Philippine Journal of Science 150 (1): 193-199, February 2021 ISSN 0031 - 7683 Date Received: 06 Apr 2020

Metarhizium (=) rileyi (Farlow) Samson from Spodoptera exigua (Hübner) Cross Infects , Spodoptera frugiperda (J.E. Smith) (Lepidoptera: ) Larvae

Melissa P. Montecalvo* and Marcela M. Navasero

National Crop Protection Center, College of Agriculture and Food Science University of the Philippines Los Baños 4031 College, Laguna, Philippines

Mycobiocontrol is a promising management strategy in mitigating the fall armyworm Spodoptera frugiperda (J.E. Smith) infestation in the Philippines. An isolate of (=Nomuraea) rileyi (Farlow) Samson from onion or beet armyworm, S. exigua, which induced high mortality to this pest, was assessed against different larval instars of S. frugiperda. Surface-sterilized corn leaves were treated with different conidial concentrations and fed to S. frugiperda larvae. Cross infection of this entomopathogenic to S. frugiperda was confirmed with a fungal infection that was initiated at 1–2 d post-treatment depending on the age of the larvae. Larval mortality significantly increased at 4–5 d post-treatment. Up to 100% larval mortality was recorded at 7 d post-treatment. Early larval instars (1st–3rd) were more susceptible than late larval instars (4th–6th). Higher conidial concentrations caused a higher and faster rate of larval mortality than lower conidial concentrations. The inflicted mycoses due to M. rileyi resulted 5 8 –1 in a slightly lower lethal dose (LD50) (1.44 x 10 to 9.36 x 10 conidia ∙ mL ) and shorter mean time to death (4.51–8.89 d). Mummification of the cadaver confirmed fungal infection with white fungal growth that later changed to green during sporulation. Our findings suggest the infectivity of M. rileyi and can be explored as a biological control agent against S. frugiperda.

Keywords: biological control, , fall armyworm,

INTRODUCTION Damages in different growth stages of corn were observed in incidence areas and may cause significant yield losses. In the Philippines, armyworms are major and invasive Armyworm larvae feed on leaf tissues, which can result in lepidopterous insect pests of crops. The black armyworm, severe defoliation (Business Queensland 2020). Spodoptera exempta (Walker), infested corn, rice, and sugarcane crops in Southern Tagalog and Bataan provinces Armyworms are effectively controlled by entomopathogens in 2010 (Navasero et al. 2011). The onion or beet such as nucleopolyhedroviruses (Montecalvo and armyworm, S. exigua, caused an estimated crop loss of Navasero 2019), fungi (Grijalba et al. 2018; Montecalvo PHP 1.6 B from February–April 2016 in onion production and Navasero 2020), bacteria (Moar et al. 1995), and areas – including Nueva Ecija, Pangasinan, and Ilocos Sur protozoa and nematodes (Business Queensland 2020). (Navasero et al. 2017). In 2019, the invasive species of fall Among the entomopathogens, entomopathogenic fungi are armyworm, S. frugiperda, was reported in 17 municipalities promising biological control agents against Spodoptera in 10 provinces in the Philippines (Navasero et al. 2019). species. Mycobiocontrol utilizes fungi to lower the insect density to reduce the disease-producing activity and crop *Corresponding Author: [email protected]

193 Philippine Journal of Science Montecalvo and Navasero: Metarhizium (=Nomuraea) Vol. 150 No. 1, February 2021 rileyi from Spodoptera exigua damage (Sandhu et al. 2012). Over 750 fungal species can Bioassay using various conidial suspensions. Young corn provoke fungal infections in insect populations. leaves (5-cm length) were surface sterilized, following Asi et al. (2013). Conidial concentrations were sprayed to both Metarhizium species are known entomopathogenic fungi sides of the corn leaves using a mist sprayer. Tween 80 that are widely studied because of their global distribution, (0.1%) was applied to corn leaves for the control set-up. broad host range, safety, environmental friendliness, and Four treated corn leaves were introduced to S. frugiperda ease of mass production (Aw and Hue 2017). Reported larva/e in a Petri plate with moistened cotton. For early species of Metarhizium infecting lepidopterous pests (1st–3rd) larval instars, lower conidial concentrations (1 x include M. anisopliae against S. exigua (Han et al. 2014) 103 to 1 x 108 conidia ∙ mL–1) were used. Higher conidial and S. litura (Petlamul and Prasertsan 2012), and M. concentrations (1 x 105 to 1 x 1010 conidia ∙ mL–1) were rileyi against S. frugiperda (Grijalba et al. 2018) and S. assayed for late (4th–6th) larval instars. Ten (10) 1st and litura (Rajan and Muthukrishnan 2009). Infected insects 2nd larval instars were initially cultured per plate and die due to mechanical damage resulting from tissue single cultured upon reaching 3rd larval instar. One larva invasion, depletion of nutrient resources and toxicosis, and per plate was cultured for 3rd–6th larval instars. The Petri production of toxin in the insect body (Sandhu et al. 2012). plates were sealed with Parafilm. Fresh corn leaves that In our previous research, M. rileyi was successfully were surface-sterilized were introduced in the succeeding isolated from the cadavers of S. exigua and effectively days post-treatment. Each treatment was replicated thrice caused the lethal infection to this pest in a laboratory with 10 larvae per replicate. bioassay (Montecalvo and Navasero 2020). High lethal Mortality was observed daily. Mycosis of cadavers was infection to S. exigua resulted in a low pupation rate and confirmed in blotter set-up. Cadavers were dipped in 1% adult emergence. M. rileyi is a dimorphic hyphomycete sodium hypochlorite for 1min and washed twice in sterile that causes epizootic death in insect species belonging to distilled water for 1 min. The cadavers were dried in sterile Lepidoptera and Coleoptera (Sandhu et al. 2012). filter paper and placed in a microscope slide inside a Petri Considering the virulence of this entomopathogenic fungus plate lined with moistened filter paper. against Spodoptera species, this study aimed to assess the Percentage mortality was corrected using the equation: potential of M. rileyi as a new tool to be integrated into M (%) = [(t – c) / (100 – c)] × 100, where: M = corrected pest management programs against S. frugiperda. We mortality; c = percentage mortality in controls; t = conducted this bioassay to elucidate the pathogenicity of percentage mortality in treatments (Abbott 1925). this isolate to the different larval instars of this insect pest Mean time to death was calculated using the formula: and to determine the lethal dose (LD) and time as important mean time to death (d) = [(x1y1)+(x2y2)+(xnyn)] / total parameters in determining its biocontrol efficacy. mortality, where: x = number of larvae died on a given day; y = number of days of which the observation was made considering the time when the trial was initiated (El-Hawary and Abd El-Salam 2009). MATERIALS AND METHODS

Laboratory rearing of S. frugiperda. Advanced generation Statistical Design and Analysis (F9) larvae of S. frugiperda were obtained from the The experimental set-up was arranged in CRD. The data existing culture at the Biocontrol Laboratory of NCPC, was analyzed following the general linear procedure of which was originally collected from Gonzaga, Cagayan, Statistical Tool for Agricultural Research. Treatment Philippines. Neonates were fed with fresh leaves of a means were compared by means of the analysis of variance native variety of corn (Lagkitan) until the desired stages using Tukey's honestly significant difference test. LD of larvae. Bioassays were conducted when the appropriate values were calculated using PriProbit ver 1.63. age/instar of test insects was reached. Preparation of conidial suspensions. M. rileyi isolate from S. exigua was revived and subcultured in PDA for at least 14 d. This entomopathogenic fungus was isolated from RESULTS mummified larvae of S. exigua collected from the infested The entomopathogenic fungus M. rileyi isolated from S. field of bunch onion from San Jose City, Nueva Ecija. exigua caused the lethal infection to S. frugiperda larvae Conidia were harvested from the cultures and suspended (Figure 1). Various larval instars of S. frugiperda have in 0.1% Tween 80 solution. Conidia in the suspension a different reaction to M. rileyi infection. Early larval were counted using a haemacytometer. Various conidial instars were more susceptible than late larval instars. concentrations (1 x 103 to 1 x 1010 conidia ∙ mL–1) were Fungal infection was initiated at 1–2 d after treatment prepared by diluting the stock in 0.1% Tween 80 solution.

194 Philippine Journal of Science Montecalvo and Navasero: Metarhizium (=Nomuraea) Vol. 150 No. 1, February 2021 rileyi from Spodoptera exigua

Figure 1. Cumulative mortality of Spodoptera frugiperda larvae treated with different conidial concentrations of Metarhizium (=Nomuraea) rileyi.

depending on the age of the larvae. Larval mortality significantly increased at 4–5 d post-treatment. Higher conidial concentrations caused higher and faster larval mortality than lower conidial concentrations. Conidial concentrations of 1 x 107 and 1 x 108 conidia ∙ mL–1 killed more than 50% of 1st and 2nd larval instars at 6 and 5 d post-treatment, respectively. The conidial concentration of 1 x 1010 conidia ∙ mL–1 caused 50% mortality to 4th and 5th larval instars at 4 d post-treatment. However, mortality in 3rd and 5th larval instars peaked at 7 d post-treatment. Lower conidial concentrations (1 x 103 to 1 x 106 conidia Figure 2. Mean mortality of early larval instars of Spodoptera ∙ mL–1) caused slower larval mortality. frugiperda infected with Metarhizium (=Nomuraea) rileyi at 7 d post-treatment. Zero mortality in Control. Fungal infection in early and late larval instars at 7 d post-treatment is presented in Figures 2 and 3. M. rileyi caused significantly different mortalities against different larval instars. The virulence of M. rileyi was dependent on conidial concentrations and age of larval instars. Concentrations of 1 x 106 to 1 x 108 conidia ∙ mL–1 caused 50–100% mortality in 1st and 2nd larval instars. Late larval instars succumbed to fungal infection when exposed to higher conidial concentrations. Bodies of the dead larvae infected with M. rileyi were firm. During mummification, the cadavers were covered with white fungal growth through the intersegmental membranes (Figure 4). The presence of green sporulation Figure 3. Mean mortality of late larval instars of Spodoptera frugiperda infected with Metarhizium (=Nomuraea) rileyi was evident 1 d after white fungal growth was observed. at 7 d post-treatment. Zero mortality in control.

195 Philippine Journal of Science Montecalvo and Navasero: Metarhizium (=Nomuraea) Vol. 150 No. 1, February 2021 rileyi from Spodoptera exigua

Figure 4. Mummification of Spodoptera frugiperda cadavers infected with Metarhizium (=Nomuraea) rileyi: light fungal growth in the ventral surface of the larva (a), profuse white fungal growth (b), sporulation of M. rileyi in the cadaver with greenish fungal growth (c), and sporulation of cadavers in blotter set-up (d).

Table 1. LD estimates and mean lethal time of Metarhizium (=Nomuraea) rileyi isolate against Spodoptera frugiperda. Mean time to LD Larval instar of Spodoptera frugiperda 50 Chi-square Slope larval mortality* (conidia ∙ mL–1) (d) 1st 1.44 x 105 6.318 0.869 4.51–7.51 2nd 5.20 x 105 8.035 0.950 4.90–8.89 3rd 3.17 x 106 13.742 0.857 6.55–8.17 4th 7.89 x 106 25.300 0.868 5.53–8.89 5th 9.36 x 108 25.195 0.415 6.37–8.12 6th 1.20 x 108 2.774 0.308 5.13–7.64 *Range of mean time to larval mortality of different conidial concentrations.

Further incubation of the mummified cadavers in blotter DISCUSSION set-up encouraged the sporulation of M. rileyi. Results on the virulence of M. rileyi isolate from S. exigua Probit analysis of mortality provided LD values corroborate with other studies regarding its bioefficacy (Table 1). Based on the values, increasing conidial against S. frugiperda. M. rileyi is native in maize fields concentrations of M. rileyi infected late larval instars in Cuba and it was successfully isolated from samples from 1.44 x 105 to 1.20 x 108 conidia ∙ mL–1 for 1st–6th of S. frugiperda larvae collected from maize fields in larval instars. The mean time to death varied among the Quivican municipality, Mayabeque province, Cuba different larval instars ranging from 4.51–8.89 d. LT50 (Alvarez et al. 2018) and Mexico (Lezama-Gutierrez et was earliest in 1st larval instar (4.51 d) followed by 2nd al. 2001), suggesting that M. rileyi is naturally occurring larval instar (4.90 d). in the field and could indicate a potential for increasing biological control in cornfields. Bosa et al. (2004) reported the efficacy of M. rileyi isolates in Colombia, causing 73–100% mortality in 2nd instar of S. frugiperda.

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M. rileyi has the ability to cause epizootic in several levels of conidial germination, and rapid ecdyses wherein economically vital lepidopterous pests of different crop conidia were removed before their germ tubes penetrated ecosystems (Ingle 2014). This entomopathogenic fungus the host hemolymph. In addition, chances of fungal infects 60 species of Lepidopteran infesting cotton, infection may be due to loss of inoculum by molting; sunflower, corn, and soybean (Fronza et al. 2017). Several however, this process does not always result in an escape bioassays confirmed its pathogenicity to other Spodoptera from infection (Meekes 2001). species. Montecalvo and Navasero (2020) documented its rd pathogenicity against 3rd larval instar of S. exigua with 73– Similar LD50 values for 3 instar were observed in our 6 –1 100% mortality. Rajan and Muthukrishnan (2009) assayed assay against S. frugiperda (3.17 x 10 conidia ∙ mL ) 6 different M. rileyi isolates against S. litura and discovered with our previous bioassay against S. exigua (1.49 x 10 –1 that the isolates had different degree of virulence against conidia ∙ mL ) (Montecalvo and Navasero 2020). The different instars of S. litura with 73.40–83.40% mortality virulence of our M. rileyi isolate can also be compared in 1st instar, 70.60–76.60% mortality in 2nd instar, and with other bioassays. In the bioassays conducted by Bosa 63.40–70.60% mortality in 3rd instar. The virulence of et al. (2004), an M. rileyi isolate has lethal concentration 3 –1 M. rileyi is attributed to the production of enzymes, (LC) 50 of 9.8 x 10 conidia ∙ mL while the other isolate 4 –1 secondary metabolites, and large amounts of extracellular was 1.2 x 10 conidia ∙ mL . Grijalba et al. (2018) polysaccharide during its growth that may have a role in documented that emulsifiable concentrate of M. rileyi st 4 adhesion to the host cuticle and mummification process against 1 instar of S. frugiperda was 1.17 x 10 conidia –1 (Fronza et al. 2017). ∙ mL under glasshouse conditions. Vimala Devi (1994) 6 –1 recorded an LC50 of 9.3 x 10 conidia ∙ mL of M. rileyi Our observation on the susceptibility of early larval instars against S. litura 2nd instar larvae. Three M. rileyi isolates conformed with the results of Rajan and Muthukrishnan tested by Rajan and Muthukrishnan (2009) recorded st 7 7 (2009) wherein they observed that M. rileyi was highly higher LC50 values for 1 instar (3.18 x 10 to 4.50 x 10 infective to early instars of S. litura than late instars. In conidia ∙ mL–1), 2nd instar (5.15 x 107 to 5.68 x 107 conidia this study, we observed the possible increase in resistance ∙ mL–1), and 3rd instar (15.00 x 107 to 18.27 x 107 conidia –1 to fungal infection as the larvae of S. frugiperda mature. ∙ mL ) of S. litura. Lower LC50 values are desired since Early larval instars were more susceptible than late larval fewer conidia or smaller quantity of formulated products instars, which can be explained by the composition is needed to maintain efficient control of the pest, which of its integument that allowed effective penetration of minimizes production and application costs (Grijalba et al. the fungus and resulted in higher mortality than late 2018). LT50 values also corroborate with previous studies larval instars (Bosa et al. 2004). The possible defense with 4.17–7.83 d for S. exigua (Montecalvo and Navasero mechanism present in the larval stages of insects can 2020). Bosa et al. (2004) recorded 6.2–7 d LT50 for M. be attributed to the cuticular composition when fungal rileyi isolates against 2nd instar S. frugiperda. Fronza et penetration occurred and resistance of the insect to toxic al. (2017) noted that genetic diversity and characteristics metabolites produced by the fungi (Watanabe 1987). of insect cuticle are the main known causes of differences in the pathogenicity of M. rileyi. We also observed that the virulence of M. rileyi was dependent on the conidial concentrations, similar to the observation of Montecalvo and Navasero (2020) in their bioassay of M. rileyi isolate against S. exigua. This finding CONCLUSION also conforms with the results of El-Hawary and Abd El-Salam (2009) and Han et al. (2014) that exposure to M. rileyi from S. exigua was infective to all larval higher conidial concentration leads to shorter lethal time. instars of S. frugiperda, which are related species. Thus, Han et al. (2014) observed that larval mortality due to M. this entomopathogenic fungus is a potential biological anisopliae FT83 and Isaria fumosorosea FG340 differed control agent against the two armyworm species. The significantly at different conidial concentrations with susceptibility of other lepidopterous pests to this M. rileyi increased mortality in S. exigua with increasing conidial isolate and its performance in screenhouse and field should concentration. M. anisopliae was also found pathogenic also be determined. Characterization of the fungal isolate to S. frugiperda (Romero-Arenas et al. 2014). will aid in further product development such as mass production and formulation. Low conidial concentrations could result in less mortality and shorter period for fungal infection, hence a possibility to escape the fungal infection by molting and pupation. Kim and Roberts (2012) noted that fungal infection was evaded by early nymphal stages of cotton aphids due to the low number of conidia attached to their cuticle, low

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