Eco-friendly biopesticide of Beauveria bassiana to control sweet potato weevil Cylas formicarius (Coleoptera: Curculionidae)

Cylas formicarius is a major pest of sweet potato in various countries, with yield losses reaching 100%. Control technology using chemical pesticides is not successful. This study aims to examine the efficacy of the entomopathogenic fungus Beauveria bassiana compared to chemical pesticides against C. formicarius, which was conducted in Lumajang Regency, East Java, from May to September 2021. The results showed that the highest tuber productivity occurred in innovative technology, reaching 43 t/ha with only 1% tuber damage. While the productivity of the existing technology is only 20 t/ha with a tuber damage rate of 21%. The advantages of innovative technology can also be seen in the abundance of both predatory an parasitoid insects. The application of B. bassiana to control the tuber borer at the base of the stem is safer for the survival of useful insects, especially predators, and parasitoids. In addition, the tubers obtained are more organic, so the selling price of these commodities is more expensive than conventional ones. The entomopathogenic fungus B. bassiana has a great opportunity to be used as a potential biopesticide candidate to be developed as a technological innovation to replace the efficacy of chemical pesticides.


Introduction
The widest sweet potato production centers in East Java are; Mojokerto, Malang, Blitar, Lumajang, and Banyuwangi Regencies.The average productivity at each of these locations is still relatively low, only around 15 t/ha, while research results by applying new technological innovations can reach above 30 t/ha.There are several limiting factors at the farm level in efforts to increase sweet potato productivity.However, these limiting factors still have a huge opportunity to be improved in order to obtain optimal productivity, such as the adoption of new superior varieties of technology innovations which have high productivity.Sweet potato superior varieties which has a high yield potential of up to 30 t/ha, among others; Beta 1, Beta 2, Antin 2, and Antin 3, which contain high beta carotene and anthocyanins [1].Another limiting factor is the presence of plant-disturbing organisms (pests and diseases), which are very detrimental to sweet potato farmers [2].
The main pest that attacks sweet potatoes is the tuber borer caused by the Cylas formicarius beetle [3][4].In general, adult female C. formicarius lays eggs at the base of the stem, and then the eggs hatch into larvae, and all stages of the larvae drill and cause tuber damage.Loss of tuber yield due to being bitten by C. formicarius larvae reaches 100% because the tubers that have been drilled are no longer 1312 (2024) 012025 IOP Publishing doi:10.1088/1755-1315/1312/1/012025 2 suitable for consumption.The condition of these tubers has been accumulated by furan terpenes compounds and coumarins, which can cause liver cancer, breast cancer, etc.Farmers generally only rely on the efficacy of chemical pesticides to save tubers, but the application of synthetic insecticides is considered less successful because it only kills the adult stage on the surface of the plant [6], [7].Meanwhile, the larval phase that causes tuber damage is in the soil, so farmers must understand insect behavior [8][9][10][11].
A control technology innovation that has prospects for development is the application of biopesticides from the entomopathogenic fungus Beauveria bassiana [12][13][14].The results of the latest research by [15] and [16] show that the entomopathogenic fungus B. bassiana is quite effective as a biological agent for suppressing and controlling the tuber borer C. formicarius in endemic lands.The efficacy of the B. bassiana fungus is characterized by its ability to produce the chitinase enzyme, which is able to hydrolyze the cuticle so that it is toxic in killing target insects.The advantages of the B. bassiana fungus are also its toxins, such as beauvericin, bassianolide, bassiacridin, bassianin, and oosporein.The oosporein toxin is capable of killing egg-stage insects, and this excess character is not available by other control technologies, especially chemical insecticides.The entomopathogenic fungi that are ovicidal have great potential and are very effective in suppressing the development of pest populations in the field.Therefore, B. bassiana is a type of entomopathogenic fungus that is more widely used as a biological agent to control various types of pests.The host of B. bassiana is wide enough to kill various types of pests in food crops, horticulture, and plantation crops [17][18][19].This study aims to determine the efficacy of the application of the entomopathogenic fungus B. bassiana and chemical pesticides in controlling the tuber borer C. formicarius.

Study site
The research was conducted in Pasrujambe Village, Pasrujambe District, Lumajang Regency, East Java, which is geographically located between 112 o 50'-113 o east longitude and 7 o 62'-8 o 23 south latitude.This research starts from May to December 2021.The object of this research activity only uses insects that damage sweet potatoes Cylas formicarius and not animals, so it does not require ethical clearance.

Experinmental Set Up
This research is the development of an innovative technology that uses biological agents of entomopathogenic fungi compared to (existing) farmer's technology using chemical pesticides.Each technology was tested using three hectares of farmer's land.The number of farmers involved in each sweet potato cultivation technology is 10 people, with a land area of about 2.500 m 2 for each farmer and those used as replicates.The development of innovative technology introduced to farmers is the control of the main pest, namely the sweet potato weevil caused by Cylas formicarius.The control technology used to control tuber borer pests is the application of the entomopathogenic fungus Beauveria bassiana biopesticides by soaking the cuttings before planting, followed by the application at the age of 1.5, 2, 2.5, and 3 months after planting (MAP).
The bunds were covered with silver-black plastic mulch and traps filled with sex pheromone.To suppress the occurrence of scab disease due to infection with the fungus Sphaceloma batatas, an application was made by spraying the extract of the botanical pesticide (Allium cepa).The control components are arranged in an innovative technology package that is compared to the technology normally developed by farmers with the application of the chemical pesticide with the active ingredient deltamethrin to control tuber borer and mancozeb fungicide to control the disease (Table 1).

Resaerch Implementation
Land is processed optimally with innovative technology, while minimal processing is done with existing technology.Furthermore, the soil is mixed with organic fertilizer and then made mounds with a height of 40 cm using innovative technology and 30 cm of existing technology.The bunds on the innovation technology are covered with plastic mulch, and the existing technology uses straw mulch.
Each sweet potato cutting was soaked in Allium cepa approximately 15 minutes before planting in the innovative technology, while in the existing technology, it was soaked in the chemical fungicide mancozeb.Inorganic fertilizers were given twice at the time of planting and 1.5 months after planting (MAP).Hoarding was carried out at the age of 2 MAP using existing technology, but the innovation technology was not covered because the bunds were covered with plastic mulch.Weeding was carried out twice at the age of 1 and 2 MAP on the existing technology, while the innovation technology was without weeding.The tubers were harvested at 4.5 MAP after that, the tubers were cleaned using water, and the tubers were observed for destructive damage.

Observation
The observed variables are; (1) The yield of tubers (t/ha) taken from 10 plant clumps randomly from each farmer as a replicate, (2) The degree of damage to tubers that were bitten by C. formicarius larvae (%) was taken from 10 plant clumps showing symptoms of skin grinding tubers by cutting the tubers, (3) Tubers fit for sale or fit for consumption t/ha by collecting tubers that were not damaged by C. formicarius larvae (healthy) obtained from 10 clumps in each farmer used as replicates, (4) Number of C. formicarius insects infected and colonized by entomopathogenic fungi B. bassiana from the number of trapped insects, (5) The number of tubers obtained per plant, and (6) The abundance of natural enemy populations obtained from sweeping by swinging the sweeper net five times with a single swing and the catch using traps containing pheromones sex.Caught the natural enemies from each farmer are taken as much as five points diagonally, then the insects are put into bags and then taken to the laboratory.Meanwhile, insects caught by traps were then incubated in petri dishes covered with sterile filter paper with >90% humidity to ensure infection with the entomopathogenic fungus B. bassiana.

Data Analysis
All data that has been collected is then analyzed by t-test using the Minitab 16 program.If there is a significant difference between treatments, proceed with Duncan's multiple range test at a significance level = 0.05.

Level of tuber damage
Observation of the level of tuber damage was carried out by cutting the tuber, which indicated a wound caused by C. formicarius larvae on the tuber skin and dark blue spots on the outer tuber skin.The highest level of tuber damage occurred in the existing technology on the four varieties tested, which ranged from 19-21%, while the tuber damage on the four varieties in the innovative technology was only around 1-2% (Figure 1).The highest tuber damage occurred in the existing technology, reaching 21.55% at Antin 2 variety, while the damage to the innovation technology in the same variety was only 2.35% (Figure 1).The level of tuber damage in Beta 2, Antin 3, and Beta 1 varieties was also quite high, 21.20%, 20.30%, and 19.80%, respectively.Meanwhile, tuber damage was lower in the same three varieties, namely 2.91%, 2.50%, and 1.75%.The results of this technology test indicate that innovative technology is needed to protect tubers from tuber borer attacks when compared to chemical technology using deltamethrin.This phenomenon can be seen from the superiority of the innovative technology applied to protect against tuber borer attacks on the four superior sweet potato varieties released by the Ministry of Agriculture, which were still attacked by C. formicarius, although with different levels of damage.Therefore, control technology to maintain tuber yield is needed, such as the application of innovative technology using the entomopathogenic fungus B. bassiana in this study.The success of controlling tuber borer pests using the fungus B. bassiana was also reported by [20] then [21], which indicated that the application of biopesticides from the entomopathogenic fungus B. bassiana was more effective and efficient in protecting tuber damage due to attack by C. formicarius.Furthermore, [22] confirmed that the biopesticidal efficacy of the entomopathogenic fungus B. bassiana combined with plastic mulch to cover bunds was very significant in suppressing tuber damage by up to 99% of attacks by C. formicarius larvae.The results of a study conducted by [23] indicated that the efficacy of B. bassiana biopesticides applied to suppress tuber damage due to borer attack could be seen from the number of insect deaths, the number of eggs that did not hatched, and the weight of tubers that were suitable for consumption [24].Meanwhile, according to [25], to increase and maintain the efficacy of the entomopathogenic fungus B. bassiana in killing target insects, the fungus must have an abundance of chitinase enzymes.Chitinase is a key enzyme for entomopathogenic fungi, which is used to degrade insect cuticle walls in the process of penetration and infection of pathogens, causing the death of host insects [26], [27], [28].
Tuber damage by C. formicarius (%) The results of [29] showed that the content of the chitinase enzyme was affected by each isolate of the entomopathogenic fungus B. bassiana.The more chitinase enzymes produced by these isolates, the more virulent and more pathogenic in killing target insects with a high insect mortality rate due to fungal infections [30][31][32].

Insect mortality
The average number of total mortality of C. formicarius that died as a result of the application of the B. bassiana treatment was observed three times starting at the age of 40, 60, and 80 days after planting (DAP).All C. formicarius insects that died from each weed in each treatment were collected, then incubated in a petri dish with humidity reaching 90% to ensure the efficacy of the fungus that was applied.The results showed that the highest total number of adults of C. formicarius mortality occurred in the existing technology using Beta 2 and Beta 1 varieties, 129 and 113, respectively (Figure 2).All insects that died in both varieties were due to the efficacy of the deltamethrin insecticide application.Meanwhile, the mortality rate for the two varieties, namely Beta 1 and Beta 2, from the innovative technology was lower, so that there was a real difference when compared to the existing technology.The average number of C. formicarius insects that died in the two varieties, both Antin 2 and Antin 3, did not show a significant difference between the innovative technology and the existing technology.The Antin 2 variety used the existing technology with a mortality of 66 individuals C. formicarius, but this mortality was not significantly different from the innovation technology, namely 59 individuals.Meanwhile, the Antin 3 variety at innovation technology showed insect mortality reaching 66 individuals, but the mortality of C. formicarius also was not significantly different from the existing technology, namely 69 individuals.A significant difference between the dead C. formicarius that occurred in the innovative treatment technology and the existing technology on cadavers is that there are indications of B. bassiana mycelium colonization which is white all over the body so that it looks like a mummified (Figure 3).Colonization of the fungus indicates that B. bassiana has high efficacy in killing the tuber borer C. formicarius.The high efficacy of the B. bassiaa fungus in killing C. formicarius, causing mortality reaching 90%, was also reported by [33] Innovation Existing and [34].Even the B. bassiana fungus has more advantages when compared to other entomopathogenic fungi because it is more pathogenic to various types of other important insect pests such as the whitefly Bemisia tabaci [35][36][37][38].In addition to being more pathogenic and having a wider range of hosts for various types of major pests, the colonization rate on cadavers is very fast so that the epizootic process occurs more easily to suppress the development of population explosions [39][40].formicarius in innovative technology.

Tuber Weight
The results showed that the highest tuber weight occurred in all varieties in the innovation technology except the Antin 2 variety, which occurred in the existing technology.The highest tuber yield occurred in the innovative Beta 2 variety, which reached 45.75 t/ha but was not significantly different from the tuber yield of the existing technology, which was 45.15 t/ha (Figure 4).The tuber weight yield on the Antin 3 variety was also high, reaching 36.10 t/ha on the innovation technology, but it was significantly different from the tuber yield on the existing technology, which was 35.95%.Furthermore, the tuber yield of the innovation technology was also higher in the Beta 1 variety, which was 35.25 t/ha, which was significantly different from the tuber yield of the existing technology, which was 34.95 t/ha.Meanwhile, the weight yield of tubers of the Antin 2 variety on the existing technology was higher, reaching 26.58 t/ha when compared to the innovative technology, which was 24.58 t/ha.However, not all of the tubers that have been obtained using the existing technology can be consumed because some of the tubers have also been damaged by C. formicarius larvae, so the tubers are contaminated with toxic compounds.The results of this study indicated that the innovative technology had significant success in protecting tubers from attack by the tuber borer C. formicarius.This fact is shown from the tuber yields obtained that almost match the yield potential of each variety, namely Beta 1 (35.7 t/ha), Beta 2 (34.7 t/ha), Antin 2 (37.1 t/ha), and Antin 3 (30.6 t/ha) [41].

Tubers suitable for consumption
The condition of tubers suitable for consumption is tubers that are not touched by C. formicarius larvae.
The condition of the tubers that have been drilled by C. formicarius larvae has accumulated furan terpene and coumarin compounds so that they can become toxic to consumers.The results showed that innovative technology is better at protecting from tubers borers when compared to existing technologies because tubers that are fit for consumption are quite high, ranging from 24-43 t/ha (Figure 5).This fact can be seen from the four superior varieties that were tested consistently with higher yields than tubers in the existing technology.The highest tuber yields suitable for consumption reached 43 t/ha in Beta 2 varieties, followed by Antin 3, Beta 1, and the lowest Antin 2, respectively 35 t/ha, 34 t/ha, and 24 t/ha.The performance of the tuber conditions of each superior variety tested, both Beta 1, Beta 2, Antin 2, and Antin 3 using the innovative technology was significantly different so that there were no visible injuries caused by C. formicarius larvae on the tubers (Figure 6) when compared to the existing technology.The tuber yields obtained from the existing technology were significantly lower for each superior varieties tested, respectively 28.30 t/ha, 35.57t/ha, 20.32 t/ha, and 28.57t/ha.The decrease in tuber weight suitable for consumption in the existing technology was caused by the total weight of harvested tubers being picked up by C. formicarius larvae, which ranged from 19-21%.The tubers that have been damaged by the larvae of C. formicarius already contain poison and can cause poisoning if it accumulates in the consumer's body at high doses.According to [42] that the two elements are protein compounds containing ethylene with a very high molecular weight, causing necrotic lesions in the tuber tissue and harmful for consumers.

The abundance of natural enemies populations
Natural enemy populations were determined by taking insects using sweep nets in each treatment for each repetition using five single swings.In addition, natural enemies were also observed from insect traps containing sex pheromones.Observations were made twice at the age of 60 and 90 days after planting, and then the insects were identified using a binocular stereo microscope type XT 60 -J3C 20x/40x, and the key of determination was referred to [43], [44].
The results showed that populations of natural enemies, both predators and parasitoids, were found to be more abundant in innovative technology compared to existing technology.The highest population of natural enemies was found predator Entomobrya sp.namely reaching 163 individuals, while these predators in the existing technology are only found under 10 individuals.According to [45], [46] that Entomobrya sp. is a group of predators from the order Collembola as inhabitants of the soil surface, however, this type of predator has high predation on insect pests from the aphids group that are on the plant surface.Furthermore, the abundance of natural enemy populations can also be seen in two types of parasitoids, Eretmocerus eremicus and E. mundus, 132 and 102 individuals, respectively (Table 2).and nymphs [47], [48].Furthermore, [49] that the parasitoid E. mundus was able to suppress the development of B. tabaci biotype B which is known to be difficult to control despite the application of chemical pesticides because it has experienced resistance to most chemical pesticide formulations.
The population of predators, especially canopy dwellers such as Coccinella sp., Ichura senegalensis, Agriocnemis femina, and A. pygmaea, were also more abundant due to the innovation technology of 86 individuals, 79 individuals, 67 individuals, and 46 individuals.Meanwhile, the population of the four types of predators, according to existing technology, is very limited to only 2-3 individuals.There are two types of predator populations that inhabit the canopy and the ground surface, namely Paederus fuscipes and Oxyopes javanus, each with 81 and 33 individuals.Both types of predators are gregarious predators, so they have high cruising abilities and are quite effective in finding prey.In addition to its high cruising ability, it is also capable of predating various types of prey, so it has the potential to be used as a pest control agent with a high personality.The research results of [50] indicated two types of predators with high potential to suppress B. tabaci populations in the field were Coccinella sp. and Paederus fuscipes because every 24 hours, they are able to predate up to 41 B. tabaci nymphs while the predation ability of imago is around 10-17 individuals.Meanwhile, [51] stated that the potential predators to suppress B. tabaci populations were Coccinella sp.The cause of the low population of natural enemies, both predators, and parasitoids, in existing technology is because the natural enemy are more susceptible to chemical pesticides.In addition, the predators P. fuscipes and Coccinella sp. have very active behavior so that in existing technology, where there are generally many farmer activities such as weeding, bunding reversal, and so on, the existence of these two types of predators hides or moves to agriculture.

Conclusion
The average tuber yield for four superior varieties tested reached 35 t/ha for both innovative and existing technologies, with 2% tuber damage for innovative technology, while for existing technology, it reached 21%.The cultivation technology to protect against tuber borer of C. formicarius is by soaking the cuttings before planting them in conidia suspension of the entomopathogenic fungus B. bassiana, followed by four applications starting at the age of 1.5, 2, 2.5, and 3 MAP at the base of the stem or at the soil surface.The efficacy of controlling tuber borer using the entomopathogenic fungus B. bassiana can reduce tuber yield loss by up to 19%.The advantages of the entomopathogenic fungus B. bassiana are; toxic to all stages of C. formicarius, is ovicidal or kills the egg stage, and is more environmentally friendly because it can maintain the survival of both predators and parasitoids when compared to existing technology.The entomopathogenic fungus B. bassiana can be recommended as a biopesticidal candidate for the control of the C. formicarius and as an alternative to the efficacy of chemical pesticides.

Figure 1 .
Figure 1.The level of tubers damage by C. formicarius larvae in four superior sweet potato varieties with innovation and existing technologies (Lumajang dry season, 2021).

Figure 2 .
Figure 2. Average number of C. formicarius insects that died in four superior sweet potato varieties on innovation and existing technologies (Lumajang dry season, 2021).

Figure 3 .
Figure 3. Mummification of the entomopathogenic fungus B. bassiana on the cadaver of C. formicarius in innovative technology.

13thFigure 4 .
Figure 4. Average tuber weight obtained in the four superior varieties of sweet potato on the innovation and existing technologies (Lumajang, dry season 2021).

Figure 5 .
Figure 5. Average tuber weight of four superior sweet potato varieties that are fit for consumption on the innovation and existing technologies (Lumajang, dry season 2021).

Table 1 .
Development of environmentally friendly C. formicarius tuber borer control technology using biopesticides of entomopathogenic fungus B. bassiana Note: MAP (month after planting)

Table 2 .
The abundance of insect populations of natural enemies (predators and parasitoids) on innovative and existing technologies of 90 DAP