Effect of entomopathogenic fungi against invasive pest Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) in maize

Ten indigenous entomofungal strains of Beauveria bassiana, Metarhizium anisopliae, and M. rileyi were evaluated against 2nd instar larvae of the maize fall armyworm (FAW) Spodptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) in a laboratory bioassay. Among the ten strains tested, M. anisopliae ICAR-NBAIR Ma-35 caused 67.8% mortality, followed by B. bassiana ICAR-NBAIR Bb-45 with 64.3%, and ICAR-NBAIR Bb-11 with 57.1% mortality. Rest of the isolates showed 10.7–28.6% mortality. ICAR-NBAIR Ma-35 showed LC50 of 1.1 × 10 7 spores/ml and LT50 at 1 × 10 spores/ml is 86.04 h and ICAR-NBAIR Bb-45 showed LC50 of 1.9 × 10 7 spores/ml and LT50 at 1 × 10 8 spores/ml is 88.30 h. Field evaluation with these two promising strains were conducted against maize fall armyworm for 2 years (2018 and 2019) at ICAR-National Bureau of Agricultural Insect Resources experimental farm, Bengaluru, Karnataka, India. Field trial results indicated 68 and 69% reduction of FAW infestation and 55 and 62% increase in yield in the plots treated with M. anisopliae ICAR-NBAIR Ma-35/B. bassiana ICAR-NBAIR Bb-45, respectively, during 2018. In 2019, 70 and 76% reduction of FAW infestation and 44 and 55% increase in yield were observed in the plots treated with these two entomofungal pathogens, respectively.


Background
The maize fall armyworm (FAW), Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) was reported for the first time in India in 2018. It was widely distributed in Karnataka regions like Chikkaballapur, Hassan, Shivamogga, Davanagere, Bangalore, and Chitradurga during July-August 2018 (Shylesha et al. 2018). The FAW is native of the tropical and sub-tropical regions of North, Central, and South America (Chittenden 1901;Luginbill 1928). In Africa, first report of fall armyworm on maize plants was made in 2016 (Goergen et al. 2016). It has become invasive and threatened the food security in Africa causing more than 200 million people whose staple crop is maize. In the absence of proper control methods, S. frugiperda has the potential to cause maize yield losses of 8.3 to 20.6 million tonnes per annum in Africa. The value of crop losses is estimated between US$ 2.5 and 6.2 billion ). In addition, the pest is known to be highly polyphagous, spreading to other crops from the invaded regions (Goergen et al. 2016). The application of chemical insecticides is unsustainable because it develops insecticide resistance, cost-effective, effects natural enemies, and environmental hazards as well as health risks. Therefore, it is important to minimize the use of insecticides and need to develop sustainable IPM technologies against S. frugiperda for safe, ecofriendly management practices in India Yu et al. 2003). FAW larvae are susceptible to entomopathogenic microorganisms, such as bacteria, fungi, nematodes, viruses, and protozoa (Molina-Ochoa et al. 2003;Ríos-Velasco et al. 2010). Native strains of entomopathogenic fungi as an alternative to chemical insecticides have to be evaluated against maize fall armyworm.
The present study was undertaken up to evaluate the entomofungal pathogens against S. frugiperda under laboratory and field-level conditions.

Insect culture
The second instar larvae and egg patches of Spodoptera frugiperda was obtained from mass production unit of ICAR-NBAIR, Bangalore, Karnataka, India, were used for the bioassay.

Laboratory bioassay Fungal spore suspension
Each fungal isolate was grown on broken rice for laboratory bioassay. Spore suspension was prepared by taking 1 g of 15 days old conidiated rice in 9 ml of sterile distilled water containing 0.01% Tween 80. The suspension was filtrated through three layers of muslin cloth to get hyphal-free spore suspension, and the spore concentration was adjusted to various concentrations like (1 × 10 4 , 1 × 10 5 , 1 × 10 6 , 1 × 10 7 , and 1 × 10 8 spores/ml) using Neubauer' s improved hemocytometer.

In vitro screening
The 2nd instar larvae (10 larvae/replication-3 replications) of S. frugiperda were dipped in the suspension containing 1 × 10 8 spores/ml for 15 s. The treated larvae were transferred into plastic containers and supplied with fresh detached maize leaves. Then the larvae were maintained under controlled conditions of 25 ± 2°C temperature and 70 ± 5% relative humidity for 10 days. Fresh maize leaves were provided as a food to the larvae in 24 h interval. Controls were treated with sterile water containing 0.01% Tween 80. The observation on mortality due to fungal infection was carefully noted and recorded. The percent mortality of the larvae for each  isolate was calculated using Abbott's formula (Abbott, 1925). The data were subjected to one-way analysis of variance (ANOVA) using statistical software SPSS windows version 20.0. Further studies were carried out using promising isolates.
Corrected%mortality ¼ 1 − n in T after treatment n in Co after treatment Ã100 where n = insect population, T = treated, Co = control The egg patches of S. frugiperda laid on maize leaves were used for bioassay studies of two promising strains (30 eggs/replication-5 replications). Each egg patch containing 30 eggs were dipped in the spore suspension containing 1 × 10 8 spores/ml for 15 s and the treated eggs were maintained on maize leaves under controlled conditions of 25 ± 2°C temperature and 70 ± 5% relative humidity until they hatched. Similarly, the eggs in the untreated control were treated using sterile distilled water and maintained on maize leaves. Observations on egg hatching were recorded carefully at 24 intervals for a period of 5 days.

Concentration and time mortality studies
Two promising isolates were subjected to dose and time mortality studies. Five spore concentrations (1 × 10 4 , 1 × 10 5 , 1 × 10 6 , 1 × 10 7 , and 1 × 10 8 spores −1 ) were used to study LC 50 and bioassays were carried out as described above. In case of time mortality studies, single concentration (1 × 10 8 spores −1 ) was used to determine LT 50 . The dose and time to kill 50% of the population (LC 50 and LT 50 ) were determined by probit analysis (Finney 1971). Statistical analysis was done using SPSS windows version 20.0.
The field trials were laid out in completely randomized block design with 3 treatments and eight replications using maize hybrid variety (BRMH-1) obtained from Karnataka State Seeds Corporation, India, for this study. The maize seeds were sown manually in the experimental plot with a plot size of 5 m × 6 m for each treatment and spacing of 60 cm × 30 cm were maintained and irrigated regularly. All the agronomic practices with recommended doses of fertilizers were followed to maintain good plant health till harvest of crop as per package of practice by UAS, Bengaluru, Karnataka, India, and harvesting was done manually. The talc formulations of these isolates were applied at the dose of 5 g/l (1 × 10 8 Cfu/g) during 20, 30, and 40 days after sowing (10 days interval) of maize seeds in the experimental field during both the seasons as soon as pest incidence was observed. Pre-and postobservations on number of plants infested with FAW were recorded after 5 days of treatment. The plant infestation with FAW was recorded based on the number of plants infested by FAW out of 120 plants (15 plants per replication in each treatment, total 120 plants). The percent reduction in plants infestation by FAW was calculated. Yield was recorded at the time of crop harvest for each treatment. The data was subjected to statistical analysis for drawing inferences using SPSS windows version 20.0. Significant level was set at 0.05 and the means were separated by Turkey test.

Conclusion
Based on 2-year field trials conducted in FAW infested plots in Karnataka state of India, M. anisopliae ICAR-NBAIR Ma-35 and B. bassiana ICAR-NBAIR-Bb-45 were recommended as potential isolates for management of FAW in maize crop, as the plots treated with this biocontrol agent showed minimum infestation levels of FAW and considerable increases in the yield than the untreated control.