STUDY OF Beauveria bassiana AND Paecilomyces sp. FILTRATES AGAINST Spodoptera litura (F.)

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1 STUDY OF Beauveria bassiana AND Paecilomyces sp. FILTRATES AGAINST Spodoptera litura (F.) Deni Zulfiana *, Ni Putu Ratna Ayu Krishanti, and Apriwi Zulfitri Research Center for Biomaterials, Indonesian Institute of Sciences, Cibinong Science Center, Cibinong-Bogor 16911, Indonesia * Corresponding author: zulfiana@yahoo.com Abstract The continuous use of pesticides to overcome the attack of armyworm (Spodoptera litura F.) on vegetable crops in Indonesia has been causing bad impact on health and the environment. Today, the development of biological agents as an alternative to chemical pesticides in order to reduce the use of pesticides have been done, one of them is the utilization of entomopathogenic fungi. However, studies on how the effect of the fermented filtrates of entomopathogenic fungi especially Beauveria bassiana and Paecilomyces sp. toward S. litura larvae is still unknown. This study aims to determine the effectiveness of the extract of entomopathogenic fungi, B. bassiana and Paecilomyces sp. against the 3 rd larval stage of S. litura. The filtrate was produced through the fermentation process using a combination of Czapex-dox broth with 10% yeast extract. Bioassay on S. litura had been done by spray and leaf dip methods. The mortality rate of S. litura larvae that treated with the extract of B. bassiana using both methods was higher than treated with Paecilomyces sp. ( 60% and 30-40%, respectively). B. bassiana filtrates had significant difference to S. litura larval mortality compared to control, while Paecylomyces sp. filtrates did not (α = 0.05). Furthermore, the use of different method showed no significant difference either in B. bassiana or Paecylomyces sp. strain. Therefore, S. litura larvae mortality was suspected due to the activity of toxin and enzyme excreted in the growth media. Keywords: Beauveria bassiana, fermentation, mortality, Paecilomyces sp., Spodoptera litura Introduction Armyworm (Spodoptera litura) is one of destructive pests causing economy loss in the agroecosystem (Embriani, 2014). Larvae of armyworm feed most of plant parts such as fruit and leaf, only leaving the midrib. The population of armyworm reaches the highest in dry season when humidity is high, thus the attack is becoming more intensive. The larvae are nocturnal while during the day hiding in grassy vegetation, under the leaf or in the soil cavity. This pattern makes it difficult to control armyworm (Kalshoven, 1981). There are several methods of controlling S. litura such as physical / mechanical, or the most common method is the use of synthetic chemical pesticides. Concerns about the negative impacts of synthetic chemical pesticides generates the development of biocontrol agents as an alternative in order to reduce the danger of excessive usage of pesticides on human health and the environment (Hazraet al. 1998). One is the use of Myco-biocontrol, which is using entomopathogenic fungi to control these pests. The use of entomopathogenic fungi has many advantages, namely: are naturally present in the soil therefore more environmentally friendly; has a fairly complex mechanism so that the possibility of resistance is low; and is selective against the target pest (Sandhu et al., 2012). Currently, the use of entomopathogenic fungus has been considered as an important strategy in reducing pest insect populations including S. litura. Some of well known entomopatogen fungus are Metarhizium anisopilae (Metchnikoff) Sorokin, Beauveria bassiana (Balsamo) VUILLEMIN dan 213

2 Paecilomyces fumosoroseus (Wize) Brown & Smith (Wanida & Poonsuk, 2012; Shoaib et al., 2012; Meikle et al., 2005). The most common application of entomopathogenic fungi is using the conidia that can attack the insect directly by damaging the cuticle, while application using fermented filtrate producing secondary metabolites such as toxins and enzymes compound from entomopathogenic fungi is still unknown. This study aimed to evaluate the potential of fermented filtrate of two isolates entomopathogenic fungi B. bassiana and Paecylomyces sp. as a pest control agent of S. litura. Materials and Methods Fungal strain and submerged fermentation The isolates of B. bassiana and Paecilomyces sp. were obtained from Bogor Agricultural University culture collection. Each fungal strain that had been cultured on PDA media for 7 days was cultured in conical flasks containing autoclaved czapex-dox liquid medium with 10% yeast extract. The flasks were then agitated at 120 rpm in room temperature for eight days. The fungal filtrates were harvested by separating its fungal residue using Whatman filter papers. The filtrates were subsequently used in the bioassay tests. Bioassays of entomopathogenic fungi against the larvae of Spodoptera litura The bioassays were carried out using spray and leaf dip methods. The spray method was conducted by spraying 10 ml of fungal extract on the integument of the 3 rd larval stage of S. litura. Afterward, the diet (taro leaf) was given daily to the treated larva. The other infection approach used in this research was leaf dip method. Taro leaf in 4x4 cm size was dipped for 5 minutes into the fungal filtrates and baited to the 3 rd larval stage of S. litura which had been previously fasted for 6 hours. Fresh leaf was provided daily after the leaf treated diet were completely eaten. Each bioassay using spray and leaf dip methods was performed on three replicates. Each replicate consisted of 10 larvae of S. litura. The larva and the diet sprayed and dipped in sterile distilled water were used as control. The mortality of larvae was counted every 24 hours within 72 hours of observation. Further observation was done macroscopically on dead larvae to gain symptoms of larval mortality caused by entomopathogenic fungus. Statistical analysis The data was subjected to ANOVA and the difference between means was analyzed using LSD test at the 0.05% level. Results and Discussion The pathogenicity of Beauveria bassiana and Paecilomyces sp. towards S. litura s larva using spray and leaf dip methods are displayed in Figure 1 and 2 below. S. litura larval mortality using leaf dip method The average percentage of larval mortality by filtrate Paecylomyces sp., B. bassiana, and control were 40%, 63.3% and 16.67% respectively (Figure 1). B. bassiana filtrate showed higher pathogenicity than Paecylomyces sp. filtrate, this might be due to different specificity between both strains. Paecylomyces sp. was more likely to infect a host at the adult stage (moth) so it may be less effective in the larval stage (Rai et al., 2014). High host specifity possessed by B. bassiana might supported its pathogenicity against S. litura larvae (Rai et al., 2014). 214

3 Larval mortality (%) a 20 ab Paecilomyces Beauveria Kontrol Treatment b Figure 1. Mortality rate of S. litura larvae after treated with the filtrate of entomopathogenic fungi by leaf dip method. Means followed by different letters are significantly different from each other according to LSD test (α: 0.05) Based on ANOVA and LSD test results, mortality rate of S. litura larvae treated with B. bassiana filtrate was significantly different than those of the control at 95% confidence level (Table 1). This showed that filtrate B. bassiana was effective against S. litura larvae. Meanwhile, mortality rate of S. litura larvae treated with Paecylomyces was not significantly different from either B. bassiana treatment or control treatment. Table 1. Analysis of variance (ANOVA) of S. litura larval mortality using leaf dip method Source of variation Degrees of freedom Sums of Mean F f-test Treatment * Error Total 8 46 *f test< F means variance between the means of two populations is significantly different Mortality rate of S. litura larvae treated with entomopathogenic fungi resulted in higher percentage than those of control. However, S. litura larvae mortality rate due to Paecylomyces sp. treatment did not significant different compared to control, statistically. The death of larvae after feeding on leaf treated with entomopathogenic fungi might be caused of toxic secondary metabolites produced by the fungus such as destruxin, beauvericin, bassianolide or beauverolides (Sandhu et al., 2012). Fernandes et.al., (20212) said the role of a series of enzymes excreted on the fungi filtrate such as esterase, pectinase, kaseinase, cellulase, amylase and protease may also have a role in the fungus pathogenicity. This is also supported by Shahid et al., (2012), the enzymes in the fungal filtrates degrade integument degradation and other parts of the larvae thus causing death. S. litura larval mortality using spray method Mortality rate of S. litura sprayed with B. bassiana filtrate resulted in higher percentage (60%) compared to those sprayed with Paecylomyces sp. (20%) and control (13.335). (Figure 2). 215

4 Larval mortality (%) a b b Paecilomyces Beauveria Kontrol Treatment Figure 2. Mortality rate of S. litura larvae after sprayed with filtrate of entomopathogenic fungi. Means followed by different letters are significantly different from each other according to LSD Test at P = 0.05 Based on statistical analysis, ANOVA and LSD test at 95% confidence level 95% (Table 2), mortality rate of S. litura larvae treated with B. bassiana was significantly different compared to control and Paecylomyces sp. treatment. However, larvae sprayed with Paecylomyces sp. did not significantly different to control. This might be due to higher activity of pectinase enzyme contained in B. bassiana filtrate to degrade larval cuticle (Fernandes et al., 2012). Degradation of larvae cuticle is an initial important step in fungal pathogenicity to allow access for other enzymes and toxin entering the larvae body (Reddy et al., 2013). Fungi that excretes enzymes, toxin and other metabolites causes faster death to larvae compared to those who does not produce secondary metabolites as explained by Kumari et al., (2014). Table 2. Analysis of variance (ANOVA) of S. litura larval mortality using leaf dip method Source of variation Degrees of freedom Sums of Mean F f-test* Treatment Error Total *f test< F means variance between the means of two populations is significantly different ANOVA analysis between leaf dip and spray method Mortality rate of S. litura larvae resulted in leaf dip and spray method treated was subjected to ANOVA, to find information whether there was any different statistically in both methods (Table 3 and Table 4). The result shows there was no significant difference between those methods, either in B. bassiana or Paecilomyces sp., in other words, both methods were quite effective against S. litura larvae. Table 3. Analysis of variance (ANOVA) of S. litura larval mortality using leaf dip and spray method treated with B. bassiana Sum of df Mean square F Sig.* Between Groups Within Groups Total * Sig. level (.643) > α = 0.05 the results are not statistically significant 216

5 Table 4. Analysis of variance (ANOVA) of S. litura larval mortality using leaf dip and spray method treated with Paecilomyces sp. Sum of df Mean square F Sig. Between Groups Within Groups Total * Sig. level (.196) > α = 0.05 the results are not statistically significant Macroscopic observation of larval mortality symptoms Observation on S. litura mortality symptoms was also done besides counting of mortality percentage (Figure 3). Mortality symptoms on larvae trated with both fungal filtrates showed similar symptoms. Behavior of larvae before dead phase also did not show significant difference such as decreasing food consumption level, slowed movement as well as contact responses. A B C Figure 3. Mortality visualization of infected S. Litura larvae by entomopathogenic fungi (A) control (distilled water), (B) dead larva of S. litura infected by B. bassiana (C) dead larva of S. litura infected by Paecilomyces sp. In order to find out mode of action of toxin produced by the fungal filtrates responsible for S. litura larval mortality, morphological observation was done macroscopically on infected and control larvae. Figure 3 shows fungal infected larvae shrank thus smaller, darker compared to control larvae, the body become dry and stiff. From those symptoms, toxin produced while fermentation process was expected as mode of action causing larval mortality. Furthermore, according to Sandhu et al. (2012) the process of cell destruction with cytotoxins may occur before penetration of hyphae, this was fit to this research finding that no mycelial growth beyond the larval dead body. B. bassiana has been identified contains several kind of toxin such as beauvericin, bassianolide, or beauverolides. Another type of toxin obtained from other fungi such as Metarhizium anisopliae such as destruxin and Cytochalasin. The toxin will cause paralysis of the larvae that led to the death of the larvae. However, to determine the specific toxin responsible for S. litura larval mortality in this study, further studies to characterize the toxin contained in the larval body need to be conducted. In addition to a toxin, an enzyme produced by fungi can also cause death in armyworm. At the beginning of the treatment, there may be a protease enzyme and esterase, followed by the production of chitinase and lipase enzymes which have the capability to decrease the immune system of the armyworm (Pedrini, 2006). Conclusion The fermented filtrate of B. bassiana had significant effect on mortality of larvae of S. litura, while filtrate fungi Paecylomyces sp. showed insignificant effect (α = 0.05). There were no significant differences between application methods, leaf dip or spray method. Mode of action on pathogenicity of both fungal strains might be caused of the toxin and enzyme produced in the filtrate. Acknowledgement The authors would like to say thank you to DIPA 2016 for funding this study and also Redian Fikri Mayo and Wahyu Setyaji Dwiantara from Bandung Institute of Technology for technical assistance in the laboratory. 217

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