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Lecanicillium attenuatum strain

A technology of attenuating leukocytosis and strains, applied in fungi, biocides, plant growth regulators, etc., can solve problems such as no systematic reports, and achieve high-efficiency chitinase, high parasitic ability, and high enzymatic activity. Effect

Inactive Publication Date: 2012-05-02
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Lecanococcus is widely distributed in the world and parasitizes insects. There is no systematic report on the biological control of nematodes by this fungus.

Method used

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  • Lecanicillium attenuatum strain
  • Lecanicillium attenuatum strain
  • Lecanicillium attenuatum strain

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Embodiment 1. Isolation of the Lecanococcus attenuata bacterial strain of the present invention

[0040] (1) Medium: Selective medium (Kerry B R. An assessment of progress toward microbial control of plant parasitic nematode. Journal of Nematology, 1990, 22(4S): 621-631.): corn flour medium CMA , 17g; NaCl, 17.5g; Triton-100 (emulsifier), 3ml; Rose Bengal, 75mg; streptomycin sulfate, 50mg, add water to 1000ml. When the medium is about 45°C, add streptomycin and rose bengal, shake well and pour into a 9cm diameter Petri dish for use.

[0041] (2) Separation method: wash the soybean roots collected from Anda, Heilongjiang Province, infected with cyst nematode with water to remove sediment, pick fresh cysts under a Nikon SMZ1000 dissecting microscope, and disinfect the cysts with 2% NaClO surface. Place on the prepared medium, each dish contains 5 cysts, then place the dish in a constant temperature incubator at 27°C for cultivation, observe regularly, pick the growing my...

Embodiment 2

[0042] Example 2. Colony traits culture and morphological identification of the present invention

[0043] (1) Cultivation of colony traits: Transfer the strains stored in the refrigerator at 4°C to a CMA plate with a diameter of 9 cm, activate at 27°C for 5 days, use a puncher with a diameter of 6 mm to cut off the bacterial block at the edge of the colony, and transfer it to a new PDA Plates were cultured in the dark at 27°C and repeated 3 times. From the 2nd day, the colony shape, color and colony growth were recorded until the colonies covered the plate.

[0044] The colony grows slowly on the CMA medium. After 12 days of culture, the colony diameter can reach 3.3-3.4cm; figure 1 , E), the back is initially white, and after 10d it becomes pale yellow (see figure 1 , F).

[0045] (2) Morphological identification of bacterial strains: using the glass slide culture method, the CMA medium is divided into thin slices of 5 mm × 5 mm, picked on the glass slide, and then the ac...

Embodiment 3

[0047] Example 3. Molecular Biology Identification, ITS Sequence Alignment and Phylogenetic Analysis of the present invention

[0048] (1) Synthesis of amplification primers: synthesized by Shanghai Sangon Bioengineering Service Co., Ltd.;

[0049] Universal PCR amplification primers:

[0050] ITS1-F: 5'-CTTGGTCATTTAGAGGAAGT-3'

[0051] ITS4-R: 5'-CCTCCGCTTATTGATATGC-3'

[0052] (2) Genomic DNA extraction: the DNA of Lecanococcus attenuata was extracted by CTAB method;

[0053] (3) PCR reaction system (20 μl): 1 μL of template DNA, 10 μL of PCR Master Mix (Thermo Fisher Scientific), 1 μL of upstream and downstream primers, supplemented with deionized water to 20 μL. Reaction conditions: 94°C for 4min pre-denaturation; 94°C for 1min, 55°C for 30s, 72°C for 90s, 35 cycles; 72°C for 10min;

[0054] (4) Determination of the gene sequence of the Lecanococcus attenuata bacterial strain: the PCR product was detected by 1% agarose gel electrophoresis, and the results are shown in ...

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Abstract

The invention discloses a lecanicillium attenuatum strain with the preservation number of CGMCC (China General Microbiological Culture Collection) NO.5328. The strain can allow meloidogyne ova and female ova to parasitize in the strain. The activities of chitin degradative enzyme and excision enzyme are respectively measured by applying an N-acetyl glucosamine method and a paranitrobenzene methodto be 16.34U / h ml and 0.59U / h ml respectively; and higher activities of the generated chitin degradative enzyme system at different temperatures of 27-50DEG C and different pH values of 3.5-6.0 can be respectively detected. According to the measurement of the activity of chitin enzyme by electrophoresis, five bands are measured with the molecular weights of 79.6kDa, 65.6kDa, 54.1kDa, 42.1kDa and 32.9kDa respectively. The relative hatching inhibition rate of chitin enzyme coarse liquid generated by the lecanicillium attenuatum to the meloidogyne ova is measured to be 83.17 percent under the invitro condition. The lecanicillium attenuatum is applied to biological control over meloidogyne diseases of crops, particularly provides an effective path for controlling the meloidogyne diseases seriously harming melon and fruit vegetables in protected land in northern area of China as well as has remarkable biological benefit, economic value and social value.

Description

technical field [0001] The invention relates to the biological control of crop diseases, in particular to an attenuate wax scale fungus which efficiently produces chitinase and has a parasitic effect on root-knot nematode eggs and females, and belongs to the field of microorganisms and their application. Background technique [0002] Root-knot nematode is an important plant parasitic nematode disease that occurs worldwide. In recent years, root-knot nematodes have caused serious losses to agricultural production in my country, especially the Solanaceae and Cucurbitaceae vegetables grown in protected areas in the north (Shen Di, Li Xixiang, Feng Lanxiang, Wang Haiping, Song Jiangping, Yang Cuirong, Gong Huizhi. Cucurbitaceae Resistance evaluation of vegetable germplasm resources to root-knot nematode incognita. Journal of Plant Genetic Resources, 2007, 8(2): 340-342. Dong Daofeng, Cao Zhiping, Wang Xiuhui, Hu Ju, Maria Lodovica Gullino. Effects on tomato growth and yield. Acta...

Claims

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Application Information

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IPC IPC(8): C12N1/14A01N63/04A01P5/00C12R1/645
Inventor 武侠刘爱华胡东青林森王凤龙
Owner QINGDAO AGRI UNIV
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