CN105052499A - Method for quickly establishing phialophora fungus and crop mutualism system - Google Patents
Method for quickly establishing phialophora fungus and crop mutualism system Download PDFInfo
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- CN105052499A CN105052499A CN201510511768.XA CN201510511768A CN105052499A CN 105052499 A CN105052499 A CN 105052499A CN 201510511768 A CN201510511768 A CN 201510511768A CN 105052499 A CN105052499 A CN 105052499A
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- fungi
- saksenaea
- phialophora
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
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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
- A01G7/00—Botany in general
- A01G7/06—Treatment of growing trees or plants, e.g. for preventing decay of wood, for tingeing flowers or wood, for prolonging the life of plants
Abstract
The invention relates to a method for quickly establishing a phialophora fungus and crop mutualism system and belongs to the fields of applied microbiology, plant nutrition and ecology. According to the method, phialophora fungi are cultivated by means of corn grits subjected to high-temperature sterilization, then the phialophora fungi are mixed with a sterile culture medium to serve as an inoculant, sterile crop seeds are sown on the inoculant and germinate, and then the phialophora fungus and crop mutualism system is established quickly in the seedling growing period. The method specifically comprises the steps of phialophora fungus cultivation, inoculant preparation, seed surface sterilization, mutualism system establishment, mutualism system microscopic examination and fungal isolation and reinspection. The method has the advantages that phialophora fungus inoculation can be conducted while crop seedling culture is conducted, phialophora fungus colonization can be achieved in the seedling stage, operation is easy, and seedling culture time is shortened greatly. The method can be applied to researches on phialophora fungus and crop relation and has high theoretical and practical significance in large-area crop cultivation.
Description
Technical field
The invention belongs to microbiology, Plant Nutrition and field of ecology.Be specifically related to a kind of method setting up Saksenaea fungi and crops symbiosis cultivating system fast.
Background technology
Research discovery Saksenaea fungi (
phialophoraspp.) be one of the most dominant groups of root system of plant endogenetic fungus, belonging to dark color has every endogenetic fungus (DarkSepatateEndophytes, DSE) category.Along with going deep into of research, find that these DSE fungies can play the Ecological Functions of similar arbuscular mycorrhizal fungi, it is in plant nutrition absorption (particularly N, P), resistance, all play an important role in disease resistance, such as Saksenaea fungi (
phialophorasp.I-52) to the biological control etc. of take-all (Take-alldiseaseofwheat).In addition, Saksenaea fungi has host range widely, can with non-mycorrhizal plants as crucifer Chinese cabbage, arabidopsis etc. form good symbiotic relation, and it is studied, application prospect is very wide.
Existing Saksenaea fungi-crops symbiosis cultivating system is mostly by arbuscular mycorrhizal fungi research method, or inoculate again at crop growth certain phase, the general inoculation time of such as tomato was two leaf one heart stages, and it exists, and elapsed time is long, efficiency is low, be unfavorable for the shortcomings such as spread.
Summary of the invention
The object of the invention is to the deficiency overcoming prior art, and a kind of method setting up Saksenaea fungi and crops symbiosis cultivating system is fast provided.
The method setting up Saksenaea fungi and crops syntaxial system fast of the present invention, its concrete steps are as follows:
(1) Saksenaea fungal culture: after the Saksenaea fungi of freezing on test tube slant is activated, transfer on PDA culture medium flat plate, be inverted cultivation 20 days in 28 DEG C of constant incubators; Φ 6mm bacterium block is got respectively for subsequent use along colony edge;
(2) Inoculant prepares: get ordinary student corn grits 6g and be placed in 250ml triangular flask, adds water and no corn grits and leached excessive moisture after soaking 2 hours, be cooled to room temperature, inoculate 3 pieces of diameter 6mm bacterium blocks after sterilizing, and 28 DEG C of cultivations 20 days are for subsequent use as Inoculant;
(3) the surface of the seed sterilization: be 70% ethanol disinfection 5min through volume ratio by crop seeds, aseptic water washing 3 times, then the hypochlorite disinfectant 5min using volume ratio 3%, aseptic water washing 3 times, disinfects rear for subsequent use;
(4) foundation of syntaxial system: get 300g sterilization matrix and mix with cultured above-mentioned Inoculant, and be evenly sprinkling upon the flowerpot upper strata containing 2/3 volume medium matter, thickness 2cm, then sow the crop seeds 30 through surface sterilization; Be placed in illumination box, condition of culture is: luminous intensity 2000Lux, light application time 12 hours, and temperature is 25 DEG C, cultivates 20 days;
(5) syntaxial system microscopy: after 20 days, extract Saksenaea Fungal colonization situation in 5 crops seedling microscopy root systems, be specially: seedling selected by complete taking-up, wash the also long root segment of clip foundation portion 3cm 5 and put into test tube, add mass ratio 10%KOH solution to the complete submergence of root sample, dissociate in 90 DEG C of water-baths transparent to root sample, acid fuchsin again through routine dyes, lactic acid neutralizes and film-making after lactic acid glycerin decoloring, examine under a microscope Saksenaea Fungal colonization situation, dark septate hypha is had when observing in root, the typical Saksenaea fungi such as Microsclerotia feature structure, preliminary judgement is grown successfully surely.
(6) divide bacterium to recheck: to choose the preliminary crops root segment judging successfully surely to grow Saksenaea fungi, endophyte of plant separating method routinely detects whether root is decided at the higher level but not officially announced grows fungi by being connect bacterial classification; The root segment chosen is placed on culture of isolated fungi on PDA flat board through surface sterilization, and inoculate bacterium by being separated the fungi that obtains and carry out morphological feature with former and compare, and verifies that Saksenaea fungi-crops syntaxial system sets up situation further.
Saksenaea fungal culture PDA used in above-mentioned steps (1) is conventional medium, and its formula is: potato 200g, glucose 20g, and agar 15g, adds water and be settled to 1L, PH nature.
In above-mentioned steps (2), corn grits sterilising conditions is 121 DEG C of high-temperature sterilizations 30 minutes.
In above-mentioned steps (4), culture matrix is common loess: humus soil: perlite is by volume: 2:2:1, sterilising conditions is 121 DEG C of high-temperature sterilizations 2 hours, repeats 3 discontinuous sterilizations, every minor tick 48 hours.
In above-mentioned steps (6), Crop Root section surface conditions for sterilization is: the alcohol immersion 1min of volume ratio 70%, sterile water wash 3 times, and volume ratio 10% clorox soaks 2min, sterile water wash 3 times.
The inventive method Saksenaea originated from fungus used is in Yunnan University Micro Biological Key Laboratory.
Beneficial effect of the present invention is: can namely carry out the inoculation of Saksenaea fungi while crops nursery, Saksenaea fungi successfully can be surely grown in seedling stage, simple to operate, greatly shorten seedling raise period, can be applied to Saksenaea fungi and crops interrelationship study, the commerial growing for crops has most important theories and practical significance.
Embodiment
Be below specific embodiment of the invention case, protection scope of the present invention is not limited to described case.
Embodiment Saksenaea originated from fungus used is in Yunnan University Micro Biological Key Laboratory.Embodiment method therefor is with described in summary of the invention part.
Embodiment 1: Saksenaea (
phialophorasp.) fungi-tomato syntaxial system is cultivated
1, Saksenaea fungal culture: after Saksenaea fungi K36, A204, Z48, L45a bacterial strain of freezing on test tube slant is activated, be incubated at respectively on conventional PDA culture medium flat plate, be inverted cultivation 20 days in 28 DEG C of constant incubators; Getting diameter along colony edge is respectively that 6mm bacterium block is for subsequent use.
2, Inoculant prepares: get ordinary student corn grits 6g and be placed in 250ml triangular flask, add water and do not have corn grits and leached excessive moisture after soaking 2 hours, after 121 DEG C of high-temperature sterilization 30min, cool to room temperature, respectively access 3 pieces of diameter 6mm bacterium blocks, 28 DEG C cultivate 20 days for subsequent use as Inoculant.
3, the surface of the seed sterilization: be 70% ethanol disinfection 5min through volume ratio by tomato seeds, aseptic water washing 3 times, then the hypochlorite disinfectant 5min using volume ratio 3%, aseptic water washing 3 times, disinfects rear for subsequent use.
4, the foundation of syntaxial system: by culture matrix by common loess: humus soil: perlite is by volume: 2:2:1 mixes, 121 DEG C of high-temperature sterilizations 2 hours, totally three times, every minor tick 48 hours.After cool to room temperature, respectively get 300g aseptic substrate to mix with cultured above-mentioned corn grits respectively, and the flowerpot upper strata be evenly sprinkling upon separately containing 2/3 volume medium matter, thickness 2cm, then the tomato seeds 30 of surface sterilization evenly sowed by every basin, is placed in illumination box, condition of culture is: luminous intensity 2000Lux, light application time 12 hours, temperature is 25 DEG C, cultivates 20 days.
5, syntaxial system microscopy: after 20 days, Saksenaea Fungal colonization situation in each extraction 5 tomato seedling microscopy root systems, be specially: seedling selected by complete taking-up, wash the also long root segment of clip foundation portion 3cm 5 and put into test tube, add mass ratio 10%KOH solution to the complete submergence of root sample, dissociate in 90 DEG C of water-baths transparent to root sample, acid fuchsin again through routine dyes, lactic acid neutralizes and film-making after lactic acid glycerin decoloring, examine under a microscope Saksenaea Fungal colonization situation, dark septate hypha is had when observing in root, the typical Saksenaea fungi such as Microsclerotia feature structure, preliminary judgement is grown successfully surely.
6, bacterium is divided to recheck: random selecting tentatively judges successfully surely to grow the tomato root segment 5 of Saksenaea fungi, through the alcohol immersion 1min of volume ratio 70%, sterile water wash 3 times, volume ratio 10% clorox soaks 2min, and sterile water wash 3 times, is placed in suck dry moisture on aseptic filter paper, be placed on PDA flat board, 28 DEG C of constant temperature culture 20 days, are separated and obtain the Saksenaea fungi identical with former Inoculant form and anatomical features, show that Saksenaea fungi-tomato syntaxial system is successfully established further.
Embodiment 2: Saksenaea (
phialophorasp.) fungi-cucumber syntaxial system is cultivated
The present embodiment Saksenaea fungi used is numbered K36, A204, Z48, L45a, host plant is cucumber, concrete implementation step is with embodiment 1, cultivate and observe dark septate hypha, Microsclerotia by OLYPUS-BX51 sediments microscope inspection after 20 days, and obtain the Saksenaea fungi identical with former Inoculant form and anatomical features respectively after being rechecked by point bacterium, show that Saksenaea fungi-cucumber syntaxial system is successfully established.
Claims (4)
1. set up the method for Saksenaea fungi and crops syntaxial system fast for one kind, utilizing aseptic corn grits to cultivate Saksenaea fungi is inoculation material, inoculate the crop seeds through surface sterilization, set up Saksenaea fungi-crops symbiosis cultivating system fast at nursery stage, its concrete operation step is as follows:
(1) Saksenaea fungal culture: after the Saksenaea fungi of freezing on test tube slant is activated, transfer on PDA culture medium flat plate, be inverted cultivation 20 days in 28 DEG C of constant incubators; Φ 6mm bacterium block is got respectively for subsequent use along colony edge;
(2) Inoculant prepares: get ordinary student corn grits 6g and be placed in 250ml triangular flask, adds water and no corn grits and leached excessive moisture after soaking 2 hours, be cooled to room temperature, inoculate 3 pieces of diameter 6mm bacterium blocks after sterilizing, and 28 DEG C of cultivations 20 days are for subsequent use as Inoculant;
(3) the surface of the seed sterilization: be 70% ethanol disinfection 5min through volume ratio by crop seeds, aseptic water washing 3 times, then the hypochlorite disinfectant 5min using volume ratio 3%, aseptic water washing 3 times, disinfects rear for subsequent use;
(4) foundation of syntaxial system: get 300g sterilization matrix and mix with cultured above-mentioned Inoculant, and be evenly sprinkling upon the flowerpot upper strata containing 2/3 volume medium matter, thickness 2cm, then sow the crop seeds 30 through surface sterilization; Be placed in illumination box, condition of culture is: luminous intensity 2000Lux, light application time 12 hours, and temperature is 25 DEG C, cultivates 20 days;
(5) syntaxial system microscopy: after 20 days, extract Saksenaea Fungal colonization situation in 5 crops seedling microscopy root systems, be specially: seedling selected by complete taking-up, wash the also long root segment of clip foundation portion 3cm 5 and put into test tube, add mass ratio 10%KOH solution to the complete submergence of root sample, dissociate in 90 DEG C of water-baths transparent to root sample, acid fuchsin again through routine dyes, lactic acid neutralizes and film-making after lactic acid glycerin decoloring, examine under a microscope Saksenaea Fungal colonization situation, dark septate hypha is had when observing in root, the typical Saksenaea fungi such as Microsclerotia feature structure, preliminary judgement is grown successfully surely,
(6) bacterium is divided to recheck: to choose the preliminary crops root segment judging successfully surely to grow Saksenaea fungi, detect according to endophyte of plant separating method that root is decided at the higher level but not officially announced whether grows fungi by being connect bacterial classification, the root segment chosen is placed on culture of isolated fungi on PDA flat board through surface sterilization, and inoculate bacterium by being separated the fungi that obtains and carry out morphological feature with former and compare, verify that Saksenaea fungi-crops syntaxial system sets up situation further.
2. a kind of method setting up Saksenaea fungi and crops syntaxial system fast according to claim 1, is characterized in that in step (2), corn grits sterilising conditions is 121 DEG C of high-temperature sterilizations 30 minutes.
3. a kind of method setting up Saksenaea fungi and crops syntaxial system fast according to claim 1, it is characterized in that in step (4), culture matrix is common loess: humus soil: perlite is by volume: 2:2:1, sterilising conditions is 121 DEG C of high-temperature sterilizations 2 hours, repeat 3 discontinuous sterilizations, every minor tick 48 hours.
4. a kind of method setting up Saksenaea fungi and crops syntaxial system fast according to claim 1, it is characterized in that in step (6), Crop Root section surface conditions for sterilization is: the alcohol immersion 1min of volume ratio 70%, sterile water wash 3 times, volume ratio 10% clorox soaks 2min, sterile water wash 3 times.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105918127A (en) * | 2016-05-20 | 2016-09-07 | 武汉理工大学 | Method for building DSE-plant symbiosis system based on DSE rapid expanding propagation |
| CN108419475A (en) * | 2018-03-21 | 2018-08-21 | 黑龙江中医药大学 | A kind of method that endogenetic fungus promotes wilsonii germination |
| CN109757331A (en) * | 2017-11-09 | 2019-05-17 | 西南科技大学 | A kind of pseudo-wild habitat Orchid Mycorrhizae fungi and spring sword seed symbiosis-based culturing method |
| CN113088458A (en) * | 2021-04-09 | 2021-07-09 | 浙江省农业科学院 | Industrial liquid fermentation medium and culture method for rice endophytic fungus fusarium culmorum |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1682584A (en) * | 2004-04-15 | 2005-10-19 | 广东省微生物研究所 | Method for establishing symboitic relationship for arbuscular nycorrhizal fungi and tomato hairy root |
| CN1961631A (en) * | 2005-11-07 | 2007-05-16 | 中国医学科学院药用植物研究所 | Application of endogenous fungus in cultivation of snow lotus seedling and snow lotus |
| CN101263778A (en) * | 2007-12-06 | 2008-09-17 | 云南大学 | Method for fast establishing DSE and plant symbiosis cultivation system and uses thereof |
| CN102715089A (en) * | 2012-07-10 | 2012-10-10 | 云南大学 | Establishing method for indoor symbiotic relationship of amanita flavipes and cyclobalanopsis glaucoides as symbiotic plant |
| CN102754596A (en) * | 2012-06-21 | 2012-10-31 | 云南大学 | Establishing method of symbiont of cyclobalanopsis glaucoides and bolete |
| CN103828722A (en) * | 2014-03-24 | 2014-06-04 | 鲁东大学 | Method for blueberries seedling and cultivating blueberries in large area by applying DSE (Dark Septate Endophyte) fungus |
-
2015
- 2015-08-20 CN CN201510511768.XA patent/CN105052499A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1682584A (en) * | 2004-04-15 | 2005-10-19 | 广东省微生物研究所 | Method for establishing symboitic relationship for arbuscular nycorrhizal fungi and tomato hairy root |
| CN1961631A (en) * | 2005-11-07 | 2007-05-16 | 中国医学科学院药用植物研究所 | Application of endogenous fungus in cultivation of snow lotus seedling and snow lotus |
| CN101263778A (en) * | 2007-12-06 | 2008-09-17 | 云南大学 | Method for fast establishing DSE and plant symbiosis cultivation system and uses thereof |
| CN102754596A (en) * | 2012-06-21 | 2012-10-31 | 云南大学 | Establishing method of symbiont of cyclobalanopsis glaucoides and bolete |
| CN102715089A (en) * | 2012-07-10 | 2012-10-10 | 云南大学 | Establishing method for indoor symbiotic relationship of amanita flavipes and cyclobalanopsis glaucoides as symbiotic plant |
| CN103828722A (en) * | 2014-03-24 | 2014-06-04 | 鲁东大学 | Method for blueberries seedling and cultivating blueberries in large area by applying DSE (Dark Septate Endophyte) fungus |
Non-Patent Citations (2)
| Title |
|---|
| 刘怀旭: "《农业现代科学技术丛书 土壤肥料》", 31 May 1985 * |
| 贺学礼等: "不同VA菌根真菌对玉米生长及抗旱性的影响", 《西北农业大学学报》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105918127A (en) * | 2016-05-20 | 2016-09-07 | 武汉理工大学 | Method for building DSE-plant symbiosis system based on DSE rapid expanding propagation |
| CN105918127B (en) * | 2016-05-20 | 2018-05-01 | 武汉理工大学 | A kind of method that DSE- plant symbiosis systems are established based on DSE fast-propagations |
| CN109757331A (en) * | 2017-11-09 | 2019-05-17 | 西南科技大学 | A kind of pseudo-wild habitat Orchid Mycorrhizae fungi and spring sword seed symbiosis-based culturing method |
| CN108419475A (en) * | 2018-03-21 | 2018-08-21 | 黑龙江中医药大学 | A kind of method that endogenetic fungus promotes wilsonii germination |
| CN108419475B (en) * | 2018-03-21 | 2020-12-18 | 黑龙江中医药大学 | Method for promoting germination of acanthopanax seeds by endophytic fungi |
| CN113088458A (en) * | 2021-04-09 | 2021-07-09 | 浙江省农业科学院 | Industrial liquid fermentation medium and culture method for rice endophytic fungus fusarium culmorum |
| CN113088458B (en) * | 2021-04-09 | 2022-11-29 | 浙江省农业科学院 | Industrial liquid fermentation medium and culture method for rice endophytic fungus fusarium culmorum |
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