Natural occurrence of Metarhizium rileyi on Spodoptera frugiperda larvae in the state of Chihuahua

M. Ordóñez-García1; J. C. Bustillos-Rodríguez1; D. I. Berlanga-Reyes2; O. J. Cambero-Campos3; M. Á. Salas-Marina4; M. O. Estrada-Virgen3; C. Rios-Velasco2

1. Doctorado en Ciencias, Centro de Investigación en Alimentación y Desarrollo, A.C. Unidad Cuauhtémoc. Cd. Cuauhtémoc, Chihuahua, México. Av. Río Conchos S/N. Parque Industrial. C.P. 31570., Centro de Investigación en Alimentación y Desarrollo, A.C., Unidad Cuauhtémoc,

<city>Cd. Cuauhtémoc</city>
, Mexico , 2. Profesor-Investigador, Centro de Investigación en Alimentación y Desarrollo, A.C. Unidad Cuauhtémoc. Cd. Cuauhtémoc, Chihuahua, México. Av. Río Conchos S/N. Parque Industrial. C.P. 31570., Centro de Investigación en Alimentación y Desarrollo, A.C., Unidad Cuauhtémoc,
<city>Cd. Cuauhtémoc</city>
, Mexico ,
3. Profesor-Investigador, Unidad Académica de Agricultura, Universidad Autónoma de Nayarit. Xalisco, Nayarit, México. Carretera Tepic-Compostela Km. 9. C.P. 63155., Universidad Autónoma de Nayarit, Unidad Académica de Agricultura, Universidad Autónoma de Nayarit,
, Mexico ,
4. Universidad de Ciencias y Artes de Chiapas, División de Ingeniería. Carretera Villacorzo-Ejido Monterrey Km 3.0. Chiapas, México. C.P. 29000., Universidad de Ciencias y Artes de Chiapas, Universidad de Ciencias y Artes de Chiapas, División de Ingeniería,
, Mexico

Correspondence: *. Corresponding Author: Claudio Rios Velasco. Centro de Investigación en Alimentación y Desarrollo, A.C. Unidad Cuauhtémoc. Cd. Cuauhtémoc, Chihuahua, México. Av. Río Conchos S/N. Parque Industrial. C.P. 31570. E-mail: E-mail:


The fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith), is the main pest of maize (corn). Its control depends mainly on the use of chemical insecticides, which cause adverse effects on the environment and the health of agricultural workers. A viable alternative to these products is the use of biological control agents, such as entomopathogenic microorganisms, highlighting fungi among others. The fungus Metarhizium rileyi (Farlow) Kepler (Hypocreales: Clavicipitaceae), plays an important role in the population regulation of lepidopteran pests, causing natural epizootics when the conditions for its growth are optimal. The aim of the study was to determine the natural occurrence of M. rileyi fungus on S. frugiperda larvae in a corn plot. Four hundred fifty-two FAW larvae from all instars were collected from an infested maize (corn) plot from Cusihuiriachi municipality, Chihuahua in August and September 2017, where the number of larvae with mycosis was recorded. The fungi M. rileyi and Beauveria bassiana (Balsamo) Vuillemin (Hypocreales: Cordycipitaceae) were identified according to their macroscopic and microscopic morphological characters. Temperature, relative humidity (RH) and accumulated precipitation were obtained from the weather stations of Unifrut and INIFAP. The highest percentage (48 %) of mycosed larvae by M. rileyi was obtained during the sample performed in August, where temperature and RH average were 16.76 °C and 80.1 %, respectively, and an accumulated precipitation of 218 mm. These results suggest that the climatic conditions were optimal for the development of the fungal epizootic on field.

Received: 2017 December 19; Accepted: 2018 May 18

revbio. 2020 Jun 1; 5(spe1): e444
doi: 10.15741/revbio.05.nesp.e444

Keywords: Key words: Corn, fall armyworm, biological control, entomopathogenic fungi.


The fall armyworm (FAW) Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) is the most important pest of maize in America (Portillo et al., 1991). Its conventional control involves the application of chemical insecticides. However, the incorrect use of these pesticides has caused multiple problems of intoxication towards farmers and generated resistance by the pest (Tinoco & Halperin, 1998; Ahmad et al., 2007). Given to above, alternatives to the use of chemical insecticides have been searched for, fostering the use of S. frugiperda biological control agents, such as parasitoids, predators, nematodes, fungi, bacteria and viruses among others (Molina-Ochoa et al., 2003; Negrisoli et al., 2010; Rios-Velasco et al., 2011). Entomopathogenic fungi play an important role in decreasing pest populations, causing epizootics when agroclimatic conditions are optimal for fungus growth, as well as pest population densities (Thorvilson, 1984; Sujeetha & Sahayaraj, 2014). Metarhizium genus has been reported as a entomopathogenic genus causing natural epizootics in various pest insects (Vázquez-Moreno & Elósegui-Claro, 2011). The biological activity of this genus has been widely studied due to its agricultural importance (Zhang et al., 2016; Mochi et al., 2017). Although the Metarhizium genus is well defined, the delimitation of the species is difficult due to the great variation in the host range and in conidia size (White et al., 2003). In 2014 a phylogenetic analysis indicated that fungal isolates identified as Nomuraea rileyi (Farlow) Samson (Hypocreales: Clavicipitaceae) belonged to the Metarhizium genus (Kepler et al., 2014; Baral, 2017). Metarhizium rileyi (Farlow) Kepler frequently causes natural epizootics in lepidopteran populations, especially from the Noctuidae family, in different subtropical and temperate agroecosystems, reducing larval populations, which makes it an excellent biocontroller for its high virulence (Boucias et al., 2000; Inglis et al., 2001; Devi et al., 2003; Suwannakut et al., 2005). The aim of the study was to determine the natural occurrence of M. rileyi on S. frugiperda larvae in corn plots from Chihuahua, Mexico.

Material and Methods

Sampling area

Samplings were carried out in a corn (Zea mays, L.) plot, free from the use of chemical insecticides in the locality of Ojo de Agua (28º12’57’’N; 107º1’48’’W, 2,129 masl) belonging to Municipality of Cusihuiriachi, Chihuahua, Mexico. Plants were randomly chosen at 70 and 100 days after sowing (physiological stages 2-4), respectively.

Larvae collection

FAW larvae from all instars of S. frugiperda were collected on two sampling dates (August 26th and September 22nd, 2017) (Table 1). Larvae were transferred to the Laboratory of Postharvest Physiology, Plant Pathology and Biological Control of the Centro de Investigación en Alimentación y Desarrollo, A.C. (CIAD), Cuauhtémoc Unit, Chihuahua, Mexico and were individually maintained under controlled conditions (26 ± 2 °C, with a RH > 70 % and a 12:12 L:D photoperiod) in 1 oz plastic cups (Solo, Dart Containers) containing an artificial diet (Southland Products Incorporated). Larvae were systematically checked every 24 hours until they showed mycosis, parasitism or until they reached adulthood.

Table 1.

Incidence of natural enemies of Spodoptera frugiperda, emphasis on entomopathogenic fungi.

Plot Date (Day
and month)
Total number
of larvae
Parasitized larvae
(Meteorus sp.)
Entomopathogenic fungi Adults Unknown
causes of
M. rileyi B. bassiana
Ojo de
26-Ago 250 12 120 9 74 35
22-Sep 202 13 59 10 90 30
Total 452 25 179 19 164 65

Obtaining parasitoids

Emerged parasitoids were collected and maintained in 70 % ethanol and then they were classified according to the taxonomic keys of Wharton et al. (1997), under a stereoscope (Leica G26). The percentage of parasitism was calculated based on the number of parasitized larvae divided by the total of collected larvae × 100 (Pair et al., 1996).

Isolation of entomopathogenic fungi

FAW larvae that showed mycosis were placed in humid chambers to induce mycelial growth and sporulation. Subsequently, larval mycelium was taken and inoculated in Petri dishes that contained potato dextrose agar (PDA-Bioxon®) culture medium and were incubated at 28 ± 2 °C (Precision Scientific) until its growth, colonies with typical characteristics of the fungi Metarhizium and Beauveria were purified by means of the hypha point technique.

Morphological identification of entomopathogenic fungi

Fungi mycelia were mounted, stained with lactophenol blue, observed under an optical microscope (AxioScope, Carl Zeiss) at 1,000x magnifications and were identified according to their macro- and microscopic characters, using the taxonomic keys of Barnett & Hunter (2006).

Record of climatic conditions

Temperatures and RH recorded during the sampling period were taken from the weather station “La Capilla Cusihuiriachi” Unifrut (Unión Agrícola Regional de Fruticultores del Estado de Chihuahua). The accumulated precipitation of August and September 2017 was obtained from a weather station of the Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP) near the experimental area (INIFAP, 2017; UNIFRUT, 2017).

Results and Discussion

Four hundred fifty-two S. frugiperda larvae were collected, 198 (43.8 %) of these larvae showed mycosis caused by M. rileyi and B. bassiana, 25 larvae were parasitized by Meteorus sp. (5.5 %) and 65 (14.38 %) died from unknown causes. Meteorus has been reported as a parasitoid of some pest lepidopterans of the Noctuidae family such as S. frugiperda, S. litoralis, S. exigua, Agrotis ipsilon, among others, with different parasitism rates (Caballero et al., 1990; Caballero et al., 1992; Estrada-Virgen et al., 2013). In S. frugiperda larvae, parasitism rates recorded in Mexico have also been variable. In this regard, Cortez-Mondaca et al. (2010) recorded 11 % in Sonora, a higher percentage (4.4 times) than 2.5 % found by García-Gutiérrez et al. (2013) in the valleys of Durango, Mexico.

In the August sampling, the number of larvae infected by M. rileyi and B. bassiana (Balsamo) Vuillemin (Hypocreales: Cordycipitaceae) was of 120 (48.0 %) and 9 (3.6 %), respectively, while, in September, 59 (29.2 %) larvae infected by M. rileyi were found and another 10 (5.0 %) by B. bassiana (Table 1). Metarhizium rileyi, has been reported infecting larvae of nocturnal lepidopteran species, such as Anticarsia gemmatalis Hübner, Helicoverpa zea (Boddie), Spodoptera eridania (Cramer), Spodoptera sunia (Guenee), Trichoplusia brassicae (Hiibner) and S. frugiperda (Lepidoptera: Noctuidae), in various agroecosystems (Vázquez-Moreno & Elósegui-Claro, 2011; Namasivayam et al., 2013).

In August, an average temperature of 16.76 °C, 80.1 % RH, and an accumulated rainfall of 218 mm were recorded. While, in September there was an average temperature (16.7 ° C) equal to the one of August; however, the RH and the accumulated rainfall were lower, with 68.1 % and 55 mm, respectively. The percentage of specimens of S. frugiperda mycosed by M. rileyi was higher in the first sampling, attributed to the high RH and rainfall that may have triggered the occurrence of epizootics. In addition, the high incidence of M. rileyi on S. frugiperda larvae was possibly due to its pathogenicity and virulence and/or to the lower hydrophobicity of their conidia, which allow them to remain adherent to plants foliage, an area in which S. frugiperda spends most of its larval stage, which increases the probability of being infected. On the contrary, B. bassiana conidia are more hydrophobic, so they are more easily dragged towards the ground by the action of rain or wind and, therefore, the probability of making contact with the larvae decreases (Boucias & Pendland, 1998).

In this regard Ignoffo & García (1985) and Kish & Allen (1978) mentioned that some of the factors that favor M. rileyi growth and that cause the occurrence of epizootics are periods of high RH (≥ 70 %) and the presence of wind inducing the dispersion of infective conidia. Another determining factor is rain, which allows conidia to be dragged through the soil and the plant, promoting infection, together with the characteristics of the host, the pathogenicity and virulence of entomopathogens, and the impact of anthropogenic activities, particularly in agroecosystems (Kish & Allen, 1978; Kubicek & Druzhinina, 2007). Inglis et al. (2001) emphasized that physiological and morphological factors also influence the susceptibility of insect pests to entomopathogenic fungi, among them, their population density, developmental stage, nutrition and exposure to injuries caused by mechanical, chemical or non-microbial (predators and parasitoids) agents. Naturally, M. rileyi has been found causing epizootics in various lepidopterans species. Rios-Velasco et al. (2010) found M. rileyi causing epizootics in S. frugiperda larvae in corn in the state of Coahuila, Mexico. Duarte-da Costa et al. (2015) reported the natural occurrence of this fungus in Helicoverpa armigera (Hübner) larvae in Brazil, causing 33.1 % of mortality. Ruiz-Nájera et al. (2013) and Ordoñez-García et al. (2015) reported M. rileyi causing mycosis in S. frugiperda larvae in Chiapas and Chihuahua, Mexico, however incidence rates were 3.05 % and 8.6 %, respectively. At the beginning of the infection, S. frugiperda larvae infected by M. rileyi were stiff previously to the whitish initial mycosis, which finally became green due to spores production (Figure 1). It should be noted that entomopathogenic fungi that cause natural epizootics persist in the soil as mycelium or conidia inside insect cadavers (Charnley & Collins, 2007). According to Nicholls (2008), after 5 to 6 days post-infection, hyphal bodies were developed in the hemocele, blood and adipose cells, causing the death of the insect, finally, these hyphal bodies invade the cadaver and give rise to the production of conidiophores, phialides and conidia (Figure 1, 2).

[Figure ID: f1] Figure 1.

Infective development of the entomopathogenic fungus Metarhizium rileyi on Spodoptera frugiperda larvae. a) rigid dead larva; b) appearance of mycelium; c) complete mycosis; d) initial sporulation; e) production of spores.

[Figure ID: f2] Figure 2.

Infection of Spodoptera frugiperda by Metarhizium rileyi, a) mycosed larva; b) macroscopic morphology of the fungus grown in PDA medium; c) and d) microscopic morphology of the fungus showing phialides and conidia at 1,000x magnifications.

The macroscopic characteristics of the growth of M. rileyi on infected larvae and in the culture medium were similar, showing a whitish coloration at the beginning and olive green at the end due to the formation of spores (Figure 1). The isolates produced grayish-green, short-chain phialides and ovoid conidia (Figure 2) (Humber, 1997; Bosa et al., 2004).


The highest incidence of the M. rileyi fungus occurred in the sampling performed in August 2017, which could be favored by the climatic conditions, especially the high rainfall and an RH greater than 80 %, contributing to dispersion and favoring the growth of the fungus. These conditions could cause new epizootics during the rainy season with high populations of the pest.

fn1Cite this paper: Ordóñez-García, M., Bustillos-Rodríguez, J. C., Berlanga-Reyes, D. I., Cambero-Campos, O. J., Salas-Marina, M. Á., Estrada-Virgen, M. O., Rios-Velasco, C. (2018). Natural occurrence of Metarhizium rileyi on Spodoptera frugiperda larvae in the state of Chihuahua Revista Bio Ciencias 5(nesp1), e444. doi:


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