Browsing by Author "DEVAKI, K"
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ThesisItem Open Access ISOLATION, IDENTIFICATION AND MOLECULAR CHARACTERIZATION OF Beauveria bassiana (Balsamo) Vuillemin STRAINS EFFECTIVE AGAINST Spodoptera litura (Fabricius)(Acharya N G Ranga Agricultural University, Guntur, 2019) SREESIRI, N; DEVAKI, KA roving survey was carried out in various regions of Andhra Pradesh during rabi, 2018-2019 for the natural occurrence of entomopathogenic fungi, Beauveria bassiana on Lepidopteran caterpillars. Soil samples were also collected from the surveyed fields and a few fungal infected caterpillars of Spodoptera litura, S. frugiperda, Helicoverpa armigera and Bombyx mori were collected from radish, groundnut, castor, maize fields of RARS Tirupati, Chintapalli, Palamaneru, Yagili, Kuppam. A total of 13 infected insect cadavers were collected and from which 10 were identified as B. bassiana of which one isolate was from infected silkworm larvae and three were Nomuraea rileyi isolates. From 140 soil samples in cropped area and virgin soils of Kurnool and Chittoor districts, 34 B. bassiana isolates were collected from Mahanandi, Alinagaram, Atmakur, Kalyani dam, Kovelakuntla villages. Sixteen strains were selected for laboratory bioassay against second instar S. litura at five different concentrations i.e., 1×1012, 1×1010, 1×108, 1×106 and 1×104 spores ml-1. Among the sixteen strains, AT recorded 93.33, 83.33, 70.00 and 56.67 per cent larval mortality at 1×1012, 1×1010, 1×108, 1×106 spores ml-1 after 120 h of treatment, whereas at 1×104 spores ml-1 MB recorded highest mortality of 50.00 per cent at 144 h after treatment. Regarding LC50 the strain MB registered the lowest LC50 (3.1×104) and AT has the lowest LC90 (3.41×1011) value in terms of spores ml-1. The LT50 value for all 16 strains of B. bassiana were recorded at 1×1012×1010, 1×108, Similarly, AT registered the least LT50 value of 148.75 h followed by MT strain (163.67 h). xv Among the eighteen random primers screened for variability studies in the native strains of B. bassiana, five primers (OPA-13, OPA-15, OPB-10, OPC-11 and OPE-18) produced polymorphic bands and 100 per cent polymorphism was observed among the strains in the present collection. Jaccard’s similarity co-efficient between the seventeen B. bassiana isolates produced 100 per cent genetic variation in strains i.e. MB and AT, MB and KD, Y and HAT-3, Y and MB, Y and MT, MTe and HAT-3, MT and MTe, KK and Y, KK and MTe, KK and N, MR and AT,MR and KD, MR and Y, MR and MTe, MR and KK, HAT-2 and Y, H2 and MTe, HAT-2 and N, A and MR, R and HAT-3, R and MB, R and MT, R and KK, R and MR, R and HAT-2, Ye and MB, Ye and MR, HAT-1 and MB, HAT-1 and Y, HAT-1 and MTe, HAT-1 and N, HAT-1 and MR, HAT-1 and R, Kup and AT, Kup and HAT-3, Kup and MB, Kup and Y, Kup and MTe, Kup and N, Kup and KK, Kup and MR, Kup and HAT-2, Kup and A, Kup and R, Kup and Ye, NBAII and Y, NBAII and KK, NBAII and HAT-2, NBAII and A, NBAII and R. In the resulted dendrogram AT, KD, Y, MTe, KK, A, R, Ye and HAT-1 isolates form one cluster and HAT-3, MB, MT, N, MR and HAT-2 form another cluster. However, the two isolates NBAII and Kup were separated from the two clusters.ThesisItem Open Access MOLECULAR CHARACTERIZATION OF Bacillus thuringiensis cry GENES WITH INSECTICIDAL ACTIVITY AGAINST Spodoptera litura IN GROUNDNUT(Acharya N.G. Ranga Agricultural University, 2017) DEVAKI, K; MURALI KRISHNA, TA total of 925 soil samples representing Chittoor, Kadapa, Nellore districts of Andhra Pradesh covering different ecosystems was collected to isolate bacterial cultures. These bacterial cultures were subjected to Gram staining, endospore staining and crystal staining for identification of Bacillus thuringiensis. Out of 324 Gram positive isolates, 227 isolates were able to produce endospores and maximum number of endospore producing isolates were observed in soil samples of forest ecosystem (95.77%), compared to other soil samples collected from Nellore, Chittoor and Kadapa districts. About 203 crystal staining positive Bt strains were identified. Soil samples from cultivated fallow harboured maximum crystal positive isolates (39.82%). Study on crystal morphology revealed that spherical crystals (26.11%) were most dominant, followed by irregular (24.14%) and bipyramidal (13.30%). A combination of bipyramidal and cuboidal (6.40%), cuboidal and spherical (4.43%) and bipyramidal and spherical (1.97%) were observed in 13, 9 and 4 isolates, respectively. Most of the effective isolates were observed with bipyramidal, cuboidal crystals against S. litura. In laboratory bioassay 21 isolates (C44, C33, C59, C63, C79, C92, C97, C105, C134, C212, K18, N3, N30, N44, N48, N58, N115, F287, F468, F493 and F504) were found effective against third instar S. litura larvae with 76-100 per cent mortality. The isolate from Talakona forest area (F493) was effective with 100 per cent mortality, followed by F468 (86.67%) from Bhakarapet Ghats and F287 (76.67%) from Talakona forest area and F504 (76.67%) from S.V. Zoo park area. Twenty one effective isolates were further studied for determining lethal concentrations to arrive 50 per cent mortality (LC50) and time to kill 50 per cent xvii larval population (LT50). LC50 values were in the range of 9.59 104 to 1.88 106 and HD-1 recorded lowest LC50 value, followed by F493 (9.76 104 ) and N30 (1.90 105 ). Lowest LT50 of 61.99h was observed in treatment with HD-1 followed by F493 (78.52h). Ninety two Bt strains were characterized for the presence of various cry genes by using primers viz., cry1Aa, cry1Ab, cry1Ac, cry1C, cry1Da1, cry1Ea1, cry1F, cry1Fa1, cry 1I, cry2, cry2Aa1, cry8, cry9Aa1, cry9Ca1, cry18 and cry20. Among the nine cry1 genes analyzed in the present study, cry1I was the predominant gene and present in 35 isolates (38.46%), followed by cry1Aa in 30 Bt isolates (31.87%), cry1Ac in 26 isolates (28.57%), cry1C in 18 isolates (19.78%) and cry1Fa1 in 17 isolates (18.68%), whereas, cry1Ab gene was observed in only one isolate i.e. C36. In case of cry2 genes, cry2 was observed in 14 (15.38%) isolates and cry2(a)1 was observed in 19 (20.88%) isolates. Among the two cry2 genes, cry2A(a)1 was dominant compared to cry2 in Chittoor, Nellore and forest ecosystems, whereas cry2 positive isolates were more in Kadapa district Bt samples. Among the two cry9 family genes, cry9Ca1 was dominant in 22 Bt isolates (24.18%) and 13 isolates were observed with cry9Aa1 (14.29%). In Chittoor (10 isolates), Nellore (4 isolates) and forest ecosystem (7 isolates) cry9Ca1 gene positive isolates were more compared to Kadapa district samples, where cry9Aa1 (4 isolates) samples were high compared to cry9Ca1. Eight cry genes (cry1Aa, cry1Ac, cry1Fa1, cry1I, cry2, cry2A(a)1, cry8, cry9Ca1) were observed in F493, a isolate from Talakona forest area, which was away from human interference and observed with high organic matter. This isolate harboured cry gene belongs to cry1, cry2, cry8 and cry9 groups. Similarly, isolate C67 observed with 66.67 per cent also amplified with eight cry primers (cry1Aa, cry1Ac, cry1C, cry1Da1, cry1Ea1, cry1Fa1, cry2, cry2A(a)1) followed by C134 with 7 cry genes (cry1Ac, cry1C, cry1Da1, cry1Fa1, cry1I, cry2A(a)1, cry8). These types of strains might be resulted in multifunctional insecticide activity, which is useful for control of several groups of insect pests. C134 (83.33%) consisting 7 cry genes. While, C68 (50.00%), F323 (50.00) and F504 (76.67%) were observed with 6 cry genes. Some of the isolates C63 (76.67%), K18 (86.67%) and N48 (76.67%) which were effective in bioassays, did not show amplification with any one of the cry genes screened in the present study. Sequencing of 16s ribosomal RNA results of three Bt strains (F493, F504, N115) confirmed that, these three strains are B. thuringiensis strains with high insecticidal activity. Blast analysis of these strains showed 99, 97 and 96 per cent similarity with the existing Bt gene sequences available in NCBI, GenBank and these three strains were deposited in NCBI, GenBank with Accession Nos. MF487790, MF487791 and MF197874. Field evaluation of solid and liquid formulations of Bt isolates revealed that, solid formulations were comparatively more effective in some of the isolates. Larval population/ m row at 3 and 7 days after spray, foliar damage due to S. litura at 7 and 14 days after spray was low in treatments with F493, F504 which were comparable with standard check HD-1 in both solid and liquid formulations. Highest pod yield was recorded in HD-1, F493 and F504 treated plots.