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Lecanicillium psalliotae strain

A technology of Lecanococcus spp. and bacterial strains, applied in the direction of fungi, biocides, plant growth regulators, etc., can solve the problems of lack of anti-root-knot nematodes, no safe and effective control of root-knot nematodes, etc., and achieve efficient production of several Efficacy of tetratinase, large application potential, high enzyme activity

Inactive Publication Date: 2012-04-18
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Root-knot nematodes have a wide range of hosts, and there is still a lack of commercial crop varieties resistant to root-knot nematodes, and measures to rotate crops with non-host plants are difficult to implement in protected areas (Esmenjaud D, Voisin R, Van G C, Bosselut N, Lafargue B, Di Vito M, Dirlewanger E, Poёssel ​​J L, Kleinhentz M. Genetic dissection of resistance to root-knot nomatodes Meloidogyne spp. in plum, peach, almond and apricot from various segregating interspecific Prunus progenies. Tree Genetics and Genomes, 2009, 5: 279-289 . Dube B, Smart G C. Biological control of Meloidogyne incognita by Paecilomyces lilacinus and Pasteuria Penetrans. Journal of Nematology, 1987, 19(2): 222-227.)
The use of traditional fumigation or non-fumigation chemical nematodes to control root-knot nematodes has been or will be banned due to soil residues and environmental damage. So far, there are no safe and effective measures to control root-knot nematodes in production.

Method used

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  • Lecanicillium psalliotae strain
  • Lecanicillium psalliotae strain
  • Lecanicillium psalliotae strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1. Isolation of the lecanococcus bacterial strain of the present invention

[0035] (1) Culture medium: use water agar medium (WA), heat to dissolve, cool to about 45°C, add streptomycin (0.1mg / ml), shake well and pour into a 9cm diameter petri dish for use.

[0036] (2) Separation method: wash the loofah roots collected from Chengyang District, Qingdao City infected with root-knot nematode with water to remove sediment, pick fresh egg masses under a Nikon SMZ1000 dissecting microscope, shake and dissociate in 2% NaClO, and dissolve them at 500 Collect the eggs on a mesh sieve, and then sterilize the surface with 2% NaClO, then spread the egg suspension on the prepared WA medium, each dish contains about 50 eggs, and then put the culture dish in a constant temperature incubator at 27°C Cultivate, observe regularly, pick the grown hyphae to PDA medium, and store them at 4°C after the mycelium grows to be observed and identified later.

Embodiment 2

[0037] Embodiment 2. The bacterial colony character cultivation and the morphological identification of the lecanococcus spp. strain of the present invention

[0038] (1) Cultivation of colony traits: Transfer the strains stored in the refrigerator at 4°C to a PDA 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 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. The colony of Lecanococcus cerevisiae grows quickly, and its diameter is 56-61 mm in PDA medium for 10 days, and it is white cotton-like when viewed from the front (see figure 1 G), the back of the colony is red or purple, and the pigment can be produced in 3-5 days, and the red or pink pigment often diffuses into the agar (see figure 1 h).

[0039] (2) Morphologi...

Embodiment 3

[0041] Example 3. Molecular biology identification, rDNA-ITS sequence alignment and phylogenetic analysis of the present invention's Lecanococcus cerevisiae strain

[0042] (1) Synthesis of amplification primers: synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0043] Universal PCR amplification primers:

[0044] ITS1-F: 5'-CTTGGTCATTTAGAGGAAGT-3'

[0045] ITS4-R: 5'-CCTCCGCTTATTGATATGC-3'

[0046] (2) Extraction of genomic DNA: the DNA of Lecanococcus cerevisiae was extracted by CTAB method;

[0047] (3) PCR reaction system (20 μl): template DNA 1 μl, PCR Master Mix (Thermo Fisher Scientific) 10 μl, upstream and downstream primers 1 μl each, supplemented with deionized water to 20 μl. Reaction conditions: pre-denaturation at 94°C for 4min; 35 cycles at 94°C for 1min, 30s at 55°C, and 90s at 72°C; extension at 72°C for 10min.

[0048] (4) Gene sequencing of Lecanococcus spp.: The PCR product was detected by 1% agarose gel electrophoresis, and th...

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Abstract

The invention discloses a lecanicillium psalliotae strain with the preservation number of CGMCC NO: 5329. The strain can colonise spawns, two-year-old larvae and female adults at different life stages of rootknot nematode. Activations of chitinous substance degradative enzyme and excisionenzyme generated by the strain can be respectively determined through an N-acetyl glucosamine method and a p-nitrobenzene method, the enzymatic activities thereof can reach 3.98 U / h.mL and 0.38 U / h.mL, and higher activity of the produced chitinous substance enzyme can be detected at different temperature (27 DEG C-75 DEG C) and different pH (3.0-7.0); and the influence of chitinous substance enzyme extract produced by the strain on eclosion of rootknot nematode spawns is determined in vitro, and the inhibition ratio and the destruction ratio of enzyme solution on relative eclosion of the spawns are respectively 94.52 percent and 84.32 percent. The lecanicillium psalliotae strain disclosed by the invention is applied to biological control of crop rootknot nematodiasis, provides an effective way for protecting sweet potatoes, fruits, vegetables in north China and particularly controlling destructiverootknot nematodiasis, and has obvious ecological benefit, economic value and social value.

Description

technical field [0001] The invention relates to biological control of crop diseases, in particular to a lecanococcus sp. that efficiently produces chitinase and has a parasitic effect on root-knot nematodes, 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 (Kery B R. An assessment of progress toward microbial control of plant parasitic nematode. Journal of Nematology, 1990, 22(4S): 621-631. Stirling G R. Biological control of plant parasitic nematodes: progress, problems and prospects. Wallingford: CABI, 1991: 106-108.). 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 evaluat...

Claims

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Application Information

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IPC IPC(8): C12N1/14A01N63/04A01P5/00C12R1/645
Inventor 武侠刘爱华曹君正王凤龙
Owner QINGDAO AGRI UNIV
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