50 min, stirring to make the temperature uniform;
(2) sowing seeds of fructus aurantii: according to the using amount of inoculating 100-300 spores to 10 bitter orange seeds, firstly spreading the burkeslea flavedo FS-1-106 microbial inoculum prepared in the step (I) on a sowing substrate, then covering the substrate with a thickness of 2-3 cm, sowing the bitter orange seeds obtained after the detoxification treatment in the step (1) in the substrate, then covering river sand with a thickness of 1-2 cm, sowing and germinating, wherein 8-10 leaves are formed, and the bitter orange with a plant height of 10-15 cm is a mycorrhiza seedling; wherein the matrix is obtained by mixing fresh soil and river sand according to the mass ratio of 1:1 and sieving the mixture by a 20mm multiplied by 20mm hole sieve.
Transplanting the obtained fructus aurantii mycorrhiza seedlings to a special citrus seedling cup containing a matrix, wherein the matrix in the seedling cup is obtained by mixing 2 parts of fresh soil, 1 part of chaff, 1 part of sawdust and 0.5 part of cow dung according to the volume part ratio, and the cultivation management is carried out after transplanting the special seedling cup, so that the seedling cup is used for scion grafting of a citrus variety navel orange taking fructus aurantii as a stock; the cultivation management is to shade the transplanted seedlings of the mycorrhiza of the bitter orange for 15-20 days, and manage the seedlings of the bitter orange according to normal conditions after 30 days; then, spraying 50% Hoagland nutrient solution every 2 weeks; after 90 days, 1/1000 high-nitrogen low-phosphorus compound fertilizer (N: P: K: 21: 7: 12) is sprayed every 2-3 weeks.
2.2 the application of the Leidectomyces flavum FS-1-106 microbial inoculum prepared in the step 2.1 in the production of citrus mycorrhiza is used as an experimental group (AM); the control group (CK) was prepared by the same procedure as in 2.1 except that no inoculum of Ledebakella flavedo FS-1-106 was added.
Results and analysis:
compared with the plants of the control group (CK), the seedlings of the mycorrhiza of the bitter orange in the experimental group (AM) show vigorous growth vigor, the leaves of the plants are dark green, the plants are robust, and the extremely obvious difference is achieved in the height, the diameter of the stems, the number of the leaves and the content of chlorophyll of the plants (figure 2 and figure 3).
The fructus aurantii is used as a rootstock, the navel orange is used as a scion for grafting, and the plant growth potential of the experimental group (AM) which takes the mycorrhiza seedlings of the fructus aurantii as the rootstock is obviously higher than that of a control group (as shown in figure 7).
And (3) detecting the root system mycorrhiza infection rate of the root system of the bitter orange mycorrhiza seedlings:
randomly extracting 5 seedlings from an experimental group (AM) and a control group (CK) for mycorrhiza infection detection 3 months after the fructus aurantii mycorrhiza seedlings are potted and planted:
the root system is washed clean with tap water and the filter paper absorbs dry water. Cutting the root system to about 1cm long, placing into a tissue embedding box, adding 20% KOH solution to completely soak the root system, carrying out water bath at 90 ℃ for 0.5h, washing with clear water for 3 times, and draining off water. Then adding alkaline H2O2(10％H2O210mL, 1mL of ammonia water and 189mL of distilled water) for 2 hours at room temperature, washing for 3 times by using clear water after the decoloration is finished, and draining the water. Then adding 5% glacial acetic acid, acidifying at room temperature for 2h, pouring off glacial acetic acid solution, and loading the root system into a centrifuge tube. Dyeing with Quink pure black ink (5% glacial acetic acid 95mL + ink 5mL), dyeing in a water bath at 66 ℃ for 1h, pouring out the dye solution after dyeing is finished, rinsing with clear water for several times, and soaking in clear water for more than 12h for decolorization for later use. And (3) selecting the root segments subjected to the decolorization treatment on a glass slide, placing 4 root segments in parallel on each glass slide, adding 2-3 drops of lactic acid into the root system to perform the decolorization effect, covering a 24mm multiplied by 50mm cover glass, and flattening the root segments with fingers by a little force. Observation and photography were carried out using an Olympus BX53-32p02 microscope.
The infection detection of the root system of the mycorrhizal plant of the bitter orange shows that the root system is obviously infected by inoculating the < i > Redkillella flavedo </i > -FS-1-106 </i > microbial inoculum, the structure is rich, the root system of the mycorrhizal seedling plant can be observed to have obvious hypha, arbuscular and vesicle structures (figure 4 and figure 5), and the mycorrhizal infection is not detected by not inoculating the < i > Redkillella flavedo </i > -FS-1-106 </i > microbial inoculum, so that the microbial inoculum can establish a good reciprocal symbiotic relationship with the bitter orange, and the colonization force is strong.
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 college of autonomous region agro-sciences
XIAMEN University
NATURAL MEDICINE INSTITUTE OF ZHEJIANG YANGSHENGTANG Co.,Ltd.
<120> method for producing mycorrhiza seedlings of citrus rootstock and bitter orange
<130> JC
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 542
<212> DNA
<213> Redeckera fulvum
<400> 1
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ttgaacttcg ggatttgctc attggtcagg cttcactgtc tgtactggtg taacgaattc 120
ctaccttctg atgaatcttc atgccattca cttggtgcgt aggggaatca ggactgttac 180
tttgaaaaaa ttagagtgtt taaagcaggc tattgctcga atacattagc atggaataat 240
agaataggac ggttggttct attttgttgg tttctaggat caccgtaatg attaataggg 300
atagttgggg gcattagtat tcaattgtca gaggtgaaat tcttggattt attgaagact 360
aactactgcg aaagcatttg ccaaggatgt tttcattaat caagaacgaa agttagggga 420
tcgaagacga tcagataccg tcgtagtctt aaccataaac tatgccgact agggatcgga 480
cgaagttaat ttttaaatga ctcgttcggc accttacggg aaaccaaagt tttttgggtt 540
ca 542
Claims (8)
1. The method for producing the mycorrhiza seedlings of the citrus rootstock bitter orange is characterized by comprising the following operation steps of:
(1) detoxification treatment of the seeds of the bitter orange;
(2) sowing seeds of fructus aurantii: according to the using amount of inoculating 100-300 spores to 10 bitter orange seeds, firstly spreading a burkesler flavedo FS-1-106 microbial inoculum on a sowing substrate, then covering the substrate, sowing the bitter orange seeds obtained after detoxification treatment in the step (1) in the substrate, then covering river sand, sowing and germinating, wherein the bitter orange with 8-10 leaves is obtained after sowing and germinating, and the bitter orange with the plant height of 10-15 cm is mycorrhizal seedlings; the preparation method of the dacgaspora xanthioides FS-1-106 microbial inoculum comprises the following steps:
(a) taking clover as a host, inoculating spores of the Sorangium flavum FS-1-106 into root systems of the seedlings of the clover, wherein the inoculation amount is 1000-1500 spores per pot of the clover; planting the seeds in a culture medium, and performing illumination culture for 12-16 weeks to obtain the fulvia fulvidraco filaments; a potted plant (780mm multiplied by 350mm multiplied by 250mm) production mode is adopted, and the planting density of the clovers is 50-80 plants/pot; the Classification and nomenclature of the Clarkey fulvia fulva FS-1-106 is Classification and nomenclature of the Clarkey fulvia fulva (Redeckara fulvum) FS-1-106, the nucleotide sequence table is shown as SEQ ID NO:1, and the preservation unit is: china general microbiological culture Collection center, preservation Address: the microbiological research institute of western road No.1, 3, national academy of sciences, north-kyo, the rising area, the preservation date: year 2020, month 05, day 25, accession number: CGMCC No. 19902;
(b) stopping watering the clovers cultured for 12-16 weeks in the step (a), removing stem and leaf parts after the plants are dried, reserving root systems, obtaining hypha in a culture medium, the culture medium and the reserved root systems of the clovers, namely the burkesleya flavedo FS-1-106 microbial inoculum, wherein the spore amount in the microbial inoculum is 80-120 spores/g, and the culture medium contains spores of the burkeslea flavedo besides the hypha.
2. The method of claim 1, wherein: the detoxification treatment of the seeds of the bitter orange in the step (1) is to pretreat the seeds of the bitter orange in water bath at 40 ℃ for 10 minutes and then soak the seeds of the bitter orange in warm water at 53 ℃ for 50 minutes.
3. The method of claim 1, wherein: the matrix in the step (2) is obtained by mixing fresh soil after being sieved with river sand according to the volume ratio of 1:1, firstly spreading a burkeslea flavedo FS-1-106 microbial inoculum on a seeding matrix, then covering the seeding matrix with a matrix with the thickness of 2-3 cm, seeding the fructus aurantii seeds obtained after detoxification treatment in the step (1) in the matrix, and then covering the matrix with river sand with the thickness of 1-2 cm.
4. The method of claim 1, wherein: the culture medium in the step (a) is obtained by mixing river sand and zeolite in a volume ratio of 3:1, and the mixed culture medium is sterilized for 1h under high-pressure steam at 121 ℃.
5. The method of claim 1, wherein: 300-500 mL of Hoagland nutrient solution with the concentration of 50% is poured every 2 weeks during the culture in the step (a).
6. Use of the fruit of citron mycorrhiza seedlings produced by the method as described in any one of claims 1 to 5, wherein: transplanting the trifoliate orange mycorrhiza seedlings obtained in the step (2) to a citrus seedling cup containing a matrix, and performing cultivation management to graft scions of citrus varieties taking trifoliate orange as stocks.
7. Use according to claim 6, characterized in that: the cultivation management is to shade the seedlings of the mycorrhiza of the bitter orange for 15 to 20 days after the transplantation, and manage the seedlings of the bitter orange according to normal conditions after 30 days; then, spraying 50% Hoagland nutrient solution every 2 weeks; after 90 days, 1/1000 high-nitrogen low-phosphorus compound fertilizer (N: P: K: 21: 7: 12) is sprayed every 2-3 weeks.
8. Use according to claim 6, characterized in that: the matrix in the citrus seedling raising cup containing the matrix is obtained by mixing 2 parts of fresh soil, 1 part of chaff, 1 part of sawdust and 0.5 part of cow dung according to the volume part ratio.
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