OCCURRENCE OF ENTOMOPATHOGENIC FUNGI AND PARASITIC NEMATODES ON SPODOPTERA FRUGIPERDA (LEPIDOPTERA: NOCTUIDAE) LARVAE COLLECTED IN CENTRAL CHIAPAS, MÉXICO

Headnote

Abstract

Fall armyworm larvae (FAW), Spodoptera frugiperda (J. E. Smith) were collected from whorl-stage corn, Zea mays (L.), between the V and V stages, in 22 localities of Central, Chiapas, México, called "La Frailesca" during late Jun 2009 to determine the occurrence of native entomopathogens and parasitic nematodes, and to determine the most parasitized instars. A total of 1,247 larvae were examined in search of native biological control agents. Overall total larval mortality was 16.36%. The percent larval mortality due to entomo- pathogens and parasitic nematodes was 12.99%. Hexamermis sp., probably albicans Siebold (Mermitha: Mermithidae) were isolated from 105 FAW larvae (8.42%) with highest incidence from the 4th, 5th, 6th and 3rd instars, respectively. A hyphomycete, Nomuraea rileyi Farlow (Samson), was recovered from 38 larvae (about 3.05% parasitism) with highest incidence in the 5th, 3rd, 4th, and 6th instars, respectively. Lowest percentage of FAW larval parasitism was caused by unidentified microsporidian or microsporidium (1.52%), infecting 19 larvae in the 6th, 5th and 3rd instars, respectively. First and 2nd instars did not show parasitism. This information is useful in designing future biological control programs.

Key Words: Fall armyworm, natural enemies, mermithid nematodes, entomopathogenic microorganisms, corn

Resumen

Se recolectaron larvas de gusano cogollero (FAW), Spodoptera frugiperda (J. E. Smith) en maizales, Zea mays (L.) en etapa vegetativa de verticilio, entre las etapas V y V en 22 localidades de la región central de Chiapas, México, conocida como "La Frailesca" a finales de Junio de 2009, con la finalidad de determinar la presencia de entomopatógenos y nematodos parásitos, y para determinar los estadios larvarios más parasitados. Un total de 1247 gusanos cogolleros fueron examinados para agentes de control biológico nativos. La mortalidad total fue de 16.36%. El porciento de mortalidad larval debido a entomopatógenos y nematodos parásitos fue de 12.99%, nematodos mermítidos probablemente de la especie Hexamermis albicans Siebold fueron aislados de 105 larvas (8.42%) provenientes de los estadios del 3° al 6°. Entre tanto, un hongo Hyphomycete, Nomuraea rileyi Farlow (Samson), se recuperó de 38 FAW larvas entre los estadios del 3° al 6° causando alrededor de 3.05% de parasitismo. El porcentaje más bajo de parasitismo larvario fue causado por un microsporido no identificado infectando a 19 larvas entre los estadios 6°, 5° y 3° (1.52%). En este inventario, los nematodos mermítidos, N. rileyi y un microsporidio no identificado fueron los parásitos y patógenos más frecuentes. Las larvas de primer y segundo estadios no mostraron parasitismo. Esta información es útil para el diseño futuro de programas de control biológico.

Palabras Clave: Gusano cogollero del maíz, enemigos naturales, nematodos mermítidos, microorganismos entomopatógenos, maíz

The fall armyworm (FAW), Spodoptera fru- giperda (J. E. Smith) (Lepidoptera: Noctuidae) is well known in the Western Hemisphere as a voracious insect pest of multiple agricultural crops (Clark, 2007; Murúa et al. 2009). It is an important economic pest of corn, Zea mays (L.) (Poales: Poaceae), other grain crops and forage sorghums, peanuts, cotton, soybeans, alfalfa, and occasionally other crops (Sparks 1986; Lu & Adang 1996).

Corn is one of the most valuable field crops in the United States of America (USA). The FAW is one of the most destructive insects attack- ing corn; FAW feeds on tender leaves and stalks causing severe damage in every stage of plant development, resulting in limited production or complete destruction of whorl-stage plants (Wiseman et al. 1967, 1996). In Latin America maize losses in grain production are about 90% (Andrews 1988; Hruska & Gould 1997). Chemi- cal control of FAW is a common practice; however, the adverse effects of the use of insecticides lead to a search for alternatives (Aktar et al. 2009). Microbial control is an environmentally sound and valuable alternative for controlling the FAW. FAW larvae are susceptible to entomopathogenic microorganisms such as bacteria, fungi, nema- todes, viruses, and protozoa (Gardner & Fuxa 1980; Hamm et al. 1986; Patel & Habib 1988; Lezama-Gutiérrez et al. 1996; Molina-Ochoa et al. 1999; Molina-Ochoa et al. 2003; Ríos-Velasco et al. 2010).

The natural enemy complex, particularly pathogens, is poorly known in México, and there is an increased interest in developing biological control methods for FAW. Most concentrated ef- forts for biological control appear to be directed towards the "rear and release" augmentation, followed by importation and thirdly by conserva- tion; Lewis et al. (1997) suggested a reversed or- der of priorities. There is a need to understand, promote and maximize the effectiveness of in- digenous populations of natural enemies, and based on the knowledge and results of these, we should supplement any gaps by importation. The information on the naturally occurring natural enemy complex of the FAW larvae in "La Frail- esca" located in central-western Chiapas, México is scarce, so we surveyed whorl-stage corn to de- termine the occurrence, and parasitism rates of entomopathogenic microorganisms and parasitic nematodes of FAW larvae, as well as to deter- mine which of the larval instars were the most parasitized.

matERials aNd mEthods

This survey was conducted during late Jun 2009 in the region known as "La Frailesca" locat- ed in the central-western of the state of Chiapas (municipalities of Villaflores, Villacorzo, La Con- cordia and Ángel Albino Corzo) located between the coordinates 15°45' and 16°30' N latitude, and between 92°30' and 93°45' W longitude; eleva- tion ranging between 700 and 800 m asl (Anu- ario Estadístico de la Producción Agrícola 2008). The region has an annual mean temperature of 22 °C, and annual rainfall between 700 to 1200 mm (Table 1).

During late Jun 2009 collections of FAW lar- vae were made from whorl-stage corn in the phe- nological stages Vto V. (Ritchie et al. 1992). For each locality, at least 274 larvae were sampled. Each sample consisted of different FAW instars. FAW larvae were individually placed into 250 mL plastic cups, covered on top with a paper lid, held in the laboratory (25 ± 3 °C, 80% RH, and 12:12 h L:D) to record infected larvae. Larvae were fed with fresh corn leaves, each about 20 cm2/cup, re- plenished every 36 h until pupation. Corn leaves were disinfected by immersion for 2 min in a 0.5% sodium hypoclorite solution, then washed with sterile, distilled water (Sánchez & Bellotti 1997).

Mermithid nematodes that emerged from lar- vae were placed in 2 mL crystal vials with 70% ethanol, and were identified according Nickle (1972). Dead larvae showing signs of fungus in- fection were placed in plastic Petri dishes (60 x 10 mm) lined with filter paper moistened with sterile distilled water until the fungus sporulated on the insect (Fargues & Rodríguez-Rueda 1980). Nomuraea rileyi Farlow (Samson) (Moniliales: Moniliaceae) was isolated from dead larvae on medium composed of 200 mL of "V8" vegetable juice, 3 g CaCO 5 g glucose, 2 g yeast extract, 15 g agar, and 800 mL distilled water (Fargues & Rodríguez-Rueda 1980). Fungal identification was according to Brady (1979) and Barnett & Hunter (1998). Larvae showing signs of infection by microsporidia were examined microscopically. REsults aNd discussioN

Out of 1,247 larvae collected from corn in 22 locations of "La Frailesca" Chiapas, 204 larvae were found to be infected by mermithid nema- todes, entomopathogenic fungi and microsporid- ia. Total mortality by these agents was 12.99%. Larval mortality by unknown causes was about 3.37% (42).

Mermithid nematode species Hexamermis probably albicans Siebold (Nematoda: Mermithi- dae) caused the highest mortality rate of 8.42% (105 larvae), followed by N. rileyi causing 3.05% mortality (38 larvae), and an unidentified micro- sporidia causing 1.52% (19 dead larvae).

Thus mermithid nematodes were the most important natural enemies of FAW larvae. In the four locations, the 4th, 5th, and 3rd FAW instars were most parasitized by the mermithid Hexa- mermis sp. with 34, 25, and 22 larvae or 2.73%, 2.00%, and 1.76% parasitized, respectively (Table 2).

Hexamermis sp. has a wide distribution range, having been reported infesting FAW larvae in Honduras, Brazil, Nicaragua, and Argentina (Van Huis 1981; Valicente 1989; Wheeler et al. 1989; Vera et al. 1995). In México, Hexamermis sp. was reported in the states of Colima (Leza- ma-Gutiérrez et al. 2001), Nayarit and Veracruz (Molina-Ochoa et al., 2003) causing percentage mortality ranging from 0.0-14.9%, 0.0-3.33% and 0.0-15.05%, respectively. Hexamermis sp., has been also reported attacking other insect pests such as Diatraea saccharalis (F.) (Lepidoptera: Crambidae), and Hypsipyla grandella (Zeller) (Lepidoptera: Pyralidae) in Colombia (Vergara 2004).

Nomuraea rileyi was the only entomopatho- genic fungus species isolated from dead FAW larvae, mostly 3rd to 6th instars. Twelve 3rd in- stars (0.96%), ten 4th and ten 5th instars (0.80%, respectively), and six 6th instars (0.48%) were infected by this fungus in the 4 locations (Table 2); larvae were fully covered with whitish hyphae, dusty green mass of N. rileyi spores, conidia as well as phialides (Bosa et al. 2004). The high- est infection percentage caused by N. rileyi was exhibited by 3rd instars at 0.96%. We did not observe 1st and 2nd instars parasitized by N. ri- leyi; however, Bosa et al. (2004) emphasized that 2nd instar FAW was the most susceptible, but we found that the 5th instars suffered the high- est mortality with 0.40% at Villaflores (Table 2). However, Molina-Ochoa et al. (2003), and Leza- ma-Gutiérrez et al. (2001) reported 44.4% and 16.7% for a single location in the state of Jalisco. Nomuraea rileyi caused an epizootic in Coahuila, México, and the number of rainy days and the rainfall amount were possibly the most important environmental factors favoring the growth of the fungus (Ríos-Velasco et al. 2010).

An unidentified microsporidia that infected FAW larvae was the third most frequent cause of FAW total larval mortality (1.52%) (Table 2). Mor- tality ranged from 0.00 to 0.48%. Similar mortal- ity percentages were reported by Lezama-Gutiér- rez et al. (2001) in the Mexican states of Colima, Jalisco and Michoacán. In these studies FAW lar- vae showed no obvious symptoms prior to their death, but after death the cadavers were often dry and fragile, resembling the ash of a cigarette. The identification was not verified by electron micros- copy. Of the total larval mortality 4.24% was at- tributed to unknown causes (Table 2).

Regardless of location, the highest mortality rates caused by entomopathogens and parasitic nematodes were observed on the 3rd, 4th, 5th and 6th FAW instars (Table 2). Similar results were reported by Alcocer & Méndez (1965), who asserted that FAW last instar larvae allow the development of entomopathogens and parasitic nematodes. Gross & Pair (1991), stated that a parasitoid Ophion flavidus Brulle (Hymenoptera: Ichneumonidae), parasitized 4th, 5th, and 6th FAW instars with equal success, but was mini- mally successful in completing development on late 6th instar larvae.

Ruiz-Nájera et al. (2007) stated that the para- sitoid complex, as part of the native natural en- emy complex in corn fields of Chiapas can cause a reduction in the FAW larval populations. We also found that the distribution of the entomopatho- gens indicates a potential for biological control.

Results of this survey suggest a need for more taxonomic studies of microsporidia and parasitic nematodes in Mexico, particularly for the mermi- thid species of Hexamermis in central Chiapas. We speculate that the augmentation and release of N. rileyi in La Frailesca, Chiapas corn fields during the rainy season would reduce FAW infes- tations and their damage to the crop.

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