CN108541514B - Method for cultivating orange Rumex Lactarius mycorrhiza seedlings - Google Patents
Method for cultivating orange Rumex Lactarius mycorrhiza seedlings Download PDFInfo
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- CN108541514B CN108541514B CN201810235925.2A CN201810235925A CN108541514B CN 108541514 B CN108541514 B CN 108541514B CN 201810235925 A CN201810235925 A CN 201810235925A CN 108541514 B CN108541514 B CN 108541514B
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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
- A01G18/00—Cultivation of mushrooms
- A01G18/60—Cultivation rooms; Equipment therefor
- A01G18/64—Cultivation containers; Lids therefor
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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
- A01G18/00—Cultivation of mushrooms
- A01G18/20—Culture media, e.g. compost
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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
- A01G18/00—Cultivation of mushrooms
- A01G18/60—Cultivation rooms; Equipment therefor
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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
- A01G9/00—Cultivation in receptacles, forcing-frames or greenhouses; Edging for beds, lawn or the like
- A01G9/02—Receptacles, e.g. flower-pots or boxes; Glasses for cultivating flowers
- A01G9/021—Pots formed in one piece; Materials used therefor
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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
- A01G9/00—Cultivation in receptacles, forcing-frames or greenhouses; Edging for beds, lawn or the like
- A01G9/02—Receptacles, e.g. flower-pots or boxes; Glasses for cultivating flowers
- A01G9/029—Receptacles for seedlings
- A01G9/0293—Seed or shoot receptacles
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Abstract
A method for cultivating orange Rumex Lactarius mycorrhiza seedlings, which is characterized in that: the method comprises the following steps: 1) Preparing a seedling raising device: 2) Culturing aseptic seedlings; 3) Preparing a microbial inoculum; 4) Inoculating; 5) Adding seedling substrate; 6) Installing a water guide device and a shading, rain shielding and dust preventing cover; 7) Cultivating mycorrhizal seedlings; the device designed by the patent is used for cultivating the orange Rumex Lactarius mycorrhiza seedling, the mixed bacteria infection rate is low, and the puffball infection rate is 0.
Description
Technical Field
The invention belongs to the technical field of plant seedling raising, and particularly relates to a method for cultivating Rumex citrifolia mycorrhizal seedlings.
Background
Mycorrhizal edible fungi such as bolete, truffle, lactarius deliciosus and the like are important species in edible fungi, and are symbiotic on the root system of trees to form exogenous mycorrhiza, absorb photosynthetic products of plants, and simultaneously supply nutrients absorbed by the bolete, truffle, lactarius deliciosus and the like to the trees for utilization and are in a symbiotic relationship. At present, the common practice of artificially cultivating mycorrhizal edible fungi is to firstly cultivate symbiotic sapling in a sterilized culture medium, inoculating mycelium or spore and other propagules as a microbial inoculum to the root system of the aseptic seedling, then hardening off the inoculated mycorrhizal seedlings (some are not hardened off) in open air after the mycorrhizal seedlings are cultivated in a laboratory or a greenhouse for a period of time, and transplanting the mycorrhizal seedlings to a production site for fruiting after the mycorrhizal seedlings reach the requirements. However, the main problems of the prior art are: (1) The mycorrhizal seedling cultivation can only be cultivated in a greenhouse or a laboratory, is difficult to produce in large scale, has low production operability, and has large facility investment and high mycorrhizal seedling production cost. (2) The mycorrhizal seedling cultivation period is long, a large amount of clean water is needed, tap water after high-temperature sterilization is mainly used under the laboratory condition at present, and the cost is high. Tap water or rainwater is adopted in large-scale production, and non-target mycorrhizal fungus infection exists. (3) The current common methods for inoculating aseptic seedlings include root dipping inoculation of a microbial inoculum, sowing inoculation of the microbial inoculum and perforation filling inoculation of the microbial inoculum. The microbial inoculum root dipping and microbial inoculum broadcast seeding methods are both used for transplanting aseptic seedlings, the seedling root system is easy to damage in the transplanting seedling recovery period, the seedlings are extremely easy to die when the weather is improper, the seeding failure is caused, the conventional punching filling microbial inoculum seeding method has little influence on the seedlings, the direct contact opportunity of the inoculated microbial inoculum and the seedling root system is small, the growth speed of inoculated strains is very slow, and the strains are not easy to survive for a long time, so that the mycorrhiza infection rate is reduced. (4) After inoculation of the sterile seedlings with the inoculant, especially three months after inoculation, the moisture management of the culture medium is very difficult and difficult to master accurately, often resulting in reduced mycorrhiza infection rates and even seedling decay and death. (5) The container for culturing mycorrhizal seedlings used at present is a common seedling culturing container, needs to be placed in a greenhouse or under indoor conditions, needs fluorescent lamps to provide light sources, and is high in energy consumption and cost, and not beneficial to large-scale cultivation of mycorrhizal seedlings. Insufficient illumination under greenhouse conditions influences photosynthesis and mycorrhiza synthesis effects of the seedlings. (6) When the seedlings inoculated with mycorrhizal fungi are directly placed in an open air condition by adopting a common seedling raising container, the seedling raising substrate is easy to lose water, and the natural non-target mycorrhizal fungi spores and other propagules (can be transmitted into the substrate through rainwater, wind or underground soil), so that the non-target mycorrhizal fungi (such as puffball, abdominal fungus and the like) are infected, pollution is very easy to cause, and the cultivation of the target mycorrhizal fungi fails.
Disclosure of Invention
The invention aims to solve the problems in the prior art and aims to provide a method suitable for cultivating orange Rumex Lactarius mycorrhiza seedlings in open air. By designing the special device, the root system part of the mycorrhizal seedling grows and develops under the relatively closed condition in the whole growth process, the non-target fungus propagules are difficult to invade the root system of the seedling to infect the seedling, and the target mycorrhizal fungus is easy to survive after artificial inoculation, so that the qualified mycorrhizal seedling can be cultivated in a large scale and at low cost.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a method for cultivating orange Rumex Lactarius mycorrhiza seedlings, which is characterized in that: the method comprises the following steps:
1) Preparing a seedling raising device: the device comprises a container body, a shading, rain shielding and dust preventing cover and a water guiding device;
the side wall of the container body is provided with an air hole and an inoculation hole, a step is inwards arranged at the joint of the side wall of the container body and the lower bottom surface, the step is provided with a pore plate in a matched manner, and the side wall of the container body between the lower bottom surface and the pore plate is provided with a drain hole;
the upper end of the shading, rain shielding and dust preventing cover is provided with an elastic tying, the lower end of the shading, rain shielding and dust preventing cover is provided with a water outlet, and binding ropes are matched with the elastic tying; the light-shielding, rain-shielding and dust-preventing cover is made of black-and-white reflective film materials, and the white color is outwards and the black color is inwards;
the water guide device consists of a hollow bent pipe and a tripod guide bracket fixedly connected with the bent pipe, and a water guide groove is arranged below the tripod guide bracket; an inoculation hole cover is arranged on the inoculation hole in a matched mode, and an inner buckle and an outer buckle used for fixing the inoculation hole cover on the container body are arranged at the edge of the inoculation hole cover;
2) Sterile seedling cultivation: culturing a pinus massoniana aseptic seedling in the container body;
3) And (3) preparation of a microbial inoculum: preparing an orange liquid lactobacillus preparation;
4) Inoculating: inoculating in autumn and winter for 10-11 months, wherein the inoculation method comprises the following steps: after the aseptic seedlings of masson pine have lateral roots for development, taking down an inoculation hole cover, obliquely inserting a puncher into the inoculation hole of the container body downwards for punching by 5-10cm, and inoculating 5-10ml of orange lactarius fluid inoculant into each hole; fixing the inoculation hole cover at the inoculation hole through an inner buckle and an outer buckle, sealing, and uniformly sprinkling about 300-500ml of nutrient solution to the seedling substrate from the upper part of the container;
5) Adding seedling raising matrix: adding the sterilized seedling substrate again above the seedling substrate sprayed with the nutrient solution until the container body is full, and keeping the surface of the substrate slightly steamed bread-shaped;
6) Installing a water guide device and a shading, rain shielding and dust preventing cover: placing a water guide device on a seedling raising substrate which is slightly steamed bread-shaped, requiring an elbow pipe to be abutted against the stem part of the aseptic seedling, covering a light-shielding, rain-shielding and dust-proof cover on the container body, enabling the rhizome of the aseptic seedling and the elbow pipe of the water guide device to pass through an elastic tying and be positioned at the outer side of the light-shielding, rain-shielding and dust-proof cover, and tying a tying rope at the elastic tying part by using the tying rope;
7) Culturing mycorrhizal seedlings: the mycorrhizal seedlings of the device provided with the water guide device and the light shielding, rain shielding and dust preventing cover are placed in the open air for cultivation; watering is not carried out for 2 weeks after inoculation, then water is irrigated according to the water content condition of the matrix, and the water content of the matrix is maintained to be 60-70% for culture; the irrigation water source is from underground deep well water.
The method for cultivating the orange Lactarius rupestris mycorrhiza seedling is characterized in that the cultivation method of the pinus massoniana aseptic seedling in the step 2) comprises the following steps: soaking Pinus massoniana seeds in tap water, draining, removing inferior seeds, soaking in 0.1% potassium permanganate for 1 hr for surface sterilization, washing with sterile water for 3 times, seeding in a container body filled with seedling substrate, keeping the water content of the substrate at 60-70%, and culturing in a greenhouse or greenhouse; the water source is deep well water; the seedling substrate needs to be sterilized for 1h at 121 ℃ and 0.1 MPa.
The method for cultivating the orange Rumex lactiflora seedlings is characterized by comprising the following steps of: the formula of the seedling substrate is vermiculite: sandy loam=1:1 (volume ratio), natural PH.
The method for cultivating the orange Rumex volvacea mycorrhiza seedling is characterized in that the preparation method of the orange Rumex volvacea liquid microbial inoculum in the step 3) comprises the following steps:
1) Separating strain tissue: collecting orange Lactarius rupestris fruiting body, taking back to laboratory for tissue separation, collecting mung bean size tissue at joint position of fungus cover and fungus handle, culturing on solid culture medium for one week, and collecting mycelium for purification culture;
2) And (5) propagation of strains: taking cultured strains, putting the strain blocks into a liquid culture medium, shaking the liquid culture medium for culture at 25 ℃ at the rotating speed of 140r/min for 25 days for later use;
3) Preparing a liquid microbial inoculum: filtering the bacterial liquid in the step 2) to obtain bacterial balls, adding a liquid culture medium, and mixing the bacterial balls and the liquid culture medium according to a volume ratio of 1:1; then adding sodium alginate into the mixed solution, and crushing and uniformly stirring to prepare a liquid microbial inoculum; the sodium alginate is added according to the amount of 3-5g added to each 1L of the mixed solution.
The method for cultivating the orange Rumex lactiflora seedlings is characterized by comprising the following steps of: the formula of the liquid culture medium comprises the following components: 200g of potato is boiled and filtered to obtain juice, 20g of glucose, 5g of brown sugar, 1.5 g of monopotassium phosphate, 1g of magnesium sulfate, 1g of ammonium chloride, 1g of peptone, 0.5ml of 1% ferric citrate, 1ml of 0.1% zinc sulfate, 1 mg of vitamin B and water are added to fix the volume to 1000ml; the solid culture medium is prepared by adding 20g of agar into 1L of the liquid culture medium.
The method for cultivating the orange Rumex lactiflora seedlings is characterized by comprising the following steps of: the hole puncher in the step 4) is composed of a punching end and a handle end, wherein the punching end is flat and sharp, has a length of 5-6cm, a width of 1.5-1.8cm, the handle end is cylindrical, a length of 8-12cm and a diameter of 1.5-1.8cm.
The method for cultivating the orange Rumex lactiflora seedlings is characterized by comprising the following steps of: the distance between the lower bottom surface of the container body and the pore plate is 1-2cm, namely the height of the step is 1-2cm.
The method for cultivating the orange Rumex lactiflora seedlings is characterized by comprising the following steps of: the deep well water is derived from underground water with the depth of less than 15 m.
The method for cultivating the orange Rumex lactiflora seedlings is characterized by comprising the following steps of: the inoculation holes on the side wall of the container body are circular, the diameter of each hole is 1.5-2cm, the number of the inoculation holes is 5, the inoculation holes are uniformly distributed in a ring shape, and the inoculation holes are arranged at the middle upper part of the container body.
Compared with the prior art, the invention has the following beneficial effects: (1) The device designed by the invention has simple structure, is convenient to use, avoids facilities such as a greenhouse, can cultivate target exogenous mycorrhizal seedlings in a large scale, and greatly reduces the infection of non-target mycorrhizal fungi (such as puffball, abdominal bacteria and the like). (2) The container body is made of a black opaque hard plastic material and plays a role of a supporting framework, and the black material provides a dark environment for mycorrhizal fungi and root systems. An inner space of 1-2cm is arranged between the lower bottom surface of the container body and the pore plate, so that the container body is prevented from being in direct contact with a nursery land, and the infection of indigenous mycorrhizal seedlings to a seedling raising matrix is prevented; the side wall is provided with the air holes which can meet the breathing requirements of mycorrhizal fungi and root systems. The side wall is provided with an inoculation hole which is convenient for punching and inoculating the target microbial inoculum. (3) The light-shielding, rain-shielding and dust-preventing cover is a soft black-and-white reflective plastic film, the white color is outwards, the black color is inwards, a small opening is reserved at the upper end of the light-shielding, the elastic tying rope is adopted at the root stem of the seedling, the phenomenon that non-target mycorrhizal fungi are infected by media such as rainwater or wind is avoided, meanwhile, the black-and-white reflective plastic film can reflect illumination to the leaf of the seedling, photosynthesis is increased, and meanwhile, the damage to mycorrhizal fungi and root systems caused by the loss of moisture in the matrix and the damage to the mycorrhizal fungi and the root systems caused by the overhigh temperature in the matrix due to the direct irradiation of the illumination to the matrix are reduced. (4) The inoculation method adopts the perforation filling microbial inoculum, and adds nutrient solution to induce the microbial inoculum to continue growing, prolongs the survival period of strains, adopts a sharp puncher to properly cut off lateral roots or hurt cortical tissues, is beneficial to hypha infection root systems, does not influence the growth of seedlings, and can greatly improve the mycorrhiza infection rate. (5) The water guide device designed by the invention has the functions of watering mycorrhizal seedlings on one hand and supporting the shading, rain shielding and dust preventing covers on the other hand, and the bent pipe of the water guide device is connected with a drip irrigation water pipe, namely, the drip irrigation is adopted for watering thoroughly, watering is not needed for a long time, the water content of seedling substrates is well maintained, the stability of the growth environment of the root systems of the seedlings is maintained, the influence of moisture change on mycorrhizal fungi and the root systems is reduced, and especially, three months after inoculation. (6) The invention saves water, on one hand, the use of the shading, rain shielding and dust preventing cover reduces the water evaporation of seedling raising matrix, and on the other hand, the invention adopts groundwater to supply, thereby solving the difficult problem that a large amount of sterile water is needed in production. (7) Compared with the conventional seedling culture container for culturing mycorrhizal seedlings, the invention obviously reduces the mixed bacterial infection rate and greatly improves the synthesis rate of target mycorrhizal. The invention can cultivate mycorrhizal seedlings without greenhouse facilities or indoor conditions, namely under open air conditions, solves a series of technical problems of large-scale, low-cost and high-quality cultivation of mycorrhizal seedlings, can also be used for nursery seedling in arid areas, can greatly save water and improve the survival rate of seedlings.
Drawings
FIG. 1 is a schematic diagram of the structure of a mycorrhizal seedling cultivated according to the present invention;
FIG. 2 is a schematic cross-sectional view of a mycorrhizal seedling cultivated in accordance with the present invention;
FIG. 3 is a schematic view of the structure of the present invention without the light-shielding, rain-shielding and dust-proof cover;
FIG. 4 is a schematic cross-sectional view of a container body according to the present invention;
FIG. 5 is a schematic view of a water guiding device according to the present invention;
FIG. 6 is a schematic view of the structure of the access port cover of the present invention;
FIG. 7 is a schematic view of a hole puncher according to the present invention;
FIG. 8 is a three-view of the hole punch of the present invention;
FIG. 9 is a morphology of Rumex citri-Marathogen
In the figure: 1-a container body; 2-a shading, rain shielding and dust preventing cover; 3-a water guiding device; 1-1-ventilation holes; 1-2-inoculation holes; 1-3 steps; 1-4-drainage holes; 1-5-well plate; 1-6-inoculation hole cover; 1-7, inner and outer buckles; 2-1, tightening and tying; 2-2-water outlet; 2-3-tying rope; 3-1-bending pipe; 3-2-tripod guide bracket; 3-3-a water guiding groove; 4-1-punching a hole end; 4-2-handle end.
Detailed Description
The invention will be described in further detail with reference to figures 1-9 and the detailed description.
Example 1
The invention relates to a device suitable for cultivating mycorrhizal seedlings under open air conditions, which mainly comprises the following three parts: a container body 1 for cultivating aseptic seedlings and mycorrhizal seedlings, a light-shielding, rain-shielding and dust-proof cover 2 for covering the container body, and a water guide device 3 for injecting water into the mycorrhizal seedlings.
According to the container body 1 designed by the invention, as shown in fig. 4, the ventilation holes 1-1 are formed in the side wall of the container body 1, so that better ventilation of seedling substrate plant root systems is realized, and the mycorrhizal fungi and root system respiratory requirements are met; the side wall of the container body 1 is also provided with an inoculation hole 1-2 which is convenient for punching and inoculating the target microbial inoculum. In order to avoid direct contact between the bottom of a seedling raising container and a nursery land and avoid infection of indigenous mycorrhizal seedlings to seedling raising matrixes, a step 1-3 is inwards arranged at the joint of the side wall of a container body 1 and the lower bottom surface, a pore plate 1-5 is arranged on the step in a matched mode, a drain hole 1-4 is arranged on the side wall of the container body between the lower bottom surface and the pore plate, 1-2cm of inner space is kept between the lower bottom surface and the pore plate, namely, the height of the step 1-3 is 1-2cm, when the seedling raising matrix is positioned above the pore plate 1-5, redundant moisture in the matrixes flows out into the inner space between the lower bottom surface and the pore plate through the hole on the pore plate 1-5, and then flows out of the container body through the drain hole 1-4 on the side wall. The inoculation holes 1-3 on the side wall of the container body can be circular, the diameter of the holes is 1.5-2cm, 3-6 inoculation holes, preferably 5 inoculation holes are uniformly distributed in a ring shape and are arranged at the middle upper part of the container body. In order to facilitate inoculation and seal the inoculation hole after inoculation, the invention is provided with an inoculation hole cover 1-6 in a matched manner on the inoculation hole 1-3, the edge of the inoculation hole cover 1-6 is provided with an inner buckle 1-7 and an outer buckle 1-7 for fixing the inoculation hole cover on the container body, the inner buckle and the outer buckle are preferably arranged in 3 pairs and are divided into an inner buckle and an outer buckle (see figure 6), the inner buckle is buckled on the inner side of the side wall of the container body, and the outer buckle is buckled on the outer side of the side wall of the container body.
The shading, rain shielding and dust preventing cover 2 designed by the invention is preferably made of black and white reflective film materials, and white faces outwards and black faces inwards. The upper end of the device is provided with an elastic tying 2-1, the lower end is provided with a water outlet 2-2, and the elastic tying 2-1 is matched with a binding rope 2-3; the binding ropes 2-3 are preferably elastic ropes and are tied at the root and stem parts of the seedlings, the tightness is suitable for avoiding the inflow of excessive loose rainwater and also avoiding the adverse effect of excessive tightening on plant growth. Because the cover body wraps the container body, a gap (namely a water outlet 2-2) is reserved on the side edge of the base of the container body for water drainage, the root system and the seedling culture substrate are prevented from being in direct contact with the nursery land, and the indigenous mycorrhizal seedlings are spread into the seedling culture substrate. The design of the shading, rain shielding and dust preventing cover avoids the infection of non-target mycorrhizal fungi by the medium such as rainwater, wind, nursery soil and the like, and simultaneously, the black and white reflective plastic film can reflect the illumination to the leaf parts of the nursery stock, thereby increasing photosynthesis, and simultaneously reducing the influence of the illumination on the direct irradiation of the matrix on the water loss in the matrix and the damage of the excessive temperature in the matrix on mycorrhizal fungi and root systems.
The water guide device 3 designed by the invention is shown in fig. 5, the water guide device 3 is composed of a hollow bent pipe 3-1 and a three-leg guide bracket 3-2 fixedly connected with the bent pipe, and a water guide groove 3-3 is arranged below the three-leg guide bracket 3-2. When the drip irrigation device is used, the water inlet end of the bent pipe 3-1 is connected with a drip irrigation water pipe, namely, permeable drip irrigation water is adopted, water seepage irrigation water flow is adopted, and the influence on root system and hypha development is small. The seedling substrate is thoroughly irrigated once, irrigation is not needed for a long time, the water content of the seedling substrate is well maintained, the stability of the growth environment of the root system of the seedling is maintained, the influence of moisture change on mycorrhizal fungi and the root system is reduced, and especially three months after inoculation. The structure plays the effect of watering the mycorrhizal seedlings on one hand, and on the other hand plays the role of supporting the shading, rain shielding and dust preventing cover to prevent rainwater from flowing backwards into the container.
The cultivation method of mycorrhizal seedlings by using the device comprises the steps of preparing a seedling substrate, preparing a mycorrhizal fungicide, cultivating aseptic seedlings, inoculating the aseptic seedlings, cultivating the mycorrhizal seedlings and the like.
The method mainly comprises the following steps:
1) Culturing aseptic seedlings: thoroughly soaking plant seeds for seedling raising with tap water, draining, removing inferior seeds, soaking for 1 hr with 0.1% potassium permanganate for surface sterilization, flushing with sterile water for three times, sowing into a container body filled with seedling raising matrix, culturing under the condition of 60-70% matrix humidity, and irrigating with sterile water; the seedling substrate needs to be sterilized for 1h at 121 ℃ and 0.1 MPa. This stage is cultivated in a greenhouse, greenhouse or laboratory. Of course, when the field is tense in a greenhouse, a greenhouse or a laboratory, after the sterile seedlings grow to a certain height, the sterile seedlings can be sleeved with a light-shielding, rain-shielding and dust-preventing cover and moved to the outdoor for cultivation;
2) And (3) preparation of a microbial inoculum: preparing a microbial inoculum with mycelium as an inoculum or with spores as an inoculum; the microbial inoculum can be solid microbial inoculum or liquid microbial inoculum, wherein the liquid microbial inoculum has better effect. The preparation method of the microbial inoculum taking mycelium as an inoculum comprises the following steps: after mycelium is propagated by liquid, the mycelium is filtered to obtain mycelium pellets, the mycelium pellets and nutrient solution are uniformly mixed according to the volume ratio of 1:1, 3-5g of sodium alginate is added into each 1000ml of mycelium pellet nutrient solution, and the mycelium pellets and nutrient solution are uniformly stirred;
3) Aseptic seedling inoculation: after the lateral roots of the aseptic seedlings develop, the inoculation hole cover 1-6 is taken down, a puncher is adopted to punch holes at the inoculation hole 1-2 of the container body, then a microbial inoculum is inoculated, the inoculation hole cover 1-6 is fixed at the inoculation hole through the inner buckle 1-7 and the outer buckle 1-7, namely, the inoculation hole is sealed and fixed on the container body, and then nutrient solution is poured from the upper part of the container to the surface of the substrate; then, adding sterilized seedling raising matrix again into the container body 1 until the container body is filled and the seedling raising matrix is slightly steamed bread-shaped, then placing a water guide device 3 on the seedling raising matrix, requiring an elbow pipe 3-1 to be abutted against the stem part of the aseptic seedling, covering a shading, rain-shielding and dust-proof cover 2 on the container body 1, enabling the overground part of the aseptic seedling and the elbow pipe 3-1 of the water guide device 3 to pass through an elastic tying 2-1 and be positioned at the outer side of the shading, rain-shielding and dust-proof cover 2, and tying a tying rope 2-3 at the position of the elastic tying 2-1 by using the tying rope 2-3; see fig. 1-3. The hole puncher (see fig. 7-8) used in the invention is designed as follows: the puncher consists of a punching end 4-1 and a handle end 4-2, wherein the punching end is flat and sharp wedge-shaped (similar to an axe), the length is 5-6cm, the width is 1.5-1.8cm, the handle end is cylindrical, the length is 8-12cm, and the diameter is 1.5-1.8cm; the adoption of a sharp puncher can properly cut off lateral roots or hurt cortical tissues, which is beneficial to hyphae to infect root systems. The inoculation method is not affected on the growth of the nursery stock, and can also greatly improve the mycorrhiza infection rate;
5) Cultivation of mycorrhizal seedlings: the inoculated aseptic seedlings are moved out of a greenhouse or a greenhouse, are cultured under natural environment conditions, and are injected with aseptic water through an inlet of a water guide device 3; the sterile water is derived from groundwater having a depth of 15m or less. When in use, the water inlet end of the bent pipe 3-1 is connected with a drip irrigation water pipe, namely, permeable drip irrigation is adopted for watering.
The invention is suitable for symbiotic seedling culture of mycorrhizal edible fungi such as bolete, lactarius deliciosus, truffle and the like, and the main technical links are as follows: (1) Selecting proper symbiotic tree species such as Phlebopus portentosus and Lactarius pinnatifida according to the characteristics of mycorrhizal edible fungi, wherein the symbiotic tree species are wetland pine, masson pine and the like; the symbiotic host plant of the truffle is hickory nut, chinese chestnut, yunnan pine and the like; (2) The container body of the container is adopted for aseptic seedling cultivation, the substrate is sterilized at high temperature, the water source is from groundwater below 15m, and the container is cultivated in a greenhouse or a room until the seeding requirement is met; (3) When the aseptic seedlings reach the inoculation requirement, a puncher is used for punching and filling the microbial inoculum, and nutrient solution is added to induce the microbial inoculum to continue growing, so that the contact opportunity and time of root systems and strains are increased; (4) The culture device of the invention is used, and the culture method and the operation steps after inoculation are correctly used; (5) The aseptic seedling is cultivated in a room or a greenhouse in a cultivation period, a shading, rain shielding and dust preventing cover and a water guiding device are covered after inoculation, and full illumination cultivation is performed under an open air condition. Compared with the common container for cultivating mycorrhizal seedlings, the invention can greatly reduce the dip-dyeing of the powerful mycorrhizal fungi such as puffball and the like, obviously reduce the infection rate of mixed fungi, greatly improve the synthesis rate of target mycorrhizal fungi and provide technical guarantee for producing high-quality mycorrhizal seedlings in large scale and low cost.
Example 2
The device of example 1 was used to inoculate and culture a symbiotic seedling of Pinus massoniana of Lactarius rupestris, as follows:
1. aseptic seedling cultivation
Radix seu herba Pinus MargaritiferaPinus massonianaLamb.) seed is fully soaked in tap water, drained, inferior seeds are removed, the seeds are soaked in 0.1% potassium permanganate for 1 hour to carry out surface disinfection, the seeds are sown in a container body filled with seedling culture matrixes after being washed three times by sterile water, the matrixes are cultured under the humidity of 60-70%, and the watering is deep well water; the seedling substrate needs to be sterilized for 1h at 121 ℃ and 0.1 MPa. The cultivation season of the aseptic seedlings is 5-6 months. Culturing in a greenhouse or greenhouse. The formula of the seedling substrate is vermiculite: sandy loam=1:1 (volume ratio), natural PH.
2. The preparation method of the microbial inoculum comprises the following steps of
1) Separating strain tissue: orange Lactarius is collected in Jinyun county ocean mountain forest region of Lishui city(Lactarius akahatsu) The sporophore is brought back to a laboratory for tissue separation, mung bean size tissue at the joint position of the fungus cover and the fungus handle is taken, and the sporophore is placed on a special solid culture medium for culturing for one week, and mycelium is selected for purifying and culturing for later use. The special solid culture medium comprises the following formula: 200g of potato is boiled and filtered to obtain juice, 20g of glucose, 5g of brown sugar, 1.5 g of monopotassium phosphate, 1g of magnesium sulfate, 1g of ammonium chloride, 1g of peptone, 0.5ml of 1% ferric citrate, 1ml of 0.1% zinc sulfate, 1 mg of vitamin B, 20g of agar and water are added to fix the volume to 1000ml;
2) And (5) propagation of strains: taking cultured strain, and cutting with a surgical knife to 1cm 2 Culturing 5 strain blocks in a liquid shake flask, wherein the formula of a liquid culture medium comprises: 200g of potato is boiled and filtered to obtain juice, 20g of glucose, 5g of brown sugar, 1.5 g of monopotassium phosphate, 1g of magnesium sulfate, 1g of ammonium chloride, 1g of peptone, 0.5ml of 1% ferric citrate, 1ml of 0.1% zinc sulfate, 1 mg of vitamin B and water are added to fix the volume to 1000ml;
adopting 500ml shake flask, wherein the liquid loading amount is 300ml, the culture temperature is 25 ℃, the rotating speed is 140r/min, and the culture is 25d for standby;
3) Preparing a liquid microbial inoculum: the mycelium liquid microbial inoculum is prepared by adding 3-5g sodium alginate into each 1000ml of mycelium filtered mycelium pellets and nutrient solution (the formula is the same as the liquid culture medium in the step 2) according to the volume ratio of 1:1, and crushing and uniformly stirring.
3. Inoculating: inoculating in autumn and winter for 10-11 months, wherein the inoculation method comprises the following steps: after the aseptic seedlings of masson pine have lateral roots for development, taking down an inoculation hole cover 1-6, obliquely inserting a puncher into the inoculation hole 1-2 of the container body downwards for punching by 5-10cm, and inoculating 5-10ml of orange liquid fungus agent into each hole; the inoculation hole cover 1-6 is fixed at the inoculation hole by the inner and outer buckles 1-7 to seal, and the nutrient solution is evenly poured into the seedling substrate from the upper part of the container by about 300-500ml.
4. Adding seedling raising matrix: and adding the sterilized seedling substrate again above the seedling substrate sprayed with the nutrient solution until the container body is full, and keeping the surface of the substrate slightly steamed bread-shaped.
5. Installing a water guide device and a shading, rain shielding and dust preventing cover: placing a water guide device on a seedling raising substrate which is slightly steamed bread, requiring an elbow pipe to be abutted against the stem part of the aseptic seedling, then covering a light-shielding, rain-shielding and dust-proof cover on the container body, enabling the rhizome of the aseptic seedling and the elbow pipe of the water guide device to pass through the elastic tying and be positioned at the outer side of the light-shielding, rain-shielding and dust-proof cover, and tying a tying rope at the position of the elastic tying 2-1 by using the tying rope.
Culturing mycorrhizal seedlings: the mycorrhizal seedlings of the device provided with the water guide device and the light shielding, rain shielding and dust preventing cover are placed in the open air for cultivation; watering is not carried out for 2 weeks after inoculation, then water is irrigated according to the water content condition of the matrix, and the water content of the matrix is maintained to be 60-70% for culture; the irrigation water source is from underground deep well water. The deep well water is preferably derived from groundwater having a depth of 15m or less. Culturing for 6-12 months, inoculating Miao Xi surface mycelium sleeves of Pinus massoniana, and culturing in the form of two forks, stick shape, coral shape and reddish brown, as shown in figure 9.
The infection of mycorrhiza of nursery seedling under open air condition is shown in table 1. The result shows that the common container is adopted for seedling cultivation, the infectious rate of the mixed bacteria is up to 41.1 percent, and the infectious rate of puffball is up to 30.6 percent. The infectious rate of the device used in the patent is 0.96%, and the infectious rate of puffball is 0. The infection rate of the mixed bacteria is obviously reduced, and the technical guarantee is provided for producing high-quality masson pine mycorrhizal seedlings in a large-scale and low-cost manner.
TABLE 1 infection of mycorrhiza for nursery seedlings under open air condition
Claims (9)
1. A method for cultivating orange Rumex Lactarius mycorrhiza seedlings, which is characterized in that: the method comprises the following steps:
1) Preparing a seedling raising device: the device comprises a container body (1), a shading, rain shielding and dust preventing cover (2) and a water guiding device (3);
the side wall of the container body (1) is provided with ventilation holes (1-1) and inoculation holes (1-2), a step (1-3) is inwards arranged at the joint of the side wall of the container body (1) and the lower bottom surface, an orifice plate (1-5) is arranged on the step in a matched mode, and a drain hole (1-4) is arranged on the side wall of the container body between the lower bottom surface and the orifice plate;
the upper end of the shading, rain shielding and dust preventing cover (2) is provided with an elastic tying (2-1), the lower end of the shading, rain shielding and dust preventing cover is provided with a water outlet (2-2), and binding ropes (2-3) are matched with the elastic tying (2-1); the shading, rain shielding and dust preventing cover (2) is made of black and white reflective film materials, and the white color is outwards and the black color is inwards;
the water guide device (3) is composed of a hollow bent pipe (3-1) and a three-leg guide bracket (3-2) fixedly connected with the bent pipe, and a water guide groove (3-3) is arranged below the three-leg guide bracket (3-2); an inoculation hole cover (1-6) is arranged on the inoculation hole (1-3) in a matching way, and an inner buckle (1-7) and an outer buckle (1-7) for fixing the inoculation hole cover on the container body (1) are arranged at the edge of the inoculation hole cover (1-6);
2) Sterile seedling cultivation: culturing pinus massoniana aseptic seedlings in the container body (1);
3) And (3) preparation of a microbial inoculum: preparing an orange liquid lactobacillus preparation;
4) Inoculating: inoculating in autumn and winter for 10-11 months, wherein the inoculation method comprises the following steps: after the aseptic seedlings of masson pine have lateral roots for development, taking down an inoculation hole cover (1-6), adopting a puncher to downwards insert 5-10cm holes at the inoculation hole (1-2) of the container body, and inoculating 5-10ml of orange liquid fungus agent into each hole; fixing the inoculation hole cover (1-6) at the inoculation hole through the inner and outer buckles (1-7) for sealing, and uniformly sprinkling the nutrient solution to the seedling substrate from the upper part of the container by about 300-500ml;
5) Adding seedling raising matrix: adding the sterilized seedling substrate again above the seedling substrate sprayed with the nutrient solution until the container body (1) is full, and keeping the surface of the substrate slightly steamed bread-shaped;
6) Installing a water guide device and a shading, rain shielding and dust preventing cover: placing a water guide device (3) on a seedling raising substrate which is slightly steamed bread-shaped, requiring an elbow pipe (3-1) to be abutted against the stem part of the sterile seedling, covering a shading, rain-shielding and dust-proof cover (2) on a container body (1), enabling the rhizome of the sterile seedling and the elbow pipe (3-1) of the water guide device (3) to pass through an elastic tying (2-1) and be positioned at the outer side of the shading, rain-shielding and dust-proof cover (2), and tying a tying rope (4) at the position of the elastic tying (2-1) by using the tying rope (2-3);
7) Culturing mycorrhizal seedlings: the mycorrhizal seedlings of the device provided with the water guide device and the light shielding, rain shielding and dust preventing cover are placed in the open air for cultivation; watering is not carried out for 2 weeks after inoculation, then water is irrigated according to the water content condition of the matrix, and the water content of the matrix is maintained to be 60-70% for culture; the irrigation water source is from underground deep well water.
2. The method for cultivating the orange lactiferous mushroom mycorrhizal seedlings according to claim 1, wherein the cultivation method of the pinus massoniana aseptic seedlings in the step 2) is as follows: soaking Pinus massoniana seeds in tap water, draining, removing inferior seeds, soaking in 0.1% potassium permanganate for 1 hr for surface sterilization, washing with sterile water for 3 times, sowing in a container body (1) filled with seedling substrate, maintaining the water content of the substrate at 60-70%, and culturing in a greenhouse or greenhouse; the water source is deep well water; the seedling substrate needs to be sterilized at 121 ℃ and 0.1MPa for 1h.
3. A method for cultivating Rumex citri mycorrhiza seedlings according to claim 2, wherein: the formula of the seedling substrate is vermiculite: the volume ratio of the sandy loam is 1:1, and the natural PH value is achieved.
4. The method for cultivating Rumex citri mycorrhiza seedlings according to claim 1, wherein the preparation method of the Rumex citri liquid microbial inoculum in the step 3) is as follows:
1) Separating strain tissue: collecting orange Lactarius rupestris fruiting body, taking back to laboratory for tissue separation, collecting mung bean size tissue at joint position of fungus cover and fungus handle, culturing on solid culture medium for one week, and collecting mycelium for purification culture;
2) And (5) propagation of strains: taking cultured strains, placing strain blocks into a liquid culture medium, shaking the liquid culture medium for culturing at 25 ℃ at a rotating speed of 140r/min, and culturing at 25d for later use;
3) Preparing a liquid microbial inoculum: filtering the bacterial liquid in the step 2) to obtain bacterial balls, adding a liquid culture medium, and mixing the bacterial balls and the liquid culture medium according to a volume ratio of 1:1; then adding sodium alginate into the mixed solution, and crushing and uniformly stirring to prepare a liquid microbial inoculum; the sodium alginate is added according to the amount of 3-5g per 1L mixed solution.
5. The method for cultivating Rumex citri mycorrhiza seedlings according to claim 4, wherein: the formula of the liquid culture medium comprises the following components: boiling potato 200g, filtering to obtain juice, adding glucose 20g, brown sugar 5g, potassium dihydrogen phosphate 1.5 g, magnesium sulfate 1g, ammonium chloride 1g, peptone 1g,1% ferric citrate 0.5ml,0.1% zinc sulfate 1ml, and vitamin B1 10mg, adding water to volume to 1000ml; the solid culture medium is prepared by adding agar 20g into the liquid culture medium according to each 1L.
6. A method for cultivating Rumex citri mycorrhiza seedlings according to claim 1, wherein: the hole puncher in the step 4) is composed of a hole punching end (4-1) and a handle end (4-2), wherein the hole punching end is flat and sharp, has a length of 5-6cm, a width of 1.5-1.8cm, a handle end is cylindrical, a length of 8-12cm and a diameter of 1.5-1.8cm.
7. A method for cultivating Rumex citri mycorrhiza seedlings according to claim 1, wherein: the distance between the lower bottom surface of the container body and the orifice plate (1-5) is 1-2cm, namely the height of the step (1-3) is 1-2cm.
8. A method for cultivating Rumex citri mycorrhizal seedlings according to claim 1 or 2, characterized in that: the deep well water is derived from underground water with the depth of 15m or less.
9. A method for cultivating Rumex citri mycorrhiza seedlings according to claim 1, wherein: the inoculation holes on the side wall of the container body are circular, the diameter of each hole is 1.5-2cm, the number of the inoculation holes is 5, the inoculation holes are uniformly distributed in a ring shape, and the inoculation holes are arranged at the middle upper part of the container body.
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