INTRODUCTION
Sweet potato (Ipomoea batatas (L.) Lamb) is one of 20 foodstuffs producing carbohydrate energy. This diversifying food also supports the government program as an alternative to rice (Chafid, 2016). One limiting factor in increasing the productivity and quality of sweet potato tubers is the presence of sweet potato weevils (Capinera, 2012). It is commonly referred to as “Lanas pest”
caused by weevils (Cylas formicarius Fabricius).
This pest is widely found in tropical regions of Africa, Asia, and North & South America (Talekar,
1991). The pest also attacks sweet potato plants during the dry season (Indiati & Saleh, 2010), and this pest is harmful when in the field, even as a storage pest (Kalshoven, 1950). The weevil attacks cause a decrease in the quantity and quality of sweet potatoes. The most significant reduction in the rate of sweet potatoes occurs in larval and imago pest stages (Anitha, K., Anitha, G., Hirur, Suresh, Nayak, 2021). Symptoms of the damage caused are the formation of terpenoid compounds, which are compounds that cause a bitter taste so that the tubers cannot be consumed (Jansson &
ARTICLE INFO Keywords:
Conidial density Germination rate Lecanicillium lecanii Lethal Concentration (LC50) Mortality rate
Article History:
Received: October 26, 2021 Accepted: September 18, 2022
*) Corresponding author:
E-mail: lutfiafifah@staff.unsika.ac.id
ABSTRACT
The growth of entomopathogenic fungi Lecanicillium lecanii is strongly related to the media in increasing the fungi sporulation, enhancing its effectiveness in infecting target pests. This study aims to obtain L.
lecanii propagation media with the highest conidia production, and the increased virulence on Cylas formicarius. The first stage of alternative media selection consists of: Potato Dextrose Agar; maize, rice; bran.
The second stage of infectivity of the best alternative media consists of 5 treatments in 5 replications: synthetic insecticide (C+); distilled water (C-); 107 conidia/ml; 108 conidia/ml; 109 conidia/ml. The results show that the best colony growth rate is (1.15 mm) on Maize media, and the highest conidia density is (4.2 x 10⁸ conidia/ml) on Maize media is not significantly different from PDA (1.3 x 10⁸ conidia/ml). The best germination rate is (74.31%) on Maize media, and the highest media weight (1.10 g) on Rice media is not significantly different from maize (1.45 g). The infectivity of L.lecanii affect the mortality of C.
formicarius (74%) substantially at a concentration of 109 conidia/ml.
The LC50 value obtained is 2.6 x 10⁷ conidia/ml. Thus, maize media can be an alternative medium for mass propagation of L. lecanii.
ISSN: 0126-0537Accredited First Grade by Ministry of Research, Technology and Higher Education of The Republic ofIndonesia, Decree No: 30/E/KPT/2018
Cite this as: Afifah, L., Aena, A. C., Saputro, N. W., Kurniati, A., Maryana, R., Lestari, A., Abadi, S., & Enri, U. (2022).
Maize media enhance the conidia production of entomopathogenic fungi Lecanicillium lecanii also Its effective to control the weevil Cylas formicarius (Fabricius) (Coleoptera: Brentidae). AGRIVITA Journal of Agricultural Science, 44(3), 513- 525. http://doi.org/10.17503/agrivita.v44i3.3605
Maize Media Enhance the Conidia Production of Entomopathogenic Fungi Lecanicillium lecanii also Its Effective to Control the Weevil Cylas formicarius (Fabricius) (Coleoptera: Brentidae)
Lutfi Afifah1*), Aulia Corry Aena1), Nurcahyo Widyodaru Saputro1), Anik Kurniati2), Rosalia Maryana2), Ani Lestari1), Slamet Abadi3) and Ultach Enri4)
1) Department of Agrotechnology, Faculty of Agriculture, Universitas Singaperbangsa Karawang,
Indonesia
2) Forecasting Center for Plant Pest Organisms, Karawang, West Java, Indonesia
3) Department of Agribusiness, Faculty of Agriculture, Universitas Singaperbangsa Karawang, Indonesia
4) Department of Information Technology, Faculty of Computer Science, Universitas Singaperbangsa Karawang, Indonesia
Raman, 1991). This damage resulted in a decrease in the selling price of sweet potatoes and the low feasibility of consuming sweet potatoes (Anitha, K., Anitha, G., Hirur, Suresh, Nayak, 2021).
The farmer’s effort in controlling the attack of weevil pests is the application of synthetic insecticides. Until now, synthetic insecticides have not been effective in suppressing attacks of weevil pests because insects attack the stem and inside the tubers (Nonci, 2005). Around 20% of sweet potato farmers in Central and East Java use chemical insecticides to control C. formicarius (Supriyatin, 2001). According to Soetopo & Indrayani (2007), using synthetic insecticides that are not carried out wisely in the long term can cause environmental problems. The problems include increasing resistance of target pests, an explosion of the resurgence of non-target pests, killing of natural enemies and other valuable insects, pollution of soil and water sources, decreased biodiversity, and human health hazards when in direct contact with synthetic pesticides.
Many negative impacts appear from synthetic insecticides, such as insecticide resistance, resurgence, and the occurrence of secondary pests. Therefore it is necessary to implement Integrated Pest Management (IPM), one of which is by controlling with the use of biological agents. One group of pathogenic biological agents that can be used is entomopathogenic fungi (Trizelia, Armon, &
Jailani, 2015). Some entomopathogenic fungi have been developed to control various plant pests, for example, using entomopathogenic fungi (Balls.) Vuill in testing the efficacy of C. formicarius pests (Bayu & Prayogo, 2016; Ratissa, 2011; Saputro, Prayogo, Rohman, & Alami, 2019; Supriyatin, 2001;
Tantawizal, Inayati, & Prayogo, 2015), the use of Metarhizium anisopliae (Metch.) Sorokin to control cocoa pod-sucking pests (Erdiyanto, Purnomo, Wibowo, & Yasin, 2013). Besides, Lecanicillium lecanii (= Verticillium lecanii) was first reported by Viegas in 1939 to attack aphids (Shinde, Patel, Purohit, Pandya, & Sabalpara, 2010).
The host distribution of L. lecanii is quite broad, and the fungus is cosmopolitan, which is easy to find in areas with tropical and subtropical climates, resulting in a relatively high diversity of isolates. In addition, the fungus L. lecanii is used for pest control and plant disease control (Harni et al., 2015). Anggarawati (2014) explains the use of the
fungus L. lecanii to reduce Helopeltis spp. at the 3rd instar nmph stage, a pest mortality percentage of 96.2% and a 106 conidia/ml density. The percentage of brown ladybug (Riptortus linearis) eggs that did not hatch after being infected with L. lecanii reached 80%. While the eggs that hatched formed first instar nymphs but could not develop into second instar nymphs because they failed to molt and die (Prayogo & Bayu, 2020). The denser the conidia formed, the faster the fungus infects and kills A.
glycines (Martin, Li, Ma, Feng, & Lu, 2021).
In using entomopathogenic fungi as biocontrol agents, it is necessary to know how to produce conidia in large numbers and in a short time. So, it is required to use alternative media to obtain conidia with high density and good viability.
Good quality conidia will be accepted by getting the right alternative media for mass propagation of entomopathogenic fungi. Entomopathogenic fungi are now in many countries used as mycoinsecticides.
Efficient and economical mass production needs to be a significant concern so that the control can suppress pest populations effectively in the field (Jaronski, 2023). Propagation of fungi with the best substrates can increase the efficacy of fungi against pests or plant diseases. Tantawizal, Inayati,
& Prayogo (2015) state that the increase in the effectiveness of entomopathogenic fungi against target organisms can be found in media that boost the high level of spore germination and high conidia density. In addition, insect stage, time application, the method of application, and the frequency of application to the target pests are expected to be considered. Research on the entomopathogenic fungus L. lecanii using various alternative media with starch-rich substrates has the potential to be developed to determine the fungus’s growth and its effectiveness on C. formicarius mortality. This study aims to obtain the best alternative media for the propagation of L. lecanii that could accelerate colony growth, produce the most biomass, and have the highest virulence against the tuber borer C. formicarius.
MATERIALS AND METHODS
The study was carried out from February – September 2019 and was conducted in 2 (two) stages. Stage one was the alternative media selection test, and stage two was the best alternative media concentration test on C. formicarius.
Phase I. Alternative Media Test
The research method used in the first stage was the Experimental Method with a Completely Randomized Design (CRD) pattern. Alternative media test with 4 treatments repeated 5 times so that 20 experimental units were obtained. The following 4 types of alternative media treatments for the propagation of L. lecanii fungi: A= Potato Dextrose Agar (PDA) (control), B= Maize, C= Rice, D= Bran.
Preparation of Alternative Media
Maize and rice washed and steamed for 25 minutes, then dried. Dried maize and rice were put in plastic bags of 30 g each/petri dish and then sterilized in an autoclave. Then the Inoculum of L.
lecanii was inoculated to sterilize bran, maize, and rice. Inoculation was carried out in laminar airflow (LAF) to avoid contamination. The culture will be incubated for 21 days at 20-23oC. As check control, a PDA medium was used.
Colony Diameter of L. lecanii
Colony diameter measurements were carried out every day until the media were 21-d-old (days old). Then, the diameter was measured using a ruler by taking 4 points (Fig. 1) and calculating the average diameter (Lestari & Jajuli, 2017).
Calculation of Conidia Density and Germination Rate After the fungus was 21 and 42-d-old, conidia density and germination were observed using a haemocytometer. First, a conidia suspension was taken as much as 0.2 ml using a type pipette at a dilution of 10-1. Next, a conidia suspension was slowly dropped on the haemocytometer canal. Finally, conidia density contained in the haemocytometer was calculated after 10 hours- incubation, with a magnification of 400x. The measurement was taken twice in each observed field, followed by calculating the germination percentage.
Phase II. Alternative L. lecanii Concentration Test The research method used in the second stage was the Experimental Method with a Completely Randomized Design (CRD) pattern.
Testing with 5 treatments repeated 5 times to get 25 units of the experiment. Each unit of the experiment contained 10 test insects. The concentration test: C+ (control +) = synthetic insecticide; C- (control -) = Distilled water; L.
lecanii = 107 conidia / ml; L. lecanii = 108 conidia / ml; L. lecanii = 109 conidia / ml.
Fig. 1. Colony diameter sampling of entomopathogenic fungi
Table 1. The average diameter of L. lecanii colonies in several alternative growing media
Type of Media Colony diameter (cm)
d-old3 6
d-old 9
d-old 12
d-old 15
d-old 18
d-old 21
d-old
PDA 3.80a 7.18a 9.00a 9.00a 9.00a 9.00a 9.00a
Maize 2.40b 5.50b 7.26b 7.89b 8.27a 8.60a 8.83a
Rice 1.60c 3.82c 5.85c 7.52b 8.31a 8.70a 8.76a
Barn 1.70c 3.09d 3.87d 4.54c 5.13b 5.62b 5.81b
Remarks: The number followed by the same letter in the same column shows not significant according to LSD 5% after transformation √ (Log x)
Data Analysis
Data from observations of alternative media selection tests and L. lecanii concentration tests were statistically analyzed using Analysis of variance (ANOVA) to determine whether they were significantly different. If the F test results differ, proceed with the Least Significant Difference test (LSD) at a significant level of α = 5% (Gomez &
Gomez, 1995). In addition, the LC50 mortality test on C. formicairus was analyzed using probit analysis with the PoloPlus version 1.0 program.
RESULTS AND DISCUSSION Colony Diameter of L. lecanii
Grains, rice husks, vegetable waste, rice and wheat washing water, rice soaking water, sawdust, and liquid media such as coconut water are various agricultural products and by-products that are often used for the mass production of entomopathogenic fungi (Sahayaraj & Namasivayam, 2008). In this study, we looked at multiple media types for the propagation of L. lecanii. The average colony diameter growth on PDA media is higher than on maize, rice, and bran media. Aini & Rahayu (2015) stated that the longer incubation time will lead to a greater diameter of the fungus colonies. The highest growth was achieved at 9 days, with an average of 9.00 cm on PDA media (Table 1). Therefore, the colony in PDA media can be halted, which may be the diameter of L. lecanii fungus can be more comprehensive if it is not grown on a 9 cm petri dish. In addition, this case also suspected L. lecanii fungus had reached the stationary phase. PDA media is a commonly used medium for culturing microorganisms, not only bacterial cultures but also fungal cultures. Based on this study’s results, mycelia’s growth in PDA media was wider than in other media. In addition, PDA media contained simpler nutrients than maize, bran, and rice culture media (Sinaga, 2015).
Further tests show that the average diameter colony in maize (8.27; 8.60; 8.83 cm) and rice (8.31; 8.70; 8.76 cm) media show no significant differences at the age of 15-d-old to 21-d-old. (Table 1). It was to be proven that the rice and maize media
could be used as the alternative media to propagate the entomopathogenic fungi L. lecanii. However, there were different results on the type of bran media, which showed an increased colony but did not significantly differ. This colony’s slow growth is likely because the bran media contains low or less suitable nutrients during L. lecanii incubation, so that the fungus is difficult to utilize the nutrients or absorb them during the growth phase. The results are in line with Astuti, Sudarsono, & Prabowo (2005) wheat bran medium did not produce the spread of mycelium fungus Spicaria sp., resulting in uneven distribution.
Colony Growth Rate of L. lecanii
There is uniformity in the time of the initiation start of L. lecanii growth in each media. The optimal growth rate of L. lecanii fungi was seen in alternative media such as maize and rice. Soetopo & Indrayani (2007) suggest that the optimal development of B.
bassiana fungi is faster achieved in rice media than in maize media. The growth characteristics of L.
lecanii mycelium in rice media are more compact throughout all rice seeds, whereas mycelium in maize media only grows on the surface (Fig. 2).
Taurisia, Proborini, & Nuhantoro (2015) states that the composition of each media is different, so it causes differences in the growth of fungi. The media used to grow entomopathogenic fungi greatly determines the rate of colony growth and conidia production during development.
The regression analysis in Fig. 3 represents the relationship between colony growth rate and incubation time. This research performed a regression analysis based on observational data on colony growth rates up to the age of 21-d-old. R2 values in all treatments showed a range of 0.79 - 0.93; this was considered good, 79 - 93% reduced growth rate of L. lecanii was influenced by the length of incubation time, while other factors influenced the rest. All P-values showed a number <0.05, which means a significant effect; the incubation time of entomopathogenic fungi affects the growth rate of L. lecanii colonies. Thus, it can be concluded that the longer the incubation time, the growth rate of the fungus colonies will decrease.
Fig. 2. Appearance of L. lecanii colonies on Maize, Bran, PDA and Rice media on (a) 3-d-old (b) 12-d-old and (c) 21-d-old
Fig. 3. Regression of growth rates of L. lecanii colonies in different media
Conidia Density and Germination Rate of L.
lecanii
LSD further test results at 5% indicate that alternative media types significantly affect conidia density. Maize media gives conidia density values that are not substantially different from PDA media but are pretty different from rice and bran media (Table 2). Media with the right type and composition significantly affect the growth of fungi. Sanjaya, Tobing, & Lisnawati (2018) argue that the kind of media for fungal growth largely determines the rate of colony growth and the number of conidia produced during development. The number of conidia will assess the effectiveness of entomopathogenic fungi in controlling host insects.
The data shows that the highest conidia density was obtained in the maize media (4.2x10⁸), while the conidia density value in rice media (2.0x10⁷) does not give optimal results (Table 2). It is suspected that the L. lecanii fungi used more nutrients to form hyphae during incubation. It is because rice media
generally contains high carbohydrates compared to protein. High carbohydrates, especially sugar, help fungal metabolism in hyphal growth. Rice contains 85% - 90% starch, pentose ranges from 2.0-2.5%
and sugar 0.6-1.4% (Smith & Dilday, 2002). The statement above is confirmed by Sanjaya, Tobing, &
Lisnawati (2018) that the substrate which contains high carbohydrates and a little protein will stimulate the fungus to form hyphae and produce less toxin.
Conidia are produced by conversion of hyphal elements or borne on sporogenous cells on or within specialized structures termed conidiophores and participate in the dispersal of the fungus.
The media will support the growth of the fungi, the suitable media will produce high quantities of conidia. Several factors made the difference conidia produced by using different media: nutrient content of media, the humidity, contaminant, the amount of media used, incubation time of entomopathogenic fungi, etc (Afifah, Desriana, Kurniati, & Maryana, 2020)
Fig. 4. The shape of conidia L. lecanii at 40 x 10 times magnification
Table 2. Average density and Germination Rate of L. lecanii conidia in several alternative growing media Type of Media Conidia density (spore/ml) Germination Rate (%)
PDA 1.3x10⁸ a 45.23 b
Maize 4.2x10⁸a 74.31 a
Rice 2.0x10⁷b 45.74 b
Bran 2.1x10⁷b 28.67 c
Remarks: The average value denoted by the same letter in the same column shows no significant difference according to LSD 5% after transformation √ (Log x)
The results are in line with Afifah & Saputro (2020) research on maize media (1.06 x 10⁸ conidia/
ml). Conidia density was not significantly different from PDA media (1.39 x 10⁸ conidia/ml). However, this is different from Mutmainnah (2015), who states that maize media has the lowest conidia density compared to tofu pulp, rice, and bran media, which is 36.33 x 106 conidia/ml. Meanwhile according Afifah & Saputro (2020) stated that B. bassiana conidia density on maize media (1.06 x 108 conidia/
ml) was significantly different from conidia density on rice media, i.e., 4.49x107 conidia/ml). Sanjaya, Tobing, & Lisnawati (2018) also state that there are differences in the number of conidia produced on each substrate, depending on the nutrient content contained in the medium. In addition, some alternative media have different compositions that
show further growth and virulence. The following is a histologic form of L. lecanii conidia (Fig. 4).
Germination is the ability of conidia in the early stages of fungal growth before being applied to host insects (Prayogo & Bayu, 2020). Sprouts also indicate the ability of conidia that can grow and develop if environmental conditions support it (Afifah, Desriana, Kurniati, & Maryana, 2020).
Maize media showed the highest germination rate of 74.31%, while the germination rate on PDA and Rice showed no significant difference. The lowest germination was found in Bran media at 28.67%
(Table 2). (Kassa, 2003) states that the effective germination of entomopathogenic fungi to infect a host is 80%, but the study results do not align with that statement. The percentage of L. lecanii germination after 10 hours of incubation in maize media was quite high at 74.31% (Table 2).
Fig. 5. L. lecanii tube sprouts at 10 hours after incubation (a) sprout tube emergence and (b) sprout tubes that develop into hyphae
Table 3. The average weight of L. lecanii media before and after incubation in several alternative growing media
Types of media Weight before incubation (g) Weight after incubation (g) Weight difference (g)
PDA 120.14 118.85 1.89 b
Maize 101.34 99.99 1.35 c
Rice 99.89 98.89 1.00 c
Barn 138.89 137.56 1.33 a
Remarks: The average value is denoted by the same letter in the same column and shows no significant difference according to LSD 5%
The low germination capacity of L. lecanii isolates is thought to be caused by isolates cultured more than once, thereby reducing the germination percentage. The germination will most likely affect the infectivity of fungi against C. formicarius mortality. This is in line with Herlinda, Utama, Pujiastuti, & Suwandi (2006) who state that the propagation of fungi in vitro has constraints such as decreased quality of spore density and viability and affects virulence. Repeated subcultures and culture media, if not enriched with a medium derived from infected host insects or artificial insect starch, will result in less virulence. Herlinda, Utama, Pujiastuti, & Suwandi (2006) also showed that the B. bassiana subculture cultured only on Saborroud Dextrose Broth (SDB) media continued to decrease conidial density and viability compared to SDB media enriched with cricket flour.
Spore viability is influenced by the media’s level of spore density and food nutrition. The source of nutrients needed for spore germination is protein, but a high amount of protein does not guarantee the ability of conidia to germinate. So there is a need for compatibility between carbohydrates, proteins, starches, and glucose, which also determines the viability of conidia to germinate. Verlag, Jentzsch- cuvillier, & Tefera (2006) also added that the germination rate would reach 95 to 100% if enough protein were available. Here is a picture of L. lecanii conidia germinating after 10 hours of incubation (Fig.
5). Other research needs to be done to improve the performance of entomopathogenic fungi so that they are more adaptive to challenging environmental conditions, formulations, longer shelf life, ease of application, pathogen virulence, and spectrum of action (Maina, Galadima, Gambo, & Zakaria, 2018).
Media Weight of L. lecanii
LSD further test results at the 5% level showed that alternative media types significantly affected the weight of alternative media before and after L. lecanii inoculation. The smallest media weight difference was achieved in the rice media of 1.00 g which were not significantly different from the maize media (1.35 g) and substantially different from the PDA media (1.89 g) and bran media (1.33 g) (Table 3).
The highest decrease in media weight was also obtained in maize media which was thought to be influenced by the corresponding carbohydrate and protein content during the growth phase of L. lecanii. Carbohydrates are the primary source of energy in the form of starch and sugar (mono
and disaccharides), these sugars are food for fungi. Safavi et al. (2007) stated that the nutritional requirements for each fungal species differ, but the main energy sources needed are carbohydrates and proteins, which affect viability, virulence, and pathogenicity.
C. formicarius Mortality with L. lecanii Entomopathogenic Fungi Application
The subject research on entomopathogenic fungi as crucial biological control agents has been intensively carried out in various parts of the world for the past 100 years. Effects on the host can occur at the epizootic or enzootic level (Mora, Castilho,
& Fraga, 2017). L. lecanii, a mitosporic fungus, is commonly used worldwide and capable of infecting Lepidoptera, Hemiptera, Coleoptera, and Diptera (Altinok, Altinok, & Koca, 2019).
This mortality test on C. formicarius using L.
lecanii used the propagation from maize media with the highest conidia. The LSD further test at the 5%
level showed that the mortality due to insecticide application was 100% significantly different from the application of L. lecanii 10⁹ conidia/ml suspension, which was 74%. However, the application of L.
lecanii suspension with a density of 10⁸ conidia/
ml was not significantly different from 10⁷ conidia/
ml with a mortality of 30% and 26% (Table 4). The results of C. formicarius were reported by Ahdiaty (2013) that mortality due to B. bassiana application reached 45% at a conidia density of 10⁷ conidia/ml at 36 days of culture. Prayogo & Bayu (2019) also reported that application of B. bassiana 10⁷ conidia/
ml suspension on the soil surface at 2, 4, 6, 8 10 and 12 weeks after planting can suppress the C.
formicarius population on tubers and reduce tuber damage by 48%.
Table 4. The average percentage of total mortality of C. formicarius by treating various concentrations of L. lecanii
Conidia Density
(spore / ml) Mortality
(%) Synthetic insecticide (C+) 100 a
Distilled water (C-) 8 d
10⁷ 26 c
10⁸ 30 c
10⁹ 74 b
Remarks: The average value denoted by the same letter in the same column shows no significant difference according to LSD 5% after transformation Arcsin
Fig. 6. C. formicarius (F.) that died from L. lecanii suspension application (a) the insect covered with mycelium of L. lecanii, (b) Zoom in the mycelium of L. lecanii
Fig. 7. Relationship between conidia density and mortality of C. formicarius
In general, the application of synthetic insecticides provides immediate results on the mortality of the target pests compared to the application using biological agents. The effect of the chemical content of the active ingredient Permethrin is thought to cause a knockdown effect on the tested insects quickly. In contrast to C. formicarius, shortly after the application of L. lecanii conidia suspension, it still did not show a significant decrease in insect mortality. The higher the concentration applied, the faster the fungus will infect and kill C. formicarius.
Sutra, Salbiah, & Laoh (2013) state that there is an increase and decrease in mortality that varies with each treatment because the fungus needs to adjust to the host insect’s body to develop and get nutrition so that daily mortality can decrease from the previous day. Therefore, the lower the concentration, it takes a long time to kill the test insects because the toxin produced is also common. In addition, other factors such as the wax coating on insect cuticles directly affect the germination and virulence of fungi (Arusyid, Saraswati, & Hestiningsih, 2016).
According to Trizelia, Armon, & Jailani (2015), the mechanism of fungal infection through the cuticle begins when the attachment and germination of conidia to the cuticle. Afterward, the mechanism occurs enzymatically and chemically, which can penetrate the cuticle and cause an increase in blood pH, blood clots, and blood circulation stops so that the test insects would die. Their way of working in infecting host insects, among others, starts from the attachment of spores to the insect cuticle, germination, cuticle penetration, and dispersal within the insect (Mora, Castilho, & Fraga, 2017).
The results showed that C. formicarius, which failed to be infected with the fungus L. lecanii showed morphological features of a stiff body, hardened, and the longer the body surface would shrink. In addition, the mycelia would come out through the body segments of the imago (Fig. 6). The fungus appeared on the 6th day after the application.
The regression graph shows the relationship between C. formicarius mortality and L. lecanii conidia density. The regression line on conidia density shows a value (R2 = 0.812), the increase in mortality of C. formicarius by 82% was influenced by the density of L. lecanii conidia, while other factors influenced the rest (Fig. 7). P-values showed a number <0.05, which means a significant effect;
the conidial density of entomopathogenic fungi L.
lecanii used affects the mortality of C. formicarius.
Thus, the entomopathogenic fungus L. lecanii with a conidia density of 109 spore/ml can be used as an alternative to biological control because its effectiveness will be faster in killing target pests.
Based on the probit test LC50 obtained 2,6 x 107 spore/ml range 4.6 x 106 < LC50 < 3.13 x 10⁹. It can be explained that the conidia density of 2.6 x 10⁷ conidia/ml of L. lecanii fungi are capable of causing 50% mortality of C. formicarius adult. According to Dodia, Patel, I., & Patel, G. (2010), lethal concentration is a value that shows the amount of poison per unit weight that can kill the insects used in the experiment. The study’s results contradicted with the research conducted by Ratissa (2011) that the LC50 value was achieved with the concentration of 1.1 x 109 conidia/ml, which tested B. bassiana against C. formicarius. Faishol (2011) also tested Metarhizium brunneum against C. formicarius with an LC50 value of 4.2 x 106 conidia/ml on the 10th day after treatment. Meanwhile, Maharani (2016) obtained an LC50 value of 4.64 x 106 conidia/ml, which tested V. lecanii against Helopeltis antonii.
Arusyid, Saraswati, & Hestiningsih (2016) stated that the difference in the LC50 value depends on the viability of the conidia and the research conditions in each test. Butt, Jackson, & Magan (2001) emphasized that the ability of pathogens to infect target insects is determined by four factors:
pathogens, host insects, environment, and time.
The type of pathogen, the amount of dose and concentration, and the method of application will affect the mortality of insects. In terms of the host, the physiological and morphological factors of each test insect affect the susceptibility of insects.
CONCLUSION
The alternative media from maize have been proven as the best media to propagate the L. lecanii. The maize media achieved the highest conidial spore (4.2 x 10⁸ conidia/ml), which is not significantly different from PDA (1.3 x 10⁸ conidia/
ml), and the highest germination (74.31%) is also achieved in maize media. There is a significant effect of conidia suspension application on the mortality of C. formicarius, which is 74% at the conidia density of 109 conidia/ml. At the same time, the LC50 value is 2.6 x 10⁷ conidia/ml. It is necessary to test other media for mass propagation of L. lecanii to obtain an entomopathogenic fungus with high sporulation and infectivity against pests.
ACKNOWLEDGEMENT
The author would like to thank the Institute for Research & Community Service (Lembaga Penelitian & Pengabdian Kepada Masyarakat [LPPM]) Universitas Singaperbangsa Karawang for funding support during research activities through the LPPM Unsika Grant scheme HIPKA UNSIKA 2021.
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