Field trial of new Bt-base bioinsecticide formula, Bashield ® , for controlling Spodoptera frugiperda J.E. Smith on maize

. Maize is one of the important staple crops. The process of cultivating corn cannot be separated from pests and plant diseases, one of the attacking pests is Spodoptera frugiperda , which can attack the growing points of plants causing a decrease in corn production. Based on previous research conducted in vitro, bioinsecticide of Bacillus thuringiensis -base (Bt-base) liquid formula (namely Bashield ® ) was able to reduce pupal weight, the proportion of larvae-pupae, and pupal becomes abnormal. Action that can be taken to control this attack is by utilizing biological agent, B. thuringiensis strain BT2, as an alternative control. This research aims to evaluate or to determine the effectiveness of a new bioinsecticide of Bt-base liquid formula (Bashield ® ), as a control agent for S. frugiperda on maize in the field. This research was prepared at the Laboratory of Plant Protection and Environment, Universitas Trunojoyo Madura, and was conducted at the Horticultural Seed Garden, Socah District, Bangkalan Regency, East Java, Indonesia (7°05'17.2"S 112°42'32.0"E). The design used was a complete randomized block design (CRBD) with 4 treatments and 6 replications. The data were analyzed using 5% ANOVA and if there was a significant effect, then a DMRT follow-up test was carried out with a 5% level. The results showed that the 15% treatment was able to kill test larvae up to 78% with a high virulence level, and reduced damage up to 100%. In conclusion, the new Bt-base liquid formula, Bashield ® , has good potential for effective biocontrol and contributed as a new biopesticide for S. frugiperda.


Introduction
The fall armyworm, Spodoptera frugiperda J.E.Smith is a new invasive pest all over the world [1].In African and European countries, the economic losses caused by this pest amount to 20.6 million tonnes per year with a total loss of US$ 2.5-6.2 billion [2].In Indonesia, which currently attacks various regions.The first reports of S. frugiperda attacks in Indonesia were recorded in 2019, precisely in Lampung [3].S. frugiperda infestation threatens successful maize production and causes extensive damage due to its existence as a new invasive pest.This pest can explore up to 100 km with wind thrust in one week, this pest is also considered a more active and more voracious pest compared to other types of armyworms [4][5].Around 60% of sweet hybrid corn in Petir Village, Dramaga District, Bogor Regency, West Java, Indonesia was damaged by S. frugiperda attacks [6].Another report, the damage caused by the S. frugiperda attack also occurred in Tuban Regency, East Java with a damage range of 58% to 100% and can result * Corresponding author: syaiful.khoiri@trunojoyo.ac.id in a decrease in corn yields of up to 18 million tons per year [7][8].
Generally, the controlling of S. frugiperda was carried out using synthetic insecticides [9] but several studies have reported that pest resistance builds up quickly and makes synthetic insecticides less effective [10].Pest resistance is caused by mutations in genes that may be resistant to pyrethroids, organic phosphates, and carbamates [11].Therefore, an alternative control is needed such us using entomopatogenic bacteria [12].An example is by utilizing entomopathogenic bacteria, Bacillus thuringiensis.The use of B. thuringiensis as a biological agent is more environmentally friendly because the crystal protein is selective [13].
B. thuringiensis strains were reported effective as biological control [14][15][16].In our previous study, we successfully isolated potensial strain, namely B. thuringiensis strain BT2, and its effective to kill S. frugiperda.This strain was formulated in liquid form, namely Bashield® [in press].Formulations can maintain population density and can increase the effectiveness of active ingredients [17][18].In addition, the right formula can assist in availability, mass propagation, storage, transportation, packaging, application, and marketing [18][19].
The Bashield® was effective in controlling S. frugiperda at laboratory and greenhouse tests.But, in the field the data is not yet available.So, this research aims to check the ability of Bashield® formula in field for controlling S. frugiperda.The contributions of this research are to evaluate new Bt-base liquid formula, Bashield®, on controlling S. frugiperda as a environmental friendly technology.

Research location
This research was conducted in the Laboratory of Plant Protection and Environment, Faculty of Agriculture, Universitas Trunojoyo Madura for the preparation of the formula and at Horticulture Seed Garden, Socah, Bangkalan, East Java, Indonesia (7°05'17.2"S112°42'32.0"E).

Field preparation and seed planting
Field preparation is carried out by loosening the soil to a depth of 30 cm using a tractor.This land preparation is carried out 1 week before planting to restore the fertility level of the soil.Next step, planting is carried out by planting corn seeds at a spacing of 80 cm x 20 cm in a plot measuring 1 m x 1 m.The planting hole is made 3 cm deep with the number of seeds per hole being 2 corn seeds.
Plant maintenance is carried out in several stages starting from replanting, watering, weeding, and fertilizing.Embroidery is done by replacing plants that die or cannot grow well, this is done so that the plants can grow uniformly and is done twice in the morning until the plants are 7 days after planting (DAP).Watering is done twice a day in the morning and evening, but when it rains and the soil is damp, no watering is done.Fertilization is carried out when the plants are 7-10 days old by giving 10g NPK per plant and when the plants are 35-40 days old, 20g NPK per plant and 10 g urea per plant.Weeding is carried out on weeds that disturb the plants when the corn plants are 3 to 4 weeks old, weeding is carried out 2 times with weeding intervals once a week.

Larvae infestation
Artificial investment of S. frugiperda is carried out by inoculating 2 nd instar larvae.Artificial investment is carried out so that the number of larvae and corn plants planted can produce uniform attacks, this is because natural attacks by S. frugiperda larvae are different.The number of larvae invested is 2 larvae per plant.The larvae are invested when the plants are 3 WAP.
Bashield® application is carried out by spraying as much as 15 mL of liquid formula bioinsecticide per plant by spraying on the leaves in the afternoon according to the concentration treatment.Application is carried out once every 1 week within 2 weeks.Observations are carried out every 2 days in the morning.Observation parameters include S. frugiperda population, percentage of S. frugiperda-infested plants, and S. frugiperda infestation intensity.
Population observation of S. frugiperda is done by calculating how many dead S. frugiperda larvae are on plant shoots.The results of the larval count are then recorded according to the observation time, sample plant number, and sample plot.The insect mortality rate is carried out using the equation 1 [20], [21]: The percentage of incidence is done by observing how many plants are attacked by S. frugiperda.Observation is carried out 3 times a week, with a period of 2 days after the spraying period.Observations were made at 2 days after application (DAA), 4 DAA, 6 DAA, 9 DAA, 11 DAA, 13 DAA.The formula used in determining the percentage of plants affected by S. frugiperda is presented in equation 2: The intensity of pest attacks is carried out to find out how much the level of pest attacks that occur or leaf feeding rates.Observation is carried out 3 times a week, with a period of 2 days after the spraying period.Observations were made at 2 DAA, 4 DAA, 6 DAA, 9 DAA, 11 DAA, 13 DAA.The intensity scale uses Davis scale measurements [22].The determination of the intensity of S. frugiperda attacks is calculated using the equation 3 [7], [23].
Where n is the number of attacked plants in i, v is the scale in i; N is the number of all observed plants; and Z is the maximum score scale.

Data analysis
Data analysis was carried out using ANOVA on SPSS software to determine the effect of treatment.If there is a noticeable difference, a further DMRT test is carried out at α=5%.

Result and discussion
Mortality is the percentage mortality value of dead larvae from the total number of observed larvae.The highest mortality value occurred in treatment with a concentration of 15% with a mortality value of 78%, while the mortality value at concentrations of 10%, 5%, and 0% with mortality values of 65%, 54%, and 19% respectively (Table 1).Salaki & Watung [24] classify the virulence level based on the mortality value of the test insects, namely high virulence if the mortality value is above 50%, moderate virulence if the mortality value is 30% -<50%, low virulence if the mortality value is <30%, not virulence if the mortality value is 0%.High virulence levels were found in treatments with concentrations of 15%, 10%, and 5% with mortality values of 78%, 65%, and 54%, while low virulence levels were found in control treatments with mortality values of 19%.The mortality of S. frugiperda larvae that occurred in this study shows that B. thuringiensis contained in Bashield ® formulas can work effectively.The dead larvae show symptoms of black color, dry out, shrivel, and gradually become destroyed (Fig. 1).The symptoms same with previous study that S. frugiperda which died after two days had a blackish body color, then a few days later the body of S. frugiperda became smaller, dried out, and became destroyed [25], [26].The death of S. frugiperda is thought to be due to the presence of protein crystals produced by B. thuringiensis which are toxins for lepidopteran.Parasporal crystals of B. thuringiensis will enter through the insect's digestive tract, these protein crystals will become δ-endotoxin protein or protoxin when activated by the alkaline environment in the digestive tract, then the insect protease enzyme will activate the protoxin to become a toxin and bind specifically to the receptor in the tract.Digestion, cry toxin which attaches to the peritropic membrane is what can cause death in insects because it is injurious and causes cytoplasmic leakage [23].Based on the recorded larval mortality data, it is known that the concentration estimation value is based on regression analysis with the equation Y = a + bX with a and b values of -0.846 and 1.319 respectively with an R 2 value of 0.951.Thus, the higher the log concentration (X), the higher the mortality percentage (Y).The more the value of R is close to number 1, the stronger the influence between the independent variable (X) and the dependent variable (Y).The results of the probit analysis, LC50 from the Bashield ® formula are 4.38%.Note: Numbers accompanied by the same letter in the same column showed no significant difference based on the DMRT follow-up test of α=5% (P = 0.05).
The results of observations of the occurrence of S. frugiperda attacks in four treatments showed that the control was significantly different from other treatments in the observations of 11 DAA and 13 DAA.The highest percentage of attacks occurred in 2 DAA of 52.78% in 5% Bashield ® treatment and the lowest percentage of attacks occurred in 11 DAA of 0% in 15% Bashield ® treatment (Table 2).Based on Table 2, each treatment experienced different decreases and increases at different times.The 15% Bashield ® treatment decreased the occurrence of attacks by 0% faster than others in 11 DAA and 13 DAA.There is a decrease in attacks and an increase in re-attacks is thought to be due to the attack of S. frugiperda larvae moving from one plant to another a b with the condition of the previous plant still in an infested state.Observations of 11 DAA showed a significant difference in P0 treatment with P2 and P3 treatment, while 13 DAA observations showed a real difference in 0% treatment (without Bashiled®) with other treatments.
The percentage of S. frugiperda pest attacks is influenced by the age of the plant, pest attacks will continue to decrease when plants enter the generative phase because plants are stronger, although attacked during the vegetative phase plants can recover with good maintenance treatments such as fertilization and tillage [27].
The high attack of S. frugiperda is related to the high population level of larvae which also affects the intensity of attacks that occur [28].The results of observations of S. frugiperda attack intensity in four treatments are presented in Table 3.The highest attack intensity occurred at 2 DAA of 27.16% in the 15% Bashield ® treatment and the lowest attack intensity occurred at 11 DAA of 0% in the 15% Bashield ® treatment.The results of data analysis showed that interconcentration treatment on the attack intensity parameter of S. frugiperda had a significant effect with observation times of 9 DAA, 11 DAA, and 13 DAA, while at observation time, 2 DAA, 4 DAA, and 6 DAA had no significant effect.Bashield ® 15% (P3) 27.16a 12.65a 3.70a 0.93a 0a 0.00a Note: Numbers accompanied by the same letter in the same column showed no significant difference based on the DMRT follow-up test of α=5% (P = 0.05).
Based on Table 3, at the beginning of the observation time, namely 2 DAA, 4 DAA, and 6 DAA did not have a significant effect, allegedly because S. frugiperda larvae still attack corn plants and dead larvae on affected plants need time to lower the attack intensity scale to restore plants on a low scale.However, the level of attack intensity at the time of this observation decreased.The observation time of 9 DAA, 11 DAA, and 13 DAA showed a significant difference in P0 treatment with other treatments, this is thought to be due to a decrease in S. frugiperda attacks due to dead larvae and affected plants that have given rise to new leaves.The effectiveness of B. thuringiensis in killing S. frugiperda is strongly influenced by the type of cry toxin, susceptibility to UV, and to reach pest for inducing toxin consumption [29][30] This study was supported in part by The Institute of Research and Community Services (LPPM), Universitas Trunojoyo Madura with contract no.203/UN46.4.1/PT.01.03/2022.

Conclusion
Based on the results, the new Bashield ® formula can kill S. frugiperda larvae with the highest mortality of 78% in 15% Bashield® treatment.The LC50 value in this study was 4.38% which shows that this bioinsecticide can effectively control the attack of S. frugiperda.The 15% Bashield® treatment is the best concentration that can reduce the occurrence and intensity of S. frugiperda attacks faster than other treatments.

Table 1 .
Mortality S. frugiperda due to the application of Bashield® with different concentrations.

Table 2 .
Effects of treatment on the incidence of S. frugiperda attacks.

Table 3 .
Effect of treatment on S. frugiperda intensity attack rates.