The Effectiveness of Red Spinach (Amaranthus tricolor L.) and Green Spinach (Amaranthus hybridus L.) Extracts for Bacillus thuringiensis var. kurstaki Protectant against UVB Radiation for the Control of Armyworm (Spodoptera litura Fab.)

ABSTRACT Spodoptera litura Fab. is an insect that damage cultivated plants in Indonesia. Efforts to control it can be done by using biological agents for example by using Bacillus thuringiensis kurstaki (Btk.). Unfortunately, the Btk. is easily degraded by UV radiation. This research aimed to study the effectiveness of red and green spinach as UVB protection for Btk. and to observe the pathogenicity of Btk. formulations against armyworm. Furthermore, the sublethal effect of Btk. against S. litura was investigated. The morphology of the endospore, protein crystal, and bacterium were observed under a contrast phase microscope. The extracts at 2% (w/v) were mixed with Btk. suspensions at 5 x 104, 5x105, and 5x106 (spores/ml), respectively. The formulations then exposed under Ultraviolet B (UVB) lights for 3, 6, and 9 hours then tested against the 3rd larval instar of armyworm. The larval mortality was observed daily and the analysis of variance was analyzed by one way anova. The sublethal effects of the treatment to the pupal and adult stages were observed when the moths emerge. The results showed that the larval mortality caused by Btk. mixed with red spinach ranged from 11.7 to 26.7%. The sublethal effects of Btk. resulted in smaller sizes of pupae and imago, darker pupae, and wings abnormality of the adult stage, compared to any control treatment. The morphological observation of the bacteria showed that extracts gave UV protection against UVB. These results suggested that red and green spinach potentially can be used as a protectant for Btk. against UVB.


INTRODUCTION
Spodoptera litura Fab. is an insect that causes damage and losses on a cultivated plant in Indonesia. The high losses caused by these insects on cabbage plantations caused efforts to control these pests become a priority. Many control measures were made to control this pest including the use of chemical, herbal, and biological agents. The chemical controls such as using chemical insecticides resulting in resistance and resurgence of the pests (Marwoto & Neering, 1992). Whereas, the use of herbal controls, for example, using frangipani (Plumeria sp.) and chickweed (Ageratum conyzoides L.). One of the biological control agent commonly used is Bacillus thuringiensis kurstaki (Btk.) (Feitelson et al., 1992).
Btk. easily degrades when exposed to ultraviolet (UV) lights. It causes the spores to be inactive (Griego & Spence, 1978). Several antioxidants can be used as additives for anti ultraviolets (El-Sharkawey et al., 2009). The use of red and white dragon fruits , aloe vera (Tarigan, 2019), and tea leaves (Ningrum, 2019) have been used for the study of Btk. protectants. Spinach contains compounds of vitamin A, vitamin C, vitamin E, flavonoids, and phenol which can be used as antioxidants (Amin et al., 2006). Therefore, in this research red spinach and green spinach extracts were used as protectants of Btk. against UV B to control armyworm. It is expected that the addition of protectants can increase the pathogenicity of Btk. against the pest.

Materials
The materials used in this study include the 3 rd instar of S. litura larvae, Btk. (Dipel WP ®, Abbot Co., IN) as a biological agent, red spinach, and green spinach extract as protectants of Btk., brain heart infusion (BHI) (Oxoid, ThermoScientific, UK), and bacteriological agar (BA) (Oxoid, ThermoScientific, UK) for culture media of Btk.

Insect Collection
Armyworm at the larval stage for the initial stock was collected from cabbage farming in Cangkringan, Sleman. It was collected directly by taking the infested cabbage leaves. As many as 60 larvae were used as parental. The collected larvae were maintained in an artificial diet at Entomology Laboratory, Faculty of Biology, Gadjah Mada University.
Artificial Diet White bean-based artificial diet (Sutanto et al., 20162018) was used for the armyworm mass rearing. As much as 125 g of white bean was soaked overnight in tap water. Then, the bean was boiled until soft. After that, it was grinded in a commercial blender by adding 500 ml of distilled water. After that, the mixture was added with 50 g agar powder, 80 g fermipan, 10 g sodium benzoic, and 750 distilled water, and then boiled. After boiled, the mixture was left at room temperature for 10 minutes until the temperature drops to ± 50°C. After that 5 g of ascorbic acid was then added to the mixture and homogenously. 25 ml of the artificial diet was poured into a 90 ml plastic cup. The cup then kept at room temperature for 45 minutes then stored in a refrigerator at 4 °C until used.
Mass Rearing of Armyworm Larvae collected from the field were transferred on to an artificial diet (Shorey & Hale, 1965) with some modifications until pupation. The seven days old pupae then collected and surface sterilized in 5% (v/ v) chlorox (Bayclin ®, SC Johnson, IN), then airdried for 30 minutes and transferred into a glass jar (d: 20 cm, h: 40 cm) for the adult emergence. After emerges, the adults were provided with 10% honey solutions for adult feeding and opac paper for egglaying substrate. The laid eggs were collected daily and transferred into an artificial diet for larval feeding. The F 2 of armyworm were used for the bioassay.
Btk. Culture on BHIA Btk. from the commercial product (Dipel WP®, Abbot Co., IN) were cultured BHIA medium in a 15 ml test tube. Btk. isolate then incubated for five days at room temperature (28°C) until the protein crystals and spores were formed. The formation of these was observed under a contrast phase microscope (Nikon BX -1, JP).

Red and Green Spinach Extraction
As much as 20 g of each of stems and leaves of red and green spinach were weighed and washed in running tap water. Then blended with a commercial blender by adding 180 ml of distilled water. The suspension then filtered using two layers of muslin cloth then stored in a refrigerator (4°C) as a 10% (w/v) stock.
Btk. Formulations Exposure under UVB Additive extracts at 2% (w/v) were used for making Btk. serial concentrations at 5x10 5 , 5x10 6 , and 5x10 7 (spores/ml). One ml of each of the formula was taken and homogenously dropped into a disposable plastic petri dish (d: 6cm, h: 1.5 cm), then exposed under UVB lights (2x 10 W Phillips tube, IN) for 3, 6, and 9 hours. After exposed, the treated suspensions then collected by adding 9 ml of autoclaved distilled water to get 5x10 4 , 5x10 5 , and 5x10 6 (spores/ml) final concentration for the bioassay. The effects of UVB to the morphology of vegetative cell, spore, and crystal protein were observed under a phase-contrast microscope.
Btk. Bioassay against the 3 rd instar of Armyworm Each of the above final concentrations of Btk. suspension was taken and homogenously added into the surface of an artificial diet provided in a disposable petri dish (d: 6cm, h: 1.5 cm), then airdried at room temperature for one hour. After that, 20 larvae of the 3 rd instar of armyworm were added carefully using a soft brush. The bioassay was carried out using 3 replicates for each treatment. The addition of autoclaved distilled water was used as a control. Mortality parameters were measured at 24, 48, and 72 hours after treatment. Sublethal effects were observed until the 10 th day after treatment.

Experimental Design and Statistical Analysis
This study was done using a completely randomized design. The effects of the treatments to the variance of means of larval mortality were done using oneway anova at 95% significance, then followed by LSD if the anova was significant. The pathogenicity prediction of the formula was predicted using LC 50 and LC 90 calculation based on probit analysis (Finney, 1949). All the analysis procedures were done by using SPSS ver. 21.

The effects of UV B to Btk. Morphology
The observation on the effects of UVB to Btk. morphology was done (Figure 1, 2, and 3). On the UVB radiation for 3 hours, spores and protein crystal were clearly can be observed. Whereas, on 6 and 9 hours UVB radiation the protein crystal and spores were degraded, and only they were remained in fewer numbers compared to controls. UV rays resulting in the loss of toxicity and cell pathogenicity. The longer exposure time to UV made the protein crystal and spores degraded (Griego & Spence, 1978) and caused the loss of pathogenicity (Khasdan et al., 2001). Spinach leaves contain vitamin A, vitamin C, vitamin E, flavonoids, and phenols as antioxidants (USDA, 2020). Carotenoids, vitamins, and anthocyanins have important roles to fight free radicals (Anbhudasan, 2014). The number of spores was more visible in the red spinach protectant. This could be caused by the red spinach by which containing higher antioxidant compounds compared to green spinach. By this, it can give more protection to the Btk. compared to green spinach. Btk. formulated with red spinach exposed to UVB; B: Btk. formulated with green spinach exposed to UVB; C: Btk. formulated with red spinach un-exposed to UVB; D: Btk. formulated with green spinach un-exposed to UVB; c: protein crystals); s: spore). Btk. formulated with red spinach exposed to UVB; B: Btk. formulated with green spinach exposed to UVB; C: Btk. formulated with red spinach un-exposed to UVB; D: Btk. formulated with green spinach un-exposed to UVB; c: protein crystals); s: spore). Figure 3. The effects of UVB exposure for 9 hours to Btk. spores and protein crystals (1,200 magnification under a contrast phase microscope) under concentrations of 5x10 6 spores/ml in red and green spinach extracts (A: Btk. formulated with red spinach exposed to UVB; B: Btk. formulated with green spinach exposed to UVB; C: Btk. formulated with red spinach un-exposed to UVB; D: Btk. formulated with green spinach un-exposed to UVB; c: protein crystals); s: spore).

Armyworm Larvae
Based on the observations of armyworm larval mortality treated with Btk. formulations which exposed to different periods of UVB were shown in Figure 4. . The mortality percentage of 3 rd instar larvae of armyworm during after by treated by Btk. formulated with red spinach and green spinach extracts which was exposed at UVB for 3 hours (A); UVB for 6 hours (B); UV B for 9 hours (C).
In the UVB treatments at 3 and 6 hours of exposures, Btk. with protective red spinach in the concentration of 5x10 6 spores/ml had a mortality of 26.7 % and 16.7%, respectively. This is because the levels of antioxidants that function to protect Btk. from UV light on red spinach are higher than green spinach. The number of spores and protein crystals in red spinach is higher than green spinach, so it is better at killing S. litura. Whereas, at 9 hours of UVB exposures, Btk. with green spinach extract at the concentration of 10 6 spores/ml had the highest mortality (13.3%), while for concentrations of 10 5 and 10 4 spores have mortality <7% in third instar larvae.
Mortality increases in parallel with periods of the treatments (Bouda et al., 2001). The larval mortality in the treatment of green spinach extract at a concentration of 10 6 spores/ml at 3 and 9 hours of UVB exposures was higher compared to the mortality at 6 hours exposure. The percentage of larval mortality in Btk. with red spinach protectant was higher than in green spinach. This can be due to the amount of protein crystals and spores in Btk. with red spinach were higher than Btk. with green spinach, thus it had higher pathogenicity. The protein crystal that enters the body of the insects passed through the insect's digestive tract and was activated by the alkaline conditions in the digestive tract to become δ-endotoxin or protoxin proteins. Protoxin will become a toxin if activated by the insect protease enzyme and is bound specifically to receptors in the digestive tract (Schunemann et al., 2014). The symptoms of sub-lethal effects to the larvae caused by Btk. are hardened of the body, stiff, blackish in color/ melanization and their size is shorter than the size before treatment (Khetan, 2001). The bacterial toxin infection may damage the digestive system of the larvae and may cause mortality (Schnepf et al., 1998). Table 1. The lethal concentrations (spores/ml) of Btk. formulated with red and green spinach treated under UVB lights for 3, 6, and 9 hours against armyworm 3 rd larval instar (Arlinda, 2019).
In this study, LC 50 and LC 90 values (Table 1) were used as the prediction of the pathogenicity for Btk. formulation. At 3 and 6 hours UVB exposures, the red spinach treatment was more pathogenic than the green spinach, with LC 50 1.5 x 10 4 and LC 90 1.9 x 10 5 (spores/ml), respectively. Red spinach gave a better UVB protection compared to green. This is possible because of antioxidant contents in red spinach is higher than green spinach.
Sub-lethal Effects of Btk. formulations exposed to UVB against armyworm larvae. Some of the treated armyworms were survived and developed into pupa and adult stages but posed abnormal morphologies (Table 2). In this study, the survived larvae were able to become pupae but are in smaller sizes, darker in color, and shorter than those control. Adults in Btk, treated also had relatively smaller sizes compared to control. Additionally, the pupae that succeeded emerge had an abnormal wing with the characteristics of curly and short wings. A similar result was observed on the studies on the effects of genetically modified maize and Bt. genes

CONCLUSION
Based on the research that has been done, it can be concluded that Btk. with red spinach extract gave more protection to Btk. when exposed under UVB compared to green spinach. The sub-lethal effects of Btk, formulations include the lesser of the growth and development of larvae, pupae, and adults, as well as wings abnormality.