Application of Gliocladium roseum in biological control of Plutella xylostella

Abstract

The present invention discloses the application of Gliocladium roseum in the biological control of Plutella xylostella. The strain is Gliocladium roseum SWFUYHL02-01. Through exploration, it is known that the Gliocladium roseum SWFUYHL02-01 provided by the present invention has toxicity and control effect on the larvae of Plutella xylostella. Its median lethal concentration (LC 50 ) for controlling the larvae of Plutella xylostella is 4.1×10 4 spores / mL, and the median lethal time (LT 50 ) is 32.4 h, providing a certain experimental and theoretical basis for the biological control of the larvae of Plutella xylostella, avoiding residues in the process of chemical drug control, reducing environmental impact and harm to human health, etc., providing new microbial resources for the biological control of Plutella xylostella, and having significant research significance and application value.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to application of Glioma pinkum in biological control of Plutella xylostella. Background Art

[0002] The diamondback moth (Plutellaxylostella L.) belongs to the family of Lepidoptera, also known as the small green caterpillar and the two-headed moth. The adult body is six to seven millimeters long, and twelve to sixteen millimeters long when the wings are unfolded. Female diamondback moths are usually smaller than male diamondback moths. The diamondback moth is most harmful to cruciferous vegetables such as cabbage and radish. It often lives in concentrated areas and harms vegetables, chewing on vegetable leaves, causing the leaves to become net-like, resulting in a decline in the quality of vegetables, affecting the sales of vegetables, and even causing a complete loss of harvest in some areas. The diamondback moth has been seriously occurring in southern provinces of my country. Its damage has caused many vegetable crops to lose their original value, causing extremely adverse effects on the economic income of vegetable farmers, making vegetable farmers miserable. It has become a major pest that vegetable farmers need to solve urgently.

[0003] Clonostachys rosea is a filamentous fungus widely distributed throughout the world. It is an important parasitic fungus in nature. It can parasitize plants, insects and nematodes. It is also an important heavy parasite that can be used to prevent and control diseases.

[0004] At present, in addition to agricultural measures and physical control, the prevention and control of diamondback moth mainly relies on its natural enemies and chemical pesticides, such as chlorantraniliprole, indoxacarb, cypermethrin, emamectin benzoate, spinetoram, etc., which are easy to cause pesticide residues and environmental pollution, affect product quality, and are not conducive to human health. It can be seen that biological control is the most efficient control method with low or no toxicity to humans and animals. Therefore, it is of great significance to screen out a biological control bacterium with a high control effect on diamondback moth. Summary of the invention

[0005] The purpose of the present invention is to provide an application of Glioma pinkum in biological control of diamondback moth, so as to solve the problems of chemical residues in the process of diamondback moth control, environmental impact and harm to human health, and provide new microbial resources for biological control of diamondback moth.

[0006] In order to achieve the above object, the present invention provides an application of Glioma pinkum in biological control, wherein the Glioma pinkum is SWFUYHL 02-01, which has high toxicity to Plutella xylostella and can be used for biological control of Plutella xylostella.

[0007] The invention provides a biological bacterial agent which can be used for biological control of diamondback moth. The biological bacterial agent is a spore suspension of pink Glioma SWFUYHL 02-01.

[0008] Preferably, the concentration of the spore suspension of the above-mentioned Gliocladium roseum SWFUYHL 02-01 is 1.0×10 8 spores / mL.

[0009] The application of Gliocladium roseum of the present invention in the biological control of Plutella xylostella solves the problems of chemical drug residues in the process of Plutella xylostella control, causing environmental impact and harming human health, and has the following advantages:

[0010] The present invention provides a biocontrol strain Gliocladium roseum SWFUYHL 02-01 that can effectively control Plutella xylostella. The spore suspension prepared from the spores of this bacterium has high virulence to Plutella xylostella larvae, and provides an efficient working concentration of 1×10 8 spores / mL. Its median lethal concentration (LC 50 ) for Plutella xylostella larvae is 4.1×10 4 spores / mL, and the median lethal time (LT 50 ) is 32.4 h. The results provided by the present invention provide a certain experimental and theoretical basis for the biological control of Plutella xylostella larvae, and provide new microbial resources for the biological control of Plutella xylostella, having significant research significance and application value. Brief Description of the Drawings

[0011] Figure 1 It is the result of Plutella xylostella larvae being infected by Gliocladium roseum SWFUYHL 02-01 in the present invention.

[0012] Figure 2 It is the plate culture result of Gliocladium roseum SWFUYHL 02-01 and the re-inoculation of the mycelium on the body surface after infecting Plutella xylostella in the present invention.

[0013] Figure 3 It is the regression relationship between the time and mortality of Plutella xylostella larvae infected by the spore suspension of Gliocladium roseum SWFUYHL 02-01 with a concentration of 1.0×10 8 in the present invention.

[0014] Figure 4 It is the regression relationship between the Plutella xylostella larvae infected by the spore suspension with different concentrations at 54 h and the mortality in the present invention. Detailed Description of the Invention

[0015] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0016] Note: All reagents and consumables not specifically described in the present invention are conventional, and all experimental operations not specifically described are conventional in the art.

[0017] The fungus used in the present invention was identified as Gliocladium roseum SWFUYHL 02-01, which was cryopreserved in the Key Laboratory of Forest Disaster Early Warning and Control in Yunnan Province. This strain has been published in "Identification and Biological Characteristics of Entomogenous Fungus Gliocladium roseum of Cephalecia chuxiongica" Acta Phytophylacica Sinica 45.03 (2018): 622-631. doi: 10.13802 / j.cnki.zwbhxb.2018.2018007.

[0018] Examples

[0019] I. Activation of the strain

[0020] The cryopreserved Gliocladium roseum SWFUYHL 02-01 was placed at room temperature for 10 min. On the ultra-clean workbench, the colonies were picked out with an inoculation needle and inoculated onto newly prepared conventional PDA media, with 30 dishes inoculated for each strain. After inoculation, the dishes were sealed with a sealing film and then sealed in a sealed bag, which was marked with a marker pen. The sealed bag with the mark was placed in a constant temperature incubator and cultured at 28 °C for 7-15 d. During the culture process, pay attention to observing the growth of the strain, and timely handle the contaminated strain culture dishes to prevent further contamination.

[0021] II. Preparation of the spore suspension

[0022] Take the culture dishes in the constant temperature incubator that have grown mycelium fully, elute them with 0.05% Tween-80 solution, stir multiple times, and filter the mycelium to obtain the spore suspension. Determine the spore concentration of the spore suspension through a hemocytometer.

[0023] Among them, the calculation formula for the spore concentration is as follows:

[0024]

[0025] III. Virulence determination

[0026] Select the pre-cultured and well-grown Gliocladium roseum SWFUYHL 02-01, elute it through the above method, determine the spore suspension concentration, and set the spore suspension concentration gradient as 1.0×10 4 、1.0×10 5 、1.0×10 6 、1.0×10 7 、1.0×10 8 spores / mL with reference to the method of Ge Wenchao et al.

[0027] Using the spraying method, the median lethal concentration was determined. Among them, spore suspensions with different concentrations were set as the experimental groups, and sterile water was used as the control group. There were 20 diamondback moth larvae in each concentration group. The treated diamondback moth larvae together with the vegetable leaves were placed in a petri dish lined with sterile water-moistened filter paper and wet cotton, and the petri dish was sealed with transparent plastic wrap (about 70 small holes were pricked with a needle), and then placed in a constant temperature incubator at 25 °C for incubation. Observation was carried out every 6 h, and the number and time of diamondback moth deaths were recorded and the mortality rate was calculated.

[0028] Among them, the formula for calculating the corrected mortality rate is as follows:

[0029]

[0030] The growth of diamondback moth was observed every 6 h, data were recorded, the infection of Gliocladium roseum on diamondback moth was obtained, and the infection rates of Gliocladium roseum at different concentrations were calculated. The results are shown in Table 1 below. Select diamondback moth larvae infected by Gliocladium roseum SWFUYHL02-01 and with hyphae growing out (as Figure 1 shown), and the surface hyphae were re-inoculated onto a new PDA medium. After culturing for 10 d, the morphology was observed. The growth of the hyphae of Gliocladium roseum SWFUYHL 02-01 under normal culture conditions and after the surface hyphae infecting diamondback moth were re-inoculated could be obtained. The comparison results are as Figure 2 shown, where a1 in the figure is the plate culture result of normal Gliocladium roseum SWFUYHL 02-01, and a2 is the plate culture result of the surface hyphae of Gliocladium roseum SWFUYHL 02-01 after infecting diamondback moth. Then, the mortality rate of diamondback moth infected by Gliocladium roseum SWFUYHL 02-01 was analyzed and compared with the control. It can be known that the median lethal concentration LC 50 and median lethal time LT 50 of the strain Gliocladium roseum SWFUYHL 02-01 against diamondback moth. Based on this, the virulence intensity of Gliocladium roseum SWFUYHL 02-01 against diamondback moth was further determined. The bacterial suspension prepared from Gliocladium roseum SWFUYHL 02-01 can be used as a biological fungicide for the biological control of diamondback moth.

[0031] Table 1 Corrected mortality rates of diamondback moths infected with Gliocladium roseum at different concentrations at different times

[0032]

[0033] The above data show that the corrected mortality rate of diamondback moth treated with a spore suspension at a concentration of 1.0×10 8 reached 58.1% at 36 h and 97.6% at 60 h, approaching 100%. That is, in this experiment, when using 1.0×10 8The Plutella xylostella was treated with the spore suspension of Gliocladium roseum SWFUYHL 02-01, and almost all the Plutella xylostella died after 60 h. The data were analyzed by regression analysis, and the concentration with the highest regression significance was selected as 1.0×10 8 spores / mL for the experimental group. The regression relationship between time and mortality was obtained as y = 0.0153x + 0.0045, and the results are as Figure 3 shown. Among them, 'y' in the formula is the corrected mortality rate, and 'x' in the formula is the infection time. From this, the median lethal time LT 50 of Gliocladium roseum SWFUYHL 02-01 against Plutella xylostella was calculated to be 32.4 h. The regression relationship of the time period with the highest regression significance is as Figure 4 shown, that is, at 54 h, y = 0.0758x + 0.15, where 'y' in the formula is the corrected mortality rate, and 'x' in the formula is the logarithm of the spore suspension concentration. The median lethal concentration LC 50 of Gliocladium roseum SWFUYHL 02-01 against the larvae of Plutella xylostella was calculated to be 4.1×10 4 spores / mL.

[0034] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be understood that the above description should not be construed as a limitation of the present invention. After those skilled in the art have read the above content, various modifications and substitutions of the present invention will be obvious. Therefore, the protection scope of the present invention should be defined by the appended claims.

Claims

1. Application of Gliocladium roseum in biological control, characterized in that, Clonostachys rosea in biological control, The name of this Clonostachys rosea is SWFUYHL 02-01. This strain has virulence against Plutella xylostella and can be used for the biological control of Plutella xylostella.

2. Application of a biological bacterial agent in biological control of Plutella xylostella, characterized in that, The biological bacterium agent described above is Gliocladium roseum Clonostachys rosea The spore suspension of SWFUYHL 02-01.

3. The application according to claim 2, characterized in that, The pink Gliocladium mold Clonostachys rosea The concentration of the spore suspension of SWFUYHL 02-01 is 1.0×10 8 spores / mL.

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