CN-115094108-B - Paecilomyces lilacinus and application thereof in production of cyclosporin C and prevention and control of bemisia tabaci

Abstract

The invention relates to the field of insect control, in particular to a paecilomyces lilacinus strain XI-5 and a method for extracting and identifying crude toxin generated by a secondary metabolite of the paecilomyces lilacinus strain XI-5 from the paecilomyces lilacinus strain XI-5. The present invention demonstrates that the secondary metabolite of Paecilomyces lilacinus strain XI-5 produces cyclosporin C. Cyclosporin C has a stomach toxic effect on bemisia tabaci adults. After 24 hours of application, the LC50 concentration of cyclosporin C on bemisia tabaci adults was 31.75 μg/ml, and the LC30 concentration of cyclosporin C resulted in a decrease in the female spawning amount/fertility of bemisia tabaci compared to the control group.

Inventors

• SHAWKAT ALI
• WU JIANHUI
• XU QIAI
• Sun Tingfei
• ZHANG JIEMING
• ZHANG XIAOCHEN
• ZHANG YOUDAN

Assignees

• 华南农业大学
• 华南农业大学
• 鹤山市农业技术推广中心
• 鹤山市农业技术推广中心

Dates

Publication Date

20260421

Application Date

20220705

Priority Date

20220705

Claims (6)

1. 1. A method for extracting a crude toxin containing cyclosporin C from paecilomyces lilacinus strain XI-5, comprising the steps of: 1) Inoculating spore suspension of Paecilomyces lilacinus strain XI-5 into CM liquid culture medium for shake culture; 2) Obtaining bacterial liquid through vacuum suction filtration, regulating the PH of the bacterial liquid to be 2.5-4.0, and adding ethyl acetate with the same volume for extraction; 3) The organic phase is retained by separating liquid, and ethyl acetate is distilled by a rotary evaporator to obtain a secondary metabolite of the strain, namely crude toxin containing cyclosporin C.
2. 2. The method for extracting toxins from Paecilomyces lilacinus strain XI-5 according to claim 1, further comprising dissolving the crude toxin with isopropanol, filtering with 0.22 μm filter membrane, and performing on-machine analysis for ESI positive and negative ion analysis.
3. 3. The method for extracting toxins from paecilomyces lilacinus strain XI-5 according to claim 1, wherein the spore suspension is inoculated into CM liquid medium by shaking culture of paecilomyces lilacinus strain XI-5 to a concentration of 1.0 x10 6 spores/mL, after which it is placed in a shaking incubator at 27 ℃,125 rpm, and shaking cultured for 2 weeks.
4. 4. A method for extracting toxins from paecilomyces lilacinus strain XI-5, according to claim 3, wherein the pre-shake culture step comprises: Inoculating Paecilomyces lilacinus strain XI-5 to PDA culture medium, culturing in an incubator with temperature of 26+ -1 deg. C, L:D=16:8 for 10D, and maturing; After sporulation, mycelium is scraped into a 0.05% Tween-80 solution by a small medicine spoon, and the mycelium is fully stirred by a magnetic stirrer to uniformly disperse the conidium, so that a suspension with the concentration of 1.0X10 8 spores/mL is finally prepared, and then the suspension is inoculated into a CM liquid culture medium.
5. 5. The method for extracting toxins from paecilomyces lilacinus strain XI-5 according to claim 1, wherein in step 2), bacterial liquid is obtained through vacuum filtration, the PH of the bacterial liquid is adjusted to 3.0, ethyl acetate with the same volume is added for extraction 24 h, and the bacterial liquid is placed in a 4 ℃ low-temperature refrigerator and fully stirred every 4: 4 h.
6. 6. The method for extracting toxins from paecilomyces lilacinus strain XI-5 of claim 1, further comprising subjecting the extracted crude toxins to LC-MS analysis.

Description

Paecilomyces lilacinus and application thereof in production of cyclosporin C and prevention and control of bemisia tabaci Technical Field The invention relates to the field of insect control, in particular to a paecilomyces lilacinus strain XI-5, and a toxin produced by a secondary metabolite of the paecilomyces lilacinus strain XI-5 extracted and identified therefrom and application thereof in controlling bemisia tabaci. Background Bemisia tabaci is a worldwide pest and causes great economic losses to agricultural production every year [1,2]. Bemisia tabaci-Nakava No. 1 (MEAM 1) (previously referred to as "biological B") poses a constant threat to global field crop production [3,4]. Whitefly management programs worldwide rely mainly on chemical pesticides, but improper use of these chemicals has led to the development of pesticide resistance [5,6]. Due to the rapid development of bemisia tabaci resistance, the general control based on biological control has recently attracted public attention [7,8]. For over a century, entomopathogenic fungi have been recognized as potential agents for biological control of different insects. Entomopathogenic fungi are diverse in variety, complex in metabolic type, and suitable for safety levels in humans and other non-target organs, which makes them attractive to other pest control strategies [7]. Fungi belonging to the genus rhodobacter (formerly the genus Paecilomyces) are pathogens of plant-borne insects and nematodes [9,10]. Paecilomyces lilacinus (Purpureocilliumlilacium) is a well known insect pathogen that has been used to control different crop pests such as aphids, whiteflies, thrips, drosophila and plant parasitic nematodes [9-11]. The secondary metabolite of Paecilomyces lilacinus produces a different secondary compound/toxin, called Paecilomyces lilacinus toxin [12]. These toxins have stomach toxic effects on insects and nematodes. Cyclosporin is a nonpolar cyclic oligopeptide produced from different fungal species belonging to the genera Beauveria, verticillium, paecilomyces lilacinus and Curvularia [12,14]. Cyclosporin is secreted by fungi to kill infected insect hosts or to damage their immune system to promote fungal growth [15-17]. Cyclosporin acts by blocking glycoprotein-related pumps and is involved in the clearance of xenobiotics from the haemolymph [14,18]. This study was aimed at extracting and characterizing cyclosporin C produced by the secondary metabolite of Paecilomyces lilacinus strain XI-5, which in our previous study was markedly pathogenic to bemisia tabaci [10]. In addition, toxicity studies were performed on cyclosporin C to observe its effect on bemisia tabaci survival and reproduction [12,13]. Reference to the literature 1. Hu, J.; De Barro, P.; Zhao, H.; Wang, J.; Nardi, F.; Liu, S.S. An extensive field survey combined with a phylogenetic analysis reveals rapid and widespread invasion of two alien whiteflies in China. PLoS ONE.2011,6, e16061. 2. Firdaus, S.; Vosman, B.; Hidayati, N.; Supena, E.; Darmo, J.; Visser, R.G.F.; van Heusden, A.W. The Bemisiatabacispecies complex: Additions from different parts of the world. Insect Sci. 2013, 20, 723–733. 3. Cuthbertson, A.G.S.; Vänninen, I. The importance of maintaining protected zone status against Bemisiatabaci.Insects2015, 6, 432–441. 4. Zhang, C.; Ali, S.; Musa, P.D.; Wang, X.M.; Qiu, B.L. Evaluation of the pathogenecity of Aschersoniaaleyrodis on Bemisiatabaci in the laboratory and greenhouse. Biocontrol Sci. Technol. 2017, 27, 210–221. 5. Cuthbertson, A.G.S.; Murchie, A.K. European red spider mite-An environmental consequence of persistent chemical pesticide application. Int. J. Environ. Sci. Technol. 2005, 2, 287–290. 6. He, S.; Wu, X.Y.; Zheng, J.Z.; Han, Z.Q.; Zhao, C.X.; Wang, Y.Y.; Chen, H.; Chen, C.; Han, J. A study of control effect of Bemisiatabaci(Gennadius) on greenhouse tomatoes using parasitoid EncarsiaformosaGahan. J. Anhui Agri. Sci. 2013, 41,6244–6248. (In Chinese) 7. Wang, X.S.; Xu, J.; Wang, X.M.; Qiu, B.L.; Cuthbertson, A.G.S.; Du, C.L.; Wu, J.H.; Ali, S. Isariafumosorosea-based-zero-valent iron nanoparticles affect the growth and survival of sweet potato whitefly, Bemisiatabaci (Gennadius). Pest Manag. Sci. 2019, 75, 2174–2181. 8. Du, C.L.; Yang, B.; Wu, J.H.; Ali, S. Identification and virulence characterization of two Akanthomycesattenuatus isolates against Megalurothripsusitatus (Thysanoptera: Thripidae). Insects 2019, 10, 168. 9. Goffré D, Folgarait PJ. Purpureocilliumlilacinum, potential agent for biological control of the leaf-cutting ant Acromyrmexlundii. J InvertebrPathol. 2015, 130, 107–115. 10. Sun, T.F.; Wu, J.H.; Ali, S. Morphological and molecular identification of four Purpureocillium isolates and evaluating their efficacy against the sweet potato whitefly, Bemisiatabaci (Genn.) (Hemiptera: Aleyrodidae). Egypt. J. Biol. Pest. Cont. 2021, 31, 27. 11. Amala U, Jiji T, Naseema A. Laboratory evaluation of local isolate of entomopathogenic fungus, Paecilomyc