ISOLATION AND CHARACTERIZATION OF ENTOMOPATHOGENIC ...

More documents

Recommendations

Info

V. DISCUSSION Insect pathogenic fungi, often not realized by man, naturally and efficiently restrict the build up of insect pests without any interference. This part of the ‘Law of Natural Balance’ is being inadvertently destroyed by the indiscriminate use of chemical pesticides. This has led to the notion that, all forms of pest control should be integrated and environmentally acceptable to the agricultural ecosystem. It is certain that entomogenous fungi will continue to increase their share very rapidly in the integrated pest management. Mycopathogens infect insects through cuticle and are therefore, the principle pathogens among sucking insects which cannot ingest other pathogens that infect through the gut wall (Hajek and Ledger, 1994). More than 750 species of entomopathogenic fungi have been described (Maddox, 1994), of which only ten species have been extensively exploited. Current research efforts were directed at selecting native entomopathogenic fungi, characterizing them assessing their virulence and developing a formulation for them. Additionally, their compatability with certain pesticides, genetic enhancement and field evaluation was conducted. The results obtained are discussed herein. 5.1 EXPLORATION OF THE NATURAL OCCURRENCE OF INSECT MYCOPATHOGENS IN NORTHERN KARNATAKA Extensive work in the area of isolation, characterization and exploitation of entomopathogenic fungi needs to be a continuous excercise. Understanding the ecology and molecular diversity of the pathogen is of immense importance in exploiting the biotechnological potential of the pathogens. Hence, the present work undertook an extensive and repeated survey covering different agro ecological niches of northern Karnataka. The present study focused on collection of insect cadavers that were presumed to be fungal infected. Extensive variation in the per cent incidence was observed. The incidence of mycopathogens collected during the survey is indicated in the Fig. 1. Wide spread incidence was observed with respect to M. anisopliae, V. lecanii, B. brassiana and F. oxysoporum. Amongst the different mycopathogens, N. rileyi had the highest natural incidence (30-80%) in the transitional belt of northern Karnataka. The natural incidence of C. sphaerospermum was higher in Dharwad and was observed to parasitize ligeid bug (18.58%). The natural incidence of M. anisopliae varied from 1.8 to 10.3 per cent and was maximum on diamond black moth, pest that prevailed on cabbage at Hirebagewadi. The incidence of C. cladosporides and A. candidus was less than 10 per cent. These results indicated a high variability of disease incidence due to mycopathogen attack. The variability observed in the disease incidence could be due to the prevalence of different agro climatic conditions. The incidence of M. anisopliae was seen across two contrasting ecological niche i.e., Dharwad which, had a relative humidity of 80-89 per cent and a temperature of 26.5-27.7°C and Gangavathi which had a lower relative humidity of 75- 78 per cent and higher temperature 30-32.5°C. The ability of these to grow at varying temperature upto 35-37°C, indeed, has been observed (Yip et al., 1992). The present result also indicate the potential to use M. anisopliae as biopesticide under contrasting environments. 5.2 ISOLATION AND CHARACTERIZATION OF ENTOMOPATHOGENIC FUNGI The cadavers of susceptible host brought to the laboratory and those presumed to be infected by M. anisopliae and V. lecanii were used for isolation of the mycopathogen. A total of seven fungi were isolated, purified and identified based on their colony characteristic and microscopic examination. The origin of the isolates and the host are shown in the (Table 5 and Fig. 1). Of the seven, four were identified to be M. anisopliae and three were assigned to V. lecanii. The identity of the fungi was later confirmed at Agarkar Research Institute, Pune.
The hyphal and conidial characters did not vary among the isolates collected from different places during survey. However, V. lecanii exhibited significant variation in the number of days taken for sporulation, sporulation, spore yield and time taken to cover the given diet for mass production. Among the different isolates of V. lecanii, Vl1 recorded highest colony diameter (52.13) but least spore yield (4.21x10 9 ). The Ma2 and Ma1 isolates recorded higher colony diameter of 42.30 mm and 38.60 mm respectively. In these two isolates, the sporulation commenced two to three days earlier than isolates Ma3 and Ma4. These results indicate variability among the isolates which need to be realised when being used for development of effective bioinoculant and mass production. 5.3 TESTING OF EFFICACY OF Metarhizium anisopliae (Ma2) AND Verticillium lecanii (Vl1) AGAINST DIFFERENT INSECT PESTS The pathogenicity of isolates Vl1 and Ma2 was tested against various insect pest (B. brassiae, A. crassivora, M. sacchari, P. latus, A. disperses, H. armigera and O. rhinoceros). The pathogenicity was highly variable (51.35 to 94.50 per cent). Vl1 caused maximum pathogenicity in case of cabbage aphid B. brassica (94.50%). It also caused extensive damage against cowpea aphid A. crassivora (84.23%). Ma2 was highly pathogenic against H. armigera and O. rhinocerous. The toxicity of M. anisopliae against O. rihoceros has been reported by Gopalkrishnan and Narayanan (1988). These results indicate the possibility of using V. lecanii on aphids (B. brassica and A. crassivora) and M. anisopliae on H. armigera and O. rhinoceros. The wide variability observed indicates further exploration could result in isolation of more potent strains. It would also help to extend the host species. One of the ways in which the population of M. anisopliae could be enhanced in culture bank would be to isolate M. anisopliae from soil on a selective medium containing 10 µm/ml of dodine (Ndodecyl guanidine monoacetate) (Liu et al., 1993). In fungal cells, dodine induces the loss of 32 P (Brown and Sisler, 1960), P1, amino acids and UV absorbing materials and increases the permeability of the cytoplasmic membrane to externally added Ni 2+ (Miller and Barran, 1977). In plant cells, dodine induces an efflux of betacyanin and total ions. Other cationic amphiphiles (namely, cetyltrimethylammonium bromide, chlorohexidine and polymyxins) have been reported to cause severe damage to the cytoplasmic membrane in several bacterial species (Newton, 1956). Using dodine in the medium, thus would enhance the probability of isolating diverse and perhaps more effective strains. 5.4 INFLUENCE OF DIFFERENT NUTRIENT SOURCES One of the key factor in enhancing the population of mycopathogens propagules in the inoculum is the availability the nutrients. Several attempts have been made to multiply these mycopathogens using semi synthetic and solid substrate primarily to cut down cost of production. Additionally, the availability of nutrients also influences the survival of these organisms in nature. Carbon and nitrogen are the most vital nutrients required for growth and sporulation (Campbell et al., 1983). Therefore, the effect of different carbohydrates and nitrogen on the growth of M. anisopliae and V. lecanii was evaluated at different concentrations in Czapeckdox broth. The nutritional requirement of the fungi in both species and strain varied. Starch supported the highest growth of M. anisopliae Ma2 (6.01 gm/250 ml) followed by fructose and sucrose. In case V. lecanii Vl1 too, the maximum biomass was obtained when grown on starch followed by sucrose. Soluble starch has been reported to be the best substrate tested for sporulation of M. anisopliae strain when grown on soypeptone based medium (Li and Holdom, 1994) and hence has been recommended to be useful carbon source in the production media. In earlier studies, the most effective carbon source for sporulation of N. rileyi were reported to be dextrose (IM et al., 1988) and Maltose (Balardrin and Loch, 1989). Edelstein et al. (2004) on the other hand found that media with potato extract induced higher growth rate.
Page 1 and 2:
ISOLATION AND CHARACTERIZATION OF E
Page 3 and 4:
Chapter No. I INTRODUCTION C O N T
Page 5 and 6:
Contd….. Table No. Title 14. Spor
Page 7 and 8:
Contd….. Table No. Title 39. Per
Page 9 and 10:
Plate No. LIST OF PLATES Title 1. M
Page 11 and 12:
I.INTRODUCTION The increased use of
Page 13 and 14:
II. REVIEW OF LITERATURE The variou
Page 15 and 16:
The entomopathogenic fungi so far i
Page 17 and 18:
Table 1. Contd….. Sl. No. Insect
Page 19 and 20:
Milner et al. (2002) studied the ef
Page 21 and 22:
25°C for sporulation and biomass p
Page 23 and 24:
Pathogencity of various strains/iso
Page 25 and 26:
Gopalkrishnan and Mohan (2000) test
Page 27 and 28:
necessary serial dilutions under ph
Page 29 and 30:
Sl. No. Table 2. Details of differe
Page 31 and 32:
3.9 EVALUATION OF DIFFERENT FORMULA
Page 33 and 34:
Fig. 1. Mycopathogens of insect pes
Page 35 and 36:
Table 5. Contd….. Month/s visited
Page 37 and 38:
Table 5. Contd….. Month/s visited
Page 39 and 40:
Table 5. Contd….. Mycosis noticed
Page 41 and 42:
Totally eight isolates of N. rileyi
Page 43 and 44:
Metarhizium anisopliae Vertucillium
Page 45 and 46:
Ma 1 (Dharwad) Ma2 (Dharwad) Ma 3 (
Page 47 and 48:
Table 8. Mortality caused by the is
Page 49 and 50:
Helicoverpa armigera Dimond Black M
Page 51 and 52:
Plate 5. The moratality of Hlelicov
Page 53 and 54:
Table 11. Total biomass production
Page 55 and 56: Table 12. Total biomass production
Page 57 and 58: Table 14. Sporulation of Metarhiziu
Page 59 and 60: Mean spore yield x 10 8 /g 25 20 15
Page 61 and 62: Among the non-grain substrates, the
Page 63 and 64: Mean spore yield x 10 4 /g 35 30 25
Page 65 and 66: Sl. No. Table 19. Effect of fungici
Page 67 and 68: Per cent inhibition 100 90 80 70 60
Page 69 and 70: Sl. No. Table 21. Effect of insecti
Page 71 and 72: Out of nine insecticides, endosulfa
Page 73 and 74: Sl. No. Table 23. Effect of weedici
Page 75 and 76: Days after storage Table 25. Persis
Page 77 and 78: Table 26. Survival of Metarhizium a
Page 79 and 80: Table 28. Survival of Metarhizium a
Page 81 and 82: Sl. No. Table 29. Survival of Metar
Page 83 and 84: Sl. No. Table 30. Survival of Metar
Page 85 and 86: Per cent germination 90 80 70 60 50
Page 87 and 88: Sl. No. Table 33. Survival of Verti
Page 93 and 94: 4.9 PATHOGENICITY OF Verticillium l
Page 95 and 96: Sl. No. Table 39. Per cent mortalit
Page 97 and 98: Culture grown on rice grains Harves
Page 99 and 100: Tr. No. Table 41. Per cent reductio
Page 101 and 102: Tr. No. Table 43. Per cent reductio
Page 103 and 104: The fungal spray at higher dosage (
Page 105: Table 46. Effect of wild types and
Page 109 and 110: In an earlier experiment, bagasse w
Page 111 and 112: M. anisopliae exhibited rapid reduc
Page 113 and 114: higher mortality of aphids. The com
Page 115 and 116: 14. Oil formulations proved better
Page 117 and 118: CHARNLEY, A.K. AND ST. LEGER, R.J.,
Page 119 and 120: IGNOFFO, C.M., MARSTON, N.L., HOSTE
Page 121 and 122: MULLER, E.J., 2000, Control of the
Page 123 and 124: ST LEGER, R.J., BUTT, T.M., STAPLES
Page 125 and 126: APPENDIX I Mean monthly weather dat
Page 127 and 128: Maharashtra Association for the Cul
Page 129: ISOLATION AND CHARACTERIZATION OF E
show all

ISOLATION AND CHARACTERIZATION OF ENTOMOPATHOGENIC ...

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?