Virulence of conidia Beauveria bassiana (Bals.) as a bioinsecticide against Crocidolomia pavonana (F.) (Lepidoptera: Pyralidae) on broccoli plants

Crocidolomia pavonana is an important pest of broccoli plants. Pest control is currently still using chemical pesticides that negatively impact the environment. It is hoped that Beauveria bassiana will become one of the environmentally friendly bioinsecticides. The research aimed to study the virulence level of conidia density of B. bassiana as a bioinsecticide against C. pavonana pests on broccoli plants. This experiment used a completely randomized design consisting of 5 treatments and four replications so that 20 experimental units were obtained. The Basic Plant Protection Laboratory and Plant Pest Science Laboratory Faculty of Agriculture, Syiah Kuala University as the research location, which was carried out from January to September 2021. The parameters observed were larval mortality, pupae formed, imago appeared, an incubation period of B. bassiana conidia, and average time of C. pavonana. The results showed that B. bassiana with conidia density of 108/mL distilled water was effective as a bioinsecticide against C. pavonana. The highest percentage of C. pavonana mortality observed at 6 Days After Application (DAA) occurred at a density of 108 conidia/mL of water (97.50%), and the lowest was at a density of 102 conidia/mL of distilled water (62.50%). The percentage of pupae formed was observed. At 7, DAA was 15% using a density of 102 conidia /mL aquadest. Produce 5%, a density of 104 conidia/mL of distilled water was used. The percentage of imago that appeared with a conidia density of 102/mL of distilled water killed at 14 DAA yielded 15%, and 5% resulted from a density of 104 conidia/mL of distilled water. The fastest incubation occurred at a density of 108 conidia/mL of distilled water with a time of 3.50 days. Death of C. pavonana at 3.03 days required conidia density of B. bassiana 102/mL of distilled water.


INTRODUCTION
Crocidolomia pavonana is an important pest on vegetable crops, such as broccoli and other vegetables [2]. This pest destroys broccoli plants in groups. The initial attack on the growing point of the leaf, then all the leaves are eaten, leaving only the leaf bones. Heavy attacks from this pest result in economic losses [15]. Pest control is still using synthetic insecticides, which have a negative impact on the environment [18]. Therefore, it is necessary to control other effective and environmentally friendly methods, one of which is through the use of biological agents [13]. The fungus B. bassiana is a biological agent that can be used as a bioinsecticide against insect pests from several insect orders, such as Lepidoptera, Coleoptera, Isoptera, Hemiptera, Orthoptera, and other orders [17]. Effectiveness B. bassiana as a bioinsecticide is determined by the density of conidia and environmental conditions. Application of conidia B. bassiana with a density of 10 8 /mL aquadest and 10 9 /mL aquadest against the insect pest H. antoni by spraying method can produce mortality up to 100% [4]. The higher the density of B. bassiana conidia applied to insect pests, the faster and higher mortality will be produced. Conidia B. bassiana to produce mortality against insect pests is also influenced by the activity of secondary metabolites such as enzymes and toxins that cause disturbances in hemolymph function, and the insect body becomes swollen and hard [19].
The research aimed to study the virulence level of conidia density of B. bassiana as a bioinsecticide against C. pavonana pests on broccoli plants. If the F hit test analysis shows a significant difference, then the data analysis is continued with the smallest significant difference test at the 5% level (LSD 0.05).

Research implementation test insect Breeding
Propagation of the test insects by collecting C. pavonana larvae from mustard plants from the East Sector, Darussalam, Aceh Besar. The collection of C. pavonana larvae was then brought to the Plant Pest Science Laboratory using plastic jars and covered with gauze. The food for C. pavonana larvae given was fresh mustard greens which were changed every day. Larvae that will enter the pupae stage are transferred into jars that have been filled with sawdust. In the container hung cotton that has been dipped in 10% honey liquid as feed for imago C. pavonana. Eggs of C. pavonana placed on mustard leaves, then transferred to a jar and left until the eggs hatch. The larvae that have come out of their eggs are then put in fresh mustard leaves as feed which is changed every day until the larvae enter the second instar phase (test insects).

PDA media preparation
PDA was weighed using an analytical balance in the amount of 3.9 g. The material is then dissolved into 100 mL of distilled water in an Erlenmeyer as a mixture; the solution is stirred until homogeneous and heated. Next, the erlenmayer was covered with cotton covered with aluminum foil, and sealed using the paper wrap. The PDA was then sterilized in an autoclave as well as the tools to be used for research such as petridish, use needles, and scalpel, which had been wrapped in HVS paper at 121 o C for 15 minutes. The sterile PDA solution was added with 125 mg of Chloramphenicol then cooled in Laminar airflow, and then put into a sterilized petridish.

Preparation of suspension of conidia B. bassiana
The conidia suspension of B. bassiana was obtained by adding 10 mL of aquadest to a petridish containing 14 days of B. bassiana culture. The conidia were taken using a loop needle until they were released from the PDA media; 1 mL of the conidia suspension was taken using a dropper and diluted into 9 mL of distilled water and homogenized. Then the conidia were counted using a haemacytometer  pavonana larvae was carried out by the contamination method by spraying a suspension of B. bassiana conidia on C. pavonana larvae for 5 seconds. Then air-dried for a few seconds and put in each jar. The mustard leaves were cut in a rectangular shape, measuring 5x5 cm, a total of 3 pieces in each treatment; then the mustard leaves were put into a jar that had been lined with straw paper. Then 10 larvae of C. pavonana instar II were added. After 24 hours, the feed was replaced with fresh leaves, as well as for other treatments.
Observed variables mortality of C. pavonana larvae Mortality of C. pavonana larvae was carried out by counting the number of dead larvae from one day after application to each treatment unit until all larvae became pupa. Larvae mortality was calculated using the formula [1].
Po= r/n x100% notes: Po = Percentage of mortality r = Number of dead larvae n = total number of larvae

C. pavonana pupa formed
The percentage of pupa was calculated from the period the larva entered the pre-pupae stage until the pupae were formed. The percentage of pupa formed is calculated using the following formula: Percentage of pupa formed=(number of pupa formed)/ (number of early larvae) x100% Imago C. pavonana appearance The percentage of imago was calculated using the following formula: Percentage of imago that appeared = (number of imago that appeared)/(number of early larvae) x 100%

Average period to death of C. pavonana
The rate of death was calculated with an interval of 1 day after application until there are treatment units that die 100%; the calculation is carried out using the following formula: Rate of death period = (Σ(period of death x number of dead larvae)/ (Number of early larvae).
The percentage of pupa was calculated from the period the larva entered the pre-pupae stage until the pupae were formed. The percentage of pupa formed is calculated using the following formula: Percentage of pupa formed = (number of pupa formed)/(number of early larvae) x 100% Imago C. pavonana Appears.

Incubation period of the fungus B. Bassiana
The incubation period is the period of period between the inoculation of the fungus B. bassiana until it causes symptoms of infection in the larvae of C. pavonana. This observation was made based on the incubation period of the fungus, whether there were any changes observed visually in the larvae. Calculations were made on the average incubation period of the fungus from one day after application until the appearance of symptoms of infection in the larvae.

RESULTS AND DISCUSSION
The results of observations on the mortality of C. pavonana larvae due to the application of B. bassiana conidia with different density levels in each treatment, and observed at 3 to 6 days after application (DAA) showed that the effect was very significant at each observation time, namely 3, 4, 5 and 6 days after application of conidia B. bassiana. The average mortality of C. pavonana larvae can be observed in Table 2. The numbers followed by the same letter in each column were not significantly different at 0.05 test LSD. Table 2 shows that the average mortality of C. pavonana larvae due to the application of B. bassiana conidia experienced a very significant change at 3,4,5 and 6 DAA observations. In general, the mortality of C. pavonana larvae occurred from 3 to 6 DAA in each treatment. Observations on 6 DAA that the highest mean mortality was found in the treatment of conidia density 10 8 (97.50%), followed by 10 6 (82.50%) and 10 4 (75%). while the lowest was found in treatment 10 2 , (62.50%). This is thought to be related to the application level of conidia density. The higher the conidia density given, the higher the  [14].
That the application of conidia B. bassiana to Plutella xylostella larvae resulted in the death of larvae up to 55.00% using a density of 108 conidia/mL aquadest which was observed 4 days after application [3]. and application of B. bassiana conidia to Spodoptera litura larvae caused 72.50% mortality with a density of 10 8 conidia / mL of distilled water and was observed 7 days after application [11].
Conidia B. bassiana can infect C. pavonana larvae, presumably due to the activity of the enzymes it products [8], when infecting the host, the conidia of the fungus B. bassiana produce enzyme compounds such as proteases, lipotics, amylase and chitinase [15]. If there is contact between the conidia and their host, the conidia will form a sprout tube and secrete enzymes to penetrate the cuticle of the larvae so that it can enter the larva's body. The fungus B. bassiana produces toxins, such as beauvericin, beauverolite, bassianolite, isorolite and oxalic acid, then B. bassiana hyphae enter the host's body through natural holes [19].
The infection entomopathogen fungi cause death in the host by absorbing nutrients and spreading toxins to the hemolymph so that it can affect the development and physiology and reproduction of insects. Visually, early symptoms of infected C. pavonana larvae were characterized by reduced feeding activity and weaker physical characteristics of the larvae and slower larval movement [7]. The larvae will experience a change in skin color to pale white then the larvae will die and harden like a mummy. Dead larvae will have black spots before the growth of mycelium on the outer skin of the insect [21]. The initial symptoms due to infection from entomopathogenic fungi are slow, silent, or inactive movements until they eventually die [10] The result of C. pavonana pupae The results of observations of C. pavonana pupae produced due to the application of B. bassiana conidia with various conidia density levels were calculated on observations 7 days after application. The results of the F test on analysis of variance showed that the treatment of conidia density of the fungus B. bassiana had a very significant effect on the percentage of pupae production at 7 days after application.
The average of C. pavonana pupae produced can be seen in table 3.  Table 3 shows that the larvae of C. pavonana that successfully pupae after the application of B. bassiana conidia were significantly different in each treatment. The highest percentage of pupae up to 100% occurred at control. 15% produce pupae occurred in the treatment of conidia 10 2 / mL aquadest of distilled water. 5% produced C. pavonana pupae after application of B. basiana conidia with conidia density of 10 4 conidia/mL of distilled water. The occurrence of differences in the quantity of individual larvae to turn into pupae is thought to be strongly related to the size of the application of different B. bassiana conidia density. At the period of application, it was suspected that not all of the conidia were attached to the cuticle of the host. so it takes a period to infect the host. The ability of B. bassiana conidia to infect the host is influenced by concentration, viability and virulence [6]. Thus, the application of low conidia density of B. bassiana took longer to infect the host.
Generally, the larvae applied to B. bassiana conidia died, but there were still larvae that became pupae and imago. The change of skin at each instar is one of the inhibiting factors for B. bassiana conidia in penetrating the cuticle of C. pavonana. That conidia density affects the percentage of mortality and the process of pupae formation. Different application of B. bassiana conidia resulted in different number of conidia that were able to attach to the pupae [22].

C. pavonana larvae that successfully become Imago
Observation of C. pavonana larvae into imago. The application of B. bassiana conidia with different conidia densities and observed Virulence of conidia Beauveria bassiana (Bals.) as a bioinsecticide … (Muhammad Sayuthi, Alfian Rusdy, Hasnah, Naila Evi Azahra) at 14 days after application, showed that it had a very significant effect on the change from successful C. pavonana larvae to imago. The average imago of C. pavonana that appeared can be observed in Table 4.  Table 4 shows that the imago of C. pavonana that was successfully transformed into imago after the application of B. bassiana conidia was significantly different between each treatment. The highest percentage of C. pavonana larvae that managed to become imago using a density of 10 2 conidia/mL aquadest, which was 15.00%, compared to the use of a density of 10 4 conidia/mL aquadest, which was 5%. and control reaches 100% to imago. The percentage of C. pavonana larvae that managed to become imago was closely related to the conidia density of B. bassiana applied. The density of the applied B. bassiana conidia is related to the opportunity for the conidia to adhere to the host cuticle. The higher the density of conidia attached to the host, the greater the amount of toxins and enzymes produced that enter the host's body. This resulted in the mortality rate of C. pavonana increasing.
The application of conidia of B. bassiana 10 8 mL/10 mL of distilled water to Bactrocera carambolae pupae showed that the percentage of pupae that succeeded in becoming imagos reached 52% [21]. If the larvae that have been infected by the fungus B. bassiana and cause symptoms of infection at the pupa stage, it is possible that symptoms will appear when entering the imago stage, marked by not being to produce eggs. This is a result of the fungus that has entered the insect's body will grow and taking nutrients from the host [4].

Incubation period of the fungus B. bassiana
The results of observations of the incubation period of the fungus B. bassiana on C. pavonana larvae with several sizes of conidia density were calculated based on the presence or absence of changes observed visually in the larvae. The results of the F test on analysis of variance showed that the incubation period of the fungus B. bassiana had a very significant effect on the infection activity of C. pavonana larvae. The average incubation period of the B. bassiana against C. pavonana can be observed in Table 5. 3.50d LSD 0.244 The numbers followed by the same letter in each column were not significantly different in the 0.05 test LSD Table 5 shows that the fastest incubation period for the fungus B. bassiana occurred in the conidia 10 8 /mL Aquades treatment, which was 3.50 days, and the 10 4 /mL aquadest and 10 6 /mL aquadest treatments showed that the average incubation period was 4.75 and 4, respectively. 50 days and the longest incubation period occurred in treatment 10 2 , 5.75 days. The difference in incubation period in each treatment was thought to be related to the level of the different conidia density in each treatment. This is because the higher the conidia density of the fungus, the easier it is for the fungus to infect the host. Likewise, if the conidia density is low, the host infection process takes long.
Visual observation of C. pavonana larvae infected with the fungus B. bassiana was indicated by decreased feeding activity of larvae, slow movement of larvae. Larvae infected with the fungus B. bassiana will experience a process of changing the color of the insect's body to become pale white and harden, then the larva's body will change color to black-brown, then the mycelia will appear out through natural holes that will penetrate the integument of the C. pavonana larvae. The fungus will absorb the body fluids of the host, so that the integumental surface of the infected host's body looks dry, wrinkled, and dark in color and hardens like a mummy [14].
B. bassiana conidia inoculation process is strongly influenced by insect body structure. The fungus B. bassiana on larvae with a thin cuticle layer and relatively soft larvae body Virulence of conidia Beauveria bassiana (Bals.) as a bioinsecticide … (Muhammad Sayuthi, Alfian Rusdy, Hasnah, Naila Evi Azahra) structure causes the fungus to more easily infect and penetrate. The results of research by stated that the growth of B. bassiana colonies showed a faster growth rate from the second day to the fourth day, on the sixth day the colonies grew the most, in addition to genetic factors produced was also influenced by external factors, namely temperature and pH [17].

Mean Time to Death of C. pavonana.
The results of observations of the average time of death of C. pavonana due to the application of B. bassiana with different conidia density levels significantly affected the time of death of C. pavonana larvae and can be observed in Table 6.  Table 6 shows that the application of B. bassiana conidia to C. pavonana larvae had very significant differences in each treatment. The fastest average time of death occurred in treatment 10 2 , which was 3.03 days. While the longest occurred in treatment 10 6 conidia/mL aquadest (5,05 days). This is presumably because the time required to cause insect death varies depending on the virulence and resistance properties of the host. In treatment 10 2 conidia/mL aquadest the average time of death was found to be faster this is because there are individual larvae that have successfully entered the pupae stage. The longest average death time occurred in the 10 6 conidia/mL aquadest treatments because the highest average larvae mortality occurred at Treatment of conidia density of 10 2 /mL aquadest to produce larval death of C. pavonana took 4.20 days while 10 8 conidia/mL aquadest was needed to produce larvae death it took 4.08 days. The difference in mean time of death required for C. pavonana larvae is thought to be strongly related to time and application and conidia density of B. bassiana. Conidia from B. bassiana to infect the host takes 2 to 14 days after application, and its effectiveness is strongly influenced by environmental factors such as temperature, humidity and light. [9]. The morphological condition of the host affects the process of conidia infection in the host body until it enters the host's body [12].

CONCLUSION
The results showed that the density of conidia aquadest B. bassiana 10 8 /mL had a positive effect on its virulence against C. pavonana pests. The highest mortality of C. pavonana larvae was found in the density of B. bassiana conidia 10 8 /mL of distilled water, which was 97.50% at observation 6 days after application, compared to the density of 10 2 conidia/mL of distilled water. that is 62.50%. C. pavonana larvae successfully pupaed 15% at a conidia density of B. bassiana 10 2 /mL and 5% at a density of 10 4 conidia/mL aquadest which were observed 7 days after application. The percentage of C. pavonana larvae that were successfully transformed into imago at a conidia density of 10 2 /mL was 15%, and 5% at a conidia density of 10 4 /mL of distilled water, which were observed 14 days after application. The fastest incubation time occurred at a density of 10 8 conidia/mL aquadest, which was 3.50 days. The fastest average time of death occurred at a density of 10 2 /mL aquadest, which was 3.03 days