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CN108112298A - A kind of breeding method of Mycorrhizal Fungi of Pinus massoniana nursery stock - Google Patents

A kind of breeding method of Mycorrhizal Fungi of Pinus massoniana nursery stock Download PDF

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CN108112298A
CN108112298A CN201711383456.0A CN201711383456A CN108112298A CN 108112298 A CN108112298 A CN 108112298A CN 201711383456 A CN201711383456 A CN 201711383456A CN 108112298 A CN108112298 A CN 108112298A
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seedling
nursery stock
soil
fungi
culture
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辜夕容
倪亚兰
江亚男
贾豪
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Southwest University
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    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01GHORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G17/00Cultivation of hops, vines, fruit trees, or like trees
    • A01G17/005Cultivation methods
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01CPLANTING; SOWING; FERTILISING
    • A01C1/00Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01CPLANTING; SOWING; FERTILISING
    • A01C1/00Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
    • A01C1/08Immunising seed
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01GHORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G7/00Botany in general
    • A01G7/06Treatment of growing trees or plants, e.g. for preventing decay of wood, for tingeing flowers or wood, for prolonging the life of plants
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14Fungi; Culture media therefor

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Abstract

The invention discloses a kind of breeding methods of Mycorrhizal Fungi of Pinus massoniana nursery stock, comprise the following steps:Seed treatment, soil treatment prepare Applying Ectomycorrhizal Fungi bush mycorrhiza agent, are inoculated with using Applying Ectomycorrhizal Fungi bush mycorrhiza agent, seedling management, outplanting, cultivate Mycorrhizal Fungi of Pinus massoniana nursery stock.Ectomycorrhizal fungi-inoculated of the present invention significantly improves upgrowth situation, the Nutrient Absorption of masson pine, promotes the growth of seedling, especially promotes the absorption of phosphorus element.

Description

A kind of breeding method of Mycorrhizal Fungi of Pinus massoniana nursery stock
Technical field
The invention belongs to the cultivation fields of Mycorrhizal Fungi of Pinus massoniana nursery stock, specifically, are related to a kind of Mycorrhizal Fungi of Pinus massoniana seedling The breeding method of wood.
Background technology
Exotrophic mycorrhiza (Ectomycorrhizae, ECM) is the most important mycorrhizal type of forest, is Applying Ectomycorrhizal Fungi The homobium of (Ectomycorrhizae Fungi, ECMF) and plant nutrient root.In this homobium, ECMF absorbs nutrient It is supplied with moisture needed for host, at the same the plant supply necessary carbohydrate of ECMF and other nutriments, the two interdependence And high unity.The plant in the whole world about 97% is mycorrhizal plants, and wherein arbor accounts for the overwhelming majority, such as Pinaceae (Pinaceae), Cupressaceae (Cupressaceae), Salicaceae (Salicaceae), Betulaceae (Betulacele), Fagaceae (Fagaceae) the over one hundred platymiscium of 34 sections such as, and these seeds are mostly important commerical tree species.Therefore, exotrophic mycorrhiza is improving plant Very big influence is also generated while nutrition to forest ecosystem.Early in the fifties in last century, (the Harley such as Harley J.L,McCready C.C.Uptake of phosphate by excised mycorrhizal roots of beech [J].New Phytol,1950,49:388.) find that Va Mycorrhiza Seedling is stronger 2.3-8.9 than non-Va Mycorrhiza Seedling to the absorbability of phosphorus Times, more and more researchs afterwards find that some mycorrhizal fungis can promote absorption and utilization (Hodge A.The of the host to phosphorus plastic plant:root responses to heterogeneous supplies of nutrients[J].New Phytologist,2004,162:9-24.)。Soleimanzadel(Soleimanzadeh.H.Response of Sunflower(Helianthus annuus L.)To Inoculation with Mycorrhiza Under Different Phosphorus Levels[J].American-Eurasian J.Agric.&Environ.Sci.,2012,12(3):337- 341.) to think, mycorhiza can substitute chemical phosphatic ferfilizer and be used for organic agriculture, and inoculation in the seedbed ECMF can improve seedling quality, Improve the surrival rate of afforestation.
Masson pine (Pinus massoniana Lamb.) belongs to gymnosperm, arbor, Pinaceae Pinus.It is Southwestern China The main reproducting tree species in area forest land.Mainly using field seedling raising mode, but since soil-borne disease seriously causes which nursery Survival rate is low, slow-growing.And it is very big with seedling quantity to afforest, and current seedling-raising technique link is weak, therefore improve masson pine children The yield and quality of seedling is the task of top priority of nursery work.Such as effective Applying Ectomycorrhizal Fungi is accessed in masson pine seedling stage (ectomycorrhizal fungi, ECMF) can facilitate, is economical, being effectively facilitated absorption and profit of the nursery stock to nutrients such as phosphorus With promotion seedling growth improves resistance, then improves artificial afforestration survival rate.At present, Mycorrhizal technology is by the U.S., method The states such as state, Canada, Australia, Norway are classified as the Modern Forestry biotechnology carried out extensively.
The content of the invention
In view of this, the present invention is directed to the problem of above-mentioned, provides a kind of breeding method of Mycorrhizal Fungi of Pinus massoniana nursery stock.
In order to solve the above-mentioned technical problem, the invention discloses a kind of breeding method of Mycorrhizal Fungi of Pinus massoniana nursery stock, including Following steps:
Step 1, seed treatment:The uniform masson pine seed of no disease and pests harm, full grains is selected, is in mass concentration It is clean with pure water rinsing after 25-35min progress surface sterilizations are shaken in the hydrogen peroxide of 25%-35%, it is placed in 35-45 DEG C of warm water It is middle immersion 20-28 it is small when, be then placed into the culture dish equipped with perlite and carry out vernalization, broadcast after more than 90% seed shows money or valuables one carries unintentionally Kind;
Step 2, soil treatment:Acid cold sand yellow earth under masson pine forest is selected to try soil as confession, soil is through 2% formaldehyde Solution disinfection is sterilized, and film covering 7d takes off film and teds and dries, spare after 7d;
Step 3 prepares Applying Ectomycorrhizal Fungi bush mycorrhiza agent;
Step 4, the inoculation of Applying Ectomycorrhizal Fungi bush mycorrhiza agent:After seedbed spy is flattened before sowing, paving 15cm it is thick through 2% Formaldehyde sterilizes and soil under the masson pine forest of sieving;By inoculum with through 2% formaldehyde disinfection soil be mixed into paste, then with process The masson pine seed of vernalization is mixed thoroughly, is sprinkled into nursery soil, and every square metre is broadcasted sowing 200 masson pine seeds;After planting again at it One layer of fine earth is covered on surface, is advisable with loseing seed;Then seedbed is spilt permeable;
Step 5, seedling management:Seedling management is with reference to GB6001-1985《Technical regulations for cultivation of tree seedlings》It performs;
Step 6, outplanting cultivate Mycorrhizal Fungi of Pinus massoniana nursery stock.
Further, it is as follows for the basic physical and chemical of examination soil in step 2:The content of N is 1.14g/ in Total Nutrient The content of kg, P are 0.16g/kg, and the content of K is 19.09g/kg;In available nutrient the content of N be 69.75mg/kg, the content of P For 4.55mg/kg, the content of K is 71.21mg/kg;The content of Ca is 3.45cmol/kg in exchangeability salt, and the content of Mg is The content of 0.34cmol/kg, K are 0.14cmol/kg.
Further, in step 3 to prepare Applying Ectomycorrhizal Fungi bush mycorrhiza agent specific as follows:
The separation and culture of step 3.1, Applying Ectomycorrhizal Fungi:Choose fresh, tender, the non-parachute-opening of children, no disease and pests harm it is complete With 75% cotton ball soaked in alcohol surface sterilization, after the volatilization of surface alcohol, fructification stem cap junction is broken into two with one's hands for fructification, The fungus block of 0.2cm-0.3cm sizes is taken inside the fructification broken into two with one's hands with the scalpel that alcohol and flame disinfection are crossed, PDA is connected on and puts down On plate;Tablet is put into 25 DEG C of cultures in growth cabinet;Pollution is checked whether there is after 3 days, according to Applying Ectomycorrhizal Fungi mycelia The features such as colony morphology characteristic that whether body directly grows from inoculation block, the speed of growth and fungi of the same race are formed is consistent, just Step judges whether separated strain is Applying Ectomycorrhizal Fungi pure culture, and its growth feature is observed in detail;Screening is without dirt The good each separation strain of dye, colony morphology characteristic further carries out switching purifying in PDA culture medium, per 7d once, until Its growth is stablized;Preservation is spare at 4 DEG C;
The exotrophic mycorrhiza strain isolated and purified in step 3.1 is transferred to Pachlewski solid mediums by step 3.2 On, light culture 2 weeks under constant temperature are spare as female bacterium;
Step 3.3, inoculum culture:Under super-clean bench, the strain of activation is transferred to punch method after high-temperature sterilization Pachlewski fluid nutrient mediums on, female bacterium fungus block 4 that every bottle of access area is 5mm × 5mm, quiescent culture 2 at room temperature Obtain mycelium in week;After the completion of culture, by mycelium, low speed breaks up 1min in beater, collects mycelium suspended liquid, as connecing Kind body.
Further, the PDA culture medium is prepared by the following method to obtain:After 200g potatos are cleaned peeling chopping Water 1000ml is added to boil 30min, with after filtered through gauze again plus glucose 20g, agar 20g fully dissolve, 121 DEG C of high-temperature sterilizations It is taken out after 30min spare.
Further, the formula of the Pachlewski solid mediums is:Include ammonium tartrate per 1L culture mediums 0.5g, potassium dihydrogen phosphate 1.0g, magnesium sulfate 0.5g, glucose 20g, vitamin B10.001g, agar 20g, trace element mixing Liquid 1mL;Every liter of micro-mixed liquor contains H3BO3 8.45mg、MnSO4 5mg、FeSO4 6mg、CuSO4 0.625mg、ZnCl2 2.77mg and (NH4)2MoO4 0.27mg。
Further, Applying Ectomycorrhizal Fungi is one kind in Pt 715, Ld 2 or Ld3 bacterium.
Further, seedling management reference GB6001-1985 in step 5《Technical regulations for cultivation of tree seedlings》It performs, is specially:
Step 5.1, seedbed management:Watering in due course keeps seedbed moistening;Irrigation and drainage facility is carried out simultaneously, ensures that water can be uniformly Each cell seedbed is assigned to, and without using any fungicide during experiment;
Step 5.2, each seedling stage points for attention:
A) sprouting stage is planted:It after planting arrives seed and spits bud, it is necessary to which discharging raticide prevents the plague of rats;Control is irrigated, and seedbed keeps wet Profit;
B) the sprout phase:I.e. chitting piece is unearthed, but plants shell and not yet come off, and pays attention to anti-bird pest;Take a small amount of repeatedly irrigation Measure;
C) the leaf phase:I.e. seedling apical grows strip protophyll, to be loosened the soil, weeding;Few irrigation of volume is taken to arrange It applies;
Step 5.3, thinning:In order to reduce water consumption, seedling is promoted to grow up strong and sturdy, each cell growth of seedling can be waited steady It is fixed, i.e., after seedling goes out after homogeneous month, between overstocked thin and delicate seedling is dredged, while benefit cultivation is carried out to excessively sparse place;Often Square metre retain 140-160 plants of nursery stock;
Step 5.4, disease control:Pinus Massoniana Young Seedlings were unearthed in two months, since soil and air humidity increase, easily by Germ is infected.Phase most common disease is samping off, damping-off;Therefore frequently to observe in this period, promptly prevent;Once hair The existing state of an illness, sick seedling is pulled out burn immediately, is then sprayed with the Bordeaux mixture of 0.5%-1.0%, per 7d once;
Step 5.5, pest control:Dendrolimus punctatus is the masson pine pest of most serious, and eating pine needle seriously affects pine forest Growth;It is more convenient using chemical prevention and control method during nursery, i.e., 50% DDVP is diluted 2000 times;Or 50% Folithion Emulsion dilutes 1500 times.
Further, outplanting is specially in step 6:
Step 6.1, lifting:Lifting before luxuriant is not yet sprouted in spring, lifting need to be taken using scoop before, Ensure seedling root with place soil, it is impossible to directly with hand-lifting, accomplish:Hinder lateral root, fibrous root less, keep root system than more complete and not Fracture seedling stem, does not hinder terminal bud;Then root is packed with film;
Step 6.2, classification:Determine whether nursery stock is qualified according to Comprehensive Control condition, root system, ground diameter and height of seedling;Comprehensive control Condition processed is:Without quarantine object pest and disease damage, seedling stem leads to that straight, color and luster is normal;Qualified seedling is divided into I, II two grade;Seedling sorting Giving shade lee side;
Step 6.3, quality inspection:Seedling quality detection will carry out in a seedling batch after lifting, take random sampling Method carries out;Vernier caliper measurement ground diameter, accuracy 0.05cm;Ruler measures height of seedling and root system, accuracy 1cm;
Step 6.4, temporary planting:The place that good, the leeward cool place of draining to be selected cannot such as be transplanted after lifting in time carries out vacation It plants.
Step 6.5, packaging:Nursery stock weight per package is no more than 30kg, and often bag needs labelled;
Step 6.6, transport:Transportational process will take moisturizing, ventilative measure, prevent from exposing to the weather, and long-distance transport nursery stock will It is wrapped up with straw or straw bag.
Further, the nursery stock grade classification in step 6.2 see the table below:
Compared with prior art, the present invention can be obtained including following technique effect:
1) ectomycorrhizal fungi-inoculated of the present invention significantly improves upgrowth situation, the Nutrient Absorption of masson pine, promotes children The growth of seedling especially promotes the absorption of phosphorus element.
2) Ld2 growth-promoting effects are best in the present invention, can be as the preferable strain of later stage mycorrhizal seedling raising, to improve afforestation matter Amount and forest felling license accelerate the paces of China forest greening.
Certainly, implement any of the products of the present invention it is not absolutely required to while reach all the above technique effect.
Description of the drawings
Attached drawing described herein is used for providing a further understanding of the present invention, forms the part of the present invention, this hair Bright schematic description and description does not constitute improper limitations of the present invention for explaining the present invention.In the accompanying drawings:
Fig. 1 is the Mycorrhizal Infection Incidence of masson pine of the present invention;Different lowercase letter indication differences are notable (p < 0.05) in figure, Similarly hereinafter;
Fig. 2 is influence of the Applying Ectomycorrhizal Fungi of the present invention to Pinus Massoniana Young Seedlings phosphorus content;
Fig. 3 is influence of the Applying Ectomycorrhizal Fungi of the present invention to Pinus Massoniana Young Seedlings potassium content;
Fig. 4 is influence of the Applying Ectomycorrhizal Fungi of the present invention to Pinus Massoniana Young Seedlings calcium content;
Fig. 5 is influence of the Applying Ectomycorrhizal Fungi of the present invention to Pinus Massoniana Young Seedlings content of magnesium;
Fig. 6 is influence of the Applying Ectomycorrhizal Fungi of the present invention to Pinus Massoniana Young Seedlings iron content.
Specific embodiment
Carry out the embodiment that the present invention will be described in detail below in conjunction with embodiment, thereby to the present invention how application technology hand Section can fully understand and implement according to this to solve technical problem and reach the realization process of technical effect.
The invention discloses a kind of breeding methods of Mycorrhizal Fungi of Pinus massoniana nursery stock, comprise the following steps:
Step 1, seed treatment:The uniform masson pine seed of no disease and pests harm, full grains is selected, is in mass concentration It is clean with pure water rinsing after 25-35min progress surface sterilizations are shaken in the hydrogen peroxide of 25%-35%, it is placed in 35-45 DEG C of warm water It is middle immersion 20-28 it is small when, be then placed into the culture dish equipped with perlite and carry out vernalization, broadcast after more than 90% seed shows money or valuables one carries unintentionally Kind;
Step 2, soil treatment:Disappear for examination soil for acid cold sand yellow earth, soil under masson pine forest through 2% formalin Bacterium is killed, film covering 7d takes off film and teds and dries, spare after 7d.1 is shown in Table for examination physiochemical properties of soil.
The basic physical and chemical of 1 soil of table
Step 3 prepares Applying Ectomycorrhizal Fungi bush mycorrhiza agent:
The separation and culture of step 3.1, Applying Ectomycorrhizal Fungi:
Applying Ectomycorrhizal Fungi is one kind in Pt 715, Ld 2 or Ld3;
Isolation and Culture uses PDA culture medium:After 200g potatos are cleaned peeling chopping water 1000ml is added to boil 30min with again glucose 20g, agar 20g being added fully to dissolve after filtered through gauze, takes out spare after 121 DEG C of high-temperature sterilization 30min. All mask works are completed on superclean bench.Fresh, tender, the non-parachute-opening of children, the complete fructification of no disease and pests harm are chosen, is used 75% cotton ball soaked in alcohol surface sterilization, after surface alcohol volatilization after, fructification stem and cap junction are broken into two with one's hands, with alcohol with The scalpel that flame disinfection is crossed takes the fungus block of 0.2cm-0.3cm sizes inside the fructification broken into two with one's hands, is seeded on PDA plate. Tablet is put into 25 DEG C of cultures in growth cabinet.Pollution is checked whether there is after 3 days, according to Applying Ectomycorrhizal Fungi mycelium whether Directly from inoculation block on grow, the speed of growth and fungi of the same race formed colony morphology characteristic it is consistent the features such as, preliminary judgement Whether separated strain is Applying Ectomycorrhizal Fungi pure culture, and its growth feature is carried out to observe and record in detail.Screening is without dirt The good each separation strain of the features such as dye, colonial morphology further carries out switching purifying in PDA culture medium, per 7d once, directly Stablize to its growth.Preservation is spare at 4 DEG C.
Step 3.2, female bacterium culture:The exotrophic mycorrhiza strain isolated and purified in step 3.1 is transferred to Pachlewski to consolidate On body culture medium, light culture 2 weeks under constant temperature are spare as female bacterium.Pachlewski solid culture based formulas is shown in Table 2.
Table 2Pachlewski solid mediums form
Note:Every liter of micro-mixed liquor contains H3BO3 8.45mg、MnSO4 5mg、FeSO4 6mg、CuSO4 0.625mg、 ZnCl22.77mg and (NH4)2MoO4 0.27mg。
Step 3.3, inoculum culture:Under super-clean bench, the strain of activation is transferred to punch method after high-temperature sterilization Pachlewski fluid nutrient mediums on, female bacterium fungus block 4 that every bottle of access area is 5mm × 5mm, quiescent culture 2 at room temperature Obtain mycelium in week.After the completion of culture, by mycelium, low speed breaks up 1min in beater, collects mycelium suspended liquid, as connecing Kind body.
Step 4, bush mycorrhiza agent inoculation:Nursery is carried out March, after seedbed spy is flattened before sowing, paving about 15cm is thick Through 2% formaldehyde sterilize and sieve masson pine forest under soil,
Masson pine inoculation is using dressing methods.Pt 715, Ld 2, Ld3 inoculum mycelia are broken up, with being sterilized through 2% formaldehyde Soil be mixed into paste, then mix thoroughly, be sprinkled into respectively in corresponding nursery soil, every square with the masson pine seed Jing Guo vernalization Rice broadcasts sowing about 200 masson pine seeds.One layer of fine earth is after planting covered on its surface again, is advisable with loseing seed.Then seedbed is spilt It is permeable.
Step 5, seedling management:Mycorrhizal seedlings seedling management is with reference to GB6001-1985《Technical regulations for cultivation of tree seedlings》It performs.
Step 5.1, seedbed management:Watering in due course keeps seedbed moistening.Irrigation and drainage facility is carried out simultaneously, ensures that water can be uniformly Each cell seedbed is assigned to, and without using any fungicide during experiment.
Step 5.2, each seedling stage points for attention:
A) sprouting stage is planted:It after planting arrives seed and spits bud, it is necessary to which discharging raticide prevents the plague of rats.Control is irrigated, and seedbed keeps wet Profit.
B) the sprout phase:I.e. chitting piece is unearthed, but plants shell and not yet come off, and pays attention to anti-bird pest.Take a small amount of repeatedly irrigation Measure.
C) the leaf phase:I.e. seedling apical grows strip protophyll, to be loosened the soil, weeding.Few irrigation of volume is taken to arrange It applies.
Step 5.3, thinning:In order to reduce water consumption, seedling is promoted to grow up strong and sturdy, each cell growth of seedling can be waited steady It is fixed, i.e., after seedling goes out after homogeneous month, between overstocked thin and delicate seedling is dredged, while benefit cultivation is carried out to excessively sparse place.Often Square metre retain 140-160 plants of nursery stock.
Step 5.4, disease control:Pinus Massoniana Young Seedlings were unearthed in two months, since soil and air humidity increase, easily by Germ is infected.Phase most common disease is samping off, damping-off.Therefore frequently to observe in this period, promptly prevent.Once hair The existing state of an illness, sick seedling is pulled out burn immediately, is then sprayed with the Bordeaux mixture of 0.5%-1.0%, per 7d once.
Step 5.5, pest control:Dendrolimus punctatus is the masson pine pest of most serious, and eating pine needle seriously affects pine forest Growth.It is more convenient using chemical prevention and control method during nursery, you can 50% DDVP is diluted 2000 times;Or 50% kill snout moth's larva Loose emulsion dilutes 1500 times.
Step 6, outplanting:
Step 6.1, lifting:Lifting before luxuriant is not yet sprouted in spring.It needs to be taken using scoop before lifting, Ensure seedling root with place soil, it is impossible to directly with hand-lifting, accomplish:Hinder lateral root, fibrous root less, keep root system than more complete and not Fracture seedling stem, does not hinder terminal bud.Then root is packed with film, so can also while effectively root tissue water status being avoided to evaporate It carries a large amount of mycorhiza soil to afforest, seedling-slowing stage is short after afforestation, and the speed of growth is fast.
Step 6.2, classification:
A) determine whether nursery stock is qualified according to Comprehensive Control condition, root system, ground diameter and height of seedling.Comprehensive Control condition is:Nothing Quarantine object pest and disease damage, seedling stem lead to that straight, color and luster is normal.
B) qualified seedling is divided into I, II two grade, nursery stock grade classification is shown in Table 3.
3 seedling quality table of grading of table
C) seedling sorting must give shade lee side, and grade mark is carried out after classification.
Step 6.3, quality inspection:Seedling quality detection will carry out in a seedling batch after lifting, take random sampling Method carries out.Each cell of this research is randomly selected 30 plants and is detected.Vernier caliper measurement ground diameter, accuracy 0.05cm;Ruler Measure height of seedling and root system, accuracy 1cm.
Step 6.4, temporary planting:The place that good, the leeward cool place of draining to be selected cannot such as be transplanted after lifting in time carries out vacation It plants.
Step 6.5, packaging:Nursery stock weight per package is no more than 30kg, and often bag needs labelled, and label substance includes:Seeds, Nursery stock species, seedling age, standard code, credit rating, quantity, lifting date.
Step 6.6, transport:Transportational process will take moisturizing, ventilative measure, prevent from exposing to the weather, and long-distance transport nursery stock will It is wrapped up with straw or straw bag.
Embodiment 1
Step 1, seed treatment:Masson pine seed is provided by Chongqing City achievements in forest-tree seedling station, selects no disease and pests harm, full grains Uniform masson pine seed, it is clean with pure water rinsing after 30min progress surface sterilizations are shaken in 30% hydrogen peroxide, it is placed in 40 Impregnated in DEG C warm water 24 it is small when, be then placed into the culture dish equipped with perlite and carry out vernalization, after most of seed shows money or valuables one carries unintentionally Sowing.
Step 2, soil treatment:It is the acid cold sand yellow earth of Chongqing City's Pinus Massoniana At Jinyun Mountain, Sichuan Province hayashishita for examination soil, soil is through 2% Formalin disinfection, film covering 7d, take off film and ted and dry, it is spare after 7d.1 is shown in Table for examination physiochemical properties of soil.
Step 3 prepares Applying Ectomycorrhizal Fungi bush mycorrhiza agent:
The separation and culture of step 3.1, Applying Ectomycorrhizal Fungi:Applying Ectomycorrhizal Fungi is Pt 715;
Isolation and Culture uses PDA culture medium:After 200g potatos are cleaned peeling chopping water 1000ml is added to boil 30min with again glucose 20g, agar 20g being added fully to dissolve after filtered through gauze, takes out spare after 121 DEG C of high-temperature sterilization 30min. All mask works are completed on superclean bench.Fresh, tender, the non-parachute-opening of children, the complete fructification of no disease and pests harm are chosen, is used 75% cotton ball soaked in alcohol surface sterilization after the volatilization of surface alcohol, fructification stem cap junction is broken into two with one's hands, with alcohol and fire The scalpel that flame sterilized takes the fungus block of 0.2cm-0.3cm sizes inside the fructification broken into two with one's hands, is connected on PDA plate.It will be flat Plate is put into 25 DEG C of cultures in growth cabinet.Pollution is checked whether there is after 3 days, it is whether direct according to Applying Ectomycorrhizal Fungi mycelium From inoculation block on grow, the speed of growth and fungi of the same race formed colony morphology characteristic it is consistent the features such as, preliminary judgement divides Whether it is Applying Ectomycorrhizal Fungi pure culture from strain, its growth feature is carried out to observe and record in detail.Screen pollution-free, bacterium Fall the good each separation strain of the features such as form and switching purifying is further carried out in PDA culture medium, until its life per 7d once It is long to stablize.Preservation is spare at 4 DEG C.
Step 3.2, female bacterium culture:The exotrophic mycorrhiza strain isolated and purified in step 3.1 is transferred to Pachlewski to consolidate On body culture medium, light culture 2 weeks under constant temperature are spare as female bacterium.Pachlewski solid culture based formulas is shown in Table 2.
Step 3.3, inoculum culture:Under super-clean bench, the strain of activation is transferred to punch method after high-temperature sterilization Pachlewski fluid nutrient mediums on, female bacterium fungus block 4 that every bottle of access area is 5mm × 5mm, quiescent culture 2 at room temperature Obtain mycelium in week.After the completion of culture, by mycelium, low speed breaks up 1min in beater, collects mycelium suspended liquid, as connecing Kind body.
Step 4, bush mycorrhiza agent inoculation:Nursery is carried out March, after seedbed spy is flattened before sowing, paving about 15cm is thick Through 2% formaldehyde sterilize and sieve masson pine forest under soil,
Masson pine inoculation is using dressing methods.By inoculum with through 2% formaldehyde disinfection soil be mixed into paste, then with process The masson pine seed of vernalization is mixed thoroughly, is sprinkled into respectively in corresponding nursery soil, and every square metre is broadcasted sowing about 200 masson pine seeds. One layer of fine earth is after planting covered on its surface again, is advisable with loseing seed.Then seedbed is spilt permeable.
Step 5, seedling management:Mycorrhizal seedlings seedling management is with reference to GB6001-1985《Technical regulations for cultivation of tree seedlings》It performs.
Step 5.1, seedbed management:Watering in due course keeps seedbed moistening.Irrigation and drainage facility is carried out simultaneously, ensures that water can be uniformly Each cell seedbed is assigned to, and without using any fungicide during experiment.
Step 5.2, each seedling stage points for attention:
A) sprouting stage is planted:It after planting arrives seed and spits bud, it is necessary to which discharging raticide prevents the plague of rats.Control is irrigated, and seedbed keeps wet Profit.
B) the sprout phase:I.e. chitting piece is unearthed, but plants shell and not yet come off, and pays attention to anti-bird pest.Take a small amount of repeatedly irrigation Measure.
C) the leaf phase:I.e. seedling apical grows strip protophyll, to be loosened the soil, weeding.Few irrigation of volume is taken to arrange It applies.
Step 5.3, thinning:In order to reduce water consumption, seedling is promoted to grow up strong and sturdy, each cell growth of seedling can be waited steady It is fixed, i.e., after seedling goes out after homogeneous month, between overstocked thin and delicate seedling is dredged, while benefit cultivation is carried out to excessively sparse place.Often Square metre retain 140-160 plants of nursery stock.
Step 5.4, disease control:Pinus Massoniana Young Seedlings were unearthed in two months, since soil and air humidity increase, easily by Germ is infected.Phase most common disease is samping off, damping-off.Therefore frequently to observe in this period, promptly prevent.Once hair The existing state of an illness, sick seedling is pulled out burn immediately, is then sprayed with the Bordeaux mixture of 0.5%-1.0%, per 7d once.
Step 5.5, pest control:Dendrolimus punctatus is the masson pine pest of most serious, and eating pine needle seriously affects pine forest Growth.It is more convenient using chemical prevention and control method during nursery, you can 50% DDVP is diluted 2000 times;Or 50% kill snout moth's larva Loose emulsion dilutes 1500 times.
Step 6, outplanting:
Step 6.1, lifting:Lifting before luxuriant is not yet sprouted in spring.It needs to be taken using scoop before lifting, Ensure seedling root with place soil, it is impossible to directly with hand-lifting, accomplish:Hinder lateral root, fibrous root less, keep root system than more complete and not Fracture seedling stem, does not hinder terminal bud.Then root is packed with film, so can also while effectively root tissue water status being avoided to evaporate It carries a large amount of mycorhiza soil to afforest, seedling-slowing stage is short after afforestation, and the speed of growth is fast.
Step 6.2, classification:
A) determine whether nursery stock is qualified according to Comprehensive Control condition, root system, ground diameter and height of seedling.Comprehensive Control condition is:Nothing Quarantine object pest and disease damage, seedling stem lead to that straight, color and luster is normal.
B) qualified seedling is divided into I, II two grade, nursery stock grade classification is shown in Table 3.
C) seedling sorting must give shade lee side, and grade mark is carried out after classification.
Step 6.3, quality inspection:Seedling quality detection will carry out in a seedling batch after lifting, take random sampling Method carries out.Each cell of this research is randomly selected 30 plants and is detected.Vernier caliper measurement ground diameter, accuracy 0.05cm;Ruler Measure height of seedling and root system, accuracy 1cm.
Step 6.4, temporary planting:The place that good, the leeward cool place of draining to be selected cannot such as be transplanted after lifting in time carries out vacation It plants.
Step 6.5, packaging:Nursery stock weight per package is no more than 30kg, and often bag needs labelled, and label substance includes:Seeds, Nursery stock species, seedling age, standard code, credit rating, quantity, lifting date.
Step 6.6, transport:Transportational process will take moisturizing, ventilative measure, prevent from exposing to the weather, and long-distance transport nursery stock will It is wrapped up with straw or straw bag.
Embodiment 2
Step 1, seed treatment:The uniform masson pine seed of no disease and pests harm, full grains is selected, is 25% in mass concentration Hydrogen peroxide in shake 35min carry out surface sterilization after, it is clean with pure water rinsing, be placed in 35 DEG C of warm water impregnate 28 it is small when, so It is placed into afterwards in the culture dish equipped with perlite and carries out vernalization, sowed after more than 90% seed shows money or valuables one carries unintentionally;
Step 2, soil treatment:Disappear for examination soil for acid cold sand yellow earth, soil under masson pine forest through 2% formalin Bacterium is killed, film covering 7d takes off film and teds and dries, spare after 7d.1 is shown in Table for examination physiochemical properties of soil.
Step 3 prepares Applying Ectomycorrhizal Fungi bush mycorrhiza agent:
The separation and culture of step 3.1, Applying Ectomycorrhizal Fungi:Applying Ectomycorrhizal Fungi is Ld 2;
Isolation and Culture uses PDA culture medium:After 200g potatos are cleaned peeling chopping water 1000ml is added to boil 30min with again glucose 20g, agar 20g being added fully to dissolve after filtered through gauze, takes out spare after 121 DEG C of high-temperature sterilization 30min. All mask works are completed on superclean bench.Fresh, tender, the non-parachute-opening of children, the complete fructification of no disease and pests harm are chosen, is used 75% cotton ball soaked in alcohol surface sterilization after the volatilization of surface alcohol, fructification stem cap junction is broken into two with one's hands, with alcohol and fire The scalpel that flame sterilized takes the fungus block of 0.2cm-0.3cm sizes inside the fructification broken into two with one's hands, is connected on PDA plate.It will be flat Plate is put into 25 DEG C of cultures in growth cabinet.Pollution is checked whether there is after 3 days, it is whether direct according to Applying Ectomycorrhizal Fungi mycelium From inoculation block on grow, the speed of growth and fungi of the same race formed colony morphology characteristic it is consistent the features such as, preliminary judgement divides Whether it is Applying Ectomycorrhizal Fungi pure culture from strain, its growth feature is carried out to observe and record in detail.Screen pollution-free, bacterium Fall the good each separation strain of the features such as form and switching purifying is further carried out in PDA culture medium, until its life per 7d once It is long to stablize.Preservation is spare at 4 DEG C.
Step 3.2, female bacterium culture:The exotrophic mycorrhiza strain isolated and purified in step 3.1 is transferred to Pachlewski to consolidate On body culture medium, light culture 2 weeks under constant temperature are spare as female bacterium.Pachlewski solid culture based formulas is shown in Table 2.
Step 3.3, inoculum culture:Under super-clean bench, the strain of activation is transferred to punch method after high-temperature sterilization Pachlewski fluid nutrient mediums on, female bacterium fungus block 4 that every bottle of access area is 5mm × 5mm, quiescent culture 2 at room temperature Obtain mycelium in week.After the completion of culture, by mycelium, low speed breaks up 1min in beater, collects mycelium suspended liquid, as connecing Kind body.
Step 4, bush mycorrhiza agent inoculation:Nursery is carried out March, after seedbed spy is flattened before sowing, paving about 15cm is thick Through 2% formaldehyde sterilize and sieve masson pine forest under soil,
Masson pine inoculation is using dressing methods.By inoculum with through 2% formaldehyde disinfection soil be mixed into paste, then with process The masson pine seed of vernalization is mixed thoroughly, is sprinkled into respectively in corresponding nursery soil, and every square metre is broadcasted sowing about 200 masson pine seeds. One layer of fine earth is after planting covered on its surface again, is advisable with loseing seed.Then seedbed is spilt permeable.
Step 5, seedling management:Mycorrhizal seedlings seedling management is with reference to GB6001-1985《Technical regulations for cultivation of tree seedlings》It performs.
Step 5.1, seedbed management:Watering in due course keeps seedbed moistening.Irrigation and drainage facility is carried out simultaneously, ensures that water can be uniformly Each cell seedbed is assigned to, and without using any fungicide during experiment.
Step 5.2, each seedling stage points for attention:
A) sprouting stage is planted:It after planting arrives seed and spits bud, it is necessary to which discharging raticide prevents the plague of rats.Control is irrigated, and seedbed keeps wet Profit.
B) the sprout phase:I.e. chitting piece is unearthed, but plants shell and not yet come off, and pays attention to anti-bird pest.Take a small amount of repeatedly irrigation Measure.
C) the leaf phase:I.e. seedling apical grows strip protophyll, to be loosened the soil, weeding.Few irrigation of volume is taken to arrange It applies.
Step 5.3, thinning:In order to reduce water consumption, seedling is promoted to grow up strong and sturdy, each cell growth of seedling can be waited steady It is fixed, i.e., after seedling goes out after homogeneous month, between overstocked thin and delicate seedling is dredged, while benefit cultivation is carried out to excessively sparse place.Often Square metre retain 140-160 plants of nursery stock.
Step 5.4, disease control:Pinus Massoniana Young Seedlings were unearthed in two months, since soil and air humidity increase, easily by Germ is infected.Phase most common disease is samping off, damping-off.Therefore frequently to observe in this period, promptly prevent.Once hair The existing state of an illness, sick seedling is pulled out burn immediately, is then sprayed with the Bordeaux mixture of 0.5%-1.0%, per 7d once.
Step 5.5, pest control:Dendrolimus punctatus is the masson pine pest of most serious, and eating pine needle seriously affects pine forest Growth.It is more convenient using chemical prevention and control method during nursery, you can 50% DDVP is diluted 2000 times;Or 50% kill snout moth's larva Loose emulsion dilutes 1500 times.
Step 6, outplanting:
Step 6.1, lifting:Lifting before luxuriant is not yet sprouted in spring.It needs to be taken using scoop before lifting, Ensure seedling root with place soil, it is impossible to directly with hand-lifting, accomplish:Hinder lateral root, fibrous root less, keep root system than more complete and not Fracture seedling stem, does not hinder terminal bud.Then root is packed with film, so can also while effectively root tissue water status being avoided to evaporate It carries a large amount of mycorhiza soil to afforest, seedling-slowing stage is short after afforestation, and the speed of growth is fast.
Step 6.2, classification:
A) determine whether nursery stock is qualified according to Comprehensive Control condition, root system, ground diameter and height of seedling.Comprehensive Control condition is:Nothing Quarantine object pest and disease damage, seedling stem lead to that straight, color and luster is normal.
B) qualified seedling is divided into I, II two grade, nursery stock grade classification is shown in Table 3.
C) seedling sorting must give shade lee side, and grade mark is carried out after classification.
Step 6.3, quality inspection:Seedling quality detection will carry out in a seedling batch after lifting, take random sampling Method carries out.Each cell of this research is randomly selected 30 plants and is detected.Vernier caliper measurement ground diameter, accuracy 0.05cm;Ruler Measure height of seedling and root system, accuracy 1cm.
Step 6.4, temporary planting:The place that good, the leeward cool place of draining to be selected cannot such as be transplanted after lifting in time carries out vacation It plants.
Step 6.5, packaging:Nursery stock weight per package is no more than 30kg, and often bag needs labelled, and label substance includes:Seeds, Nursery stock species, seedling age, standard code, credit rating, quantity, lifting date.
Step 6.6, transport:Transportational process will take moisturizing, ventilative measure, prevent from exposing to the weather, and long-distance transport nursery stock will It is wrapped up with straw or straw bag.
Embodiment 3
Step 1, seed treatment:The uniform masson pine seed of no disease and pests harm, full grains is selected, is 35% in mass concentration Hydrogen peroxide in shake 25min carry out surface sterilization after, it is clean with pure water rinsing, be placed in 45 DEG C of warm water impregnate 20 it is small when, so It is placed into afterwards in the culture dish equipped with perlite and carries out vernalization, sowed after more than 90% seed shows money or valuables one carries unintentionally;
Step 2, soil treatment:Disappear for examination soil for acid cold sand yellow earth, soil under masson pine forest through 2% formalin Bacterium is killed, film covering 7d takes off film and teds and dries, spare after 7d.1 is shown in Table for examination physiochemical properties of soil.
Step 3 prepares Applying Ectomycorrhizal Fungi bush mycorrhiza agent:
The separation and culture of step 3.1, Applying Ectomycorrhizal Fungi:Applying Ectomycorrhizal Fungi is Ld3;
Isolation and Culture uses PDA culture medium:After 200g potatos are cleaned peeling chopping water 1000ml is added to boil 30min with again glucose 20g, agar 20g being added fully to dissolve after filtered through gauze, takes out spare after 121 DEG C of high-temperature sterilization 30min. All mask works are completed on superclean bench.Fresh, tender, the non-parachute-opening of children, the complete fructification of no disease and pests harm are chosen, is used 75% cotton ball soaked in alcohol surface sterilization after the volatilization of surface alcohol, fructification stem cap junction is broken into two with one's hands, with alcohol and fire The scalpel that flame sterilized takes the fungus block of 0.2cm-0.3cm sizes inside the fructification broken into two with one's hands, is connected on PDA plate.It will be flat Plate is put into 25 DEG C of cultures in growth cabinet.Pollution is checked whether there is after 3 days, it is whether direct according to Applying Ectomycorrhizal Fungi mycelium From inoculation block on grow, the speed of growth and fungi of the same race formed colony morphology characteristic it is consistent the features such as, preliminary judgement divides Whether it is Applying Ectomycorrhizal Fungi pure culture from strain, its growth feature is carried out to observe and record in detail.Screen pollution-free, bacterium Fall the good each separation strain of the features such as form and switching purifying is further carried out in PDA culture medium, until its life per 7d once It is long to stablize.Preservation is spare at 4 DEG C.
Step 3.2, female bacterium culture:The exotrophic mycorrhiza strain isolated and purified in step 3.1 is transferred to Pachlewski to consolidate On body culture medium, light culture 2 weeks under constant temperature are spare as female bacterium.Pachlewski solid culture based formulas is shown in Table 2.
Step 3.3, inoculum culture:Under super-clean bench, the strain of activation is transferred to punch method after high-temperature sterilization Pachlewski fluid nutrient mediums on, female bacterium fungus block 4 that every bottle of access area is 5mm × 5mm, quiescent culture 2 at room temperature Obtain mycelium in week.After the completion of culture, by mycelium, low speed breaks up 1min in beater, collects mycelium suspended liquid, as connecing Kind body.
Step 4, bush mycorrhiza agent inoculation:Nursery is carried out March, after seedbed spy is flattened before sowing, paving about 15cm is thick Through 2% formaldehyde sterilize and sieve masson pine forest under soil,
Masson pine inoculation is using dressing methods.By inoculum with through 2% formaldehyde disinfection soil be mixed into paste, then with process The masson pine seed of vernalization is mixed thoroughly, is sprinkled into respectively in corresponding nursery soil, and every square metre is broadcasted sowing about 200 masson pine seeds. One layer of fine earth is after planting covered on its surface again, is advisable with loseing seed.Then seedbed is spilt permeable.
Step 5, seedling management:Mycorrhizal seedlings seedling management is with reference to GB6001-1985《Technical regulations for cultivation of tree seedlings》It performs.
Step 5.1, seedbed management:Watering in due course keeps seedbed moistening.Irrigation and drainage facility is carried out simultaneously, ensures that water can be uniformly Each cell seedbed is assigned to, and without using any fungicide during experiment.
Step 5.2, each seedling stage points for attention:
A) sprouting stage is planted:It after planting arrives seed and spits bud, it is necessary to which discharging raticide prevents the plague of rats.Control is irrigated, and seedbed keeps wet Profit.
B) the sprout phase:I.e. chitting piece is unearthed, but plants shell and not yet come off, and pays attention to anti-bird pest.Take a small amount of repeatedly irrigation Measure.
C) the leaf phase:I.e. seedling apical grows strip protophyll, to be loosened the soil, weeding.Few irrigation of volume is taken to arrange It applies.
Step 5.3, thinning:In order to reduce water consumption, seedling is promoted to grow up strong and sturdy, each cell growth of seedling can be waited steady It is fixed, i.e., after seedling goes out after homogeneous month, between overstocked thin and delicate seedling is dredged, while benefit cultivation is carried out to excessively sparse place.Often Square metre retain 140-160 plants of nursery stock.
Step 5.4, disease control:Pinus Massoniana Young Seedlings were unearthed in two months, since soil and air humidity increase, easily by Germ is infected.Phase most common disease is samping off, damping-off.Therefore frequently to observe in this period, promptly prevent.Once hair The existing state of an illness, sick seedling is pulled out burn immediately, is then sprayed with the Bordeaux mixture of 0.5%-1.0%, per 7d once.
Step 5.5, pest control:Dendrolimus punctatus is the masson pine pest of most serious, and eating pine needle seriously affects pine forest Growth.It is more convenient using chemical prevention and control method during nursery, you can 50% DDVP is diluted 2000 times;Or 50% kill snout moth's larva Loose emulsion dilutes 1500 times.
Step 6, outplanting:
Step 6.1, lifting:Lifting before luxuriant is not yet sprouted in spring.It needs to be taken using scoop before lifting, Ensure seedling root with place soil, it is impossible to directly with hand-lifting, accomplish:Hinder lateral root, fibrous root less, keep root system than more complete and not Fracture seedling stem, does not hinder terminal bud.Then root is packed with film, so can also while effectively root tissue water status being avoided to evaporate It carries a large amount of mycorhiza soil to afforest, seedling-slowing stage is short after afforestation, and the speed of growth is fast.
Step 6.2, classification:
A) determine whether nursery stock is qualified according to Comprehensive Control condition, root system, ground diameter and height of seedling.Comprehensive Control condition is:Nothing Quarantine object pest and disease damage, seedling stem lead to that straight, color and luster is normal.
B) qualified seedling is divided into I, II two grade, nursery stock grade classification is shown in Table 3.
C) seedling sorting must give shade lee side, and grade mark is carried out after classification.
Step 6.3, quality inspection:Seedling quality detection will carry out in a seedling batch after lifting, take random sampling Method carries out.Each cell of this research is randomly selected 30 plants and is detected.Vernier caliper measurement ground diameter, accuracy 0.05cm;Ruler Measure height of seedling and root system, accuracy 1cm.
Step 6.4, temporary planting:The place that good, the leeward cool place of draining to be selected cannot such as be transplanted after lifting in time carries out vacation It plants.
Step 6.5, packaging:Nursery stock weight per package is no more than 30kg, and often bag needs labelled, and label substance includes:Seeds, Nursery stock species, seedling age, standard code, credit rating, quantity, lifting date.
Step 6.6, transport:Transportational process will take moisturizing, ventilative measure, prevent from exposing to the weather, and long-distance transport nursery stock will It is wrapped up with straw or straw bag.
Illustrate the technique effect of the present invention with reference to specific experimental data:
After cultivating Pinus Massoniana Young Seedlings 4 months according to above-mentioned nursery stock way to manage, random 30 plants of seedling taking wood is often handled per district's groups, Its Mycorrhizal Infection Incidence is detected, measures growth indexes:Root weight, Miao Chong, root/shoot ratio height of seedling and ground diameter measure Nutrient Absorption:P、K、Ca、 Mg、Fe.The data obtained carries out variance analysis with SPSS17.0 softwares, and SSR methods make the Multiple range test between data, and the level of signifiance is set For 0.05.Chart is made using Microsoft Office Excel 2007;Wherein, Pt 715, Ld 2 and Ld3 are corresponded to respectively Embodiment 1, embodiment 2 and embodiment 3;CK is is not added with bush mycorrhiza agent, remaining step is the same as embodiment 1.
First, the mycorhiza infection rate of Pinus massoniana Seedlings
After culture 4 months, 30 plants of seedling is taken to detect its Mycorrhizal Infection Incidence at random.Mycorrhizal Infection Incidence is according to the children with mycorhiza Seedling radical accounts for total percentage for extracting radical to calculate.
As shown in Figure 1,3 kinds of strains testeds to the infection rate of Pinus Massoniana Young Seedlings all up to more than 80%, reach aobvious compared with CK Write difference (p < 0.05).And different strains are different to the infection rate of Pinus Massoniana Young Seedlings, Ld2 highests, up to 90.41%, Pt715 times It, is that 89%, Ld3 is minimum, is 83.5%.And the control group of non-Arbuscular Mycorrhizal Fungi bacterium, non-bacterium germination root infect.Therefore 3 kinds of strains testeds Infection ability size be followed successively by Ld2 > Pt715 > Ld3.
2nd, the growth effects of mycorrhizal seedlings
As known from Table 4, it is inoculated with and is affected to Pinus massoniana Seedling Growth, in addition to height of seedling difference is not notable, compares control Group, the root weight of seedling, Miao Chong, root/shoot ratio, ground diameter dramatically increase after inoculation, and difference reaches the level of signifiance (p < 0.05).It connects The biomass of seedling influences maximum after kind, and significantly promotes the growth of seedling under ground portion, makes its root system more flourishing:Root weight is more right According to adding 47%-138%, amplification size is:Pt715 > Ld2 > Ld3;Miao Chong adds 37%-60%, amplification compared with control Size is:Ld2 > Pt715 > Ld3.The root/shoot ratio of seedling adds 28%-81% after inoculation, and amplification size is:Pt715 > Ld2 > Ld3, this illustrates that the formation of mycorhiza generates larger impact to the Bomass allocation of seedling part above and below the ground.With it is right Photograph ratio, height of seedling variation is not notable after inoculation, but the height of seedling for being inoculated with relatively control averagely improves 14.81%, has a big increase It is small to be:Ld3 > Pt715 > Ld2;The ground diameter of seedling averagely adds 25% after inoculation, and amplification size is:Ld2 > Ld3 > Pt715。
To sum up ectomycorrhizal fungi-inoculated is with obvious effects to masson pine growth-promoting, and 3 kinds of bacterial strain growth-promoting effect aggregate performances are Pt715 > Ld2 > Ld3.
Table 4 is inoculated with influence of the different Applying Ectomycorrhizal Fungis to Growth of Masson Pine
Note:It is notable (p < 0.05) with different lowercase letter indication differences in a line in table
3rd, mycorrhizal seedlings are to the sink effect of nutrient
P, K, Ca, Mg, Fe nutrient content of Pinus Massoniana Young Seedlings is as shown in figures 2-6 after ectomycorrhizal fungi-inoculated.Inoculation pair Nutrient content influences notable in Pinus Massoniana Young Seedlings body, and influence of the different vaccination processing to nutrient content is also not quite similar, and is inoculated with The nutrient content of Pinus Massoniana Young Seedlings is all remarkably higher than control afterwards.
Phosphorus (Fig. 2):Phosphorus is the indispensable element of plant growth, and the in vivo phosphorus 85% of plant exists in the form of organophosphor, such as Accounting, protein, phosphatide etc..Phosphorus participates in photosynthesis, participates in various enzymatic reactions, and plant normal activities are deep by its shadow It rings.Southwest belongs to acid soil, and available phosphorus is relatively low, and this severely limits the growths of plant.After inoculation the phosphorus content of seedling with CK, which is compared, reaches significant difference (p < 0.05).And different strains are different to the phosphorus increment of Pinus Massoniana Young Seedlings, Ld2 highests reach 112.68%, Ld3 take second place, and are that 54.89%, Pt715 is minimum, are 23.55%.The suction phosphorus capacity of water of 3 kinds of strains testeds is successively For:Ld2 > Ld3 > Pt715.
Potassium (Fig. 3):Potassium is one of a great number of elements needed for forest after nitrogen.Potassium In Plants element concentrates on vital movement Vigorous position can not only promote the transfer and transport of photosynthate in plant, also be played in terms of plant resistance to environment stress is improved Indispensable effect.After being inoculated with Ld2, seedling potassium content dramatically increases (p < 0.05) compared with other processing.Ld3 and Pt715 connect The seedling potassium content of kind is not notable (p < 0.05) with control group difference, but compares control group, and potassium content is still risen Height is respectively 3.03%, 2.2%.On the whole, inoculation adds plant potassium content.
Calcium, magnesium, iron (Fig. 4-Fig. 6):After inoculation, Ld3 and Pt715 add the calcium content of Pinus Massoniana Young Seedlings, and amplification compared with Greatly;And absorption unobvious of the Ld2 to calcium are inoculated with, instead 2.29% is reduced than control.Magnesium, iron are that plant growth is necessary micro- Secondary element participates in various metabolic activities in plant.Pinus Massoniana Young Seedlings magnesium, iron content are all remarkably higher than and are not inoculated with nursery stock, and Ld2 Amplification is still maximum.
4th, conclusion:
The strains tested of seedling growth test of the present invention:Pt 715 (Pisolithus tinctorius 715) is picked up from Sichuan Xichang eucalyptus (Eucalyptus sp.) hayashishita;Ld 2、Ld3:For delicious lactarius (Lactarius deliciosus (L.Fr.) Gray two strains), separation obtains from the acid yellow soil (pH value 3.9~4.3) under Jinyun Mountain, chongqing masson pine forest.It will After hypha separation pure culture, Mycorrhizal seedling is cultivated in a manner of dressing seed with masson pine seed.By to Pinus Massoniana Young Seedlings mycorhiza The research of the effect of change, upgrowth situation and Nutrient Absorption, the results showed that ectomycorrhizal fungi-inoculated can dramatically increase masson pine Mycorrhizal Infection Incidence, root weight, Miao Chong, root/shoot ratio, ground diameter, height of seedling and P, K, Ca, Mg, Fe content of plant.It is true to weigh exotrophic mycorrhiza Bacterium effect of inoculation most intuitively performance be exactly plant growing state.Illustrate that ectomycorrhizal fungi-inoculated has masson pine in growth-promoting work With, and it is with obvious effects.
Be inoculated with 3 kinds of Applying Ectomycorrhizal Fungis can form exotrophic mycorrhiza with masson pine, and infection rate is more than 80%, this explanation It can form good symbiosis with masson pine, but different strains infecting potential is different, and Ld 2 is most strong.
Inoculation can remarkably promote seedling root weight, height of seedling, root/shoot ratio, ground diameter.And the growth-promoting difference on effect of height of seedling is not notable, But inoculation processing is compared control and is still slightly increased, this may be due to the shorter early growth period growth of seedling of masson pine incubation time Speed is caused slowly, and masson pine top needle length does not have rule so that measurement inevitably causes error, therefore inoculation influences it Less.Root/shoot ratio increases after inoculation, illustrates that inoculation is more flourishing to effect bigger, the seedlings root of under ground portion.It is total to be inoculated with seedling Body growing way:Pt 715 is slightly larger than Ld 2, and Ld3 is minimum.
Plant formed mycorhiza after, mainly absorbed as mycorhiza organ extension mycelia played in plant nutrient absorption aspects it is non- Normal important role, its quantity and length is considerably beyond root hair, therefore ectomycorrhizal fungi-inoculated adds the suction of plant roots Area is received, and then adds its absorption and utilization to nutrient indirectly, it is especially particularly evident to the absorption of phosphorus.The present invention The result shows that:Inoculation causes Pinus Massoniana Young Seedlings P, K, Ca, Mg, Fe content to be higher than control, and especially the absorption of phosphorus is become apparent from, The amplification of 2 phosphorus of Ld is up to 112.68%, and root system is most strong to the absorption of phosphorus element after this illustrates inoculation.And plant absorbs amplification to K It is not it is obvious that mainly being entered in a manner of active transport from the tip of a root in plant, being not required to because potassium is inorganic cation Carry out enzymatic reaction, and mycorhiza is mainly by forming the secretion such as biological enzyme, organic acid come activated absorption difficultly soluble nutrient, therefore This absorption unobvious to potassium, and the Ca in soil exists mostly in the form of slightly solubility calcium salt, result of the present invention also indicates that, Calcium is the most apparent element of Nutrient Absorption effect after phosphorus.After inoculation seedling Mg, Fe contents higher than control, but amplification compared with It is small, this may be because its be not belong to trace element, it is natively not high in plant in-vivo content.In conclusion inoculation is external Mycorrhizal fungi adds Pinus Massoniana Young Seedlings element absorption, and the most apparent compared with other elements to the absorption of phosphorus.3 kinds of bacterial strains pair The effect of Pinus Massoniana Young Seedlings Nutrient Absorption effect is followed successively by Ld2 > Ld3 > Pt715.
Several preferred embodiments of invention have shown and described in above description, but as previously described, it should be understood that invention is not Form disclosed herein is confined to, is not to be taken as the exclusion to other embodiment, and available for various other combinations, modification And environment, and can be carried out in the scope of the invention is set forth herein by the technology or knowledge of above-mentioned introduction or association area Change.And changes and modifications made by those skilled in the art do not depart from the spirit and scope of invention, then it all should be in power appended by invention In the protection domain of profit requirement.

Claims (9)

1. a kind of breeding method of Mycorrhizal Fungi of Pinus massoniana nursery stock, which is characterized in that comprise the following steps:
Step 1, seed treatment:The uniform masson pine seed of no disease and pests harm, full grains is selected, is 25%- in mass concentration It is clean with pure water rinsing after 25-35min progress surface sterilizations are shaken in 35% hydrogen peroxide, it is placed in 35-45 DEG C of warm water and impregnates It when 20-28 is small, is then placed into the culture dish equipped with perlite and carries out vernalization, sowed after more than 90% seed shows money or valuables one carries unintentionally;
Step 2, soil treatment:Acid cold sand yellow earth under masson pine forest is selected to try soil as confession, soil is through 2% formalin Disinfection, film covering 7d, takes off film and teds and dry, spare after 7d;
Step 3 prepares Applying Ectomycorrhizal Fungi bush mycorrhiza agent;
Step 4, the inoculation of Applying Ectomycorrhizal Fungi bush mycorrhiza agent:After seedbed spy is flattened before sowing, paving 15cm it is thick through 2% formaldehyde Soil under the masson pine forest of disinfection and sieving;By inoculum with through 2% formaldehyde disinfection soil be mixed into paste, then with by vernalization Masson pine seed mix thoroughly, be sprinkled into nursery soil, every square metre is broadcasted sowing 200 masson pine seeds;After planting again on its surface One layer of fine earth is covered, is advisable with loseing seed;Then seedbed is spilt permeable;
Step 5, seedling management:Seedling management is with reference to GB6001-1985《Technical regulations for cultivation of tree seedlings》It performs;
Step 6, outplanting cultivate Mycorrhizal Fungi of Pinus massoniana nursery stock.
2. the breeding method of Mycorrhizal Fungi of Pinus massoniana nursery stock according to claim 1, which is characterized in that for examination soil in step 2 The basic physical and chemical of earth is as follows:The content of N is 1.14g/kg in Total Nutrient, and the content of P is 0.16g/kg, and the content of K is 19.09g/kg;The content of N is 69.75mg/kg in available nutrient, and the content of P is 4.55mg/kg, and the content of K is 71.21mg/ kg;The content of Ca is 3.45cmol/kg in exchangeability salt, and the content of Mg is 0.34cmol/kg, and the content of K is 0.14cmol/ kg。
3. the breeding method of Mycorrhizal Fungi of Pinus massoniana nursery stock according to claim 1, which is characterized in that the preparation in step 3 Applying Ectomycorrhizal Fungi bush mycorrhiza agent is specific as follows:
The separation and culture of step 3.1, Applying Ectomycorrhizal Fungi:Choose fresh, tender, the non-parachute-opening of children, the complete son reality of no disease and pests harm Body with 75% cotton ball soaked in alcohol surface sterilization, after the volatilization of surface alcohol, fructification stem cap junction is broken into two with one's hands, uses wine The scalpel that essence and flame disinfection are crossed takes the fungus block of 0.2cm-0.3cm sizes inside the fructification broken into two with one's hands, is connected on PDA plate On;Tablet is put into 25 DEG C of cultures in growth cabinet;Pollution is checked whether there is after 3 days, according to Applying Ectomycorrhizal Fungi mycelium Whether directly from inoculation block on grow, the speed of growth and fungi of the same race formed colony morphology characteristic it is consistent the features such as, tentatively Judge whether separated strain is Applying Ectomycorrhizal Fungi pure culture, and its growth feature is observed in detail;Screen it is pollution-free, The good each separation strain of colony morphology characteristic further carries out switching purifying in PDA culture medium, until its life per 7d once It is long to stablize;Preservation is spare at 4 DEG C;
The exotrophic mycorrhiza strain isolated and purified in step 3.1 is transferred on Pachlewski solid mediums by step 3.2, Light culture 2 weeks under constant temperature, it is spare as female bacterium;
Step 3.3, inoculum culture:Under super-clean bench, the strain of activation is transferred to punch method after high-temperature sterilization On Pachlewski fluid nutrient mediums, every bottle is accessed female bacterium fungus block 4 that area is 5mm × 5mm, at room temperature quiescent culture 2 weeks, Obtain mycelium;After the completion of culture, by mycelium, low speed breaks up 1min in beater, mycelium suspended liquid is collected, as inoculation Body.
4. the breeding method of Mycorrhizal Fungi of Pinus massoniana nursery stock according to claim 3, which is characterized in that the PDA culture medium It is prepared by the following method to obtain:After 200g potatos are cleaned peeling chopping water 1000ml is added to boil 30min, use filtered through gauze Again glucose 20g, agar 20g is added fully to dissolve afterwards, taken out after 121 DEG C of high-temperature sterilization 30min spare.
5. the breeding method of Mycorrhizal Fungi of Pinus massoniana nursery stock according to claim 3, which is characterized in that the Pachlewski The formula of solid medium is:Include ammonium tartrate 0.5g, potassium dihydrogen phosphate 1.0g, magnesium sulfate 0.5g, Portugal per 1L culture mediums Grape sugar 20g, vitamin B10.001g, agar 20g, micro-mixed liquor 1mL;Every liter of micro-mixed liquor contains H3BO3 8.45mg、MnSO4 5mg、FeSO4 6mg、CuSO4 0.625mg、ZnCl22.77mg and (NH4)2MoO4 0.27mg。
6. the breeding method of Mycorrhizal Fungi of Pinus massoniana nursery stock according to claim 3, which is characterized in that Applying Ectomycorrhizal Fungi is One kind in Pt 715, Ld 2 or Ld3 bacterium.
7. the breeding method of Mycorrhizal Fungi of Pinus massoniana nursery stock according to claim 1, which is characterized in that seedling stage manages in step 5 Reason is with reference to GB6001-1985《Technical regulations for cultivation of tree seedlings》It performs, is specially:
Step 5.1, seedbed management:Watering in due course keeps seedbed moistening;Irrigation and drainage facility is carried out simultaneously, ensures that water can evenly distribute To each cell seedbed, and without using any fungicide during experiment;
Step 5.2, each seedling stage points for attention:
A) sprouting stage is planted:It after planting arrives seed and spits bud, it is necessary to which discharging raticide prevents the plague of rats;Control is irrigated, and seedbed keeps moistening;
B) the sprout phase:I.e. chitting piece is unearthed, but plants shell and not yet come off, and pays attention to anti-bird pest;Take a small amount of multiple irrigation practice;
C) the leaf phase:I.e. seedling apical grows strip protophyll, to be loosened the soil, weeding;Take few irrigation practice of volume;
Step 5.3, thinning:In order to reduce water consumption, seedling is promoted to grow up strong and sturdy, each cell growth of seedling can be waited to stablize, i.e., After seedling goes out after homogeneous month, between overstocked thin and delicate seedling is dredged, while benefit cultivation is carried out to excessively sparse place;Every square metre Retain 140-160 plants of nursery stock;
Step 5.4, disease control:Pinus Massoniana Young Seedlings were unearthed in two months, since soil and air humidity increase, easily by germ It infects.Phase most common disease is samping off, damping-off;Therefore frequently to observe in this period, promptly prevent;Once it was found that disease Sick seedling is pulled out burn immediately by feelings, is then sprayed with the Bordeaux mixture of 0.5%-1.0%, per 7d once;
Step 5.5, pest control:Dendrolimus punctatus is the masson pine pest of most serious, and eating pine needle seriously affects pine forest life It is long;It is more convenient using chemical prevention and control method during nursery, i.e., 50% DDVP is diluted 2000 times;Or 50% Folithion breast 1500 times of dilution agent.
8. the breeding method of Mycorrhizal Fungi of Pinus massoniana nursery stock according to claim 1, which is characterized in that nursery stock goes out in step 6 Garden is specially:
Step 6.1, lifting:Lifting before luxuriant is not yet sprouted in spring, lifting need to be taken using scoop before, ensure Seedling root is with place soil, it is impossible to directly with hand-lifting, accomplish:Hinder lateral root, fibrous root less, keep root system than more complete and do not fracture Seedling stem does not hinder terminal bud;Then root is packed with film;
Step 6.2, classification:Determine whether nursery stock is qualified according to Comprehensive Control condition, root system, ground diameter and height of seedling;Comprehensive Control item Part is:Without quarantine object pest and disease damage, seedling stem leads to that straight, color and luster is normal;Qualified seedling is divided into I, II two grade;Seedling sorting is being sheltered Shady lee side;
Step 6.3, quality inspection:Seedling quality detection will carry out in a seedling batch after lifting, the method for taking random sampling It carries out;Vernier caliper measurement ground diameter, accuracy 0.05cm;Ruler measures height of seedling and root system, accuracy 1cm;
Step 6.4, temporary planting:The place that good, the leeward cool place of draining to be selected cannot such as be transplanted after lifting in time is planted.
Step 6.5, packaging:Nursery stock weight per package is no more than 30kg, and often bag needs labelled;
Step 6.6, transport:Transportational process will take moisturizing, ventilative measure, prevent from exposing to the weather, and long-distance transport nursery stock uses rice Grass or straw bag package.
9. the breeding method of Mycorrhizal Fungi of Pinus massoniana nursery stock according to claim 8, which is characterized in that the seedling in step 6.2 Wooden grade classification see the table below:
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CN114504001A (en) * 2022-01-11 2022-05-17 贵州大学 Seed coating agent of masson pine ectomycorrhizal fungi, preparation and use method thereof
CN115299264A (en) * 2022-09-02 2022-11-08 福建农林大学 Application and method of n-decane in promotion of growth of China fir
CN115299264B (en) * 2022-09-02 2023-12-19 福建农林大学 Application and method of n-decane in promoting growth of fir
CN116622553A (en) * 2023-04-07 2023-08-22 四川省食用菌研究所 Lactarius deliciosus mycorrhiza growth promoting bacterium, and preparation method and application thereof
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