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ThesisItem Open Access Evaluation of entomopathogenic fungi for the management of coleopteran pests and characterisation of pesticide tolerant strains(Department of agricultural entomology, College of agriculture, Vellayani, 2014) Anis Joseph, R; KAU; Sudharma, KDevelopment of safe and sustainable technologies for pest management is the need of the hour to counter the adversities created by the synthetic pesticides. Considering this, the research on “Evaluation of entomopathogenic fungi for the management of major coleopteran pests and characterisation of pesticide tolerant strains” was carried out in the Department of Agricultural Entomology, College of Agriculture during 2007-2014. The main objectives of the study were to assess the pathogenicity of entomopathogenic fungi Beauveria bassiana (Balsamo) Vuillimen and Metarhizium anisopliae (Metschnikoff) Sorokin against coleopteran pests, determination of LC 50 , LC 90 and LT 50, development of formulations, of field efficacy, compatibility with pesticides and development of pesticide tolerant strains and molecular characterization of the fungi. assessment The isolates PDBC Bb 5 and PDBC Ma 4 were evaluated against nine coleopteran pests viz. Aulacophora foveicollis Lucas, Basilepta fulvicorne Jacoby, Cylas formicarius F., Cosmopolites sordidus Germ., Henosepilachna vigintioctopunctata F., Lanka ramakrishnai Prathapan & Viraktamath, Metriona circumdata H., Oryctes rhinoceros Linn. and Rhynchophorus ferrugineus F. Both the fungi were pathogenic to the grubs as well as adults of all the nine insects tested. Though the symptoms produced in the test insects by both the fungi were more or less the same, differences in the behavior of the infected insects were seen. The period taken for the expression of symptoms also varied. The virulence of these two fungi was determined against these nine coleopterans. Ma 4 was found inferior to Bb 5 in its ability to infect the adults of all the test insects except to that of O. rhinoceros at the comparable dose of 10 8 spores ml -1 . Further, bioassay was conducted against the adults and grubs of all the nine test insects and the LC 50 , LC 90 and LT were worked out. From the probit analysis, it was seen that to achieve fifty per cent mortality of the adult coleopterans 50 with B. bassiana within the shortest periods, spore concentrations of 5. 27 × 10 , 6.53 × 10 8 , 4.24 ×10 9 , 5.62 × 10 7 , 3.99 × 10 8 , 6.76 × 10 8 , 7.26 × 10 9 , 1.56 × 10 and 3.76 × 10 13 spores ml -1 was essential for A. foveicollis, B. fulvicorne, C. sordidus, C. formicarius, H. vigintioctopunctata, L. ramakrishnai, M. circumdata, O. rhinoceros and R. ferrugineus respectively. The LC values were much less for the grubs when compared to the adult coleopterans and the values were 2. 15 × 10 50 , 1.87 × 10 6 , 3.83 × 10 8 , 2.94 × 10 7 , 2.79 × 10 7 , 5.72 × 10 6 , 4.96 × 10 and 4.64 × 10 11 spores ml -1 respectively for the grubs of the above mentioned insects. Corresponding LC values were also worked out. The lethal time to obtain fifty percent mortality also varied with insects. With respect to M. anisopliae 11.42 × 10 10 , 3.99 × 10 8 90 , 8.86 × 10 10 , 6.27 × 10 9 , 6.96 × 10 8 , 3.66 × 10 11 8 8 15 , 6.22 × 10 , 6.13 × 10 , 2.58 × 10 13 and 2.73 × 10 15 spores ml -1 respectively was required for the adult coleopterans and 4.91 × 10 7 , 6.09 × 10 8 , 4.81 × 10 8 , 5.83 × 10 8 , 4.95 × 10 7 , 2.42 × 10 , 4.64 × 10 9 , 3.79 × 10 8 and 10.29 ×10 13 spores ml -1 respectively for the grubs. From the LC 50 and LC values, information on the effective field doses of these two fungi against the nine coleopterans could be garnered. 90 Inorder to identify cost effective materials for the multiplication of the fungus, nine substrates were evaluated. The ideal substrates that maintained the viability of B. bassiana were cow dung, wheat bran, rice bran and neem cake. Cow dung and wheat bran supported maximum cfu of M. anisopliae. The peak sporulation of the fungi was observed in the samples drawn two months after storage. Talc based formulations of both fungi maintained the required standards of colony forming units in the formulation upto three months after storage. The cfu at third month after storage for B. bassiana and M. anisopliae were 0.2 × 10 9 cfu g -1 10 8 and 0. 24 × 10 respectively. The bioefficacy of fungi cultured in different substrates and stored for different months was also evaluated against the grubs and adults of C. formicarius. With respect to bioefficacy also, the fungi cultured in cow dung, wheat bran and neem cake proved better. 7 11 9 The results of the field experiment in banana, variety Nendran to assess the effect of spore suspensions of B. bassiana and M. anisopliae, fungi in cow dung and neem cake substrates and talc based formulations of the fungi in comparison with insecticide revealed that the best treatment was talc based B. bassiana @ 30 g l excepting the insecticide check, chlorpyrifos 0.03 per cent for the management of C. sordidus. In the succeeding crop of banana talc based B. bassiana @ 30 g l was even superior to the insecticide check, chlorpyriphos 0.03 per cent. The least number of galleries (0.63) and the least number of grubs in the rhizomes (0.29) besides the lowest number of adult C. sordidus in soil samples were seen in talc based B. bassiana @ 30 g l -1 . The B : C ratios calculated for the treatments with talc based B. bassiana @ 30 g l -1 in the main crop was 1.57 compared to 1.24 for chlorpyriphos 0.03 per cent. The B : C ratios for the treatments with talc based B. bassiana @ 30 g l -1 and spore suspension of B. bassiana 5 × 10 11 spores ml -1 in the succeeding crop of banana were 1.78 and 1.49 which were higher than that for chlorpyriphos 0.03 per cent. The results from the succeeding crop indicate the ability of these fungi to self perpetuate and bring about long lasting effect in the treated area. Field experiments were also conducted in sweet potato, variety Sree Bhadra to assess the effect of two fungi. Talc based B. bassiana @ 30 g1 was superior treatment excepting imidacloprid 0.006 per cent in reducing the galleries produced by the weevils. A similar trend was evident in the second trial also. The effect of application of B. bassiana and M. anisopliae in cow dung and neem based substrates showed moderate effect with respect to the number of galleries and number of grubs. Drenching of talc based formulation of B. bassiana @ 30 g l -1 was better than its foliar treatment. The compatibility of B. bassiana and M. anisopliae with two fungicides and six insecticides was also evaluated inorder to evaluate the suitability of integrating the fungi with pesticides in pest management programmes. Good compatibility of the insecticide, imidacloprid 0.006 per cent with B. bassiana and M. anisopliae was seen. Bioefficacy of the fungi cultured in this insecticide was also higher. Attempts were -1 -1 -1 also made to develop pesticide tolerant strains of B. bassiana and M. anisopliae. For this they were grown continuously in media with varying doses of pesticides. A total of ten passages through poison food media was made. It was seen that both the fungi tolerated high doses of pesticides though there was inhibition in growth, sporulation and bioefficacy. B. bassiana and M. anisopliae tolerated even 32 times higher the recommended field dose of imidacloprid. Subculturing reduced the spore production of both the fungi and the rate of reduction was higher for B. bassiana. Variations induced in B. bassiana and M. anisopliae after ten passages through poisoned media were analysed. Pesticides varied in their ability to induce such changes in the fungi. Polymorphism was higher for B. bassiana cultured in carbendazim. The polymorphism exhibited in B. bassiana was higher (83.19 per cent) compared to M. anisopliae (38.46 per cent). Among the ten universal fungal primers evaluated, RFu – 10 was found to give maximum polymorphism. To conclude, B.bassiana and M.anisopliae are pathogenic to the grubs and adults of all the nine test insects. Of the two fungi tested Bb 5 was more virulent than Ma 4 except to O. rhinoceros. Talc based formulation of both the fungi maintained the required standards of cfu in the formulations upto three months after storage. Cow dung, wheat bran and neem cake are ideal substrates for the multiplication of the fungi. Soil drenching of talc based formulation of B. bassiana @ 30 g l three months after planting was the best treatment for the management of C. sordidus. For the management of C. formicarius in sweet potato also soil drenching of talc based formulation of B. bassiana @ 30 g l -1 was superior. Compatibility of B. bassiana and M. anisopliae with insecticides and fungicides varied, the most compatible one was imidacloprid. Maximum tolerance to this insecticide, upto 32 times higher than the field dose was shown by B. bassiana and M. anisopliae. Pesticides induced changes in DNA, the polymorphism exhibited was higher for B. bassiana compared to M. anisopliae. Nine isolates of the fungi were also identified through ITS sequencing. The new isolates were Beauveria sp. from C. formicarius, B. bassiana -1 from B. fulvicorne, B. brongniartii from M. circumdata, Metarhizium sp. from B. fulvicorne; Metarhizium sp., Metarhizium album and Metarhizium anisopliae var majus from O. rhinoceros, Fusarium moniliformae from H. vigintioctopunctata and Paecilomyces sp. from C. sordidus and these isolates can be exploited in insect specific biocontrol programmes.