Evaluation of PDKV Bacillus thuringiensis strains against Spodoptera frugiperda (J.E. Smith)

Introduction

Maize is the third most important cereal crop in India after rice and wheat in terms of area and production. Globally it is highly valued for its multifarious use as food, feed, fodder and raw material for large number of industrial products. There are several reasons for the low productivity of maize viz., environmental factors, low mechanization, pest and diseases etc. Of these, insect pests are the major constraints as this attacking maize not only directly as borers, sap suckers, stem and root feeders etc. but also indirectly as vectors of diseases (Wayal Avinash., 2022) [18] . There are four major pests of maize prevalent in India. These are spotted stem borer Chilo partellus (Swinhoe), pink stem borer Sesamia inferens (Walker), shoot fly Atherigona spp. and fall armyworm Spodoptera frugiperda (J.E. Smith) (Suby. S.B., 2020) [17] . The fall armyworm, Spodoptera frugiperda native to America is one of the important invasive polyphagous pests. It found in Brazil, Argentina and the USA (Porwell et al., 2004; Clark et al., 2007) [14,5] . This invasive pest was first reported in West Africa in late 2016 (Goergen et al., 2016) [10] . In May 2018, this highly invasive insect pest was noticed for the first time in India on the maize crop in the Shivamogga and Davanagere districts in Karnataka state (Sharanabasappa et al., 2018) [16] and thereafter it was reported to cause major damage to economically important cultivated grasses such as rice, sorghum and sugarcane as well as 23 horticultural crops like cabbage, beet, tomato, potato and onion besides cotton, pasture grasses, peanut, soybean, alfalfa and millets (Pogue, 2002; Chapman et al., 2000; CABI, 2016) [13,6,7] . S. frugiperda, is a harmful pest, poses a significant threat to tropical crops, potentially causing up to 70% yield losses in corn. Chemical insecticides have failed to manage FAW, leading to environmental implications. Biological control is the only eco-friendly solution for managing FAW populations. Bacillus thuringiensis has been highly explored as a microbial biopesticide for the control of insect pests especially in the orders of Lepidopteran, Dipteran and Coleopteran species. It is a ubiquitous gram-positive, rod shape, motile, spore-forming entomopathogenic bacterium widely used as biocontrol agent. (Fernando et al., 2010) [9] . B. thuringiensis products are used in pest management because they are highly specific to the target organism and harmless to non-target organisms and the environment (Palma et al., 2014) [15] . Bt produce insecticidal proteins during the sporulation phase as parasporal crystals. These crystals are predominantly comprised of one or more proteins Crystal (Cry) and Cytolytic (Cyt) toxins, also called d-endotoxins. These toxins are highly specific to their target insect, are innocuous to humans, vertebrates and plants, and are completely biodegradable. Therefore, Bt is a viable alternative for the control of insect pests in agriculture and of important human disease vectors (Bravo et al., 2005) [3] . Despite of its effectiveness as a biological control of pests, at present it is necessary to identify novel Bt isolates with promising Cry proteins for greater toxicity to counter the potential resistance evolved by insects. Hence, the present work attempted at screening of some locally isolated Bt strains against S. frugiperda and comparing their toxicities with standard HD-1 strain of Bacillus thuringiensis.

Materials and Methods

Insect collection and rearing

Fall armyworm (FAW) was collected from an unsprayed maize field in the university campus and brought to a laboratory. To avoid cannibalism the larvae were placed individually in ventilated plastic jars and fed with fresh tender maize leaves till pupation. After pupation, they were placed to a mating jar covered with muslin cloth containing 10% honey solution for adult feeding and folded paper strips for egg laying. After 2-3 days, old egg batches were collected and placed in sterile plastic jars. Eggs were monitored daily for hatching. On hatching, neonates were transferred to natural diet. The third instar larvae from the laboratory culture were used for bioassay experiments. The culture was maintained under controlled laboratory conditions, i.e., at 27±1 °C and 70±5% relative humidity and a 12L:12D photoperiod.

Screening of Bacillus isolates against larvae of fall armyworm 2.2.1 Preparation of culture media

For this purpose 200 ml of distilled water was measured using glass measuring cylinder and taken in glass bottle. Luria broth (5 gm) and agar powder (3 gm) was added in bottle and heated in microwave for dissolving agar. Then the digested medium was transferred to conical flasks and mouths of the flasks were sealed with tight cotton plugs and autoclaved. After autoclaving the medium was taken into sterile bottles or test tubes. Before and after pouring of media into bottles, the conical flask containing the medium was flamed to prevent contamination. The bottles or tubes were kept open till the media solidified within the laminar hood. The bottles, tubes and petri plates used in culturing were also autoclaved at 15 Ibs pressure and 121 °C temperature for 15 minutes. After autoclaving, the material was shifted to the laminar airflow, which was sterilized by U.V. light for 30 minutes. After switching off the U.V. the airflow and illumination (after 10 min.) was switched on and the floor of the cabinet and hands were sterilized with 70% alcohol. The media bottles and other materials were placed in the cabinet. The spirit lamp was lighted and the media was poured into the bottles before it cooled down. The media in the petri plates, bottles and test tubes was allowed to solidify. These media bottles were used for further subculturing of various Bt strains used in the experiments.

Parasporal preparations

For the bioassay, Spore-crystal mixture was prepared according to the method given by Dulmage et al., (1970) [8] . Spore-crystal mixture was extracted by acetone coprecipitation method. This Spore-crystal mixture was used as a source of endotoxin for bioassay of test insect. The Bt isolates mentioned in (Table 1) were inoculated in Luria broth and incubated for 72 hrs. Until sporulation at 30 °C. Then centrifuged and pellet was obtained. The pellet is suspended in 4-6 % lactose solution, until final volume is one tenth of original solution then stirred for 15-30 min. To the suspension 4 volume of acetone is added gradually and filtered to obtain aqueous suspension of spore-crystal mixture with acetone, the process is repeated twice then is dried overnight either in vacuum desiccators or in air room temperature. This was stored at 4 °C and used further for bioassays studies.

Bioassay of selected native isolates of Bt

In order to determine the lethal concentrations of selected endotoxins from Bt isolates against Spodoptera frugiperda, diet incorporation assays were followed as described by Bhalla et al., (2005) [2] for H. armigera. The diet for Spodoptera frugiperda was prepared by the method described by Burton et al., (1969) [1] . Ten 3 rd instar larvae (8 hrs. starvation) were released in each well. A set of 10 such larvae was tested for each concentration of parasporal preparations at one time. Four concentrations, viz., 200, 300, 400 and 500 µg/ml of diet of spore crystal complex of each strain, along with a control, were taken for bioassay under controlled conditions. The plates were covered and kept in the insect culture room at 28 ± 2 °C, 60 ± 5% R and 14L:10D photoperiod. Mortality was recorded after 3 days. The experiment was replicated three times. The mortality was recorded on the 7th day after the bioassay. LC50 values for bioassays were calculated.

Data analysis

The concentration response data was analysed using probit analysis with OPSTSAT software to determine the LC50 values, standard errors, slope and 95% fiducial limits (FL). From which relative toxicities of different Bt isolates was also determined as below: LC50 of more toxic Bt isolates Relative Toxicity (RT) = LC50 of less toxic Bt isolates PDKV SGn-5 OP209988

Results and Discussion

Bioassay studies of potential Bt isolates against

Spodoptera frugiperda (J.E. Smith) Eleven PDKV Bt strains for toxicity studies viz. SA-6, SA-20, SAk-6, SAk-9, SGd-1, SBn-2, SGn-4, SGn-5, SY-4, SA-18 and I-3 were used for conducting bioassay to determine the LC50 values against the test insect i.e. Spodoptera frugiperda (J.E. Smith).

LC50 of PDKV Bt isolates against S. frugiperda

Data presented in the (Table 2) depicted in Fig 1 . indicate that the local Bt isolates PDKV-SA-20, PDKV-SGd-1 and PDKV-SY-4 recorded lower LC50 values of 172.90 µg/ml, 177.61 µg/ml and 186.17 µg/ml, respectively and found relatively more toxic than standard Btk HD-1 strain which reported LC50 of 191.41 µg/ml. The LC50 values of rest of the other Bt isolates PDKV-SAk-6, PDKV-SAk-9, PDKV-SGn-4, PDKV-I-3, PDKV-SA-6, PDKV-SA-18, PDKV-SGn-5 were seen lying within the range of fiducial limits of the standard Btk HD-1 strain, hence all these locals Bt isolates said to have equal toxicity potential to that of standard Btk HD-1 strain against S. frugiperda. As local SA-20 isolate seen to exceeds the fiducial limits of the standard Btk HD-1 strain it can be referred to as the most promising strain against Spodoptera frugiperda.

Similar study regarding LC50 was reported by M. Maheesha et al., (2021) [12] , estimated LC50 of the isolate T350 and positive control HD-1 produced 100% mortality against the neonates of S. frugiperda. In probit analysis the isolate T350 recorded LC50 value of 2.04 µg/ml as against 1.99 µg/ml in positive control HD-1 which in accordance with the LC50 range obtained in present study. Likewise, Karuppaiyan et al., (2022) [11] worked on characterization and evaluation of indigenous Bt isolate T352 against S. frugiperda. Bioassay studies revealed that 100% larval mortality was exhibited in both T352 and standard strain HD-1, were LC50 value of isolate T352 recorded 1.927 µg/ml whereas HD-1 exhibited 0.421 µg/ml of LC50. Similarly, Harika et al., (2021) [4] recorded toxicity of six native Bt isolates viz., isolate 49, 51, 52, 55, 493 and reference strain HD-1. The LC50 values ranged from (1.34×10 11 to 2.64×10 7 CFU ml-1 ). Among native Bt isolates lowest LC50 value recorded by the isolate-493 with 2.03×10 8 CFU ml-1 followed by isolate-51, isolate-55 and isolate-49 with LC50 values of 1.51× 10 10 , 4.71×10 10 and 5.38×10 10 respectively. The highest LC50 value was obtained with the isolate-52 (1.34×10 11 CFU ml-1 ) and thus found to be the least effective out of all the native isolates tested.

Conclusion

The bioassay studies of different Bt strains against Spodoptera frugiperda revealed that PDKV Bt strains SA-20, SGd-1 and SY-4 have higher toxicity than the standard Btk HD-1 strain. The three strains were found 2.08, 2.03 and 1.93-fold, respectively more toxic to Spodoptera frugiperda than lowest toxic strain i.e. SBn-2. PDKV Bt strains SAk-6, SAk-9, SGn-4, I-3, SA-6, SA-18, SGn-5 were found at par in toxicity with standard Btk HD-1 strain as the LC50 values of these Bt isolates were lying within the range of fiducial limits of the standard Btk HD-1 strain. Hence, all these local Bt isolates said to have equal toxicity potential to that of standard strain against Spodoptera frugiperda which can be used for further formulation studies and novel characterization of Bt isolates can be used for development of transgenic plants against FAW and other lepidopteran insects.

Acknowledgement

The authors are grateful to Department of Agricultural Entomology, Post Graduate Institute, Dr. PDKV, Akola, MS, India for providing all the necessary facilities for conducting the present research work.

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