Athira, G Menon
Bio-efficacy of the entomo pathogenic bacteria, photorhavdus luminescens(Thomas & Poinar) aganist Tetranychus truncatus Ehara (Prostigmata: Tetranychidae) - Vellanikkara Department of Agricultural Entomology, College of Agriculture 2024 - 97,xxiiip.
Ph.D
Spider mites pose a serious threat to economically important agricultural and horticultural crops, worldwide. The spider mite, Tetranychus truncatus Ehara is a widely distributed polyphagous species inflicting considerable damage to vegetable crops and ornamental plants, both under open and protected cultivation in Kerala. The repeated use of synthetic acaricides has led to the development of resistance in T. truncatus against novel acaricides such as fenazaquin and spiromesifen, necessitating more sustainable alternative strategies for mite pest management. The symbiotic bacterium, Photorhabdus luminescens associated with the entomopathogenic nematodes (EPNs), Heterorhabditis spp. has been reported as a potential biocontrol agent against a broad range of arthropod pests, including mites.
The research programme on ‘Bio-efficacy of the entomopathogenic bacteria, Photorhabdus luminescens (Thomas & Poinar) against Tetranychus truncatus Ehara (Prostigmata: Tetranychidae)’ was carried out during 2018-2023 with the objectives to identify the potent local strains of P. luminescens; to develop suitable formulations of P. luminescens and to evaluate the efficacy of bio-formulations of P. luminescens against T. truncatus.
Soil samples were collected from different localities across five districts of Kerala viz., Kasaragod, Malappuram, Thrissur, Ernakulam, and Idukki. In the laboratory, EPNs were isolated from 14 samples following bait trapping with Galleria mellonella larva, where the per cent infection ranged between 40 to 100 per cent. Bacteria were isolated from the EPN-infected cadavers of G. mellonella (14 isolates), as well as from the Heterorhabditis-infected Galleria cadavers received from CPCRI and NBAIR (2 isolates).
Cultural characterization of the bacterial isolates showed circular to irregular, entire, opaque and smooth bacterial colonies on nutrient bromothymol blue agar (NBTA) medium. All the isolates showed negative reactions to the Gram staining and the bacterial cells were rod shaped. The bacterial colonies showed varying degrees of red colour from pinkish red to brick red in NBTA media.
For molecular characterization, the 16S rRNA gene of the isolates was amplified, sequenced and subjected to BLASTn for homology search. One species of EPN symbiotic bacteria, Photorhabdus luminescens (2 isolates) and nine non-symbiotic/ associated bacteria viz., Stenotrophomonas maltophilia (2 isolates), Alcaligenes aquatilis (1 isolate), Brevundimonas diminuta (1 isolate), Brucella pseudointermedia (1 isolate),Ochrobactrum sp. (1 isolate), Brucella pseudogrignonensis (1 isolate), Brucella anthropi (1 isolate), Pseudomonas azatoformans (2 isolates) and Pseudomonas lactis (4 isolates) were identified. A phylogenetic tree was constructed based on the gene sequences of 16S rRNA, to validate the bacterial identity.
The two isolates of P. luminescens (ODA and CNT1) obtained in the study were used for the bio-efficacy studies on eggs and adults of T. truncatus. The Cell Suspension (CS) and Cell-Free Supernatant (CFS) of both the isolates showed significant ovicidal and adulticidal effects against T. truncatus, after 72h of treatment application. In general, the mortality rate increased with an increase in the concentration of CS/CFS treatments. At the concentration of 108 cells/ml, the CFS and CS treatments of ODA isolate recorded egg mortality of 84.00 and 44.00 per cent, while CNT1 isolate recorded 58.67 and 84.00 per cent egg mortality, respectively. At the highest concentration, ODA isolate exhibited significantly higher mortality of adult mite for both CS (98.67%) and CFS (100%). Similarly, CNT1 isolate recorded adult mortality of 100 and 98.67 per cent, respectively for CS and CFS treatments.
The quantitative assay of protein recorded 238.5 mg/ml and 148. 05 mg/ml of protein in the bacterial supernatant of ODA and CNT1 isolates, respectively. The qualitative analysis of protein was carried out using SDS PAGE. Three protein bands at 25-29 kDa and a single protein band at 42 kDa were obtained. Toxin proteins corresponding to these molecular weights were reported to possess significant insecticidal effects. The P. luminescens ODA isolate that recorded the highest mortality of T.truncatus for both CS and CFS treatments in the laboratory bioassays, as well as a higher growth rate on NBTA media was selected for the preparation of bio- formulations. The solid formulations using 1000g of starch and 3300g of talc per 1000 ml of culture media yielded perfect powder-like consistency, respectively for starch- based and talc-based formulation. Glycerol, trehalose, starch and polyvinyl propylene (PVP) were used as additives to prepare liquid formulations.
Four liquid and two solid formulations developed in the study were evaluated against eggs and active stages of T. truncatus, separately in laboratory bioassays. All the formulations of P. luminescens (2.5×108 cells/ml) showed significant ovicidal activity at 72h of treatment application. The PVP-based liquid formulation recorded significantly higher mortality of eggs (97.33 %) and adults (100 %), followed by the starch-based solid formulation (82.67 and 90.67%, respectively).
Study on the shelf life of the formulations showed that the bacterial population declined from the day of inoculation (DAI) to 90 DAI, both under refrigerated and room temperature storage conditions. In liquid formulation, the population declined from 3.6×108 cfu/ml to 3.67×105 cfu/ml and 3.6×108 cfu/ml to 2.33×106 cfu/ml, respectively under 4°C and room temperature (RT), from 0 days after inoculation (DAI) to 90 DAI. Similarly, for solid formulation, the bacterial population declined from 3.33×108 cfu/ml to 3.0×105 cfu/ml and 3.33×108 cfu/ml to 2.0×105 cfu/ml, respectively.
The two bioformulations of P. luminescens were evaluated against T. truncatus on cucumber in polyhouse. By the 7th day of treatment, the liquid formulation (99.15 %) resulted in significant reduction in the mite population comparable with that of spiromesifen (99.92%), horticultural mineral oil (99.76 %) and neem oil emulsion (98.02 %), followed by solid bioformulation (94.61 %).
The research identified two native isolates of P. luminescens possessing significant acaricidal activity against T. truncatus. The effectiveness of both liquid and solid formulations of P. luminescens brought out in the study suggests the potential of this bacterium for utilization in mite pest management.
Agricultural entomology
Photorhavdus luminescens
Entomo pathogenic bacteria
Prostigmata: Tetranychidae
632.6 / ATH/BI Ph.D
Bio-efficacy of the entomo pathogenic bacteria, photorhavdus luminescens(Thomas & Poinar) aganist Tetranychus truncatus Ehara (Prostigmata: Tetranychidae) - Vellanikkara Department of Agricultural Entomology, College of Agriculture 2024 - 97,xxiiip.
Ph.D
Spider mites pose a serious threat to economically important agricultural and horticultural crops, worldwide. The spider mite, Tetranychus truncatus Ehara is a widely distributed polyphagous species inflicting considerable damage to vegetable crops and ornamental plants, both under open and protected cultivation in Kerala. The repeated use of synthetic acaricides has led to the development of resistance in T. truncatus against novel acaricides such as fenazaquin and spiromesifen, necessitating more sustainable alternative strategies for mite pest management. The symbiotic bacterium, Photorhabdus luminescens associated with the entomopathogenic nematodes (EPNs), Heterorhabditis spp. has been reported as a potential biocontrol agent against a broad range of arthropod pests, including mites.
The research programme on ‘Bio-efficacy of the entomopathogenic bacteria, Photorhabdus luminescens (Thomas & Poinar) against Tetranychus truncatus Ehara (Prostigmata: Tetranychidae)’ was carried out during 2018-2023 with the objectives to identify the potent local strains of P. luminescens; to develop suitable formulations of P. luminescens and to evaluate the efficacy of bio-formulations of P. luminescens against T. truncatus.
Soil samples were collected from different localities across five districts of Kerala viz., Kasaragod, Malappuram, Thrissur, Ernakulam, and Idukki. In the laboratory, EPNs were isolated from 14 samples following bait trapping with Galleria mellonella larva, where the per cent infection ranged between 40 to 100 per cent. Bacteria were isolated from the EPN-infected cadavers of G. mellonella (14 isolates), as well as from the Heterorhabditis-infected Galleria cadavers received from CPCRI and NBAIR (2 isolates).
Cultural characterization of the bacterial isolates showed circular to irregular, entire, opaque and smooth bacterial colonies on nutrient bromothymol blue agar (NBTA) medium. All the isolates showed negative reactions to the Gram staining and the bacterial cells were rod shaped. The bacterial colonies showed varying degrees of red colour from pinkish red to brick red in NBTA media.
For molecular characterization, the 16S rRNA gene of the isolates was amplified, sequenced and subjected to BLASTn for homology search. One species of EPN symbiotic bacteria, Photorhabdus luminescens (2 isolates) and nine non-symbiotic/ associated bacteria viz., Stenotrophomonas maltophilia (2 isolates), Alcaligenes aquatilis (1 isolate), Brevundimonas diminuta (1 isolate), Brucella pseudointermedia (1 isolate),Ochrobactrum sp. (1 isolate), Brucella pseudogrignonensis (1 isolate), Brucella anthropi (1 isolate), Pseudomonas azatoformans (2 isolates) and Pseudomonas lactis (4 isolates) were identified. A phylogenetic tree was constructed based on the gene sequences of 16S rRNA, to validate the bacterial identity.
The two isolates of P. luminescens (ODA and CNT1) obtained in the study were used for the bio-efficacy studies on eggs and adults of T. truncatus. The Cell Suspension (CS) and Cell-Free Supernatant (CFS) of both the isolates showed significant ovicidal and adulticidal effects against T. truncatus, after 72h of treatment application. In general, the mortality rate increased with an increase in the concentration of CS/CFS treatments. At the concentration of 108 cells/ml, the CFS and CS treatments of ODA isolate recorded egg mortality of 84.00 and 44.00 per cent, while CNT1 isolate recorded 58.67 and 84.00 per cent egg mortality, respectively. At the highest concentration, ODA isolate exhibited significantly higher mortality of adult mite for both CS (98.67%) and CFS (100%). Similarly, CNT1 isolate recorded adult mortality of 100 and 98.67 per cent, respectively for CS and CFS treatments.
The quantitative assay of protein recorded 238.5 mg/ml and 148. 05 mg/ml of protein in the bacterial supernatant of ODA and CNT1 isolates, respectively. The qualitative analysis of protein was carried out using SDS PAGE. Three protein bands at 25-29 kDa and a single protein band at 42 kDa were obtained. Toxin proteins corresponding to these molecular weights were reported to possess significant insecticidal effects. The P. luminescens ODA isolate that recorded the highest mortality of T.truncatus for both CS and CFS treatments in the laboratory bioassays, as well as a higher growth rate on NBTA media was selected for the preparation of bio- formulations. The solid formulations using 1000g of starch and 3300g of talc per 1000 ml of culture media yielded perfect powder-like consistency, respectively for starch- based and talc-based formulation. Glycerol, trehalose, starch and polyvinyl propylene (PVP) were used as additives to prepare liquid formulations.
Four liquid and two solid formulations developed in the study were evaluated against eggs and active stages of T. truncatus, separately in laboratory bioassays. All the formulations of P. luminescens (2.5×108 cells/ml) showed significant ovicidal activity at 72h of treatment application. The PVP-based liquid formulation recorded significantly higher mortality of eggs (97.33 %) and adults (100 %), followed by the starch-based solid formulation (82.67 and 90.67%, respectively).
Study on the shelf life of the formulations showed that the bacterial population declined from the day of inoculation (DAI) to 90 DAI, both under refrigerated and room temperature storage conditions. In liquid formulation, the population declined from 3.6×108 cfu/ml to 3.67×105 cfu/ml and 3.6×108 cfu/ml to 2.33×106 cfu/ml, respectively under 4°C and room temperature (RT), from 0 days after inoculation (DAI) to 90 DAI. Similarly, for solid formulation, the bacterial population declined from 3.33×108 cfu/ml to 3.0×105 cfu/ml and 3.33×108 cfu/ml to 2.0×105 cfu/ml, respectively.
The two bioformulations of P. luminescens were evaluated against T. truncatus on cucumber in polyhouse. By the 7th day of treatment, the liquid formulation (99.15 %) resulted in significant reduction in the mite population comparable with that of spiromesifen (99.92%), horticultural mineral oil (99.76 %) and neem oil emulsion (98.02 %), followed by solid bioformulation (94.61 %).
The research identified two native isolates of P. luminescens possessing significant acaricidal activity against T. truncatus. The effectiveness of both liquid and solid formulations of P. luminescens brought out in the study suggests the potential of this bacterium for utilization in mite pest management.
Agricultural entomology
Photorhavdus luminescens
Entomo pathogenic bacteria
Prostigmata: Tetranychidae
632.6 / ATH/BI Ph.D