Effects of the entomopathogen concentration of Beauveria Bassiana [Bals] Vuil for biological control of Helicoverpa armigera Hubner [Lepidoptera : Noctuidae]

Beauveria bassiana is an entomopathogenic fungus found in the rhizosphere, known to affect the mortality of H. Armigera. The research was conducted at the Laboratory of Biological Control, Department of Plant Protection, Faculty of Agriculture, Andalas University, from January 2019 to March 2019. The aim of this study, therefore, is to obtain an effective concentration with the optimal characteristics of controlling H. armigera, utilizing a completely randomized design, with 4 treatments and 5 replications. In addition, the treatment required the application of B. bassiana suspension, at a concentration of [A] 5×107 conidia/ml, [B] 1×108 conidia/ml, [C] 5×108 conidia/ml and [D] control [distilled water]. The results showed the treatment with the highest concentration [5×108 conidia/ml] possessed the ability to increase the mortality rate by 68% in the larvae of H. armigera, at an LT50 of 5.82 days. Hence, a higher concentration affects the lifespan, and nature of pupae, and adult pests formed.


Introduction
The corncob borer, Helicoverpa armigera [Lepidoptera: Noctuidae], is a major pest known to attack corn plants, which normally occurs at the age of 45-65 days, along with the appearance of cob hairs, leading to a reduction in the quantity and quality of yield. The Directorate-General for Food Crops, Ministry of Agriculture established that potential loss in yields, due to attack by caterpillars reduced production by 40%, and similar events have been identified in plantations within Indonesia [1].
Efforts have been made by farmers to control H. armigera by using chemicals. However, such control is identified as unfriendly to the environment. Moreover, synthetic varieties have been speculated to initiate the occurrence of numerous negative impacts, encompassing environmental pollution by chemical residues, pest resistance, and blasting. Hence, the need for alternative control measures, including the conduction of biological approaches [2].
The biological control method requires entomopathogenic fungus, e.g. Beauveria bassiana fungus [3], of the Lepidoptera order. These possess the ability to infect insect pests, by penetrating the host cuticle and causing diseases, especially where there is direct contact between the fungal conidia.
B. bassiana fungus is a biological agent, which has been proven to possess the capacity to control various types of plant pests. Furthermore, it does not cause any form of phytotoxins [poisoning] in plants, as well as pollution [4], and the broad range of host affected has prompted its wide adoption in pest insect control [5]. This fungus has great potential as a biological pest control agent, and it is also an important component in an integrated system [6].

2.4.
Rearing of H. armigera H. armigera larvae were obtained from the cobs and corn silk, in the corn planting areas of Java Gadut, Limau Manis Village office, Padang City, then placed into plastic cups, before the laboratory procedure. Furthermore, each larva was provided with a piece of corn [1.5 cm long] as feed, which was replaced daily. At the pupae stage, they were separated into plastic containers containing sawdust. Besides, the imago that emerged from the pupa was moved into a plastic maintenance box [20 cm x 14 cm x 10 cm], with its top lid covered with gauze. Moreover, feeding at this stage requires the provision of 10% honey, infused into cotton and placed on the top of the box lid. The environmental condition was maintained until the egg production phase. The H. armigera eggs were transferred into a plastic container [height 19 cm x diameter 16 cm] before the hatching process into larvae, reserved up to the instar 3 [as a test subject], where they were fed with corn leaf. Furthermore, the categories to be treated were then placed in plastic cups, with the placement of 1 in each containment.

The making of suspension of entomopathogenic B. Bassiana fungi
Pure culture of B. bassiana placed on Saubouraud Dextrosa Agar Yeast [SDAY] media incubated for ± 21 days was provided, then 10 ml of distilled water and 2 drops of Tween 80 were added. This was performed to release the conidia fungus from the media surface, with the assistance of a small brush. Furthermore, the spores of the fungus present in the suspension were sieved, using filter paper and placed into an Erlenmeyer; therefore, the homogenization process was initiated, employing a vortex for 20-30 seconds. Besides, a serial dilution of 10 -1 and 10 -3 was conducted, followed by calculating the conidia density, using a Haemocytometer, leading to the preparation of a suspension with a density of 5x10 7 , 1x10 8 , 5x10 8 conidia/ml.

Application of B. bassiana entomopathogenic fungi in H. armigera larvae
B. bassiana entomopathogenic fungus, suspended at a density of 5x10 7 ,1x10 8 ,5x10 8 conidia/ml, was applied to H. armigera stadia larvae instar 3. This was performed by direct spraying technique, which occurred 3 times [0.21 ml] for each, while the control required the use of sterile aqua dest.

Larvae maintenance after application
The treated larvae were fed with corn [1.5 cm long] up to the pupae stage, where it is then moved to the specific container containing sawdust until it becomes an imago. These were then separated and placed in a plastic maintenance container, where cotton soaked in a 10% honey solution was used as feed to promote egg production.

Larvae mortality and LT 50 value
The number of dead larvae in each treatment after applying B. bassiana were counted daily and observed for the subsequent 12 days. Therefore, the percentage of mortality was calculated using the following formula: The lethal time value for 50% [LT 50 ] B. bassiana concentrations applied to the H. armigera larvae was calculated based on the observed mortality rate, and then probit analysis was performed to determine the specific value.

Pupae Formed [%]
The number of pupae formed was counted daily and observed for complete development. Therefore, the percentage was calculated using the following formula: Information: Pp : Percentage of pupae formed

Effect of B. bassiana concentration on the mortality of H. armigera larvae
Application of B. bassiana at different concentrations was observed to have affected the mortality rate of H. armigera larvae [P = 0.00], and the 5x10 8 conidia/ml was not significantly different from 1x10 8 conidia/ml, although there was significant dissimilarity with 5x10 7 conidia/ml. Furthermore, all treatments were experimentally distinct from the controls, and every increase in the concentration of B. bassiana provided also increased the occurrence of larvae mortality, and also accelerates the time of death. Hence, the highest rate was identified at 5x10 8   The cumulative mortality rate of H. armigera larvae after applying B. bassiana suspension is seen in Figure 1, where the death was initiated on the first day of treatment, up to day 12. Furthermore, the observations also showed a positive correlation between the increase in B. bassiana concentration and the mortality rate.  H. armigera larvae infected with B. bassiana showed abnormal external symptoms, and they also tend to die eventually, characterized by the hardening of the body, and the dead larvae were covered in white fungus hyphae [ Figure 2].

Effect of larva treatment on formed pupae and imago [%]
The application of B. bassiana to larvae caused a decline in the percentage of pupae and imago formed, which was prominent at higher concentrations. Therefore, a significant difference was also observed in the results obtained for each treatment [P = 0.00], although minimal disparity was identified between 5x10 7 and 1x10 8 conidia/ml. Furthermore, there was also a marked difference between the pupae and imago formed from the different concentrations of B. bassiana conidia in contrast with the control [ Table 2].   Furthermore, the application of B. bassiana was observed to initiate the formation of normal H. armigera pupae, although the abnormal types were also seen. Therefore, it was established that the differences in concentration administered affects the percentage of normal and abnormal [deformed] pupae formed [ Table 3].

Table 3. The percentage of pupae formed normal and abnormal [defective] after applying B. Bassiana
The numbers followed by the same lowercase letters in the same column and row not significantly different according to the LSD 5% test The perfectly formed pupae displayed normal shapes, characterized by a shiny brown coloration, and a marked movement when touched. However, the abnormally formed types do not transit when they are subjected to the same stimuli, and they tend to occur as brownish-black coloration. A covering with an overgrowth of B. bassiana hypha fungus is seen on the pupa body part as shown in Figure 3.  The application of B. bassiana caused most of the H. armigera imago formed to remain normal, although an increase in concentration reduced the percentage value obtained. Meanwhile, the number of abnormal types was not significantly different amongst treatments, except the interaction between the 1x10 8 conidia/ml concentration and the control [ Table 4]. The normal-shaped adult was observed to exhibit a perfect shape and size [ Figure 4], while the abnormal forms had imperfect development [defective], with damaged wings or legs.

Effect of treatment with B. bassiana on H. armigera adult life span
The administered B.bassiana tends to influence the longevity of adults, as the average life span of the control [no-treated larvae] was 9 days longer than those receiving treatment, which was limited to about 6.4-7.7 days [ Table 5].  Table 5 shows the lifespan during the adult phase with 1x10 8 and 5x10 8 conidia/ml treatments, which was estimated to be 6.4 days. These values are not significantly different from one another. Conversely, the lifespan of the 5x10 7 conidia/ml concentration [9 days] did not vary significantly from the control [7.7 days].
The results obtained showed a significant positive relationship between the application of B. bassiana and larva mortality, thus, an elevation in the concentration administered resulted in higher percentage death. This event is assumed to occur because of the increase in the number of B. bassiana fungus conidia sticking to the body of the larvae. Sitompul and Lazuardi [9] reported that the success of infecting fungus against insects is determined by conidia density, because direct contact with the host tends to cause infection, thus, a higher attachment percentage leads to faster infection of the target host.
Entomopathogenic B. bassiana fungi did not cause a decline in the number of pupae formed, nor did it initiate the incidence of infection. Therefore, a higher concentration tends to decrease the percentage of formation, as Herlinda et al, [3] reported that larvae treated with the B. bassiana die, while the live forms tend to develop into pupae, where some suffer damages in the form of imperfect organs [defective].
The infected pupae often die or fail to develop into adults, characterized by changes in body color to black on some surfaces, and fungal hyphae tend to appear in the affected areas. This event occurs due to the continued beauvericin toxin production process initiated by B. bassiana fungi, possessing the capability to damage the host's body tissue, and inhibit its metabolic processes, therefore, causing abnormal development. Rosmiati et al [10] also explained the increased propensity of hypha growth in the body of an insect to release toxic active compounds for this purpose.
The application of B. bassiana affects the number of adults formed, in contrast with the control. Thus, a higher concentration reduces the percentage of development. Also, the quantity present in a control group was observed to be much higher than in the treatment, as seen in the successful development of pupa into imago, as well as the normal growth pattern. Meanwhile, physical disability and death were prominent in the treatment category.
The adult formed exhibited both normal and abnormal [deformed] development properties, as seen in Figure 4. This event is assumed to occur during the growth of B. bassiana hyphae in the host body, and the production of beauvericin toxin, which leads to paralysis, subsequently damaging tissues and organs. As reported by Sodiq and Dwi [11], these fungi possess the capacity to confer damage, due to the production of chitinase, lipase and proteinase enzymes, which tend to decompose components of the insect cuticle, causing death as a result of tissue and organ impairment.
It was also established that this application shortens the life span of H. armigera adult, based on its ability to deplete the nutrients required for development, thus, disrupting development. Saleh et al. [12] stated that a high propensity for the toxin produced by entomopathogenic fungi to directly damage the main functions of a hosts' body, especially via hormone replacement, and the disruption of new skin formation processes, as a result of nutritional deficiencies. Therefore, the development does not ensure perfectly during the time where it is expected to turn into an adult, leading to a reduction in the capacity to survive longer. B. bassiana that was used previously was from the rhizosphere of corn [13]. It is thought that B.bassiana derived from corn rhizosphere can control H. armigera if it is made in the right concentration.

Conclusion
Based on the results and the discussion, it is, concluded that treatments of B. bassiana at the highest concentration [5x10 8 conidia/ml] cause mortality of about 68% in the H. armigera larvae, with LT 50 of 5.82 days. Also, a higher concentration negatively affected development based on lifespan, and the nature of pupae and adult form.