Morphological, biological, and molecular characterization of Type I granuloviruses of Spodoptera frugiperda

doi:10.21203/rs.3.rs-3863960/v1

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Morphological, biological, and molecular characterization of Type I granuloviruses of Spodoptera frugiperda

https://doi.org/10.21203/rs.3.rs-3863960/v1

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published 28 Jun, 2024

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The granuloviruses or GVs (Betabaculovirus) associated with the fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), especially those of Type I, have scarcely been studied but they might represent an alternative for the biocontrol of this insect. In this study, the native granuloviruses SfGV-CH13 and SfGV-CH28 isolated from FAW larvae were characterized for morphology, molecular traits, and insecticidal activity. The elapsed time between symptomatic infection of larvae and stop feeding as well as the weight of larvae before death or prior to pupation were also evaluated. Both granuloviruses isolates showed ovoid shape with a length of 0.4 µm. They showed the same DNA restriction profiles and their genome sizes were about 126 kb. The symptomatic infection with tested GVs mainly caused flaccidity of larva body and discoloration of integument. The integument lysis was only observed in 8% of infected larvae. Infected larvae gradually stopped feeding. Overall, these symptoms are characteristic of infections caused by Type I granuloviruses, which are known as monoorganotropic or slow-killing. The median lethal doses (LD₅₀) values for SfGV-CH13 and SfGV-CH28 isolates were 5.4 × 10² and 1.1 × 10³ OBs/larva, respectively. The median lethal time (LT₅₀) ranged from 17 to 24 d. LT₅₀ values decreased as the viral dose was increased. The elapsed time since symptomatic infection until pupation (LD₅₀) and body weight of larvae (third instar) were higher with SfGV-CH28 than SfGV-CH13. Both granulovirus isolates were able to kill the FAW larvae from the 12th day.

Baculoviridae

Biocontrol

Granulovirus

Pathogenicity

Virulence

Fall Armyworm

Entomopathogenic viruses members of the Baculoviridae family are the most widely distributed and studied worldwide as biocontrol agents due to their high specificity and virulence against some pest insects (Inceoglu et al. 2006; Williams et al. 2023). This family groups four genera, but Betabaculovirus (lepidopteran-specific granulovirus, GV), and Alphabaculovirus (lepidopteran-specific nucleopolyhedrovirus, NPV) are the most common genera of the family found in lepidopteran insects, including those of agricultural interest (Herniou et al. 2011; Jehle et al. 2006).

Betabaculovirus are classified into three types according to their action mode and time required to cause symptomatic infection and death of the host insect (Federici 1997). Type II GVs is the most studied, due to its high virulence, being able to infect several tissues (e.g., fatty body, tracheal matrix, and epidermis) simultaneously, which consequently causes the death of its host in a short time (˂ 6 days) even using low doses of occlusion bodies (OBs, ~2 × 10²OBs/larva) (Sciocco-Cap 2001). The Cydia pomonella GV (CpGV) and Epinotia aporema GV (EpapGV) are examples of Type II GVs that have successfully allowed the biocontrol of the lepidopterans Cydia pomonella L., and Epinotia aporema (Wals.), respectively (Biedma et al. 2015; Jehle et al. 2017). Type I GVs present a slow insecticidal activity due to require a long time (15-37 d, depending on dose and larval instar of host) to cause symptomatic infection or death of host. This type of viruses are known as slow-killing and can cause a horizontal transmission, which has higher efficacy for long-term control of insect pests since they can infect different larval instars, prolongs larval development, causing the kill in final larval instars with the consequent greater number of infective OBs produced (Hatem et al. 2011; Hilton and Winstanley 2008; Takahashi et al. 2015). Type I GVs firstly infect the midgut epithelium and, then, the infection migrates to the fatty tissue of the insect, with this tissue being the most important infection site (Federici 1997; Sciocco-Cap 2001). Other insect tissues are rarely damaged by Type I GVs, allowing the development of the larvae and favoring the replication of the new infective viruses in the insect host. The infections caused by Type I GVs do not generally cause integument lysis but induce flaccidity of insect body (Sciocco-Cap 2001). Type I GVs are recognized as factor of natural mortality for lepidopteran pests despite of their slow insecticidal activity.

Some studies have demonstrated that some types of GVs including Type I can to increase the insecticidal effectiveness of other GVs and NPVs (Espinel-Correal et al. 2012; Shapiro 2000). By enhancin (metalloprotease) protein present in OBs, which digest the peritrophic membrane of the insect and facilitate the access of the virions into intestinal cells (Bivian-Hernández et al. 2017; Ishimwe et al. 2015). Therefore, this mixture significantly alters the development of the symptomatic infection and might be used as new viral bioinsecticides or cocktails with other viruses (Caballero et al. 2001; Cuartas et al. 2014; Haase et al. 2015). Type I GVs and NPVs have even been documented in larval co-infection (Gómez et al. 2010). The granuloviruses of S. frugiperda have scarcely been studied (Cuartas et al. 2014; Ferrelli et al. 2018; Pidre et al. 2019), as compared to S. frugiperda NPVs (García-Banderas et al. 2020; Williams et al. 2023). To date, only three Type I granuloviruses (isolates from Colombia, Brazil, and Argentina) have been associated with S. frugiperda J. E. Smith (Lepidoptera: Noctuidae) (Cuartas et al. 2014; Pidre et al. 2019). This insect is the main pest of corn (maize; Zea mays L.) in Mexico and other countries, causing crop losses exceeding 30% (Blanco et al. 2014; Casmuz et al. 2010). Strategies aiming at mitigating the impact of S. frugiperda include physical and mechanical strategies (e.g., destruction of eggs and neonate larvae and use of pheromone for mass trapping), cultural strategies (e.g., early crop planting, weed removal and use of mineral fertilizers), botanical extracts and the application of broad-spectrum insecticides with being this the most extensively used with satisfactory results in suppressing this insect (Zhang et al. 2021). However, the excessive use of pesticides causes negative impacts such as environmental contamination and insecticide-resistant pest populations (Gutiérrez-Moreno et al. 2019; Hafez et al. 2021; Hussain et al. 2021; Ju et al. 2021). But the increasing demand for environment-friendly strategies call for exploring biological control for the management of this insect. Several insects (parasitoids and predators) and parasitic nematodes have extensively documented as natural enemies of S. frugiperda (Kenis et al. 2022; Ordóñez-García et al. 2015). Microbial biocontrol strategies for S. frugiperda have been mainly focused on the entomopathogenic fungi and bacteria (Kenis et al. 2022; Sagar et al. 2020). Another promising biocontrol strategy is baculoviruses (NPVs and GVs). Nevertheless, GVs are underestimated, especially Type 1 granuloviruses for causing slow killing in its host (Hussain et al. 2021). Thus, the objective of this work was to characterize two Type I granulovirus isolates and determine their insecticidal activity against S. frugiperda larvae.

2.1 Insect rearing and propagation of granuloviruses

The FAW larvae were obtained from a laboratory colony maintained under controlled conditions (26 ± 2 °C, >70% RH, 12:12 L: D) and fed with artificial diet (Southland Products Inc; Lake Village, AR, USA).

Two granulovirus isolates (SfGV-CH13 and SfGV-CH28) were obtained from infected FAW larvae in corn plots from Chihuahua, Mexico (latitude 28°12’44”N, longitude 106°59’45”W, and altitude 2,125 m asl; latitude 28°40’59”N, longitude 106°48’50”W and altitude 2,079 m asl for SfGV-CH13 and SfGV-CH28, respectively). These GVs were found in mixture with NPVs, co-infecting FAW larva. For this reason, the GVs were separated from NPVs by filtration using filter papers (pore sizes of 1.5 and 0.45 µm, respectively) and, then, by sucrose gradients (40 and 66%, w/w) using a gradient former (CBS Scientific, GM 200) according to Muñoz et al. (2001) and Ordóñez‐García et al. (2020). Twenty milliliters of these sucrose solutions were placed into 30 ml polypropylene tubes and then 5 ml of the viral suspension were deposited on the surface of the gradients and centrifuged (40,310 × g, 4°C, 1.5 h). The bands containing OBs were recovered using a Pasteur pipette and placed into 30 ml polypropylene tubes to be washed twice with sterile distilled water (SDW) by centrifugation (40,310 × g, 4°C, 40 min). The purification was confirmed by an optical microscope (Carl Zeiss AxioScope A1; Carl Zeiss, Gottingen, Germany) at 1,000 × magnifications. The OBs obtained from both GV isolates were propagated in fourth instar FAW larvae by the droplet feeding method (Hughes and Wood 1981). Larvae killed by GVs infection were collected and macerated in sterile mortars using SDW containing SDS (1%). The excess of larval cuticle was removed by filtration using muslin and centrifugation (8,500 × g, 4 °C, 10 min). The pellet of OBs was re-suspended in 10 ml of SDW and stored at -80 °C.

2.2 Bioinsecticidal activity

The insecticidal activity of both GV isolates was determined by estimating the median lethal dose (LD₅₀) and the median lethal time (LT₅₀) in third instar FAW larvae using the droplet feeding method (Hughes and Wood 1981). Six viral doses (1 × 10¹, 5 × 10¹, 1 × 10², 1 × 10³, 1 × 10⁴, and 1 × 10⁵ OBs/larva) were tested to estimate the LD₅₀, and three higher doses (1 × 10⁵, 1 × 10⁷, and 5 × 10⁷ OBs/larva) were used to determine the LT₅₀. These doses were selected because they caused a larval mortality greater than 90% (data not shown). Each FAW larva was starved for 12 h and, then, supplied with 0.5 μl of purified viral suspensions (Ordoñez-García et al. 2020). The OBs were mixed with Fluorella blue (0.001%, w/v) and sucrose (10%, w/v) before use. Previously, OBs were disaggregated by sonication (Branson 1510, Connecticut, USA) for 30 s and were counted in triplicate in a Neubauer chamber (Marienfeld, Germany), using a phase-contrast microscope (Carl Zeiss, AxioScope A1; Gottingen, Germany) at 1,000 × magnifications (Muñoz et al. 2001). The infected larvae were individually placed into 29.5 ml plastic cups and fed with artificial diet and maintained under the controlled ambient conditions described above. Both bioassays were performed in triplicate for each viral dose using 25 FAW larvae per replicate and another 25 larvae were used as control group (fed with Fluorella without viral inoculum) per replicate. Only larvae consuming the whole viral inoculum and showing intestinal tract with blue color, as confirmed by observation under a stereomicroscope (Leica G26), were considered in the experiment. Due to long time to cause mortality by this viral genus, the number of larvae killed by the action of tested GVs was recorded every 24 h, or until they reached the pupal stage (Barrera et al. 2011).

Additional study was performed to determine the days required for FAW larvae to reach the pupal stage and maximum weight before pupal stage using the LD₅₀ (5.4 × 10² and 1.1 × 10³ OBs/larva) for the granuloviruses SfGV-CH13 and SfGV-CH28 isolates, respectively, and LD90 (4.3 × 10⁵ and 9.5 × 10⁵ OBs/larva)were used to determine the maximum weight reached by the FAW larvae. Both LD₅₀ and LD₉₀ for both GV isolate were previously determined. From the sixth day post-infection (dpi), the weight of the experimental larvae was recorded every 24 h. The infected larvae were placed into new cups with artificial diet to check if they were still feeding. These bioassays were performed in triplicate, using a total of 25 larvae per replicate and another 25 were used as control larvae (fed with Fluorella without viral inoculum) per replicate following the methodologies and conditions described above.

2.3 Morphological characterization

The granulovirus isolates were identified according to their morphological characters using an optical microscope Carl Zeiss AxioScope A1 at 1000 × magnifications. The morphological characteristics of tested granuloviruses were determined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). For SEM analysis, one drop of the viral suspension was placed on the sample holder, dried, covered with a gold layer (Auto Sputter Coater 108; Cressington Scientific Instruments, Watford, UK), and immediately visualized by a scanning electron microscope FEI Helios Nanolab 600 DualBeam (FEI Company; Hillsboro, OR, USA). At least 50 OBs were considered for the measurement of size. For TEM analysis, the OBs were fixed using a mixture of 2.5% glutaraldehyde and 2% paraformaldehyde, and then, the samples were placed on 1% osmium tetra-oxide. The samples were dehydrated with ethanol and embedded with a resin. Ultrathin sections were cut and observed by a transmission electron microscope JEOL JEM-200CX (JEOL Ltd; Tokyo, Japan) at 80 kV.

2.4 Extraction of virions and viral DNA

The releasing of virions from OBs was performed by mixing the viral suspensions with 1 ml of 0.1 M sodium carbonate (Na₂CO₃), 1 ml of 0.1 M sodium chloride (NaCl) at pH 10.8, and 1 ml of buffer (TE) (0.01 M tromethamine (Tris) hydrochloric acid (HCl), 0.001 M ethylenediaminetetraacetic acid (EDTA) at pH 7.6. The mixture was incubated at 28 °C for 2 h under agitation (140 rpm) and then an equal volume of buffer (TE) (1 ml) was added (Ordóñez‐García et al. 2020). The released virions were purified by continuous sucrose gradients (20 and 66%, w / w) according to Muñoz et al. (2001), with modifications. Briefly, 20 ml of the formed gradient were placed into 30 ml polypropylene tubes, and immediately, 5 ml of the virion suspension were deposited on the surface and were centrifuged (40,310 × g, at 4 ° C, 1.5 h). The bands of virions were collected with a Pasteur pipette and washed twice with SDW using centrifugation (40,310 × g, at 4 °C, 40 min). The pellets with virions were re-suspended in 500 µl of SDW and stored at -20 °C until use.

For the extraction of viral DNA, the virion samples were mixed with 400 µl of buffer of proteinase K (0.01 M Tris, 0.005 M EDTA, 0.5% SDS) and incubated at 65 °C for 15 min. Then, 100 µl of proteinase K (2 mg/ml) (Invitrogen Life Technologies Corp; Carlsbad, CA, USA) were added and the reaction mixture was incubated at 37 °C for 2 h. An aliquot of 500 µl of a mixture phenol: chloroform: isoamyl alcohol (25: 24: 1) was added to the reaction and then it was centrifuged (17,000 × g, at 4 ºC, 5 min). The aqueous phase was collected in a new microtube, and a volume of isopropyl alcohol (500 µl) and 100 µl of 3M sodium acetate were added to the samples previous to be incubated at –20 ° C for 2 h. The mixture was centrifuged (17,000 × g, 4 ºC, 10 min) and the pellet was washed with 70% ethyl alcohol using centrifugation (17,000 × g, 4°C, 5 min). The pellet was re-suspended in 30 µl of sterile double distilled water (ddH₂O). The quality of viral DNA was examined by electrophoresis on 1% agarose gels. The DNA concentration was determined by a A260 NanoDrop One spectrophotometer (Thermo Fisher Scientific; MA, USA).

2.5 Restriction endonuclease analysis

Both GV isolates were digested with HindIII, BamHI, and PstI enzymes (Invitrogen Life Technologies Corp; Carlsbad, CA, USA). One microgram of viral DNA was digested with 10 U of the enzymes, at 37 °C for 2 h. The reaction was stopped by adding 2 µl of loading buffer 10X (Thermo-Fisher Scientific; Waltham, MA, USA). The obtained restriction fragments were analyzed by electrophoresis on 1% agarose gels at 25 V for 7 h, using TAE buffer (40 mM Tris-acetate, 1 mM EDTA at pH 8.0). A molecular weight marker of GeneRuler 1 kb DNA Ladder (Thermo Fisher Scientific) and 10 µl of SYBR Safe DNA gel stain (Invitrogen) were used to visualize the DNA on agarose gels using the image system (Bio-Rad ChemiDoc^TMXRS⁺; Hercules, CA, USA). The fragment sizes and number of both GVs isolates were estimated by comparing the bands with those of the molecular weight marker using the Image Lab software version 5.2.1 (Bio-Rad ChemiDoc^TMXRS⁺; Hercules, CA, USA).

2.6 Statistical analysis

The bioassays were conducted under a completely randomized design. The data on insecticidal activity were analyzed by an analysis of variance (ANOVA), and the means were separated by a Tukey´s test (p < 0.05). The LD₅₀, LD₉₀, LT₅₀ and fiducial limit values were analyzed using log-Probit regressions (Finney 1971). All data were analyzed using SAS software (SAS 2002). Mortality was corrected by the Abbott (1925) formula.

3.1 Insecticidal activity

Both granulovirus isolates caused significant larval mortality at four of the six tested doses (Fig. 1). In the larvae used as controls, not mortality was recorded. Larval mortality caused by the highest dose (1 × 10⁵ OBs/larva) of both GVs started on 12 dpi and on 15 dpi with the lowest dose (1 × 10¹ OBs/larva). The death of larvae ended on 45 dpi (data not shown). Both isolates caused the same infection symptoms (Fig. 2). GV-infected larvae gradually stopped feeding and their bodies become flabby. The integument lysis was only observed in 8% of infected larvae, which showed fatty body shedding after 20 dpi and remained alive in this condition for at least two days (Figs. 2b and 2d). These larvae also showed swelling and an atypical milky whitish color on their cuticle, with this color being quite different to that of control larvae (Fig. 2).

The estimated LD₅₀ for the SfGV-CH13 and SfGV-CH28 isolates were 5.4 × 10²and 1.1 × 10³ OBs/larva, respectively (Table 1). The LT₅₀ decreased as the viral dose was increased. LT₅₀ of 414.7 h (=17.3 d) and 476.2 (=19.8 d) were obtained with the highest dose (5 × 10⁷ OBs/larva) of SfGV-CH13 and SfGV-CH28, respectively. The lowest dose (1 × 10⁵ OBs/larva) of these isolates led to LT₅₀ of 570.3 h (=23.8 d) and 591.8 h (=24.8 d) (Table 2). The LD₅₀ and LD₉₀ of both GVs caused a delay in larval development. The maximum body weight prior to pupation and the time required by the larvae to reach pupation after infection with LD₅₀depended on isolate, with SfGV-CH28 isolate causing the highest changes in these response variables (Table 3). The times required to reach the pupal stage by control larvae and larvae treated with LD₅₀ of the SfGV-CH13 isolate were 15.17 and 15.26 days, respectively. The body weight of these larvae averaged 0.51 and 0.47 g/larva, respectively. The larvae treated with the SfGV-CH28 isolate required 2.5 days more to reach pupation and their body weight was up to 0.1 g higher than those of the other experimental groups.

The infection with tested isolates at LD₉₀ reduced the body weight of FAW larvae, according to the measurements carried out at 6, 10 and 15 dpi (Fig. 3). However, the body weight of larvae at 25 dpi was similar for all experimental groups (Fig. 3). Nevertheless, 20% of larvae infected with the SfGV-CH13 isolate showed a body weight ≥ 0.60 g between 19 and 27 dpi and 28% of the larvae infected with both isolates stopped feeding for a period of 3 to 6 days, after 14 dpi (data no shown).

3.2 Morphological characterization

OBs of both isolates showed a homogeneous ovoid shape. The length of OBs ranged from 0.36 to 0.49 µm for the SfGV-CH13 isolate and from 0.37 to 0.45 µm for the SfGV-CH28 isolate, with an average length of 0.4 µm for both GVs isolates (Figs. 4a and 4c). Both isolates showed a single virion (~0.3 µm) per OB (Figs. 4b and 4d).

3.3 Molecular characterization

Both isolates showed the same DNA restriction profiles. Thirteen, 14, and 16 restriction fragments were observed with the enzymes HindIII, BamHI, and PstI, respectively (Fig. 5 and Table 4). The DNA size for tested isolates was about 126 kb.

Tested isolates killed FAW larvae since 12^th dpi. Similar times for starting of death for insects infected by Type I GVs (7-14 dpi) have already been reported (Inceoglu et al. 2001). In our study, the larvae died from 12 to 45 dpi, with this time range being higher than that reported for nucleopolyhedroviruses (3-8 dpi) infecting the FAW larvae (Barrera et al. 2011; Ordóñez‐García et al. 2020). The data for insecticidal activity of granulovirus isolates and the symptoms observed in the infected larvae of this study demonstrated that both tested isolates were Type I, causing a monorganotropic infection and an slow kill of the insect, similar to documented by Alletti et al. (2017) who observed similar symptoms in Agrotis segetum Schiff. larvae infected with AgseGV. The symptomatic infection caused by tested isolates was similar to those documented for this type of GVs, with such symptoms differing significantly from those observed in insects infected with NPVs (Barrera et al. 2011; Ordóñez‐García et al. 2020; Pidre et al. 2019). The symptoms commonly observed in larvae infected with GVs include the cessation of feeding, larvae swelling, little or no liquefaction of insect body, and darkening of insect body (Sauer et al. 2017; Wang et al. 2008).

Tested GVs did not cause liquefaction of larvae body, probably due to tested the isolates did not infect the epidermis, with this preventing the deposition of chitinase on the peritrophic membrane (Sciocco-Cap 2001). Rohrmann (2019) observed that some NPVs contained gp37, a chitinase favoring the fusion of virions with cells of the middle intestine. The creamy-yellow appearance observed on larvae infected with tested GV isolates can be attributed to the accumulation of high quantities of OBs on the fatty body tissues of larvae (Sciocco-Cap 2001). This fatty tissue completely detached from the rest of the larva body in 8% of the larvae infected with tested GVs. Pidre et al. (2019) also observed severe lesions in the last abdominal segments (fatty tissue) of FAW larvae infected with a granulovirus isolate from Argentina. The isolate SfGV-CH13 was the one that showed the lowest LT₅₀ with the three doses tested against S. frugiperda larvae.

Cuartas et al. (2014) reported values of 4.5 × 10⁵ and 1.6 × 10⁵ OBs/mL in LC₅₀ for Brazilian (VG008) and Colombian (VG014) GVs isolates from S. frugiperda with mean times to death (MTD) of 29 (= 694 h) and 33 d (=792 h), respectively. Although the results obtained by Cuartas et al. (2014) in terms of LC₅₀ are not comparable with ours (LD₅₀), they coincide in terms of the longer time required by this type of granulovirus to kill S. frugiperda larvae. Hackett et al. (2000) observed shorter survival times (367 h - 439 h) for Helicoverpa armigera larvae infected with a H. armigera Type I granulovirus (HearGV) that those observed in our study. The low rate of larval death could be related to the tropism of Type I GVs, which only infect the midgut and fatty body of the host insect, as compared to Type II GVs and the NPVs that infect many tissues and cause a rapid death of the host (Sciocco-Cap 2001). Kumar et al. (2017) have pointed out that type I GVs are characterized by having a slow speed of kill in their hosts compared to type II GVs, even suggesting the need for a higher LD₅₀ (up to 100 times more) in type I GVs. Some insects can also delay the infection of Type 1 GVs as a first defense mechanism, blocking viral replication in cells during the early stages of infection (Hinsberger et al. 2019; Pauli et al. 2018; Wang et al. 2008). The larvae infected with the SfGV-CH28 isolate required more time to reach the pupal stage and showed a slower weight gain rate than those of the other experimental groups. Wang et al. (2008) observed that infected larvae can live more and be bigger than uninfected larvae. However, larvae infected at LD₉₀ of both isolates showed a lower weight at 15 dpi than control larvae, with this indicating that a lower viral dose (LD₅₀) can increase the survival time and body weight prior to death as compared to the highest viral dose (LD₉₀). Machado et al. (2020) founded that the infection-mediated lengthening of the developmental process of larvae, involving more time to reach pupal or adulthood stages, is favorable for the management of insect pests in the field, because the larvae remain exposed to their natural enemies for a longer time, increasing the chances that they will be parasitized, preyed on or infected by microbial biocontrol agents. This gives an advantage to the use of type I GVs. In addition to the fact that the larvae infected by this type of GVs stop feeding, therefore, they no longer cause more damage to the crop and produce a large number of new infective OBs ready to infect new healthy larvae, especially in S. frugiperda, which is characterized by overlapping generations (e.g., larvae of all stages), at the same phenological stage of the crop. Furthermore, it has been demonstrated that SfGV enhance infection caused by SfNPV (Cuartas-Otálora et al. 2019; Ferrelli et al. 2018; Hussain et al. 2021).

The ovoid shape, size, and the number of virions observed for tested OBs were similar to those observed for other OBs of lepidopteran granuloviruses (Barrera et al. 2014; Ikeda et al. 2015; Luque et al. 2001; Moscardi 1999). The size of tested GVs was into the range (0.3 × 0.5 µm) observed by Ikeda et al. (2015). It was also similar to that (0.43 µm) of S. frugiperda granulovirus (SfGV) (Cuartas et al. 2014). However, other sizes have been reported. Wang et al. (2008) and Barrera et al. (2014) observed sizes of 0.24-0.34 and 0.30 µm for Spodoptera litura granulovirus (SpltGV) and Erinnyis ello granulovirus (EeGV), respectively.

Restriction enzyme analysis allowed the molecular identification of tested isolates, which showed the same number and positions of the fragments after digestion with the enzymes HindIII, BamHI, and PstI (Fig. 5). This similarity might be related to the geographical origins of the isolates, which was similar for them. Barrera et al. (2014) did not observe differences in the restriction profiles of three granulovirus isolates due to they were genotypic variants of the same viral strain. On the other hand, Cuartas et al. (2014) found differences in the restriction fragments of S. frugiperda VG008 and VG014 granuloviruses from Colombia and Brazil. Ordóñez‐García et al. (2020) also observed small differences in the restriction patterns (HindIII and BamHI enzymes) of SfCH15 and SfCH32 NPV isolates, both obtained from nearby area in Mexico. However, tested isolates showed different virulence despite of their molecular similarity, suggesting that they were possibly different genotypes. Ali et al. (2018) stated that the genotypes of viruses differ in dose response and time required to kill the host (biological activity), as observed in our study. Tested isolates showed significant differences in mortality percentages at some doses, as well as at the LD₅₀ and LT₅₀, with SfGV-CH13 isolate being more virulent than the SfGV-CH28 isolate. The genome sizes for tested isolates were about ~ 126 kb. This size was smaller to those estimated by Cuartas et al. (2014) for VG014 (132.6 kb) and by Cuartas et al. (2015) for VG008 isolate with 140.9 kb. Pidre et al. (2019) estimated a size of at least 135 kb for an Argentinian isolate of S. frugiperda granulovirus. However, complete genome sequencing of GV isolates is necessary to obtain more accurate values.

In conclusion, based on insecticidal activity the two S. frugiperda granulovirus isolates against FAW larvae and symptoms induced in infected larvae, both isolates belong to Type I GVs known as slow-killing. They killed more than 90% of S. frugiperda larvae at 45 dpi at a dose of 1.0 × 10⁵ OBs/larva. Both isolates were genetically identical, according to their DNA restriction profiles. The LD₉₀extended two times the larval development time of S. frugiperda. The LD₅₀ values obtained with both GVs were similar to those reported for NPVs, however, their LT₅₀were lower than those previously reported for other S. frugiperda granulovirus and SfNPVs. The results suggest that both granulovirus isolates might be considered for use as biocontrol agents against S. frugiperda despite their slow insecticidal activity.

Funding:

This research was supported by the Secretaría de Agricultura, Ganadería, Desarrollo Rural, Pesca y Alimentación (SAGARPA-COFUPRO, México; No. CH1600001442).

Conflict of interest/ Competing interests:

The authors declare no conflict of interest

Availability of data and material:

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Code availability:

Not applicable.

Authors' contributions:

All authors contributed to the study conception and design. MOG designed research, conducted experiments and wrote the manuscript, JCBR conducted experiments, JJOP wrote and edited the manuscript, CHAM and MASM analyzed data and edited the manuscript, OJCC, MOEV interpreted data and edited the manuscript, MAMO interpreted data and conducted research, CRV conceived research and wrote and edited the manuscript. All authors read and approved the manuscript.

Acknowledgements

Magali Ordóñez García thanks the Consejo Nacional de Ciencia y Tecnología (CONACYT–México) for the provided PhD scholarship.

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Table 1. Median lethal dose (LD₅₀) and lethal dose ninety of tested Type I granulovirus isolates against third instar Spodoptera frugiperda larvae

Dose tested	Isolate	Dose obtained	Fiducial limits (95%)		χ²	df	Slope ± (SE)	Intercept ± (SE)
Dose tested	Isolate	Dose obtained	Lower	Upper	χ²	df	Slope ± (SE)	Intercept ± (SE)
LD₅₀	SfGV-CH13	5.4 × 10²	3.1 × 10²	9.5 × 10²	4.5	4	0.4 ± 0.04	-1.2 ± 0.13
	SfGV-CH28	1.0 × 10³	6.0 × 10²	2.0 × 10³	1.1	4	0.4 ± 0.08	-1.3 ± 0.12
LD₉₀	SfGV-CH13	4.3 × 10⁵	1.3 × 10⁵	2.2 × 10⁶	4.5	4	0.4 ± 0.04	-1.2 ± 0.13
	SfGV-CH28	9.5 × 10⁵	2.7 × 10⁵	5.8 × 10⁶	1.1	4	0.4 ± 0.08	-1.3 ± 0.12

LD₅₀: Median lethal dose expressed as OBs/larva. These values were obtained from a minimum of 6 doses (treatments) and estimated at 45 days post-infection (dpi).

Probit regressions were fitted using SAS program.

χ² = Goodness of fit test.

df = degrees of freedom

SE = Standard error

Table 2. Median lethal time (LT₅₀) of the doses of Type I granulovirus isolates tested against third instar Spodoptera frugiperda larvae.

Isolate	Doses (OBs/larva)	LT₅₀ (h)^a	Fiducial limits (95%)
Isolate	Doses (OBs/larva)	LT₅₀ (h)^a	Lower	Upper
SfGV-CH13	1 ×10⁵	570.3	555.9	585.1
	1 ×10⁷	502.2	489.9	514.5
	5 ×10⁷	414.7	403.6	425.9
SfGV-CH28	1 ×10⁵	591.8	573.2	611.0
	1 ×10⁷	540.3	522.0	558.4
	5 ×10⁷	476.2	455.9	495.7

Data correspond to the average of three replicates. ^aLT₅₀ Median Lethal Time expressed in hours

Table 3. Development of Spodoptera frugiperda larvae treated with the median lethal dose of two native Type I granuloviruses.

Isolate	Days to reach the pupal stage ± (SE)	Maximum weight (g) before pupal stage ± (SE)


SfGV-CH13	15.17 ± 0.09b	0.51 ± 0.02b
SfGV-CH28	17.82 ± 0.40a	0.62 ± 0.01a
Control	15.26 ± 0.05b	0.47 ± 0.01b

All values are arithmetic means ± SE. Values in the same column with the same letter are not statistically different according to Tukey's test (p < 0.05). SE = Standard error

Table 4. Molecular sizes of HindIII, BamHI and PstI restriction endonuclease fragments from the SfGV-CH13 and SfGV-CH28 granulovirus isolates.

Fragment	SfGV-CH13			SfGV-CH28
	Restriction size fragments
	**HindIII**	*Bam*HI	*Pst*I	**HindIII**	*Bam*HI	*Pst*I
1	20,000	20,000	20,000	20,000	20,000	20,000
2	20,000	20,000	20,000	20,000	20,000	20,000
3	19,200	14,142	15,344	20,000	14,433	16,311
4	17,340	12,871	10,416	18,434	13,035	11,073
5	14,433	11,772	9,486	15,344	11,772	9,852
6	8,774	8,879	8,879	8,985	9,093	9,312
7	6,092	7,084	7,099	6,071	7,168	6,798
8	4,893	6,602	6,602	5,026	6,651	5,873
9	4,237	5,916	5,798	4,268	5,996	4,858
10	3,802	5,111	5,621	3,781	5,083	4,438
11	3,283	3,802	4,754	3,486	3,889	4,213
12	2,376	3,147	3,553	2,405	3,202	3,634
13	2,031	2,970	2,795	2,125	3,086	2,823
14		2,852	2,180		2,866	2,247
15			1,702			1,719
16			1,461			1,468
Total (pb)	126,463	125,147	125,689	129,923	126,273	124,620

Fragment sizes were estimated by comparing the bands with those of the molecular weight marker using the Image Lab software version 5.2.1 (Bio-Rad ChemiDoc^TMXRS⁺; Hercules, CA, USA).

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Morphological, biological, and molecular characterization of Type I granuloviruses of Spodoptera frugiperda

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Abstract

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Introduction

Materials And Methods

2.1 Insect rearing and propagation of granuloviruses

2.2 Bioinsecticidal activity

2.3 Morphological characterization

2.4 Extraction of virions and viral DNA

2.5 Restriction endonuclease analysis

2.6 Statistical analysis

Results

3.1 Insecticidal activity

3.2 Morphological characterization

3.3 Molecular characterization

Discussion

Declarations

References

Tables

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