Tribolium castaneum is a major pest in wheat flour but can attack other processed foodstuffs. Control of these pests can be done in various ways, one of them by biological control using entomopathogenic fungus Beauveria bassiana. This study aims to determine the infectivity of B. bassiana entomopathogenic fungus against T. castaneum populations in the laboratory. The experiment was completed with random preparations, each preparation was repeated three times. Imago T. castaneum was sprayed with a conidia suspension of B. bassiana at a density of 106 and 107 conidia/mL on three different storage media namely PDA, rice, and corn media. Mortality was calculated based on the percentage of imago that died of B. bassiana fungi. One week observation showed the mortality of T. castaneum after the application of B. bassiana fungus was 55.33% with 107 conidia/mL in corn media. The highest spore density was found in rice media with an average of 10.53 x 106 conidia/mL. Meanwhile, the value of the results showed LC50 of 2.11 x 107 conidia/mL and the fastest LT50 in corn media was 5.31 days.
Key Words : Beauveria bassiana, Biological control, Entomopathogenic, Tribolium castaneum
Introduction
The main problem with storage warehouses is attack by warehouse pests. Loss of grain products as result of werehouse pest attack reaches 5 to 10% (Syafique et al., 2006). Warehouse pests are included in opportunistic insects, which can quickly use available resources, so they can grow and develop their population quickly, but when resources are no longer supportive these pests will turn to look for new resources or die (Harahap, 2012). One of the most found in food storage in warehouses is Tribolium castaneum. T. castaneum or red flour beetle (Coleoptera: Tenebrionidae) is a secondary pest that is cosmopolitan and includes an external feeder. Until now, pest control of T. castaneum is still very dependent on synthetic pesticides, because this method is easy in application and quickly reduces the pest
population. Also, there have not been found other alternatives that are quite effective. The application of insecticides is usually intensive once a week or 2-3 times a week (Trizelia, 2005). One way to control the T. castaneum is environmente friendly natural enemies consisting of entomopathogens. Control of insect pests with pathogens is a process of utilizing pathogens both already in the local ecosystem and by entering pathogens into the ecosystem from the outside through inoculation and inundation techniques. One type of insect pathogen that is widely found in nature and can be used to control insects is the fungus; Beauveria bassiana. The utilization of B. bassiana for pest control of T. castaneum has not been widely supported. The results of preliminary studies that have been carried out indicate that B. bassiana isolated from corn as animal feed can infect Imago and larvae from T. castaneum (Sepe et al., 2019). In other insect pests such as Crocidolomia pavonana (Lepidoptera: Pyralidae), infection of B. bassiana besides causing the death of imago and larvae, also affect thirs activity of feeding and oviposition (Trizelia 2005; Noma and Strickler (2000). Efforts to increase B. Bassiana success as agens of biocontrol T. castaneum in the field require high virulence strains, quick killing pest, and able to survive in the warehouse. The three factors refer to a reference to a controlling agent as well.
Materials and Methods
Preparation of B. bassiana: Fungi isolated from the collection of Plant Diseases Laboratory, Department of Plant Protection, Hasanuddin University, Indonesia. In order to increase its virulence, B. bassiana fungus were reintroduced to insect bodies to complete the infective phase. The host insect used was T. castaneum. Suspension of B. bassiana isolate was sprayed on T. castaneum and allowed to grow mycelium. The mycelia are then isolated on PDA media. Purified isolates on PDA media were propagated on rice and corn media for use in this study.
Testing the growth and production response of B. bassiana conidia on the media
Isolate on Rice Media: The rice used as an alternative medium was washed clean. The rice was next poured into boiling water and allowed to stand for an hour. The filtered rice was then dried on a sterile gauze-coated platic tray (50 grams) of rice were put into HDPE (High Density Polyethylene) bag to sterilize using autoclave for 35 minutes at 121 °C. After cold rice B. bassiana fungus suspension was induced on rice media in laminar airflow by spraying. Then The rice media was stored in the laboratory at room temperature for 21 days for optimal (Sutadji, 2016).
Inoculation of Isolates on Corn Media: Corn media was white corn that has been cleaned and soaked for 12 hours. The corn (50 grams) of soaked corn was washed again and put in an HDPE (High Density Polyethylene) bag and then sterilized with an autoclave at 121 °C for 1 hour. The next step was according to the rice media treatment method (Sutadji, 2016).
Application of B. bassiana conidia suspension to T. Castaneum : The application was performed by spraying a suspension of B. bassiana on the body of T. castaneum as much as 3ml/15 imago. Then, T. castaneum was fed 5 grams/unit of corn. Each container contains 15 imago.
The percentage mortality of T. castaneum: Warehouse pest mortality was seen every 24 hours with the total number of warehouse pests that die in each unit for 7 days of observation, then calculated with the formula on contributions:
Percent of mortality (%) = Total T. castaneum mortality
X 100%
Total T. castaneum tested
Statistic analysis: Statistical analysis was Analysis of Variance and the experimental design used was Completely Randomized Design, followed by Duncan's test of the parameters analyzed at a 5% level. Each treatment was carried out in 3 replications. Statistical analysis was performed using the SPSS 16.0 program.
Results and Discussion
The density of B. bassiana spores in various culture media: The results showed that the media opposed the number of conidia formed (Table 1). The highest number of B. bassiana conidia was obtained in rice media, which was 12.53 x 106 per average media. However, the number of conidia in rice media did not correlate with virulence rate when viewed from the number of T. castaneum that died below 50% (Table 2).
Table 1. The density of B. bassiana spores on various culture media/100 gram media
Replicate The density of B. bassiana spores on various culture media/100 gram media
PDA Rice Corn
1 4,.2 x 10⁶ 13.5 x 10⁶ 9.35 x 10⁶
2 5.5 x 10⁶ 14.75 x 10⁶ 14.6 x 10⁶
3 5.3 x 10⁶ 9.35 x 10⁶ 6.1 x 10⁶
Average 5 x 10⁶ a 12.53 x 10⁶ b 10.02 x 10⁶ ab
Numbers followed by the same letter (a) in the same column mean significantly different comparison variety-based Duncan's test, α = 0,05.
Data analysis showed that there were significant differences in conidial density in three media treatments namely rice media (12.53 x 106) statistically different from PDA media (5 x 106). The variation in the germination of each isolate was considered to have differences between each isolate. According to Prasad and Pal (2014), growth and conidia production in entomopathogenic fungi was highly dependent on media composition. The amount of conidia produced in alternative media was quite large and capable of tolerance to temperature, but the level of virulence decreases compared to natural media. Updates relating to the presence or absence of nutrient concentrations outside the conidia relating to the surface characteristics of the host species of the isolate were obtained. Pathogenic isolates in homopterans will germinate better in media that require emissions while pathogenic isolates in coleopterans will germinate in conditions of glucose deficiency (Trizelia, 2005).
Mortality of T. Castaneum: The results of the pathogenicity test of B. bassiana against T. castaneum showed that the total density of conidia used in this study affected the mortality rate of T. castaneum. Statistical analysis showed that there was a significant difference between T. castaneum applying B. bassiana conidia suspension based on differences in media treatment. About 55.56% T. castaneum died on corn media with conidia density 106 differing significantly from other conidia densities in the treatment of PDAs and rice media (Table 2).
Table 2. The Percentage mortality of T. castaneum (%) against B. bassiana for 7 days of monitoring (15 insects/unit)
Treatment Media Conidia density Percentage of mortality (%)
Control 2.22a
PDA 10⁶ 33.33ab
10⁷ 44.44bc
Rice 10⁶ 46.67 bc
10⁷ 44.44bc
Corn 10⁶ 55.56 c
10⁷ 53.33 bc
Numbers followed by the same letter (a) in the same column mean significantly different comparison variety-based Duncan's test, α = 0,05.
The results of the analysis of variance in the warehouse pest population showed the application of B. bassiana in corn media with 106 conidia/ml spore densities showed a real difference in Duncan's test of 5% level, (Table 2). The application of B. bassiana in corn media with a density of 106 conidia/ml results from the death of warehouse pest participation around 55.56%. This relates to the management of B. bassiana having the ability to control warehouse pest relationships. Tanada & Rich (1993) stated that virulent lethal isolates kill insects in a short time and non-virulent isolates require a long time to cause chronic infection. According to Scholte et al. (2004), the process of entomopathogenic fungus attack causes the host to die as follows: contact conidia on the integument is followed then stored and germinated and carried out using a sprout tube (appressorium), after entering into the haemocoel, the fungus forms a blastospora that circulates in the hemolymph and forms secondary hyphae to attack other tissues such as the nervous system, trachea, and digestive tract. The occurrence of nutritional deficiencies, toxins produced by fungi, and changes in damage to the body will cause paralysis and death in insects. According to research results Fuquet et al. (2004), Valendra et al. (2011), and Xu et al. (2008), the toxicity of the B. bassiana fungus are determined by the ability of the toxin contained in the movement of insects. The magnitude of trial mortality is indeed a benchmark and fungus isolates that can cause death with mortality reaching 80% are classified as very toxic. Meanwhile, Chong-Rodriguez et al. (2011) discuss the toxicity of B. bassiana fungus closely related to the type of growing media, growing media containing protein and carbohydrates containing sweet corn produce blastopora which is more toxic than B. bassiana fungus grown on media containing only protein (peptone).
The rate mortality of T. Castaneum: Virulence of B. bassiana is part of the T. castaneum imago that dies up to seven days after application. The results showed that the mortality of T. castaneum had begun to be seen 24 hours after application, but the highest was seen on PDA media with a spore density of 107 which was 11.11% on the 1st day seen. Whereas on the 7th-days view of T. castaneum mortality was highest seen in maize media at 55.56% at conidia density 106 (Figure 1).
Figure 1. The rate mortality of T. castaneum for seven days of observation
The results of this study indicate that the virulence of isolates varies greatly depending on isolates, host insects, and local ecological conditions. According to Utami et al (2014), the cause of death due to B. bassiana as a bioinsecticide takes several days after it is applied. Bioinsecticides and target insects have specific biological relationships that are highly protected by the nature of the inoculum that enters the larvae 's body which contains bioinsecticides (Pracaya, 2008).
Konnstantopoulou and Mazomenos (2004), Wang et al. (2005), Prasad and Veerwal (2010), Prayogo (2013) reported that the fungus B. bassiana produces toxic compounds that are toxic to insect nerves that cause nervous system disturbance and death in insects. Reports from several researchers indicate that entomopathogenic fungi belonging to the Hyphomycetes group can produce toxin compounds in the form of brassinolide, bassiacridin, oosperin, and cyclosporine which can kill various insect stages (Tikhonov et al., 2002; Mukhtar & Pervaz, 2003; Vega et al., 2008). Judging from the various advantages of these fungi, B. bassiana has the opportunity as an alternative for biological control agents T. castaneum and as an alternative to chemical insecticides.
The determination of LD50 and LC50:
The results of the B. bassiana fungus probit analysis conducted at 7 days after application, obtained LC50 of 2.11 x 107 conidia/mL. That is the density of B. bassiana conidia needed to cause 50% mortality of T.castaneum insects is 2.11 x 107 conidia/mL (Table 3).
Table 3 LC50 and LT50 values of B. bassiana fungi against T. castaneum.
Probit Analysis Types of media
PDA Rice Corn PDA Rice Corn
95% confidence limits for days 95% confidence limits for (days)a
LC50 2.11 x 107 0.324 x 107
LT50 106 10.18 6.05 5.31 1.008 0.782 0.725
LT50 107 7.86 6.39 5.44 0.896 0.806 0.736
LT50 value of B. bassiana obtained from three types of media at a density of 106 conidia/mL shows that the corn medium has a faster lethal LT50 of 5.31 days. Whereas on rice and PDA media they were 6.05 and 10.18 days, respectively. When compared between the density of 106 and 107 conidia/mL shows that the density of 106 conidia/mL is faster to kill T. castaneum imago both on corn and rice media. That is, at a density of 106 conidia/mL 50% of the T.casteneum imago population died within 5.31 days due to being infected by B. bassiana. Anshori (2017) reports that the probit analysis of LT50 B. bassiana values at a density of 109 conidia/mL is 4.67. That is, at a density of 109 conidia/mL 50% of the test risk population within 4.67 days of being infected by B. bassiana.
Summary
B. bassiana fungal spore density on rice media was 12.53 x 106 culture media/100 grams of media. Application of B. bassiana fungal suspension resulted in a mortality of T. castaneum at 53.56%. The mortality rate differed statistically significantly. The amount of conidia/mL needed to deactivate 50 % of T. castaneum was relatively low at 2.11 x 107 conida/mL. Whereas the time it takes about 5.44 days