Effectiveness Trichoderma and Beauveria bassiana on Larvae of Oryctes rhinoceros On Palm Oil Plant (Elaeis Guineensis Jacq.) In Vitro

O. rhinoceros horn beetle (Coleoptera: Scarabaeidae) is the main pest attacking oil palm crops in Indonesia, especially in palm oil rejuvenation areas. The study was conducted from April to August 2016 in the laboratory of the Faculty of Agriculture, Al-Azhar University, Medan. The materials used in this research are horn beetle pest larvae (O. rhinoceros) originating from PT. Socfin Indonesia, Trichoderma sp fungi originating from the Food Crops and Horticultural Fields of Medan and the B. bassiana fungi are derived from the Plant Seed Plant Protection Center (PBPPTP), aqua pro injection, Tween 80, rice and 96% alcohol. This research uses Completely Randomized Design (RAL) Non Factorial consisting of 13 treatments. The result of mortality of O. rhinoceros larvae on 1-14 DAA observation can be seen in appendix 2-43. Based on fingerprint analysis showed that the application of Trichoderma sp and B. basianna fungi on O. rhinoceros larvae had no significant effect on observation of 1 DAA to 9 DAA, but had a very significant effect on observation of 10 DAA to 14 DAA. The results of germination of Trichoderma sp and B. basianna fungus 4 hours after incubation period can be seen in appendix 61-66. Precentages mortality of the highest larvae of O. rhinoceros to Trichoderma sp fungus with a dose of 20 gr (96.67%) with the application method spread on the larvae. The highest amount of conidial density is found in Trichoderma sp fungi with doses of 60 g x 100-1 ml of aqua pro injection of 7.25 x 106 conidia/ml.Germination level of conidia mushroom highest in Trichoderma sp fungi with dose 60 gr x 100-1 ml aqua pro injection that is as much as 91%.


INTRODUCTION
Palm oil is the most productive plant with the highest production of oil per hectare from other vegetable oil producers. Indonesia is the second largest palm oil producer in the world after Malaysia. As many as 85% more world market of palm oil is controlled by Indonesia and Malaysia [39]. One of the obstacles in the cultivation of oil palm crops is the pest attack that can cause damage to the plant to result in decreased levels of palm oil production. Pests can attack palm oil from the pre-nursery stage to the producing stage [23]. O. rhinoceros horn beetle (Coleoptera: Scarabaeidae) is the main pest attacking oil palm crops in Indonesia, especially in palm oil rejuvenation areas. These insects bore oil palm shoots which resulted in stunted growth and damage to the point of growing so deadly plants. In palm oil rejuvenation areas, horn beetle attacks can result in delays in palm oil production up to a year and dead plants may reach 25% [23]. Reducing the use of pesticides in agricultural areas requires the availability of other safe and environmentally friendly methods of control, such as by utilizing natural enemies, such as entomopathogenic fungi, predatory insects, and parasitoids [41]. Groups of fungi that infect insects are called entomopathogenic fungi. The famous entomopathogenic mushrooms are Namuraea rileyi, Metarizium anisopeliae and B. bassiana. B. bassiana (Balsamo) Vuillemin (Ascomycota: Hyphocreales) is a facultative entomopathogenic fungus with a wide range of hosts, besides this fungus has the potential to control more than 70 insect pests belonging to different orders, especially lepidoptera pests [18]. In addition, the use of antagonistic fungus is also an alternative control option because this method is considered safe for both users, consumers and the environment. An antagonist fungus that has been widely used as a biological controller is Trichoderma sp. Trichoderma sp mushroom cultures in applicative media such as bran can be given to the planting area and are bio decomposers as well as bio fungicides. Trichoderma sp also has a very effective biocontrol mechanism in suppressing the development of pathogens such as mycoparasitism, antibiosis, and competition [7]. B. bassiana fungi have been tried to control the O. rhinoceros pests in oil palm with concentration of 25g ml-1 [32]. While Trichoderma sp fungus has been tried to control P. nicotianae disease on Deli tobacco plants with concentration of 20g / L. Therefore, it is necessary to do research to find out the concentration of B. bassiana and Trichoderma sp fungi which is most effective to control horn beetle pest larvae on oil palm crop. This study aims to determine the effectiveness of B. bassiana and Trichoderma sp fungi with different doses and methods of application to control horn beetle (O. rhinoceros) pest larvae in oil palm crops.

II. METHOD Place and time of research
This research will be conducted at the Laboratory of the Faculty of Agriculture, Al-Azhar University, Medan. The study was conducted from April to August 2016.

Materials and tools
The materials used in this research are horn beetle pest larvae (O. rhinoceros) originating from PT. Socfin Indonesia, Trichoderma sp fungi originating from the Food Crops and Horticultural Fields of Medan and the B. bassiana fungi are derived from the Plant Seed Plant Protection Center (PBPPTP), aqua pro injection, Tween 80, rice and 96% alcohol. The tool used in this research is haemocytometer, microscope, glass object, glass cover, hand sprayer, test tube, jar, kassa, erlemeyer, name label, beaker glass, scales, tube, aluminum foil and stationery.   (Hanafiah, 2010) which is assumed as follows:

Research methods
Y_ij = μ + N_i + + ε_ij Where: Y_ij: The results of observation of the treatment to the fungus and j-thr μ: Effect of middle value N_i: Effect of N treatment on the mushroom stage Σ_ij: Effect of errors from the treatment of fungi and jth thrust If the treatment has significant effect, the Duncan test is 5% and 1%.

Implementation of Research
Provision of larvae to be tested Horned beetle larvae (O. rhinoceros) originating from PT. Socfin Indonesia was taken and maintained at Al-Azhar University Laboratory in Medan. The captured larvae are then adjusted in size and size to each other to obtain sufficient quantities to test. The larvae to be tested are instar larvae II.

The supply of entomopathogenic fungi
Trichoderma sp mushroom comes from Balai Proteksi Tanaman Pangan dan Hortikultura (BPTPH) Medan, while the fungus B. bassiana is isolated from the pest of O. rhinoceros which is derived from Balai Besar Germination Plantation Crop Protection (BBPPTP) Medan. To anticipate the deficiency of the mushrooms that will be used then both mushrooms are propagated in the rice medium each containing 100 grams / pack and incubated at room temperature for 5-7 days. This mushroom removal is done in the incase to avoid contamination. In the event of contamination, re-purification by taking part of the uncontaminated fungus of B. bassiana and Trichoderma sp. To be grown on rice media until Trichoderma sp and B. bassiana are completely pure and uncontaminated.

Application of Entomopathogenic Fungi
Application is applied only once in a manner dispersed and sprayed with each type of fungus according to the treatment, on the control only by spraying aqua pro injection alone on instar larvae instar II. Then observed its development.

Observation Parameters
Percentage of Mortality Observation of larval mortality started one day after application (DAA) weeks 14 days after application. The percentage of larval mortality was calculated by the formula:      Table 3 it was found that in 10 DAA of Trichoderma sp and B. bassiana in T11 treatment was the highest mortality (86.67%) and the lowest was in control (20%). But the different T11 treatment was not significant with the treatments B21, B22, B31, B32, T12, T21, T22, T31, T32, B21, B22 and T11 differed markedly by control.

Growth of Conidia
Data from conidial density of entomopathogenic fungi Trichoderma sp and B. basianna can be seen in appendix 43-60. After the data were analyzed the density was known that the density of conidia from both entomopathogenic fungi showed a difference of Table 4.

Table.4: Conidial density of Trichoderma sp and B. basianna with various doses per ml of aqua pro injection
Types of fungus Doses Spore density 10 8 10 The results of germination of Trichoderma sp and B. basianna fungus 4 hours after incubation period can be seen in appendix 61-66. After the data were analyzed, it was found that germination of conidia from both entomopathogenic fungi showed differences in Table 5.

IV. DISCUSSIONS Percentage of Mortality
From Table 1 it can be seen that in observation 1 DAA has seen mortality in O. rhinoceros larvae, but the mortality is still relativelay low. This low mortality due to fungal conidia that enter and infect is still not maximal, because the fungi entering entomopathogen to infect the insects have a time span, otherwise the fungus of entomopathogen infects and deadly O. rhinoceros larvae is highly dependent on the amount of environmental conidia and nutrients that needed. As stated by [25,29] the ability of entomopathogenic fungi to kill larvae is highly dependent on environmental conditions, nutritional fit, pH, from where the fungus grows and develops. From Table 2 on observations 6 DAA to 9 DAA mortality of O. rhinoceros larvae increased even though the different treatments were not real. But the mortality of larvae is still low, both due to the fungus Trichoderma sp and B. bassiana slow development of the fungus is due to the nutrients that the body is less available. This is in accordance with the stated by [15] states that the development of entomopathogenic fungi influenced the nutritional content of growing media used. The Table 3 description of the occurrence of differences in mortality rates against O. rhinoceros larvae is caused by the number of conidia or concentration of conidia germination as well as different application methods. In this case the more the number of conidia that enter the body of the larvae and the germination of the high, then the death of the larvae will be faster. In addition, the number of conidia that enter the body of the larvae is also influenced by the method of application. Application with the spread method, then conidia that can enter into the body of the larvae will be more and more. Because the conidia that the application does not exist or very few are not on target. Whereas when the application with spray method the possibility of drift is large enough so that the conidia that concerns the body of the larvae is less. This is consistent with that stated by [32,34] also states that, sooner or later mortality in pests depends on the amount of dose used. This indicates that the application of Trichoderma sp and B. bassiana fungi with the same dosage applied sprinkled differently is not real by spraying either at the beginning of the observation or until the end of the observation. This is due to conidia applied from both types of fungi the germination and the power of the infection are both high. So the mortality of larvae is also high, as stated by [3,36,36] that the higher dose conidia used in the treatment, the larval mortality is also high. From observation 12 DAA showed that the highest mortality was in T11, T21, B21, B22 and B32 treatment (90%) and the lowest mortality was in control (20%). The treatments T11, T21, B21, B22 and B32 were not significantly different for all treatments but differed significantly with the controls. The high mortality of larvae other than influenced by the amount of conidia is also influenced by temperature/humidity. This is in accordance with the results of [32] study that the optimal temperature for Trichoderma sp growth ranges from 22°C-27°C.
Meanwhile, according to [1] that germination, growth and optimal sporulation of B. bassiana fungi occur at temperatures 25-30°C.
From observation 13 DAA showed that the highest mortality was in T11, B31 and B32 treatment (93.33%) and the lowest mortality was in control (20%). Treatments of T11, B31 and B32 were not significantly different for all treatments but differed significantly with controls. This suggests that the density of conidia effectively controls the mortality of the O. rhinoceros larvae. At a higher level of conidia density, more and more mycelium and conidia grow. This is in accordance with [37] assertion that it increases the occurrence of conidial contact with the body of the larvae, thereby providing a better chance for conidia to stick, germinate and penetrate into the body of the larvae. From the 14 DAA observations it was found that T11 and T12 (96.67%) and B11, B21, B22, B31 and B32 (93.33%) were the highest mortality and the lowest was in control (20%). However, the treatments of T11 and B11 differed significantly from the treatment of T12, T22, T22, T31, T32, B12, B21, B22, B31, B31, T11 and B11. This is because the longer the entomopathogenic fungi are in the body of the larvae, the amount of conidia increases. Damage to body tissue larvae due to fungus attacks will be more severe, so that the mortality rate of larvae will be higher. This is in line with the statement [19,38] the longer the contact time of entomopathogenic fungi and host, the more likely the entomopathogenic fungus to infect the host, resulting in an increase in larval mortality. From the observation in Fig. 9-10 it is also found that infected entomopathogenic larvae indicate a change in which the larval body becomes hard, mummification and the appearance of fungal conidia around the conidia color tube will appear in accordance with the color of the infecting conidial fungus. This is in line with [37] statement that infected insects of entomopathogenic fungi undergo mummification and after a few days will grow a colony of white-colored fungus around the body. At the beginning of death the larvae have not shown any change. While on the third day the larvae have begun to experience a change in color, where conidia mushrooms have begun to grow on the side of the larvae. On the fifth day until the larval larvae infected the body the larvae are fully satisfied by the entomopathogenic fungal conidia.

Growth of Conidia
Conidia density Table 4 shows that the highest conidial fungus, Trichoderma sp with dose of 60 gr x 100-1 ml of aqua pro injection, is 7.25 x 106 conidia / ml and the lowest conidial density is found in fungus B. basiaana at a dose of 20 gr x 100-1 ml aqua pro injection as much as 4.22 106 conidia/ ml. If seen from the number of doses can be concluded that, the higher the dose the number of conidia will also be higher. Table 5 shows that the number of conidia of Trichoderma sp fungi is higher than that of B. basianna. The conidia growth difference of each of these fungi may be caused by unequal nutrients. Each genus or fungal species requires nutrients, pH, water content in medium, optimal temperature, light, aeration, and different incubation periods for growth and development of conidia [24,25,42]. The percentage of conidial germination is also influenced by the amount of conidia and conidial germination. The higher the amount of conidia and higher germination, the faster the mortality of the larvae. As stated by [43] that the higher dose conidia used in the treatment, the more rapid the death.

Conidia germination
V. CONCLUSIONS From result of research can be concluded that: 1. Precentages mortality of the highest larvae of O.
rhinoceros to Trichoderma sp fungus with a dose of 20 gr (96.67%) with the application method spread on the larvae.

The highest amount of conidial density is found in
Trichoderma sp fungi with doses of 60 g x 100-1 ml of aqua pro injection of 7.25 x 106 conidia/ml.

Germination level of conidia mushroom highest in
Trichoderma sp fungi with dose 60 gr x 100-1 ml aqua pro injection that is as much as 91%.

VI. SUGGESTIONS
To know the effectiveness of the fungus Trichoderma sp and B. bassianna need dilakuakn further research on application fungus of Trichoderma sp and B. bassianna directly to the field.