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Effectiveness of Entomopathogenic Fungi on Immature Stages and Feeding Performance of Fall Armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae) Larvae

Simple Summary Fall armyworm (FAW), primarily endemic to the United States, has posed a severe threat to maize cultivation globally in recent decades. To prevent maize from being harmed by FAW, various control strategies are used, including synthetic pesticides. Synthetic chemicals are still the most effective and widely utilized technique; nonetheless, these chemicals are hazardous to humans, biodiversity, and the environment, necessitating a desperate search for safe and long-term solutions. Entomopathogenic fungi (EPFs) are thought to be an essential alternate control tool for this invasive pest. The goal of this study was to determine the effectiveness of five entomopathogenic fungal isolates (Aspergillus sp. BM-3 and SE-2-1, Cladosporium tenuissimum SE-10, Penicillium citrinum CTD-24, and Beauveria bassiana ZK-5) against immature stages (eggs, neonates, and larvae) and feeding efficacy of first to sixth instar S. frugiperda larvae at 1 × 106, 1 × 107, and 1 × 108 conidia mL−1. Among the five tested fungal isolates, C. tenuissimum SE-10, P. citrinum CTD-24, and B. bassiana ZK-5 showed significant effects on egg mortality and significantly reduced the early third instar feeding efficacy of FAW larvae at the highest concentration level. These potent fungal isolates could be suitable candidates for developing biopesticides in an integrated manner to control the FAW population. By decreasing the hatchability of eggs and reducing the feeding ability of early first to third instar FAW larvae, the findings of this study could assist in managing this invasive pest in China and enhance maize crop output. However, further research is needed to evaluate and validate laboratory outcomes in real-world situations. Abstract Maize is a major staple crop in China, and the sustainable productivity of this primary crop has been recently threatened by fall armyworm (FAW), Spodoptera frugiperda, invasion. The five fungal isolates, Aspergillus sp. BM-3 and SE-2-1, Cladosporium tenuissimum SE-10, Penicillium citrinum CTD-24, and Beauveria bassiana ZK-5 were assessed for their efficacy in causing mortality against first to sixth instar eggs and neonate larvae seven days post-treatment, and their effects on the feeding performance of sixth instar S. frugiperda larvae at 48 h post-treatment at three concentrations (1 × 106, 1 × 107, and 1 × 108 conidia mL−1) were also assessed. The six instar S. frugiperda larvae were not susceptible to the five tested fungal isolates. However, B. bassiana ZK-5 caused the highest egg mortality of 40, 70, and 85.6% at 1 × 106, 1 × 107, and 1 × 108 conidia mL−1, respectively, followed by P. citrinum CTD-24 (30.6, 50, and 75.6%) and C. tenuissimum SE-10 (25.6, 40, and 55.6%). In addition, B. bassiana ZK-5 caused the highest neonate mortality of 54.3% at 1 × 108 conidia mL−1. B. bassiana ZK-5 and P. citrinum CTD-24 caused cumulative mortality, including 93.3 and 83.3% mortality of eggs and neonates, respectively, at 1 × 108 conidia mL−1. Furthermore, B. bassiana ZK-5 reduced the feeding efficacy of first to third instar S. frugiperda larvae by 66.7 to 78.6%, while P. citrinum CTD-24 and C. tenuissimum SE-10 reduced larval feeding by 48.3 to 57.1% at 1 × 108 conidia mL−1. However, these fungal isolates were less potent in reducing the feeding activity of fourth to sixth instar S. frugiperda larvae (>46% with B. bassiana at 48 h post-treatment). The tested fungal isolates could play an essential role as microbial biopesticides in suppressing the S. frugiperda population in China after further investigations on their efficacy are obtained in the field.


Introduction
Maize is a significant crop in China, especially since the country has become a net importer of grain [1]. China is the world's second-largest corn producer after the U.S. However, maize production has been threatened by fall armyworm (FAW), Spodoptera frugiperda, J.E. Smith, 1797, (Lepidoptera: Noctuidae), which is highly ranked as one of the most destructive pests of the maize crop. This pest is native to the American continent, including tropical and subtropical regions [2], and has recently invaded Africa, Asia, and Australia with a highly detrimental impact on global food security in all invaded areas. FAW was first reported in West Africa (Nigeria and Ghana) [3] and later spread to other countries in sub-Saharan Africa [4]. In May 2018, FAW was observed in Karnataka in the southwestern part of India [5], and its occurrence was also reported in southeastern Asian countries, such as Thailand, Bangladesh, and Myanmar, by late 2018 [6].
FAW was first reported in China on 11 December 2018 [7], when the corn-specific strain of S. frugiperda invaded [8]. Spodoptera frugiperda invaded 26 provinces after migrating to China from Myanmar, and Guangxi and Yunnan were the major areas invaded by the pest [9]. The larvae of S. frugiperda have the potential to damage over 350 species of plants, including major staple and economic crops such as sorghum, corn, barley, rice, soybean, tobacco, tomato, and peanut [10], but show a high preference for corn, sugarcane, and sorghum in China. During the early attack of S. frugiperda larvae in 2019, extremely significant damage was caused to cornfields in Yunnan Province, China [11]. Spodoptera frugiperda was reported to cause 67 and 22% maize yield losses in Zambia and Ghana, respectively [12], while in Kenya and Ethiopia, this invasive pest caused maize yield losses of 47 and 32%, respectively [13], resulting in economic losses of 1.08 and 4.66 billion USD in Africa [14]. Spodoptera frugiperda larvae were also reported to feed on growing points and young organs in the peanut field and cause 78 and 65% damage at seedling and flowering stages, respectively [15]. Spodoptera frugiperda causes serious damage (30 to 90%) in wheat fields, while 10 to 80% damage was recorded in the barley field [16].
This invasive pest can potentially cause damage to tobacco fields if its population reaches its peak [17]. Therefore, many studies have focused on developing biopesticides as a component of integrated pest management (IPM) against insect pests, to minimize damage and increase the productivity of crops [18][19][20][21][22][23]. However, continuous applications of synthetic insecticides by farmers to control S. frugiperda have become a common practice, which is not only uneconomical but also harmful to the environment and natural ene-mies [24,25] and consequently leads to the development of pest resistance. Some pesticides, e.g., methomyl, endosulfan, methyl parathion, and lindane, have been used for S. frugiperda management and are known to be highly hazardous [26,27], and endosulfan and lindane are banned in many countries [28]. Moreover, pesticides are not safe for farmers due to their toxicity, and the majority of farmers have little information or knowledge on the precautionary measures to take during the application of these insecticides in the field. Hence, there is a strong need to develop alternative management methods to synthetic insecticides that are safe, environmentally friendly, and economical, to sustainably control S. frugiperda.
Entomopathogenic fungi (EPFs) have been recognized as comprehensive biopesticides in the management of many destructive pests, and could also be integrated as a key component of IPM strategies for FAW control [29,30] as they were found to be an efficient control practice against a variety of insect pests [31,32]. One of the perceived disadvantages of EPFs is that they do not cause quick mortality (killing speed) to insects, similar to their counterpart synthetic insecticides, due to their different modes of action, therefore insects continue to feed and cause damage to crops [33,34]. However, it is important to note that these EPFs help to minimize damage to crops (below the economic threshold) by inducing infection of host pests, which ultimately leads to a reduction in feeding, oviposition, development, mating, and other physiological traits of insects [35]. A recent study revealed that some Metarhizium anisopliae isolates showed significant mortality to S. frugiperda early immature stages [36].
However, there is little evidence on the efficiency of locally available EPFs in China that are associated with eggs and can affect the feeding performance of S. frugiperda larvae. Hence, the objective of this work was to find effective fungal isolates that can disrupt the eggs and feeding of S. frugiperda, to develop microbial-based biopesticides for use against this notorious invasive lepidopteran pest in China and abroad.

Insect Culture
Spodoptera frugiperda eggs were obtained from the already established colony at the Institute of Zoology, Guangdong Academy of Sciences, Guangdong, Guangzhou, China. Eggs were placed in a perforated rectangular plastic box (28 × 17 × 18 cm 3 ) and monitored daily until hatching. After emergence, neonate larvae were given tender and fresh maize leaves. First to third instar larvae were supplied with fresh maize leaves daily in a perforated rectangular plastic box (28 × 17 × 18 cm 3 ), while fourth to sixth instar larvae were individually placed in wells in a six-well plate to avoid cannibalism until pupation. Male and female pupae (50:50) were collected and placed in a perforated rectangular plastic box (28 × 17 × 18 cm 3 ) until adult emergence.
The newly emerged adults were transferred into adult glass cylindrical cages. The upper portion of the adult cylindrical glass cage was covered with a paper towel, while the inner wall of the adult cage was covered with white paper serving as an oviposition medium. Sterile cotton balls were placed in a plastic bottle lid soaked with 10% honey solution, and then the lid was placed at the bottom of the adult cage as a food source. The paper towel, white paper, and sterile cotton ball were replaced every 24 h to ensure homogenous egg ages. Insects were reared as described above until a sufficient population was achieved to conduct the experiment. Tenth-generation, laboratory-reared larvae were used for the present study. Spodoptera frugiperda colonies were maintained in a rearing room at 25 ± 2 • C, 50-70% relative humidity (RH), and an 18:6 (Dark:Light) photoperiod at the Institute of Zoology, Guangdong Academy of Sciences.

Fungal Isolates
Five fungal isolates belonging to four different genera, Aspergillus sp. (BM-3 and SE-2-1), Cladosporium tenuissimum SE-10, Penicillium citrinum CTD-24, and Beauveria bassiana ZK-5, were assessed against eggs and sixth instar, S. frugiperda larvae in terms of feeding performance. Information regarding their origin, host source, and year of isolation is described in Table 1. All fungal isolates were obtained from the Institute of Zoology, Guangdong Academy of Sciences, laboratory repository. All fungal isolates were cultured on potato dextrose agar (PDA) and maintained at 25 ± 2 • C in the dark. Fungal conidia were harvested from 2-to 3-week-old sporulated cultures and suspended in 10 mL of distilled water with 0.05% Tween-80 in universal bottles containing glass beads (6-9 beads 3 mm in diameter per bottle). The fungal conidial suspension was vortexed for 5 min at approximately 700 rpm to break the conidial clumps and ensure a homogeneous suspension. Conidial concentrations were quantified using a hemocytometer. Before the bioassay, the conidial suspensions were adjusted to different concentrations of 1 × 10 6 , 1 × 10 7 , and 1 × 10 8 conidia mL −1 through serial dilution.
Viability tests were conducted before experiments, where a concentration of 3 × 10 6 conidia mL −1 was prepared, 0.1 mL of the suspension was evenly spread on a PDA plate, and three sterile microscope coverslips were placed randomly on the surface of each inoculated plate. Each plate represented a replicate, and four replicates were performed for each isolate. The plates were sealed with parafilm and incubated under complete darkness at 25 ± 2 • C. Conidial germination was assessed after 18 h by counting 100 randomly selected conidia beneath each coverslip, under a light microscope [37,38]. Conidia were considered germinated when the length of the germ tube was at least twice the diameter of the conidium [39,40]. The viability of each of the fungal isolates was assessed (Table 2).

Effect of Fungal Isolates on Sixth Instar S. frugiperda Larvae
All five fungal isolates were assessed against sixth instar S. frugiperda larvae obtained from an already established insect colony, as described in Section 2.1 above. Fresh maize leaves of similar size and age, approximately 2-3 weeks old, were cut and placed individually in a perforated rectangular plastic box (28 × 17 × 18 cm 3 ). A cohort of 20 first instar S. frugiperda larvae was transferred onto maize leaves in each rectangular plastic box using a soft camel hairbrush. Each group of first instar larvae in a rectangular plastic box represented a replicate, and triplicate groups were analyzed for each isolate. The cut leaves with larvae in each rectangular plastic box were then sprayed with 10 mL of 1 × 10 6 , 1 × 10 7 , or 1 × 10 8 conidia mL −1 suspension using a spray tower. All the larvae were effectively exposed to the fungal spore suspension to prevent larval escape to the inoculum by going beneath the leaves. A sterilized paper towel was placed under the leaves to absorb the excess fungal suspension before applying the spray. The control was treated with sterile distilled water containing 0.05% Tween-80. The rectangular plastic boxes containing treated larvae were incubated at 25 ± 2 • C. Fresh maize leaves (surface-sterilized and untreated) were provided to the larvae daily as food. Larval mortality was recorded daily for 1 week [38]. All the treatments were arranged in a completely randomized design (CRD), with each treatment conducted in triplicate. A mycosis test was conducted following the approach of [36] to confirm larval mortality due to infection by the treated fungus. The same procedure was followed for second to sixth instar S. frugiperda larvae, while fourth to sixth instar larvae were placed individually in a six-well plate before treatment, to avoid cannibalism.

Effect of Fungal Isolates on Eggs and Neonate Larvae of S. frugiperda
The eggs (1 to 2 days old) of S. frugiperda laid on paper towels and white paper, as described in Section 2.1 above, were collected from the adult cages. A cohort of 30 eggs per butter paper was separated under the microscope by removing excess eggs with a camel hairbrush before treatments. The cohort of eggs was treated with 10 mL of 1 × 10 6 , 1 × 10 7 , and 1 × 10 8 conidia mL −1 suspensions of each fungal isolate by a spray tower, where a sterilized paper towel was placed under the butter paper to absorb the excess spore suspension. The controls were treated with sterile distilled water containing 0.05% Tween-80. After treatment, eggs were air-dried for 1 h under a laminar hood at room temperature (25 ± 2 • C), transferred into Petri dishes, and incubated at 25 ± 2 • C and an approximate RH of 70%.
All the treatments were arranged in a CRD with three replications per fungal isolate and concentration. The numbers of hatched and non-hatched eggs were recorded daily until 7 days after incubation. The neonate larvae that emerged from the treated egg cohort were counted, fed fresh maize leaves (approximately 2-3 weeks old, surface-sterilized, and untreated) in a perforated rectangular plastic box (28 × 17 × 18 cm 3 ) lined with moist filter paper, incubated at 25 ± 2 • C, and monitored daily. Neonate mortality due to fungal infection was recorded daily for 7 days postemergence, and a mycosis test was also conducted for the dead neonates, as described in Section 2.3 above.

Effect of Fungal Isolates on Feeding Performance of Sixth Instar S. frugiperda Larvae
Fresh maize leaves were collected separately and placed in a perforated rectangular plastic box (28 × 17 × 18 cm 3 ). The collected leaves were cut and weighed to the required quantity (45-60 g of maize leaves can feed approximately 20 to 25 larvae for 2-3 days, from our S. frugiperda-rearing experience). The leaves were placed in a rectangular box. The larvae were treated with each fungal isolate with 10 mL of 1 × 10 6 , 1 × 10 7 , or 1 × 10 8 conidia mL −1 suspension using a spray tower [36,41]. Twenty-five first instar S. frugiperda larvae were released in a rectangular plastic box containing treated leaves. A quantity of 7, 12, 20, 30, 45, and 60 g of maize leaves were provided daily to first, second, third, fourth, fifth, and sixth instar larvae, respectively. Larvae treated with sterile distilled water containing 0.05% Tween-80 were considered controls. The treatments were laid out in a CRD with triplicate samples. The insect diet (weighed maize leaves) was changed every second day post-treatment. The leaves were weighed before and after exposure to fungal isolates to assess the feeding performance of S. frugiperda larvae.

Statistical Analysis
Egg and larval mortality data were corrected using Abbott's formula [42] to correct for natural mortality, and their normality was tested using the Shapiro-Wilk test [43] before being subjected to one-way analyses of variance (ANOVA). In the case where the data were not normally distributed, they were further arcsine transformed before analysis. Whenever treatments were significantly different (p < 0.05), the means were separated using the Tukey test. All data analyses, including a reduction in the feeding of sixth instar S. frugiperda larvae, were performed using SPSS (version 16.0), and letters were calculated using Statistix 8.1 software.

Effect of Fungal Isolates on Sixth Instar of S. frugiperda Larvae
The viability of the five tested fungal isolates was >91% conidial germination after 18 h of incubation at 25 ± 2 • C, as shown in Table 2, with no significant differences among the isolates.
No statistically significant difference was observed among treatments and control across instars, and each treatment across instars within same concentration at p < 0.05. (Tukey after one-way ANOVA).

Effect of Fungal Isolates on Neonate Larvae of S. frugiperda
In addition to egg mortality, the fungal isolates induced additional mortality of the neonate larvae that emerged from the fungus-treated S. frugiperda eggs compared to the control 7 days postemergence. The neonate larvae were found to be susceptible to all the fungal isolates at 1 × 10 6 conidia mL −1 (F = 5.08; df = 20; p < 0.0058), except B. bassiana ZK-5, which caused only 10% neonate mortality compared to that of the control (0.0%) ( Figure  2; Table 4).

Effect of Fungal Isolates on Neonate Larvae of S. frugiperda
In addition to egg mortality, the fungal isolates induced additional mortality of the neonate larvae that emerged from the fungus-treated S. frugiperda eggs compared to the control 7 days postemergence. The neonate larvae were found to be susceptible to all the fungal isolates at 1 × 10 6 conidia mL −1 (F = 5.08; df = 20; p < 0.0058), except B. bassiana ZK-5, which caused only 10% neonate mortality compared to that of the control (0.0%) ( Figure 2; Table 4).     At a concentration of 1×10 7 conidia mL −1 , B. bassiana ZK-5 and P. citrinum CTD-24 isolates caused higher neonate mortalities of 25.9 and 15.6%, respectively (F = 3.81; df = 20; p < 0.0185), than the 0% in the control. However, no significant differences in mortality were observed among other fungal isolates compared to the control (Figure 2; Table 5). At a concentration of 1 × 10 8 conidia mL −1 , B. bassiana ZK-5, and P. citrinum CTD-24 isolates caused significant (F = 5.78; df = 20; p < 0.0033) mortality rates of 54.3 and 32%, respectively, compared to the 0.0% mortality of the control (Figure 2; Table 6). However, no significant differences in mortality were observed between the control and the rest of the fungal isolates. Furthermore, mycosis of the dead neonates was only observed in 5% of insect cadavers.

Effect of Fungal Isolates on Cumulative Mortality of Eggs and Neonates of S. frugiperda
The fungal isolates had significant differences in the cumulative mortality rates of egg and neonate larvae 14 days post-treatment at 1 × 10 6 conidia mL −1 (F = 33.2; df = 20; p < 0.0000). Beauveria bassiana ZK-5 and P. citrinum CTD-24 isolates caused cumulative mortalities of 52.2 and 37.8%, respectively, followed by C. tenuissimum SE-10 (32.2%), compared to the 0.0% mortality of the control. However, no significant difference in mortality was observed between Aspergillus sp. isolates BM-3 and SE-2-1 and the control (Table 4).

Effect of Fungal Isolates on Feeding Performance of Sixth Instar S. frugiperda Larvae
The fungal isolates were least effective in reducing the feeding ability of first to sixth instar S. frugiperda larvae at 1 × 10 6 conidia mL −1 . Beauveria bassiana ZK-5, P. citrinum CTD-24, and C. tenuissimum SE-10 reduced the feeding efficacy of first to third instar larvae by 33.3-29.2%, 23.8-20.8%, and 19-15%, respectively, while the feeding efficacy of fourth to sixth instar S. frugiperda larvae was reduced by 12.8, 11.7, and 9.4%, respectively. However, no significant differences were observed among Aspergillus sp. isolates SE-2-1 and BM-3 and the control for first to sixth instar S. frugiperda larvae 48 h post-treatment at the same concentration of 1 × 10 6 conidia mL −1 (Table 7). Table 7. Effects of fungal isolates on feeding performance of the sixth instar S. frugiperda larvae at 1 × 10 6 conidia mL −1 .

Discussion
Numerous pathogens, including viruses, fungi, protozoa, nematodes, and bacteria, have been associated with S. frugiperda [44], but only a few cause disease to the pest [45][46][47][48][49][50][51]. Spodoptera frugiperda nuclear polyhedrosis virus (NPV) is the most important among these pathogens and induces significant mortality to the pest and the entomopathogenic fungal species Erynia radicans, Nomuraea rileyi, and Entomophaga aulicae. Disease or natural epizooty mostly appear too late in the pest population to alleviate high levels of defoliation, regardless of causing microbial agents. Thus, any entomopathogens that might cause high infection to immature stages (eggs and neonate larvae) of S. frugiperda, before it reaches its voracious feeding stage, could significantly suppress the pest populations. Therefore, the present study focused on screening selected entomopathogenic fungal isolates that could effectively induce high infections to immature stages of S. frugiperda, as a prerequisite for developing biopesticides to use against the pest.
The results indicated that first to sixth instar S. frugiperda larvae were not susceptible to the tested entomopathogenic fungal isolates at different concentrations. Previous studies reported that even though some fungal isolates, e.g., Aspergillus sp., were found to be least virulent, B. bassiana was the most virulent to the fourth instar striped rice stem borer larvae, Chilo suppressalis (Walker) (Lepidoptera: Crambidae) [52], while another study reported that B. bassiana caused 12.99% larval mortality to S. frugiperda [53]. Similarly, although P. citrinum did not cause significant larval mortality to S. frugiperda, it was identified as the most toxic isolate, causing 98.67% mortality to the second instar cotton leafworm larvae, Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) [54]. Penicillium citrinum strains are also known to be associated with mosquito larvae [55], causing 100% mortality to the third instar southern house mosquito larvae, Culex quinquefasciatus (Say) (Diptera: Culicidae), within 2 h, using a conidial suspension at a concentration of 1 × 10 6 conidia mL −1 in a laboratory experiment [56]. Beauveria bassiana isolates caused 3.3-88.5% mortality to second instar larvae of the spotted stem borer, Chilo partellus (Swinhoe) (Lepidoptera: Pyralidae), while 30-84.4% mortality was obtained against fifth and sixth instar larvae of the maize stalk borer, Busseola fusca (Fuller) (Lepidoptera: Noctuidae) [57]. In previous studies, some fungal isolates showed high virulence to first instar larvae, while low virulence to later instar larvae was observed; for instance, Cladosporium sp. caused 54% mortality in first instar larvae but was least pathogenic to later instar of the corn earworm larvae, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) [58]. Few species in the genus Cladosporium have been reported to be pathogenic against homopteran insects such as aphids [59] and whiteflies [60]. Second instar S. frugiperda larvae were also reported to be less susceptible to the B. bassiana ICIPE 676 isolate, which caused moderate larval mortality of 30% [36]. However, B. bassiana isolates from soil caused 98.3% mortality to third instar S. frugiperda larvae, while fungi of the same strain, isolated from endophytically colonized maize plants, caused 75% mortality to third instar S. frugiperda larvae [61]. Variations in the effectiveness of B. bassiana against different instars of S. frugiperda larvae have also been reported [62]. In contrast, S. frugiperda was found to be the least susceptible of the most extensively tested insects to B. bassiana isolates when compared with other lepidopteran insects [63]. Hence, further studies are warranted to elucidate the mechanisms behind this low susceptibility of S. frugiperda larvae to the tested fungal isolates in this study. Interestingly, in contrast to the low infection rates observed, the B. bassiana strain caused 96.6% mortality to second instar S. frugiperda larvae at 1 × 10 9 conidia ml-1 , with a lethal time of 3.6 days [64]. Ramzi et al. [65] demonstrated that C. suppressalis larvae were highly susceptible to the two commercial B. bassiana isolates, BB1 and BB2. Resistance to fungal infection as the larvae mature may also be attributed to the composition of the larval integument that allowed for effective penetration of the fungus, resulting in increased mortality in early larval instars [66]. Meekes [67] reported that molting may be the reason for loss of inoculum and hence lower chances of fungal infection, although molting does not always result in the prevention of fungal infection. Furthermore, the low pathogenicity of fungal isolates could also be attributed to host-pathogen interactions between these isolates and S. frugiperda larvae, such as efficient attachment of conidia to the integument, negative impacts of integument composition with penetration tube of the fungi, and immune responses of the larvae toward conidia [52]. All these phenomena require detailed experiments to precisely elucidate the mechanism.
Beauveria bassiana ZK-5 and P. citrinum CTD-24 caused the highest cumulative mortality to eggs and neonates of S. frugiperda in the present study. Our findings are consistent with those of Akutse et al. [36], who found that B. bassiana ICIPE 621 caused a cumulative mortality of 87.5% to eggs and neonates of S. frugiperda. Gui et al. [59] found that Cladosporium aphidis fungal isolates were highly virulent to various aphid species, causing a cumulative mortality of 80% within 8 days at 1 × 10 8 spores mL −1 .
In contrast, Cheung and Grula [86] reported no significant reduction in feeding for the corn earworm larvae, Helicoverpa zea, infected with B. bassiana before death. The reduction in food consumption by infected insects due to fungi is one of the important factors in host mortality, which indicates fungal pathogen virulence and needs to be further investigated to determine the level of pathogenicity or antifeedant effects [87]. Beauveria bassiana caused a 60% reduction in food consumption to the long-horned grasshoppers, Uvarovistia zebra (Uvarov) (Orthoptera: Tettigoniidae), at a concentration of 5 × 10 6 spores mL −1 [88].

Conclusions
This is the first report on the use of local Chinese fungal isolates to infect S. frugiperda eggs, neonates, and larvae after pest invasion. The low susceptibility of sixth instar S. frugiperda larvae to the tested entomopathogenic fungal isolates could be a characteristic of the fungal strains tested or perhaps due to genetic diversity and phenotypic differences between S. frugiperda populations. This is also the first report on the impact of EPFs on the feeding efficacy of S. frugiperda, as effected larvae exhibited slowed feeding efficacy, leading to disruptions in larval physiology and ultimately leading to larval mortality (antifeedant effects). Two of the fungal isolates, C. tennuisimum and B. bassiana, significantly reduced the feeding performance of S. frugiperda larvae via indirect pathways of the fungi (different from direct contact effects). Further studies are warranted to evaluate the biopesticidal efficacy of these potent EPF isolates under field conditions before their integration into sustainable management of S. frugiperda.