Compatibility of Crown Flower Extract (Calotropis gigantea L.) and Aspergillus sp. to control Spodoptera exigua Hubner on Shallot Crops

Biopesticides are strongly recommended in pest management to mitigate the harmful effects of the chemicals. This study aimed to evaluate the combined effect of the crown flower extracts (CFE) and Aspergillus sp. in controlling Spodoftera exigua, a major pest of shallot. Three level concentrations of CFE (g/l) i.e. 0 (C0), 2.42 (C1), and 4.84 (C2), as well as Aspergillus sp., i.e. 0 % (A1), 0.5% (A1), and 1.0% (A2), were combined and set up in a Randomized Complete Block. Pest infestation and shallot yields were observed to assess the effectiveness of those biopesticides. The results showed that the application of CFE or Aspergillus sp. was more effective in suppressing the infestation of S. exigua than without biopesticides. However, the highest concentration of the combination of both biopesticides results in the best treatment, possibly suppressing the beet armyworm infestation by about 31,09 % compared to the control. It has the lowest values of the Area Under Pest Damage Curve (AUDPC) and the highest yield of shallots (5.50 t/ha). The study confirmed that a combination of CFE and Aspergillus sp. was suitable for managing S. exigua on shallot crops and supporting the integrated pest management of beet armyworms.


Introduction
In Indonesia, the shallot plant, Allium cepa L. group aggregatum, is severely harmed by the beet armyworm, Spodoptera exigua (Hübner).Beet armyworm is acknowledged as a typical pest in Indonesia, resulting in a significant shallot crop loss in the lowlands and can occasionally occur in highland shallots [1] [2].Infestation of S.exigua on shallots can cause crop losses of about 32 to 100% if the pest is not managed correctly [3].Farmers regularly use chemical control to eliminate this pest and sometimes mix several pesticides into a single spray solution and apply this mixture of insecticides at rates higher than those recommended by the product [4].
The farmers' high intensity and irrational pesticide application resulted in some detrimental effects, including increasing pest resistance to pesticides.Development of resistance of the S.exigua field populations to some insecticides with distinct modes of action, including chlorfenapyr, methomyl, and 1346 (2024) 012020 IOP Publishing doi:10.1088/1755-1315/1346/1/012020 2 emamectin benzoate not only been detected in Indonesia but also in China and Pakistan [4] [5][6] [7].Therefore, developing a more sustainable approach to managing this harmful pest is necessary.It includes a rotation of different chemicals with unique modes of action, using biopesticides or other integrated pest management strategies.
Microbial and botanical pesticides are claimed to be safer and have high efficacy against various crop pests instead of chemicals [8] [9].Among them are Aspergillus sp. and Calotropis gigantea.Both biopesticides showed insecticidal properties.For example; Aspergillus niger at the concentration of 1×106 spores/mL exhibit the highest mortality effect on the Red Cotton Bug, Dysdercus koenigii after 96 h of treatment [10].Application of A.niger has shown an antifeedant and toxic effect on Spodoptera litura.The metabolite produced by A.niger adversely affected the survival and development of S. litura.[11].Meanwhile, the extract of C. gigantea was reported to have toxic and antifedant activities thereby inhibiting the growth of S. exigua larvae [12].The leaf extract of C. gigantea had higher toxic and deterrent effects against P. xylostella than B. carambolae, N. lugens, and S. zeamais [13].
However, the compatibility of botanical and microbial pesticides against the crop pest was rarely studied.Some previous studies showed that entomopathogenic fungi combined with botanical insecticides effectively suppress the development or attack of insect pests [9] [20] [21].Therefore, this study was carried out to evaluate the effectiveness of the crown flower extracts combined with Aspergillus sp. to control the beet armyworm on shallots.

Study sites and Research design
This research was conducted at the Palu Valley shallot plantation in Langaleso Village, Dolo District, Sigi Regency in 2022.The study area is situated at 48.3 m asl.A total of 70 shallots were planted in each plot of 2x1 m.The distance between crops' dan plots' was 15x20 cm and 50 cm, respectively.The experiment was designed using a Randomized Block Design based on the shallots' size.Three-level concentrations of crown flower extracts (CFE) and Aspergillus sp. were combined as the treatments.The level of concentration were C0 = without CFE (Control), C1 = CFE 2.42 %, C2 = CFE 4.84 %, A0 = without Aspergillus sp, A1 = Aspergillus sp.0.5 g/l (1,37x108 ml -1 ), and A2 = Aspergillus sp.1.0 g/l (2,75x108 ml -1 ).All treatments were combined, resulting in nine combinations of treatments, and each with three replicates.

Biopesticides preparation and application
The leaf of the crown flower plant was extracted using ethanol 96% modified from Shahabuddin and Pasaru [12].The solid extract obtained was mixed with 5% sandovite solution and then diluted with distilled water according to the treatment concentration.Aspergillus sp.isolates were obtained from the Laboratory of Plant Pest and Disease, Faculty of Agriculture, Universitas Tadulako.The isolates were cultured on Potato Dextrose Agar medium (PDA), composed of Potato extract solution (200 g potatoes, 20 g dextrose, and 20 g agar).After the isolates were inoculated in the media, the cultures were shaken for 48 hours at 120 rpm and incubated for six days.The conidia density of 1 ml suspension of Aspergillus sp. was counted under the microscope using a hemocytometer following Sumikarsih et al. [14].Single treatment of CFE or Aspergillus sp. was applied once a week, seven days after planting (DAP).Meanwhile, the combination of CFE and Aspergillus sp. was applied alternately in the same week with 3-day intervals between both biopesticides.

Variable Observed 1. Attack Intensity
The attack intensity of S.exigua was observed in 18 crop samples in each plot following a double diagonal transect design.The arrangement of the crop sample is shown in Figure 1.Observations were made weekly from 14 to 56 days after planting (DAP).The intensity of pest attacks is calculated using the formula [15] as follows : where: IA = Intensity of pest attacks, ni = number of plants attacked with score certain, vi = pest attack score; (0: no symptoms of attack; 1: attack symptoms 1-20%; 2: attack symptoms 21-40%; 3: attack symptoms 41-60; 4: attack symptoms 61-80%; 5: attack symptoms 81-100%), N = number of plants observed, V = highest pest attack score.

Shallots yield
The dry weight of the tubers was measured seven days after harvest.Tuber weight per plot was then converted into t/ha.

Data Analysis
Pest infestation data were transformed to Arcsin before being further analyzed using an ANOVA and HSD test to evaluate the significant differences among the treatments.The effectiveness of treatment in suppressing S.exigua attacks is determined based on the Area Under Pest Damage Curve (AUDPC) value following Suryaningsih [15].The lowest score of AUDPC indicates the most effective treatment.

Beet armyworm infestation
Infestation of beet armyworm was first recorded by the presence of their eggs at the shallot crops.The eggs laid by their imago were first recorded seven days after plantation (DAP), while the larvae were found to feed on the crops since 14 DAP (Figure 2).The intensity of S. exigua attacks fluctuates with the highest attacks occurring at 21 and 28 DAP and decreasing at 35 DAP until 56 DAP.This phenomenon was found in both control plants and plants Sample with biopesticide applications (Figure 3).However, the attack intensity of the S. exigua on the shallot crops without biopesticide applications (C0A0) was higher than all other treatments.The positive effect of biopesticide application is shown by the lower intensity of S. exigua attacks on shallots with single or double treatment compared to those not applied with biopesticide.However, the lowest attack intensity was found on shallots treated with both types of biopesticides (CFE and Aspergillus sp.), particularly at the 21 and 28 DAP, where the pest infestation was high (Figure 1).The infestation of beet armyworm on the single treatment (especially, C2A0) and the combination treatment (C2A1 and C2A2) in all crop ages was not significantly different.However, this biopesticide combination with the highest concentration (C2A2) was able to suppress the beet armyworm infestation by about 31,09% at the 21 and 28 DAP compared to control and has the lowest values of the Area Under Pest Damage Curve (Table 1).

Shallots yield
The highest weight of shallot tuber, was obtained from the C2A2 treatment, followed by the C2A1, C2A0, C1A2, C1A1, C1A0, C0A2, C0A1 treatments and the lowest was C0A0 (Figure 4).These results indicate that the combination of CFE extract and Aspergillus sp. can suppress the attack of S. exigua better than other treatments, thereby influencing the shallots production.

Discussion
Spodoftera exigua or beet armyworm is an important pest of shallots and has been increasing resistance to various insecticides across the world [4][5][6] [7].Therefore, a more sustainable approach against this pest is urgently needed.This study showed that the application of the crown flower extracts (CFE) or Aspergillus sp.significantly decreased the infestation of S. exigua.However, the combination of both biopesticides is more effective in suppressing S. exigua attacks than using only one of them.Application of the CFE combined with Aspergillus give a double attack against S. exigua due to their different mode of action.CFE contains several secondary metabolites including alkaloid, tannin, phenol, flavonoid, and saponin that are toxic and have an antifeedant effect on pests [12] [13] [16], while Aspergillus spp.showed a pathogenicity against many insect pests with two possible mechanisms; parasitizing healthy insects or poisoning the insects with various toxic metabolites produced [18] [19].
The infestation level of S. exigua found in this study is quite lower compared to another study [1] [7] [12].It may related to some factors including the time of conducted study.This study was conducted during the rainy seasonis and it is estimated that the level of S. exigua infestation would be higher if the experiment were carried out in the dry season because no weather factors can prevent adults from laying their eggs to develop into larvae.This is per previous research found that the number of egg and larva populations [1], and imago [17] of S.exigua on shallots is much higher in the dry season than in the rainy season.Usually, after hatching from the egg, the young caterpillar immediately makes a hole at the tip of the leaf and then enters the leaf, so that the tip of the leaf appears hollow or cut.Caterpillars will chew on the inner surface of the leaves while leaving the outer epidermis.As a result of this attack, the onion leaves look translucent or have white spots, and eventually, the leaves become droopy.Initially, the caterpillars gathered.Once the leaves are empty, the caterpillars immediately spread and if the population is large, the caterpillars may also eat the shallot tubers [3].Overall, the infestation of S. exigua detected in the present study decreased after 28 DAP but both biopesticides were used in this study resulting in a higher effect in suppressing the infestation of this pest.A previous study reported that the highest infestation of S. exigua and plant damage on shallots occurred when the plants were 27 days old.After this age, the percentage of leaf damage decreases due to the formation of new leaves [1].The effectiveness of the CFE dan Aspergillus sp. in suppressing the population and attack level of S. exigua has a positive effect on the shallot yield.The highest weight of shallot tuber was obtained from the C2A2 treatment, followed by the C2A1, C2A0, C1A2, C1A1, C1A0, C0A2, C0A1 treatments and the lowest was C0A0.These results indicate that the combination of CFE extract and Aspergillus sp.effectively suppresses the attack of S. exigua better than other treatments, thereby influencing the shallot's production.This is in line with some previous studies that reported that entomopathogenic fungi combined with botanical insecticides are more effective in suppressing the development or attack of insect pests [9] [20] [21].
The reduction of S. exigua attacks due to the use of biopesticides supports photosynthesis well, and more assimilate is produced, thus supporting the development of shallot tuber much better.The shallot yield obtained in this study is classified as normal production as it is reported that the potential productivity of this local variety of shallots is 9.7 t/ha.However, at the farmer level, it is productivity is only around 4-5 t/ha [22].Therefore it can be concluded that the combination of CFA and Aspergillus sp. could suppress the yield losses due to S. exigua attack well, resulting in the highest tuber weight compared to other treatments.However, because in this study the shallot yields tended to increase following the biopesticide concentration, it is interesting to further test the effectiveness of this biopesticide combination at higher concentrations to reduce plant damage by S. exigua and increase shallot yields.

Conclusion
A combination of crown flower extracts and Aspergillus sp. were compatible to use as biopesticides to manage the S. exigua on shallot crops and support the integrated pest management of beet armyworm.This biopesticide combination with the highest concentration was able to suppress the beet armyworm infestation by about 31,09%, has the lowest values of the Area Under Pest Damage Curve, and results in the highest yield of shallots (5.50 t/ha).

Figure 2 .
Figure 2. The eggs (left) and larvae (right) of S. exigua (indicated by red arrow) at the shallot crops.

Table 1 .
The intensity of S. exigua infestation (%) and it is AUDPC score at different biopesticide treatments and crop age.