Metarhizium anisopliae

This ppt contains all detais about Metaehizium green muscardine fungus... its history and mass production with applied science.

1. 06/04/2018 1 Metarhizium anisopliae

2. K.K.W. COLLEGE OF AGRICULTURE, NASHIK DEPARTMENT OF AGRIL. ENTOMOLOGY

3. Course No.- AEL-ENTO-486 Course Title- Mass production of bioagents and biopesticides Topic- Metarhizium anisopliae Prepared By- Tushar Shivaji Deshmukh Under The Guidance of- Prof. T.B.Ugale (Module Incharge ) 06/04/2018 3 Metarhizium anisopliae

4. Presentation include… 06/04/2018 Metarhizium anisopliae 4 Introduction Scientific Classification History Appearance Uses Mode Of Action Toxins Produce Habitat Pests Attacked By Fungus Targeted Crops Life Cycle

5. 06/04/2018 Metarhizium anisopliae 5 12. Mycelium Structure 13. Pesticide Susceptibility 14. Climatic Effects on Fungus 15. Methods OF Mass Production 16. Field Application 17. Frequency Of Application 18. Improvement in Efficiency of Fungus 19. Formulations 20. Precautions 21. Human Infections 22. Advantages & Disadvantages 23. Details That Product Should Contain 24. Available Products In Market 25. Conclusion 26. References

6. Introduction: Metarhizium anisopliae (also known as Entomophthora anisopliae in the early 1900s). It was originally isolated from, the beetle Anisoplia austriaca . 06/04/2018 6 Metarhizium anisopliae

7. It is a fungus that grows naturally in soils throughout the world and causes disease in various insects by acting as a parasite; it thus belongs to the entomopathogenic fungi. It is a mitosporic fungus with asexual reproduction for which a teleomorph has not yet been discovered. The disease caused by the fungus is called green muscardine disease because of the green color of its spores. 06/04/2018 7 Metarhizium anisopliae

8. Kingdom: Fungi Phylum: Ascomycota Class: Sordariomycetes Order: Hypocreales Family: Clavicipitaceae Genus: Metarhizium Species: M. anisopliae Binomial name : Metarhizium anisopliae (Metchnikoff) Sorokin (1883) Scientific Classification Cockroach killed by  M. anisopliae 06/04/2018 8 Metarhizium anisopliae

9. 2700BC- CHINESE PEOPLE RECOGNISE DISEASES OF HONEY BEE AND SILKWORM. ANCIENT TIME- INDIAN LITERATURE REFERS THE DISEASES OF SAME INSECTS. SAME TIME- IN EUROPE ARISTOTLE WAS THE FIRST PERSON MENTION ABOUT THE DISEASES OF HONEY BEES. 1835 AGOSTINO BASSI- EXPERIMENT ON SILK WORM DISEASE. History 06/04/2018 9 Metarhizium anisopliae

10. The first use of  Entomphthora anisopliae  as a microbial agent against insects was in 1879, when Elie Metchnikoff used it in experimental tests to control the wheat grain beetle. Elie Metchnikoff Renamed as M. anisopliae by Sorokin in 1883. Sorokin 06/04/2018 10 Metarhizium anisopliae

11. Coudron et al. (1985) and Preen et al. (1985) cultured entomopathogenic fungi on cereals. Produces enzymes that can degrade the components of the insect cuticle. (Clarkson and Charnley, 1996). Earth BioSciences of New Haven , CT (formerly Taensa Company) submitted an application May 28, 1999 for registration of Metarhizium anisopliae strain F52 for non-food, indoor and greenhouse use. 06/04/2018 11 Metarhizium anisopliae

12. Appearance Infections of arthropods by Metarhizium species are easily recognized a few days after death, when the fungus grows out of the arthropod integument and forms reproductive structures. Initially, one only sees fungal hyphae that appear white, but, as conidia form and mature they often take on a characteristic olive green color. 06/04/2018 12 Metarhizium anisopliae

13. However, depending on the species and strain of Metarhizium , spores can range in color from white to yellow to brown and green (Tanada and Kaya 1993). 06/04/2018 13 Metarhizium anisopliae

14. Each spore ball is compost of cluster of conidiogenous cells. The conidia are single-celled, haploid, and hydrophobic. 06/04/2018 14 Metarhizium anisopliae

15. Habitat Metarhizium species are commonly thought of as soil saprophytes and are most frequently found in disturbed habitats like agricultural fields as compared to forest ecosystems (Meyling and Eilenberg 2007). These fungi form associations with plant roots in the rhizosphere (Hu and St. Leger 2002) and survive better in that environment than in surrounding potting soil over extended periods of time (Bruck 2005). 06/04/2018 15 Metarhizium anisopliae

16. This Fungi has been found in forests, agriculture, high UV locations, and very damp areas. It can be on leaves, or the insect cadaver after the spores have matured and covered the surface. It usually deposits its spores in soil and then waits for another host to come in contact with the spores on the dead insect or where the insect had decomposed or died. This means that it usually lives in areas that are not heavily populated or with a lot of industrial areas. 06/04/2018 16 Metarhizium anisopliae

17. Mode Of Action: Adhesion Germination Appressorium formation Penetration Extrusion Sporulation 06/04/2018 17 Metarhizium anisopliae

18. 06/04/2018 18 Metarhizium anisopliae

19. How Fungus Actually Acts… When these mitotic (asexual) spores (properly called conidia) of the fungus come into contact with the body of an insect host, they germinate and the hyphae that emerge penetrate the cuticle. The fungus then develops inside the body eventually killing the insect after a few days; this lethal effect is very likely aided by the production of insecticidal cyclic peptides ( destruxins ). 06/04/2018 19 Metarhizium anisopliae

20. The cuticle of the cadaver often becomes red. If the ambient humidity is high enough, white mold then grows on the cadaver that soon turns green as spores are produced. Insects can also spread the fungus through at the time of mating. 06/04/2018 20 Metarhizium anisopliae

21. Some insects have developed physiological mechanisms to reduce infection by fungi  M. anisopliae . For example, the Desert locust produces antifungal toxins, which can inhibit the germination of spores. In addition, insects can escape infection by molting rapidly or developing a new integument before the fungus can penetrate the cuticle. Desert locust 06/04/2018 21 Metarhizium anisopliae

22. Toxins Produced: Destruxin A Destruxin B Destruxin C Destruxin D Destruxin E Destruxin F 06/04/2018 22 Metarhizium anisopliae

23. Pests Attacked Metarhizium species are known to attack a wide range of arthropods: greater than 200 species in over 50 families. These include many species of agricultural, medical and veterinary importance. Metarhizium species are attack on various ticks and beetles; root weevils, flies, ants, thrips, and locusts and grasshoppers. 06/04/2018 23 Metarhizium anisopliae

24. Additionally, Metarhizium species have been developed in other countries for use against cockchafers, spittlebugs, grubs, borers, and for control of mosquitoes that vector malaria. However, this fungus may also infect and kill beneficial organisms. In laboratory assays, the thrips predator  Orius insidiosis  showed a high rate of susceptibility to  M. anisopliae . ( - Raymond A. Cloyd, University of Illinois ) Orius insidiosis 06/04/2018 24 Metarhizium anisopliae

25. Oryctes rhinocerus Red locust Thrips Leafhopper 06/04/2018 25 Metarhizium anisopliae

26. Aphids Mosquitoes Termites 06/04/2018 26 Metarhizium anisopliae

27. Grapes Sugarcane Cotton Groundnut Maize Sorghum Barley Rice Potato Soybean Oranges Vegetables and Fruit crops Target crops: 06/04/2018 27 Metarhizium anisopliae

28. Life Cycle Generally, the development of infection can be separated into three stages. First, asexual conidia come into contact with the arthropod integument. The conidia stick to the arthropod’s exoskeleton, germinate and grow a germ tube. A penetration peg grows under the appressorium, pierces the integument and enters the hemocoel. The penetration of the fungus is achieved by the production of a cocktail of hydrolytic enzymes including proteases, lipases, chitinases, and mechanical pressure. 06/04/2018 28 Metarhizium anisopliae

29. Second, single cells of the fungus, blastospores, bud off of the penetration structure, circulate in the insect hemocoel and multiply, thereby depleting host nutrients. Metarhizium species are also known to produce compounds that are toxic to arthropods and aid in killing the host, suppressing host immune defenses and fending off potential microbial competitors. 06/04/2018 29 Metarhizium anisopliae

30. Finally, after the host dies due to mycosis, the fungus will penetrate out of the integument and grow conidiophores, on which environmentally stable aerial conidia are produced. These conidia are passively disseminated into the environment and eventually infect new hosts. Conidia Of M. anisopliae 06/04/2018 30

31. 06/04/2018 31

32. Mycelium Structure of M. anisopliae 06/04/2018 32 Metarhizium anisopliae

33. Pesticide Susceptibility No significant effect of the pesticides on M. anisopliae is observed. A large number of fungicides negatively impacted conidial germination and mycelial growth in Petri dishes, there was little effect in the rhizosphere. The fungicides with the largest detrimental impact were Captan and Triflumizole. 06/04/2018 33 Metarhizium anisopliae

34. The herbicides reduced the respiratory activity of the fungus, while the fungus remains pathogenic to its insect host. These findings indicate that the action of pesticides on the fungus in soil was highly discrete, suggesting that these agents can be used in combination in agroecosystems without compromising fungal activity. (Dinalva A. Mochi; Antonio C. Monteiro; José C. Barbosa, Entomol. vol.34 no.6 Londrina Nov./Dec. 2005) 06/04/2018 34 Metarhizium anisopliae

35. M. anisopliae  is sensitive to temperature extremes; spore viability decreases as storage temperatures increase and virulence decreases at low temperatures. At 27 degrees C, conidial yield is maximum. Where the sporulation is optimized at RH >80%. Fungal spores are readily killed by solar radiation. Effect of Climate on Fungus: 06/04/2018 35 Metarhizium anisopliae

36. Methods of mass production of Metarhizium anisopliae:- The fungus is mass multiplied on PDA medium, PDB medium, & jaggary yeast medium. The fungus can be cultured on crushed grains of rice. 06/04/2018 36 Metarhizium anisopliae

37. Material required for media preparation: Potato dextrose media Jaggery yeast media MATERIAL QTY Jaggary 30 g Yeast 5 g Distilled water 1 lit. MATERIAL QTY Potato 200 g Dextrose 20 g Distilled water 1 lit pH – 7.0 06/04/2018 37 Metarhizium anisopliae

38. Potato dextrose media Jaggery yeast media 06/04/2018 38 Metarhizium anisopliae

39. Procedure for Media Preparation: Weigh 30 g jaggary+5 g yeast in separate plate Take half of distilled water(500ml) in the utensil and put on the heating gas After heating the water , dissolve the ingredients in hot water Add the remaining distilled water in that mixture & make up the final volume(1000ml) Fill the media in sterilize bottles as required(100ml/bottle ) 06/04/2018 39 Metarhizium anisopliae

40. Procedure for Media Sterilization: Plug the bottle with cotton plug Cover the cotton with plastic paper with the help of rubber band Keep the media bottle in autoclave for Sterilization After of 15 psi pressure sterilized bottles are remove from autoclave Autoclaved bottles are kept for cooling 06/04/2018 40 Metarhizium anisopliae

41. Procedure for Inoculation: The cooling bottels are transfered inoculation room & inoculation of the spore from pure mother culture of M. anisopliae into the bottles. After the inoculation bottles are transfered to growth room for incubation. 06/04/2018 41 Metarhizium anisopliae

42. On Jaggery Yeast media Selection and weighing of jaggery Weighing of yeast Dissolve jaggery and yeast in hot water

43. Fill the media into bottles Kept the bottles into autoclave for sterilization at 121 ˚ c at 15 psi Remove the bottles after sterilization and cool it.

44. Inoculation of M. anisopliae into media bottles This process is done in laminar air flow to avoid contamination. Fully growth of M. anisopliae after 8- 10 days of incubation Incubation of inoculated media for 8–10 days at 25–30 c temp.

45. Field Application: Foliar spray : 1 liter / acre in 200 liters of water i.e. 5ml per liter of water. The spray volume depends on the crop canopy. Soil application : 1 lit./acre from drip. Seed treatment: 4-5 gm per kg. of seeds as per standard wet treatment. Over dosing does not cause any harmful side effects to plants. 06/04/2018 45 Metarhizium anisopliae

46. Frequency of Application: Applications should be repeated at least once in 15-20 days for four times. For greenhouse pest problems, applications in every 10-15 days are recommended. All applications should be based on monitoring of pest populations. 06/04/2018 46 Metarhizium anisopliae

47. How to Improve Efficiency? Addition of milk @ 0.5 – 1 lit. per lit/kg of formulation prove to increase the efficiency of M. anisopliae. Drip irrigation given to field to increase the humidity which helps in getting better result. Application in low temprature at the time of morning and evening. 06/04/2018 47 Metarhizium anisopliae

48. Formulations: Solid Formulations a. Granules b. Wettable powder 2.Liquid Formulations a. Emulsifiable concentrates b. Solution 06/04/2018 48 Metarhizium anisopliae

49. Precautions to be taken during application: M. anisopliae formulation should not be spray along with any fungicide or antagonistic bio fungicide. There should not be spray of any fungicide Upto 10-15 days After and Before the spray of M. anisopliae . The spray should be taken in evening and morning time at low temprature. CAUTION 06/04/2018 49 Metarhizium anisopliae

50. Avoid inhalation and skin contact, wash contaminated skin, Do not drink and eat during application. Destroy empty containers after use. Keep in cool & dry place away from heat or open flame. 06/04/2018 50 Metarhizium anisopliae

51. Human Infections: Previously, M. anisopliae was not considered to be pathogenic to humans due to its optimal growth temperature of 27°C,however, human infection has been reported. 06/04/2018 51 Metarhizium anisopliae

52. Most of the cases have been ophthalmic infections with additional cases of fungal sinusitis and disseminated fungi. The first reported case occurred in Columbia, South America in 1997 and was a fungal keratitis in an 18-year-old man. There have now been four reported cases of fungal keratitis and two cases of sclerokeratitis. 06/04/2018 52 Metarhizium anisopliae

53. Uses Metarhizium anisopliae is being used as a biological insecticide to control a number of pests such as Grasshoppers,Termites, Thrips,caterpillars,aphids etc. And Medically as well. Its use in the control of malaria-transmitting mosquitoes is under investigation. 06/04/2018 53 Metarhizium anisopliae

54. It also produces different enzymes which are used in different rections. (Eg. Lipase in Biodiesel) Moreover, soil biological and physico-chemical properties like soil pH, available NPK, exchangeable cations, soil organic carbon and microbial biomass carbon were found to be increased after its application in the studied area. (Pranab Dutta, April 14, 2017) 06/04/2018 54 Metarhizium anisopliae

55. Advantages of M. anisopliae : M. anisopliae can be used to control harmful insect and pest without affecting crop. Avoid pollution and enviormental hazards. The lack of problem of resistance in insect control. Can also applied at harvest stage. No residual toxicity. Long term effect on pest suppression . 06/04/2018 55 Metarhizium anisopliae

56. Disa dvantages of M. anisopliae : No immediate action. Only effective to a specific group of insects. Each application may control part of the insect pests. If the other species may present they may continue to cause damage. 06/04/2018 56 Metarhizium anisopliae

57. -Product Name -Ingredient Statement -Registration Number -"Keep Out of Reach of Children“ -Signal Word (CAUTION) -First Aid Statement -Personal Protective Equipment (PPE) Requirements -Environmental Hazard Statement -Storage and Disposal Statement -Agricultural Use Requirements -Non-Agricultural Use Requirements -Directions for Use The product shall contain the Following information: 06/04/2018 57 Metarhizium anisopliae

58. Products Available In Market: 06/04/2018 58 Metarhizium anisopliae

59. Conclusion: Thus, Metarhizium anisopliae is an entomopathogenic fungus which controls different pests. It is efficient and almost non-toxic to human being. It is harmless to ecology as well as environment. The effects of this fungi on the effects of non-target organisms and the environment should also be considered before using them in the field. 06/04/2018 59 Metarhizium anisopliae

60. References Metarhizium anisopliae From Wikipedia, the free encyclopedia Mode of Infection of Metarhizium spp . Fungus and Their Potential as Biological Control Agents (Kimberly Moon San Aw and Seow Mun Hue) http://www.entomology.wisc.edu/mbcn/kyf607.html Cloyd, Raymond A. (1999). “The Entomopathogenic Fungus Metarhizium anisopliae ”. (Midwest Biological Control News VI ) 06/04/2018 60 Metarhizium anisopliae

61. Evaluation of the Entomopathogenic Fungi Metarhizium anisopliae and Beauveria bassiana against the Red Palm Weevil Rhynchophorus ferrugineus (G. Gindin ) Use of Beauveria bassiana and Metarhizium anisopliae for Fruit Fly control: A novel Approach (Jorge Toledo, Pablo Liedo) Science Direct-Medical Mycology Case Reports. Effects Of F undicides On The Developement Of The Entomopathogenic Fungus Metarhizium anisopliae Var . anisopliae (Maribel Yánez, and Andrés France) 06/04/2018 61 Metarhizium anisopliae

62. Sporulation Of Metarhizium Anisopliae Var. Acridum On Rhammatocerus Schistocercoides Under Humid And Dry Conditions. ( Bonifacio Peixoto Magalhaes ) Studies on factors influencing the viability of entomopathogenic fungi Metarhizium anisopliae in soil adapting culture dependent method. ( S. Karthick Raja Namasivayam ) 06/04/2018 62 Metarhizium anisopliae

63. 06/04/2018 63 Metarhizium anisopliae