Method for preparing conidia of lasiodiplodia theobromae

A technology of grape canker bacteria and conidia, applied in the direction of using spores, biochemical equipment and methods, microorganisms, etc., can solve the problem of pathogenicity degradation, decreased sporulation of Lasiodiplodiatheobromae strains, pathogenicity determination, pathogenicity Mechanism research difficulties and other problems, to achieve strong pathogenicity, solve the effect of reduced sporulation production and small difference in spores

Active Publication Date: 2015-10-14
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the experiment, due to the frequent transfer of mycelia on the same medium, the Lasiodiplodia theobromae strain had problems such as reduced sporulation and degraded pathogenicity, which brought difficulties to subsequent pathogenicity determination and pathogenic mechanism research.

Method used

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  • Method for preparing conidia of lasiodiplodia theobromae
  • Method for preparing conidia of lasiodiplodia theobromae
  • Method for preparing conidia of lasiodiplodia theobromae

Examples

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Embodiment 1

[0031] Embodiment 1, grape green branch screening

[0032] The present invention screens out grape varieties that are sensitive to grape canker and are suitable for producing conidia through a screening test, and experiments prove that the summer black grape is the most preferred grape variety. The specific tests are as follows:

[0033] (1) Activate the preserved grape canker sore bacteria strain (Lasiodiplodia theobromae) GX-5-5A on a 9cm PDA medium plate, and cultivate it for 3 days at 28°C, in an incubator with 12h light / 12h darkness, until the colony grows to Standby when 7cm.

[0034] (2) Use a 5mm sterilized hole puncher to punch out the mycelium block along the edge of the colony with the colony cultivated in step (1).

[0035] (3) Select the green branches of grape varieties such as queen bee, giant rose, and summer black shown in Table 1, cut into 30cm and include 2-3 sections of branches, and use 1% sodium hypochlorite surface disinfection for 1min with a mass per...

Embodiment 2

[0040] Example 2 Grape green branches induce spore production of canker sores

[0041] (1) Activate the preserved grape canker sore bacteria strain (Lasiodiplodia theobromae) GX-5-5A on a 9cm PDA medium plate, and cultivate it for 3 days at 28°C, in an incubator with 12h light / 12h darkness, until the colony grows to Standby when 7cm.

[0042] (2) Use a 5mm sterilized hole puncher to punch out the mycelium block along the edge of the colony with the colony cultivated in step (1).

[0043] (3) Select healthy summer black grape green branches and cut them into 30cm branches including 2-3 nodes, disinfect the surface with 1% sodium hypochlorite for 1min, then rinse 3 times with sterile water, and put them on the sterilized Dry on filter paper.

[0044] (4) Use a 5mm hole puncher to punch holes in the middle of the dried green branches to remove the phloem. The prepared mycelium block in step 2 is inoculated at the perforated place, wraps the mycelium block with a sealing film (...

Embodiment 3

[0046] Example 3 Preparation of spore suspension

[0047] (1) Cut the grape branch covered with small black spots among the embodiment 1 into 0.5cm×0.5cm size with blade, put into the 2mL eppendorf centrifuge tube that 1.5ml sterile water is housed.

[0048] (2) Grind the branches with a grinding gun for 1 min, and then vibrate on a shaker for 1 min to rupture the conidia and release spores.

[0049] (3) Filter with sterilized 2 layers of degreasing gauze to remove branch residues and hyphae, and the filtrate is the spore suspension of grape canker.

[0050] (4) After shaking the spore suspension evenly, pipette 100 μL and drop it on a Neubauer hemocytometer, cover with a cover glass, and calculate the concentration of the spore suspension under a microscope.

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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 [0001] The invention relates to a method for preparing conidia of grape canker fungus. Background technique [0002] Grape canker caused by fungi of the family Botrytisaceae has occurred in major grape producing areas in my country in recent years. The vigor weakened or even the whole plant died, which brought different degrees of losses to grape production (Ji-Ye Yan, Yue Xie, Wei Zhang., et al.Species of Botryosphaeriaceae involved in grapevine dieback in China.Fungal Diversity, 2013, 61( 1): 221-236.). In my country, four species of Botrytisaceae fungi can cause grape canker, among which Lasiodiplodia theobromae is the dominant species and has strong pathogenicity. premise (Yan J Y, Li X H. Occurrence of grapevine trunk disease caused by Botryosphaeria rhodina in China. Plant Disease, 2011, 95(2): 219.). In the experiment, due to the frequent transfer of mycelia on the same medium, the Lasiodiplodia theobromae strain had problems such as reduced sporulatio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N3/00
Inventor 刘梅李兴红燕继晔张玮周莹陈震
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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