Toxicology of Nuclear Polyhedrosis Virus, Botanical and Synthetic Pesticides on Mortality Rate of Crocidolomia binotalis (Zeller)

: Crocidolomia binotalis (Cb) (Zeller) is one of the main pests of cabbage which is difficult for farmers to control in the Ijen Crater in Indonesia. The demand for cabbage in Indonesia is increasing every year, so intensification efforts are continuously being made. Farmers intensively use synthetic pesticides as the primary control of Cb. The purpose of this study is to examine several control agents that can be used as an alternative to synthetic pesticides. Then, it provides recommendations to Ijen Crater farmers to adapt in reducing the use of synthetic pesticides. The biopesticide used was based on the Nuclear Polyhedrosis Virus strains of Spodoptera litura (SlNPV) and Helicoverpa armigera (HaNPV), botanical pesticides, and synthetic pesticides as a comparison treatment. SlNPv and HaNPV isolates were obtained from the Indonesian Sweetener and Fiber Crops Research Institute. The botanical pesticide formulas come from a combination of Azadiracta indica, Aglaia odorata, Ageratum conyzoides dried for one week and proposed to form flour. Then, it was extracted using 96% ethanol solvent, 0.5% tween 90 using a rotary evaporator at a temperature of 40 o C. For synthetic pesticide, the treatment used the active ingredient Permethrin 20 mgL -1 . The cb collected from the field was re-filtered in a Petri dish by providing artificial feed soaked with this material for 5 minutes. The highest mortality test results were found in the botanical pesticide formula with a value of 100% at 72 hours. Mortality in SlNPV and HaNPV tended to be stable with a maximum value of 72.02%. The results of mortality testing in the laboratory can be a recommendation for alternative control of Cb pests in cabbage plants in the field. In this case, it is an effort to reduce farmers' dependence on using synthetic pesticides.


Introduction:
Asustainable agricultural system is one of the Food and Agriculture Organization (FAO) efforts to alleviate problems of food, production, and malnutrition 1 . One of the implementations of sustainable agriculture is based on ecology, where biological control is an example of environment friendly and conservative control 2,3 . Ijen Crater is an intensive agricultural system and production center for horticultural crops such as cabbage and potatoes in Indonesia. So far, agricultural production has been distributed throughout the islands of Java and Bali. The problem faced by farmers is the attack of Crocidolomia binotalis (Cb)(Zeller).
The Crocidolomia binotalis (Cb) is the primary pest of cabbage [4][5][6][7] . Morphologically, in the imago phase, the Cb has a wingspan of approximately 20 mm, has a filiform antenna shape, and is light brown 8 . Cb attacks during the crop formation phase until harvest. The ability to lay eggs in female Cb reaches 200-300 eggs. Cb is included in complete metamorphosis so that, in the larval phase, there could beseveral changes in size known as instars. Cb has a high level of greed, so it does not take long to finish the cabbage plant. A study reports, Cb has more eating activity along with increasing phases of his life 9 .
The challenge in control today must be sustainable. Sustainabilityhas three dimensions, namely social, economic, and environmental. There are several conceptual stages in implementing sustainable agriculture. Before moving to the technical part, the transitional period of the cultivation system transition includes four aspects, including trust, infrastructure, institutional strength, and implementation methods so that these four aspects can accelerate the transition process in shifting from a cultivation system that applies the concept of sustainable agriculture 10 .
Typology in the Ijen Crater area based on Rapid Rural Appraisal (RRA) is classified as intensive agriculture. The same thing is shown based on the upstream part's behavior showing similarities in the intensive use of pesticides 11,12 . The impact of the intensive use of synthetic inputs has an impact on environmental quality degradation 13 . Synthetic pesticides will cause resistance to Cb [14][15][16] . Currently, Indonesia is making an acceleration in producing agricultural products that are safe and free from residues. One of them is through Good Corporate Governance, which has implications for sustainable corporate performance, which has four essential values: economic sustainability performance, environmental sustainability performance, and social sustainability performance 17 .
The purpose of this study is to examine the problem of Cb attack that occurred in the Ijen Crater Mountains with the control that implements a sustainable agricultural system, one of which is by using biological control agents using NPV (Nuclear Polyhedrosis Virus), botanical pesticides, and as a comparison using the usual synthetic pesticides used by farmers. NPV effectively kills target pests with specific advantages of targeting [18][19][20] . Meanwhile, botanical pesticides have properties as biodegradation, have toxic secondary metabolites, and are environmentally friendly 15,21 .

Location study
Observation and sampling of Crocidolomia binotalis were taken from cabbage farms in the Ijen Crater, East Java, Indonesia (Fig 1). This location is one of the centers for producing horticultural crops for distribution around Java and Bali islands. Geographically, the research location is located at an altitude of 1,100 meters above sea level and is at the coordinates Lat -7.996939 and Long 114.150269. The collected Cb wasbrought and refined at the Plant Pest Laboratory, Faculty of Agriculture, University of Jember. Botanical Pesticides. The botanical ingredients formula used is neem leaves (Azadiracta indica), Chinese henna (Aglaia odorata), and babandotan (Ageratum conyzoides) 21 . Based on the research, these ingredients are effective in controlling pests of the order Hemiptera and Lepidoptera. The ingredients are air-dried for about one week and then mashed using a blender to obtain a flour formulation. Then the extraction process used a rotary evaporator to obtain secondary metabolites. Each of these materials is weighed as much as 100 g, added 96% ethanol as much as 750 mL, and tween 90 as much as 0.5% 24 . After being homogeneous, it was incubated for 24 hours and extracted using a rotary evaporator at a temperature of 40˚C for approximately 2 hours 25 . The extraction results were obtained from botanical pesticides in the form of a paste [26][27][28] . There are several references in the selection of botanical materials to use. The preferences used can be seen in Table. 1.

Plutella xylustella
Ingredients are dried in an oven at 40 0 C for 48 hours. Then it is macerated at a cold temperature for 72 hours using a hexane solvent. Ginseng stems and leaves of A. indica are dried at 50 o C for three days and mixed with 70% methyl alcohol. The extraction was then evaporated using a solvent of 500 mL of ultrapure water containing 1 gL -1 Triton X-1000 [24][25][26] Synthetic pesticides. The rapid rural appraisal (RRA) survey results show that the majority of farmers in the Ijen Crater Mountains use synthetic pesticides with the active ingredient Permethrin 20 mgL -1 . Spraying is done every morning and evening at intervals of 2 days.

Experimental design and data analysis
The study was designed using a CRD (Completely Randomized Design). The experimental design can be seen in Table. 2. The method of application is by rearing third and fourth instar larvae that have been given artificial feed. Artificial feed using cabbage which is obtained from the origin from which the pest Crocidolomia binotalis was collected. Then the leaf pieces were soaked in each of the ingredients starting from NPV, botanical pesticides, synthetic pesticides, and comparative treatments for about 5 minutes. The leaves of the cabbage are cut into 2.5 × 2.5 cm sizes. The artificial feed is then put into a petridish which contains as many as 15 Crocidolomia binotalis larvae. Data analysis was done using ANOVA. If the calculation results are significantly different, then a further test is carried out using Tuckey with a confidence level of 95%. The analysis program that was used Minitab version 16.

Pathogenicity Test
Observations are made by calculating the amount of mortality (larval mortality) in Petri dish that occurs after application. A change in skin colour characterizes the dead larvae. The pathogenicity test was calculated by calculating the percentage of dead larvae with the total number of larvae in each treatment during time units ranging from 24 hours, 48 hours, 72 hours, and 96 hours.

Results and Discussion:
Cb attacked the cabbage crop head by entering the outer leaves (Fig 2). The Cb will gnaw at the base of the cabbage head. During the development phase, Cb will lay eggs between the fruit stalks to hatch and turn into the pupa phase.
Pupae have a lifespan of 7.00 ± 0.128 29 . The pupa will be protected and covered by a white netting membrane (Fig 2a). Cb eats cabbage from the base to the tip of the outer leaves. The largest population of cabbage heads will cause crop failure. Based on the rearing of Cb in Petri dishes, various treatments gave different visual symptoms (Fig 3). Observations were made using a Leica microscope with a magnification of 100 times. In Fig. 3, the codes (a, b, c) are visual symptoms that have been applied to botanical pesticides. The formula with a combination of Azadiracta indica, Aglaia odorata, Ageratum conyzoides based on test results on stink bug populations in the field is very effective in reducing population levels 23 . Therefore, the application ability of Cb Petri dish shows a brownish-yellow discoloration on the cuticle part. The position of insect mortality tends to be elongated. There are black dots along his body that are still visible. The mood of entry mechanism of botanical pesticides based on research results varies depending on the secondary metabolite compounds produced 30 . Azadirachtin compounds produced by A. indica can inhibit the activity of acetylcholinesterase in insects. This case is confirmed in the histopathological analysis and enzyme activity of Nilaparvata lugens 31 .
In NPV treatment by referring to Fig. 3 with codes (d, e, f), the symptoms of morphological changes in Cb pests applied by NPV (Nuclear Polyhedrosis Virus) curved and hung. This symptom is typical in insects infected by the virus. The NPV used comes from the Indonesian Sweetener and Fiber Crops Research Institute with strains from Spodoptera litura NPV (SlNPV) and Helicoverpa armigera(HaNPV). The advantage of NPV is that it has a specific killing power of targeting 32,18,19 .Viral protein will enter through insect digestion, causing insects to become paralyzed, the digestive system will be damaged, and the body size becomes smaller. Therefore, insect pests from the order Lepidoptera infected with the virus will be characterized by hanging and the body is wet and slimy. Whereas in the treatment of synthetic pesticide applications, the Cb experienced a greenish discoloration of almost the entire body surface. The active ingredient contained is Permethrin. The mechanism of action of the active ingredient is contact and stomach poison. The poison will spread starting from the digestion of the insects and spread throughout the body. The body changes color from green to dark black.   The ANOVA calculations on Tuckey's analysis show that the P. value is greater than the F. value (Sig) of 0.05%. So the Bonferonni analysis was carried out. The aim is to find out precisely the average between different treatments. Based on Fig.  4, the Bonferonni test results show the average mortality in each treatment against time units. The mean value based on time concluded the mean at 24 h> 48 h> 72 h> 96 h. The closer to 0, the average mortality is significant; conversely, if it is further away from the number 0, H0 is most likely accepted. Based on the calculation of the average mortality in Fig. 5, initially, at 24 hours, it can be seen that the Cb mortality rate is still low. In this case, the treatment reaction will still be accepted by the target. Meanwhile, at the 48 h and 72h, the mortality tended to increase. This is because the toxin is reacting to the insect's body. There has been a work process on insect digestion. Meanwhile, the 96 h is the climax phase of the toxin acting on the body. In the number of insect trials, the mortality rate is higher.
Treatments A and B used NPV (Nuclear Polyhedrosis Virus) using SlNPV and HaNPV. In both of these treatments, the mortality rate tends to be stable compared to other treatments. Initial detection is that NPV has a higher and relatively fast killing power compared to other treatments. The process of viral particles enters the digestive tract of insects. The gp64 gene functions as a cellbinding with the endocytosis process 38 . The highest mortality rate was 72.02% at 96 hours. Whereas in treatment D (Synthetic) with the active ingredient Simpermetrin, the symptom of mortality was deficient. Farmers often use these active ingredients. The low mortality is assumed that the Cb has experienced resistance to the active ingredient. Several factors can cause pests to become resistant to an active ingredient due to the large doses and spraying frequency that exceeds the recommendation.

Conclusion:
Based on the test results in this study, it can be concluded that the response to the use of biological control can provide a high mortality value. This study showed that Cb treated with botanical pesticides had a mortality rate of up to 100%. The test in this study can be a recommendation for Cb pest control for farmers to reduce their dependence on intensive pesticide use. The use of biological agents also minimizes pest resistance that has the potential for an outbreak.