CN104974975A - Method for preparing conidia of lasiodiplodia theobromae - Google Patents
Method for preparing conidia of lasiodiplodia theobromae Download PDFInfo
- Publication number
- CN104974975A CN104974975A CN201510475573.4A CN201510475573A CN104974975A CN 104974975 A CN104974975 A CN 104974975A CN 201510475573 A CN201510475573 A CN 201510475573A CN 104974975 A CN104974975 A CN 104974975A
- Authority
- CN
- China
- Prior art keywords
- grape
- branch
- colony
- spore
- sterilizing
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Abstract
The invention discloses a method for preparing conidia of lasiodiplodia theobromae. The method comprises the following steps: 1) inoculating lasiodiplodia theobromae onto a PDA (potato dextrose agar) culture medium to be cultured until a colony grows vigorously; 2) selecting a grape variety sensitive to lasiodiplodia theobromae, cutting healthy green grape branches into 30cm sections and sterilizing the surfaces of the sections; 3) punching holes in the green branches and removing the phloem; preparing hypha blocks from the colony with a sterilized puncher, inoculating the prepared hypha blocks in the holes, wrapping the hypha blocks with films to preserve moisture and inserting the inoculated branches in tissue culture containers filled with sterile water to be cultured at 28 DEG C; culturing for 48 hours under the condition that the relative humidity is more than 90%, and then maintaining the humidity at 70% and culturing for 5-10 days. The method has the beneficial effects that the preparation time of conidia can be shortened; prepared spore suspension has small spore difference, stable quality and strong pathogenicity and provides guarantees for pathogenicity identification, population genetic differentiation, pathogenesis analysis, and the like of lasiodiplodia theobromae.
Description
Technical field
The present invention relates to one and prepare the conidial method of grape ulcer bacterium.
Background technology
All generation is had in the grape main producing region of China in recent years by the fungus-caused grape ulcer of grape seat chamber Cordycepps, the position such as fruit, limb of pathogen infection grape causes the symptoms such as Grape stems is withered, limb ulcer, tree vigo(u)r is caused to weaken even whole strain dead, loss (the Ji-Ye Yan brought in various degree is produced to grape, Yue Xie, Wei Zhang., et al.Species of Botryosphaeriaceae involved ingrapevine dieback in China.Fungal Diversity, 2013,61 (1): 221-236.).In China, 4 kinds of grape seat chamber Cordycepps fungies can cause grape ulcer, wherein Lasiodiplodiatheobromae is sociales wherein and virulence is stronger, its spore suspension prepared is prerequisite (the Yan J Y of Identification of Species, Pathogenicity and infection mechanism research, Li X H.Occurrence of grapevinetrunk disease caused by Botryosphaeria rhodina in China.Plant Disease, 2011,95 (2): 219.).Owing to constantly transferring frequently with mycelia on same substratum in test, there is the problems such as sporulation quantity reduction, virulence degeneration in Lasiodiplodia theobromae bacterial strain, brings difficulty to follow-up Pathogenicity, pathogenesis etc.Therefore, how setting up a kind of conidium and method of preparing spore suspension of obtaining in a large number is fast and efficiently a technical problem urgently to be resolved hurrily.
Summary of the invention
The object of this invention is to provide one and prepare grape ulcer bacterium (Lasiodiplodia theobromae) conidial method, the method can improve grape ulcer bacterium (Lasiodiplodia theobromae) conidium output, to prepare spore suspension fast and effectively.
The process that method of the present invention is passed through to optimize induction Sporulation condition and prepare spore suspension is to obtain large, the virulent spore suspension of quantity fast.
Based on foregoing invention object, the conidial method of preparation grape ulcer bacterium (Lasiodiplodiatheobromae) of the present invention, comprises the following steps:
1) grape ulcer bacterium is inoculated on PDA substratum, under the condition of 11-13 h light every day, in 25-28 DEG C of incubator, is cultured to colony growth vigorous;
2) to choose the grape variety of grape ulcer bacterium sensitivity as inoculation material, green for the grape of health branch is cut into 28-32cm, first surface sterilization, then uses aseptic water washing, dry on the filter paper of sterilizing;
3) with the punch tool of sterilizing in step 2) in process green branch on punch, remove phloem; By step 1) cultivate the punch tool of bacterium colony sterilizing obtained and prepare mycelia block, by the inoculated by hypha block for preparing in punching place, with the moisturizing of sealed membrane parcel mycelia block, and by inoculated branch insert be equipped with in the tissue culture bottle of sterilized water, in 28-30 DEG C, every day 11-13 h light condition under cultivate; Wherein, cultivate 36-48 hour be greater than the condition of 90% in relative humidity under, then humidity maintains 70-80%, cultivates 5-10 days.
In aforesaid method, the described grape variety to grape ulcer bacterium sensitivity is summer black grape variety.
In aforesaid method, step 1) in, described colony growth is vigorous grows to the 7/9-8/9 of whole culture dish diameter for colony diameter; Preferably, described colony growth is vigorous for cultivating bacterial strain with the culture dish of 9cm, and colony growth diameter is 7-8cm.General, incubation time is 2-4 days, is specifically as follows 3 days.
In aforesaid method, step 1) in, described illumination condition is preferably in the condition of h light 12 h dark every day 12.
In aforesaid method, step 2) in, the punching diameter of described punch tool is 5-8mm; Punch position is along colony edge.
In aforesaid method, step 2) in, preferably green for the grape of health branch being cut into the branch that 30cm is long, is generally the branch section comprising 2-3 joint.
In aforesaid method, step 2) in, described surface sterilization is that the dense content of percent mass of described clorox is 1%-3%, and the time of sterilization is 1-3 minute with hypochlorite disinfectant.Described aseptic water washing preferably rinses three times.
In described method, step 3) in, the punching diameter of described punch tool is 5mm, and the middle part of branch is chosen in the position of punching.Like this, Spot expansion is conducive to.
In described method, step 3) in, inoculated branch is inserted and is equipped with in the tissue culture bottle of sterilized water, in 28 DEG C, every day 12 h light 12 h dark condition under cultivate.
In described method, described step 3) in sealed membrane can be plastics or resin material etc. transparent air-locked film, in order to easy to operate, the good sealed membrane of toughness etc. can be selected, as U.S. Parafilm sealed membrane.Also comprise method spore suspension being prepared by the conidium of acquisition in described method: with scissors by step 3) in be covered with pore grape branch be cut into 0.5cm size, put into the centrifuge tube that 1.5ml sterilized water is housed, tissue grinder 1-2min, shake 1min on the oscillator, pycnidium is broken and discharges spore, then cross and filter the residual body of branch and mycelia, filtrate is the spore suspension of grape ulcer bacterium.
Described tissue grinder adopts grinding rifle to carry out tissue grinder.
The method that described mistake filters the residual body of branch and mycelia adopts 2 pull-up fat filtered through gauze.
Aforesaid method, inoculating the fusiformis ulceration of inoculation position formation black in 3-5 days, branch was covered with in 5-7 days the point-pycnidium of black, microscopy observes inside has a large amount of conidiums to produce.
Method of the present invention, first grape ulcer bacterium is cultivated 2-4 days with in PDA substratum 25-28 DEG C of incubator, when mycelial growth is vigorous, mycelia block prepared by the punch tool of sterilizing, by inoculated by hypha block in through 1% clorox process on the green branch of grape of grape ulcer bacterium sensitivity, also inserting with the moisturizing of sealed membrane parcel is equipped with in the tissue culture bottle of sterilized water, 28 DEG C, 12h illumination/dark alternately, cultivate 5-7 days in the growth cabinet of relative humidity at 70%-80%, have a large amount of conidiums to produce when being covered with pore on branch.Then branch is cut into 0.5cm segment, puts into the centrifuge tube that 1.5ml sterilized water is housed, obtain the spore suspension of grape ulcer bacterium through grinding, concussion and filtration.
Method induction generation conidium required time of the present invention is short, spore quantity is many and virulence is strong.
In addition, the method is simple and easy to do, and induction generation conidium required time is short, spore quantity is many and virulence strong, solves the problem such as sporulation quantity reduction, pathogenic degeneration that mycelia succeeding transfer culture brings.From the pore that produces of morbidity branch through grinding, concussion, filter preparation spore suspension due to induction produce the spore time relatively fixing, concentrate, spore growth state is basically identical, overcome old young spore in spore suspension and there is the testing error brought simultaneously, the spore suspension spore amount difference of same (or different) batch preparation is little, and quality is more stable.And moisturizing developing medium sterilized water instead of nutritive medium or Nutrition Soil in test, Financial cost reduces.
In a word, spore is produced in the induction that method of the present invention carries out grape ulcer bacterium, a kind of simple and easy, efficient method is provided for obtaining a large amount of conidiums in the short period of time and preparing spore suspension, and the spore suspension spore difference of preparation is little, steady quality, virulence are strong, for the Pathogenicity of grape ulcer bacterium, population genetic differentiation and pathogenesis parsing etc. provide guarantee.
Accompanying drawing explanation
Fig. 1 is that spore device is produced in the green branch induction of grape.
Fig. 2 is that fusiformis scab appears in green branch pathology.
Fig. 3 is the pore-pycnidium (A is pycnidium under 30 × microscope, and B is pycnidium under 60 × microscope) at scab position.
Embodiment
For a better understanding of the present invention, below in conjunction with specific embodiment, the present invention is described further.
Material used in following embodiment, reagent, if no special instructions, all can obtain from commercial channels.
In following enforcement embodiment, grape ulcer bacteria strain used (Lasiodiplodia theobromae) GX-5-5A is disclosed in document " Ji-Ye Yan; Yue Xie; Wei Zhang.; et al.Species ofBotryosphaeriaceae involved in grapevine dieback in China.FungalDiversity.2013; 61:221-236. ", and the public can obtain from Beijing City Agriculture and Forestry Institute.
The green branch screening of embodiment 1, grape
The present invention is by shaker test, and filter out grape ulcer bacterium responsive, and be suitable for producing conidial grape variety, prove by experiment, summer Black Grape is most preferred grape variety.Concrete test is as described below:
(1) grape ulcer bacteria strain (Lasiodiplodia theobromae) GX-5-5A preserved is activated on the PDA culture medium flat plate of 9cm, 28 DEG C, cultivate 3 days, until colony growth to for subsequent use during 7cm in the incubator of 12h illumination/12h dark.
(2) punch tool of cultured bacterium colony 5mm sterilizing in step (1) is beaten along colony edge get mycelia block.
(3) after choosing the honeybee shown in table 1, the green branch of the grape variety such as huge rose, summer be black, be cut into the branch section that 30cm comprises 2-3 joint, with the clorox surface sterilization 1min that mass percentage is 1%, then use aseptic water washing 3 times, dry on the filter paper of sterilizing.
(4) with the middle part punching of punch tool on the green branch dried of 5mm, phloem is removed.By the inoculated by hypha block for preparing in step 2 in punching place, wrap up with sealed membrane (U.S. Parafilm) and insert in the nutrition pot that sterilizing vermiculite is housed, be placed in 28 DEG C, 12h illumination/dark alternately, the greenhouse of relative humidity about 80% cultivates, and within 10 days, measures scab length afterwards.
The scab length of table 1 different grape variety inoculation grape ulcer bacterium
| Kind | Scab length (cm) |
| Behind peak | 10.89±2.40c |
| Huge rose | 13.46±1.41b |
| Summer is black | 26.50±0.03a |
| Rose bella Reid | 1.89±0.60e |
| Fragrant prince wife | 1.66±0.02e |
| The seedless Cold boiled chicken heart | 9.45±1.78c |
| Chardonney | 3.26±0.53de |
| Merlot | 5.00±0.61d |
Scab length under using Deng Kenshi duncan's new multiple range method to compare 0.05 level after different grape variety inoculation grape ulcer bacterium.Result is as shown in table 1.In table 1, same letter representative does not have significant difference.Table 1 shows, after summer Black Grape kind inoculation, scab is the longest, and being significantly higher than the scab length of other grape varieties, is most preferred grape variety.
Embodiment 2 grape green branch induction grape ulcer bacterium produces spore
(1) grape ulcer bacteria strain (Lasiodiplodia theobromae) GX-5-5A preserved is activated on the PDA culture medium flat plate of 9cm, 28 DEG C, cultivate 3 days, until colony growth to for subsequent use during 7cm in the incubator of 12h illumination/12h dark.
(2) punch tool of cultured bacterium colony 5mm sterilizing in step (1) is beaten along colony edge get mycelia block.
(3) choose the healthy green branch of summer Black Grape and be cut into the branch section that 30cm comprises 2-3 joint, be the clorox surface sterilization 1min of 1% with mass percentage, then use aseptic water washing 3 times, dry on the filter paper of sterilizing.
(4) with the middle part punching of punch tool on the green branch dried of 5mm, phloem is removed.By the inoculated by hypha block for preparing in step 2 in punching place, wrap up the moisturizing of mycelia block with sealed membrane (U.S. Parafilm) and insert immediately the 240mi of 30ml sterilized water is housed tissue culture bottle in (as shown in Figure 1, wherein, wrap up mycelia block to be positioned at above the water surface).
(5) tissue culture bottle is placed in 28 DEG C, the growth cabinet that replaces of 12h illumination/dark cultivates.Before after inoculation, 48h keeps relative humidity to be greater than 90%, then maintains 70%, and 5-10 days is cultivated in moisturizing.In 3-5 days, inoculation position forms the fusiformis ulceration (Fig. 2) of black, branch is covered with in 5-7 days the point-pycnidium (as shown in Figure 3) of black, and microscopy observes inside has a large amount of conidiums to produce.
The preparation of embodiment 3 spore suspension
(1) with blade, the grape branch being covered with pore in embodiment 1 is cut into 0.5cm × 0.5cm size, puts into the eppendorf centrifuge tube of the 2mL that 1.5ml sterilized water is housed.
(2) first with grinding rifle, branch is carried out tissue grinder 1min, then earthquake device shakes 1min, conidium is broken and discharges spore.
(3) by 2 pull-up fat filtered through gauze of sterilizing, the removing residual body of branch and mycelia, filtrate is the spore suspension of grape ulcer bacterium.
(4) spore suspension is shaken up rear liquid-transfering gun to draw 100 μ L and drop on Neubauer blood counting chamber, covered, calculates the concentration of spore suspension under the microscope.
Embodiment 4 the inventive method prepares comparing of spore suspension method with routine
1) configuration of substratum
PDA substratum: the potato 200g cleaning peeling, the 1L that adds water boils half an hour, with gauze elimination potato, adds water and complements to 1L, add 20g glucose and 20g agar powder, be heated to agar powder and dissolve, pour packing in 300mL triangular flask into, high pressure moist heat sterilization, 121 DEG C, 20min.When being cooled to 40-50 DEG C, pour in sterilizing culture dish for subsequent use.
PSA substratum: the potato 200g cleaning peeling, the 1L that adds water boils half an hour, with gauze elimination potato, adds water and complements to 1L, add 20g sucrose and 20g agar powder, be heated to agar powder and dissolve, pour packing in 300mL triangular flask into, high pressure moist heat sterilization, 121 DEG C, 20min.When being cooled to 40-50 DEG C, pour in sterilizing culture dish for subsequent use.
MEA substratum: malt extract 30g, soy peptone 3g, agar 15g, adding distil water is settled to 1L, pours packing in 300mL triangular flask into, high pressure moist heat sterilization, 121 DEG C, 20min.When being cooled to 40-50 DEG C, pour in sterilizing culture dish for subsequent use.
OA substratum: rolled oats 30g, agar 20g, adding distil water is settled to 1L, pours packing in 300mL triangular flask into, high pressure moist heat sterilization, 121 DEG C, 20min.When being cooled to 40-50 DEG C, pour in sterilizing culture dish for subsequent use.
2) comparison of spore suspension concentration
By same grape ulcer bacteria strain (grape ulcer bacteria strain (Lasiodiplodiatheobromae) GX-5-5A, see embodiment 1) respectively on PDA substratum, PSA substratum, MEA substratum, OA substratum in 25 DEG C, cultivate 8-21 days under 12h illumination/12h dark condition, when having ripe pycnidium to produce on flat board, choose pycnidium and produce more position, be cut into 0.5cm × 0.5cm size with blade, put into the eppendorf centrifuge tube of the 2mL that 1.5ml sterilized water is housed.Step 2 through as embodiment 3) and 3) as described in method grinding, filter after, Neubauer blood counting chamber calculates the concentration of spore suspension.
Spore suspension concentration prepared by spore is produced in the induction of table 2 different methods
Use Deng Kenshi duncan's new multiple range method to compare the bacterial strain of different methods induction under 0.05 level and produce the significance of difference of conidium concentration, same letter representative does not have significant difference.Spore is not observed in "-" representative.
As shown in table 2, result shows, does not observe except spore except on PSA substratum, and the spore suspension concentration of grape ulcer bacteria strain preparation after different methods induction there are differences, and after the induction of its medium green branch, spore suspension concentration is up to 5.0 × 10
6individual/mL, comparatively additive method improves 10-100 doubly, is obviously better than additive method.From the product spore time, namely grape ulcer bacteria strain has a large amount of conidiums to produce on the 5th day after the induction of green branch, obviously shortens ordinary method and prepares time needed for spore suspension, improve test efficiency.
Result shows, induces the conidium of generation from the green branch of postvaccinal grape in the present invention, produces spore time Relatively centralized, and sporulation quantity is large and virulence is strong, and different batches spore suspension concentration difference is little, has the advantages such as simple and easy, efficient, stable.
Claims (10)
1. prepare grape ulcer bacterium (Lasiodiplodia theobromae) conidial method, comprise the following steps:
1) grape ulcer bacterium is inoculated on PDA substratum, under the condition of 11-13 h light every day, in 25-28 DEG C of incubator, is cultured to colony growth vigorous;
2) to choose the grape variety of grape ulcer bacterium sensitivity as inoculation material, green for the grape of health branch is cut into 28-32cm, first surface sterilization, then uses aseptic water washing, dry on the filter paper of sterilizing;
3) with the punch tool of sterilizing in step 2) in process green branch on punch, remove phloem; By step 1) cultivate the punch tool of bacterium colony sterilizing obtained and prepare mycelia block, by the inoculated by hypha block for preparing in punching place, with the moisturizing of sealed membrane parcel mycelia block, and by inoculated branch insert be equipped with in the tissue culture bottle of sterilized water, in 28-30 DEG C, every day 11-13 h light condition under cultivate; Wherein, cultivate 36-48 hour be greater than the condition of 90% in relative humidity under, then humidity maintains 70-80%, cultivates 5-10 days.
2. method according to claim 1, is characterized in that: the described grape variety to grape ulcer bacterium sensitivity is summer black grape variety.
3. method according to claim 1, is characterized in that: step 1) in, described colony growth is vigorous grows to the 7/9-8/9 of whole culture dish diameter for colony diameter.
4. method according to claim 3, is characterized in that: step 1) in, described colony growth is vigorous for cultivating bacterial strain with the culture dish of 9cm, and colony growth diameter is 7-8cm.
5. method according to claim 1, is characterized in that: step 2) in, the punching diameter of described punch tool is 5-8mm; Punch position is along colony edge.
6. method according to claim 1, is characterized in that: step 2) in, described surface sterilization is that the dense content of percent mass of described clorox is 1%-3%, and the time of sterilization is 1-3 minute with hypochlorite disinfectant.
7. method according to claim 1, is characterized in that: step 3) in, the punching diameter of described punch tool is 5-8mm, and the middle part of branch is chosen in the position of punching.
8. according to the method in claim 1-7 described in any one, it is characterized in that: in described method, also comprise method spore suspension being prepared by the conidium of acquisition: with scissors by step 3) in be covered with pore grape branch be cut into 0.5cm size, put into the centrifuge tube that 1.5ml sterilized water is housed, tissue grinder 1-2min, shake 1min on the oscillator, pycnidium is broken and discharges spore, then cross and filter the residual body of branch and mycelia, filtrate is the spore suspension of grape ulcer bacterium.
9. method according to claim 8, is characterized in that: what described tissue grinder adopted is grinding rifle.
10. method according to claim 8, is characterized in that: the method that described mistake filters the residual body of branch and mycelia adopts 2 pull-up fat filtered through gauze.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510475573.4A CN104974975B (en) | 2015-08-06 | 2015-08-06 | It is a kind of to prepare the conidial method of grape ulcer bacterium |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510475573.4A CN104974975B (en) | 2015-08-06 | 2015-08-06 | It is a kind of to prepare the conidial method of grape ulcer bacterium |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104974975A true CN104974975A (en) | 2015-10-14 |
| CN104974975B CN104974975B (en) | 2019-02-26 |
Family
ID=54271940
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510475573.4A Active CN104974975B (en) | 2015-08-06 | 2015-08-06 | It is a kind of to prepare the conidial method of grape ulcer bacterium |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104974975B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110878280A (en) * | 2019-12-12 | 2020-03-13 | 北京市农林科学院 | Method for inducing cacao trichoderma to quickly generate conidia |
| CN113373061A (en) * | 2021-06-22 | 2021-09-10 | 湖北省农业科学院果树茶叶研究所 | Method for obtaining grape-vine conidiophore in fruit tree tissue |
| CN114058508A (en) * | 2021-12-09 | 2022-02-18 | 中国农业大学 | Method for quickly separating and purifying cercospora and detecting drug resistance |
| CN114836326A (en) * | 2022-03-11 | 2022-08-02 | 山东省葡萄研究院 | Culture medium for enabling botrytis cinerea to produce spores and application of culture medium |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102676631A (en) * | 2011-03-08 | 2012-09-19 | 中国科学院微生物研究所 | Fabrication method of cover glass for conidiospore of Ophio cordyceps sinensis and dynamic counting method of produced conidiospore |
| CN103320376A (en) * | 2013-06-26 | 2013-09-25 | 北京市农林科学院 | Single spore isolation method of grape powdery mildew |
| CN104351259A (en) * | 2014-11-19 | 2015-02-18 | 广西壮族自治区农业科学院葡萄与葡萄酒研究所 | Biological control method of grape cankers |
-
2015
- 2015-08-06 CN CN201510475573.4A patent/CN104974975B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102676631A (en) * | 2011-03-08 | 2012-09-19 | 中国科学院微生物研究所 | Fabrication method of cover glass for conidiospore of Ophio cordyceps sinensis and dynamic counting method of produced conidiospore |
| CN103320376A (en) * | 2013-06-26 | 2013-09-25 | 北京市农林科学院 | Single spore isolation method of grape powdery mildew |
| CN104351259A (en) * | 2014-11-19 | 2015-02-18 | 广西壮族自治区农业科学院葡萄与葡萄酒研究所 | Biological control method of grape cankers |
Non-Patent Citations (1)
| Title |
|---|
| 姚晟伟 等: "葡萄溃疡病病菌(Botryosphaeria dothidea)的诱导产孢方法评价", 《中外葡萄与葡萄酒》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110878280A (en) * | 2019-12-12 | 2020-03-13 | 北京市农林科学院 | Method for inducing cacao trichoderma to quickly generate conidia |
| CN110878280B (en) * | 2019-12-12 | 2021-11-09 | 北京市农林科学院 | Method for inducing cacao trichoderma to quickly generate conidia |
| CN113373061A (en) * | 2021-06-22 | 2021-09-10 | 湖北省农业科学院果树茶叶研究所 | Method for obtaining grape-vine conidiophore in fruit tree tissue |
| CN114058508A (en) * | 2021-12-09 | 2022-02-18 | 中国农业大学 | Method for quickly separating and purifying cercospora and detecting drug resistance |
| CN114058508B (en) * | 2021-12-09 | 2024-03-15 | 中国农业大学 | Method for rapidly separating and purifying cercospora and detecting drug resistance |
| CN114836326A (en) * | 2022-03-11 | 2022-08-02 | 山东省葡萄研究院 | Culture medium for enabling botrytis cinerea to produce spores and application of culture medium |
| CN114836326B (en) * | 2022-03-11 | 2023-09-29 | 山东省葡萄研究院 | Culture medium for enabling grape white rot germs to generate spores and application of culture medium |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104974975B (en) | 2019-02-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103160442B (en) | Paecilomyceslilacinus strain having strong pathogenicity for diaphorina citri | |
| CN110468057B (en) | Endophytic pestalotiopsis fungus M7SB41 and application thereof | |
| CN104974975A (en) | Method for preparing conidia of lasiodiplodia theobromae | |
| CN105002097A (en) | Single spore isolation and purification method of verticillium dahliae | |
| CN105039181A (en) | Metarhizium anisopliae MAYX130921 and application thereof | |
| CN103243030B (en) | Lecanicilliumpsalliotae strain used for preventing and treating diaphorina citri | |
| CN104195064B (en) | The Oryza sativa L. endogeny rayungus of the one external efficient antagonism Pyricularia oryzae of strain | |
| CN106538610B (en) | A kind of purposes of celery stem-leaf extract | |
| CN101914474B (en) | Ectomycorrhizal helper bacteria bacillus pumilus and application thereof | |
| Hepden | Studies in vesicular-arbuscular endophytes: II. Endophytes in the pteridophyta, with special reference to leptosporangiate ferns | |
| Fernando et al. | Some factors affecting in vitro production, germination and viability of conidia of Corynespora cassiicola from Hevea brasiliensis | |
| CN104789509A (en) | Eucommia ulmoides endogenous bacillus pumilus and application thereof | |
| CN103146609B (en) | Pseudomonas fluorescens and method for preventing phytophthora capsici thereby | |
| CN102851225B (en) | Stenotrophomonas acidaminiphila and application in control of apple tree canker thereof | |
| CN108033905B (en) | The preparation method and application of compound pencolide | |
| CN102960369B (en) | Preparation method of granular paecilomyces lilacinus biological nematocide | |
| CN109266560B (en) | Alternaria alternata culture medium containing weeds | |
| CN104630072A (en) | Trichoderma atroviride Ta-9 strain and application thereof in prevention and control of rice diseases | |
| CN103250564A (en) | Artificial cultivating method for chestnut mycorrhiza fungi | |
| Paulitz et al. | Is Spizellomyces punctatum a parasite or saprophyte of vesicular-arbuscular mycorrhizal fungi? | |
| CN110628625A (en) | Separation and screening method of patchouli endophytic fungi | |
| CN105177104A (en) | Method for identifying phytophthora parasitica var nicotianae physiological strains | |
| CN104651240A (en) | Method for separating monospores of plasmodiophoromycetes and method for establishing a monospore line by host propagation | |
| CN113481108B (en) | Nutritional matrix for stimulating growth of nematode-trapping fungi on trunk, and preparation method and application method thereof | |
| CN106032523A (en) | Simple method for separating and purifying apple ring spot bacteria |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |