Pathogens and parasitic nematodes associated with populations of fall armyworm (Lepidoptera : Noctuidae) larvae in Mexico

Abstract

Larvae of fall armyworm (FAW), Spodoptera frugiperda (J. E. Smith) and soil samples were collected in six Mexican states. Larvae were collected from whorl-stage corn, grain sorghum, forage sorghum, and Sudan grass fields in 64 locations during the summer of 2000, to determine the occurrence of entomopathogens and parasitic nematodes. A total of 5591 FAW larvae from 64 locations were examined for indigenous FAW biological control agents. Overall total larval mortality was 3.935%. The larval mortality percent due to entomopathogens and parasitic nematodes was 3.524%, other causes reached 0.411% of total mortality. Three species of entomopathogenic fungi representing two classes, Hyphomycetes (Nomuraea rileyi, and Hirsutella sp.) and Zygomycetes (Entomophthora sp.) were recovered from FAW larvae, and two species of Hyphomycetes (Metarhizium anisopliae and Beauveria bassiana) were isolated from soil samples. An unidentified microsporidian was recovered from four locations in the State of Jalisco, three from Michoacan, three from Nayarit, and one from Veracruz and Colima, respectively. Mermithid nematodes were recovered from 24 FAW larvae at three locations in Nayarit and three larvae were recovered from two locations in Veracruz. Six larvae showing symptoms of viral disease were collected from Sinaloa (2), Jalisco (2), Michoacan (1), and Nayarit (1). Entomopathogenic nematodes from the genus Heterorhabditis sp. and Steinernema sp. were isolated from soil samples from Colima in one and two locations, respectively. Steinernema sp., and Heterorhabditis sp. were isolated from soil in one location in Michoacan. Steinernema sp. was recovered from two locations of Jalisco. In this survey, N. rileyi, mermithid nematodes, and microsporidia were the most frequent pathogens and parasites.

Full text

244
Florida Entomologist
86(3) September 2003
PATHOGENS AND PARASITIC NEMATODES ASSOCIATED
WITH POPULATIONS OF FALL ARMYWORM
(LEPIDOPTERA: NOCTUIDAE) LARVAE IN MEXICO
J
AIME
M
OLINA
-O
CHOA
1
, R
OBERTO
L
EZAMA
-G
UTIERREZ
1
, M
ARTIN
G
ONZALEZ
-R
AMIREZ
1
,
M
ARILU
L
OPEZ
-E
DWARDS
1
, M
ANUEL
A. R
ODRIGUEZ
-V
EGA
1
AND
F
RANCISCO
A
RCEO
-P
ALACIOS
1
1
Facultad de Ciencias Biológicas y Agropecuarias, Universidad de Colima
Apartado Postal No. 36, Tecomán, Colima 28100, México
A
BSTRACT
Larvae of fall armyworm (FAW),
Spodoptera frugiperda
(J. E. Smith) and soil samples were
collected in six Mexican states. Larvae were collected from whorl-stage corn, grain sorghum,
forage sorghum, and Sudan grass fields in 64 locations during the summer of 2000, to deter-
mine the occurrence of entomopathogens and parasitic nematodes. A total of 5591 FAW lar-
vae from 64 locations were examined for indigenous FAW biological control agents. Overall
total larval mortality was 3.935%. The larval mortality percent due to entomopathogens and
parasitic nematodes was 3.524%, other causes reached 0.411% of total mortality. Three spe-
cies of entomopathogenic fungi representing two classes, Hyphomycetes (
Nomuraea rileyi
,
and
Hirsutella
sp.) and Zygomycetes (
Entomophthora
sp.) were recovered from FAW larvae,
and two species of Hyphomycetes (
Metarhizium anisopliae
and
Beauveria bassiana
) were
isolated from soil samples. An unidentified microsporidian was recovered from four locations
in the State of Jalisco, three from Michoacán, three from Nayarit, and one from Veracruz and
Colima, respectively. Mermithid nematodes were recovered from 24 FAW larvae at three lo-
cations in Nayarit and three larvae were recovered from two locations in Veracruz. Six lar-
vae showing symptoms of viral disease were collected from Sinaloa (2), Jalisco (2),
Michoacán (1), and Nayarit (1). Entomopathogenic nematodes from the genus
Heterorhab-
ditis
sp. and
Steinernema
sp. were isolated from soil samples from Colima in one and two lo-
cations, respectively.
Steinernema
sp., and
Heterorhabditis
sp. were isolated from soil in one
location in Michoacán.
Steinernema
sp. was recovered from two locations of Jalisco. In this
survey,
N. rileyi
, mermithid nematodes, and microsporidia were the most frequent patho-
gens and parasites.
Key Words:
Spodoptera frugiperda
, biological control, occurrence, survey, maize, mermithid
nematodes, entomopathogenic microorganisms
R
ESUMEN
Larvas de gusano cogollero,
Spodoptera frugiperda
(J. E. Smith) (FAW), y muestras de suelo
se colectaron de 64 localidades en seis estados mexicanos. Las larvas fueron recogidas de
campos cultivados con maíz, sorgo para grano, sorgo forrajero y pasto Sudán, en estado de
cogollo o verticilio, con la finalidad de determinar la presencia de entomopatógenos y nema-
todos parásitos, durante el verano de 2000. 5591 larvas se colectaron para buscar agentes de
control biológico indígenos de esta plaga. En general la mortalidad total de larvas fue de
3.935%, la mortalidad larvaria provocada por patágenos y parásitos fue de 3.524%, otras
causas provocaron 0.411%. Tres especies de hongos entomopatógenos pertenecientes a dos
clases, los Hyphomycetes (
Nomuraea rileyi
e
Hirsutella
sp.) y los Zygomycetes
(
Entomophthora
sp.) fueron recuperados de larvas de gusano cogollero, y dos especies de
Hyphomycetes (
Metarhizium anisopliae
y
Beauveria bassiana
) fueron aislados de muestras
de suelos. Un microsporidio no identificado fue recuperado en cuatro localidades de Jalisco,
tres de Michoacán, tres de Nayarit, y una en Veracruz y Colima, respectivamente. Los ne-
matodos mermítidos parasitaron a veinticuatro larvas en tres localidades de Nayarit, asi
mismo, a tres larvas en dos localidades de Veracruz. Las larvas con síntomas de virosis se
presentaron en dos sitios de Sinaloa, dos en Jalisco, una en Michoacán y una en Nayarit. Los
nematodos entomopatógenos de los géneros
Steinernema
sp. y
Heterorhabditis
sp. fueron
aislados de muestras de suelo, presentándose en Colima, en una y dos localidades, respecti-
vamente; en Michoacán ocurrieron en una localidad, respectivamente, pero
Steinernema
sp.
solo se presentó en dos localidades de Jalisco. En este inventario,
N. rileyi
, los nematodos
mermítidos y el microsporidio fueron los patógenos y parásitos más frecuentes.
Translation provided by author
The fall armyworm (FAW),
Spodoptera frugi-
perda
(J. E. Smith), causes considerable economic
losses in maize, sorghum, peanuts, cotton, soy-
beans and occasionally other crops, in most of the
countries of the Western Hemisphere (Sparks
1986). Control of this pest is usually achieved

Armyworm Symposium 2002: Molina-Ochoa et al. 245
through the application of synthetic insecticides
(Hruska & Gould 1997), but their high cost, envi-
ronmental contamination, development of resis-
tance to chemicals, and pest resurgence (Colborn
1995; Crowe & Booty 1995) have encouraged the
search for alternatives more compatible with the
environment. Microbial control is an environmen-
tally sound and a valuable alternative to the use
of chemicals for controlling this pest.
Interactions between insect host, environ-
ment, insect host age (Fuxa et al. 1988; Molina-
Ochoa et al. 1996), pathogens and plant to be pro-
tected (Bergman & Tingey 1979; Hamm & Wise-
man 1986; Barbercheck 1993; Wiseman & Hamm
1993; Molina-Ochoa et al. 1997, 1999) determine
the strategies for using pathogens in microbial
control (Hamm 1984). FAW larvae are susceptible
to entomopathogenic bacteria, fungi, nematodes,
protozoa, and viruses (Gardner & Fuxa 1980;
Agudelo-Silva 1986; Hamm et al. 1986; Patel &
Habib 1988; Richter & Fuxa 1990; Lezama-Guti-
errez et al. 1996; Molina-Ochoa et al. 1996; Mo-
lina-Ochoa et al. 1999). The insect host age,
habitat and soil type, pesticide use, agricultural
practices, and location, influence the natural dis-
tribution of biological control organisms (Croft &
Brown 1975; Fuxa 1982; Agudelo-Silva 1986;
Hamm et al. 1986; Sosa-Gomez & Moscardi 1994;
Vanninen 1996; Chandler et al. 1997; Mietkiew-
ski et al. 1997; Molina-Ochoa et al. 2001).
As a result of economic and environmental
concerns, surveys for natural enemies of the FAW
occurring in Mexico have been conducted to de-
velop a better understanding of the pathogen
complex, parasitic nematodes and parasitoids
(Lezama-Gutierrez 2001, Molina-Ochoa 2001,
Molina-Ochoa et al. unpublished). These two sur-
veys conducted in four Mexican states reported
the occurrence of the fungi
Beauveria bassiana
,
Nomuraea rileyi
, and
Hirsutella
sp., an unidenti-
fied microsporidian, mermithid nematodes, and
an ascovirus affecting FAW larvae. The occur-
rence of the bacterium
Bacillus thuringensis
and
steinernematid and heterorhabditid nematodes
are reported from soil samples.
This paper reports on the presence of ento-
mopathogens and parasitic nematodes in FAW
larval populations and recovered from soil sam-
ples of corn, grain sorghum, forage sorghum, and
Sudan grass fields from six Mexican states, dur-
ing the summer of 2000.
M
ATERIALS
AND
M
ETHODS
Isolation of Entomopathogens from FAW Larvae
During August and September of 2000, FAW
larvae were collected from whorl-stage corn,
grain and forage sorghum, and Sudan grass fields
in 64 locations in the Mexican states of Sinaloa,
Nayarit, Jalisco, Colima, Michoacán, and Ver-
acruz. Concurrently, four soil samples were ob-
tained from each location in all of the states.
Location 43 comprised a combination of collec-
tions from adjacent corn and grain sorghum field
in whorl-stage. Sample size ranged from 33 to 119
FAW larvae per field, but most often sample size
was about 90. The number collected was corrected
by subtracting the number that died from injury
or unknown causes during the first days after col-
lection. Collection data and percent infection by
entomopathogens and parasitic nematodes is pre-
sented in Table 1. Larval mortality due to insect
parasitoids is reported elsewhere (Molina-Ochoa
et al., in press).
Larvae were placed individually in 30 cc plas-
tic cups with regular pinto bean diet (Burton &
Perkins 1989) and maintained in the laboratory
to record the larvae infected by entomopathogens
and parasitic nematodes. Mermithid nematodes
that emerged from larvae were collected and
placed in crystal vials containing 2 ml of 70% eth-
anol. Dead FAW larvae showing signs of fungal
infection were placed in plastic Petri dishes (60
×
10 mm) lined with a piece of 5.5 cm-diameter fil-
ter paper (Whatman No. 1) moistened with sterile
distilled water until the fungus sporulated on the
insect surface. A medium composed of 200 ml of
V8 vegetable juice, 5 g glucose, 2 g yeast extract,
3 g CaCO
3
, 15 g agar, and 800 ml distilled water
(Fargues & Rodriguez-Rueda 1980) for isolating
the fungus
Nomuraea rileyi
was used. A Sabou-
raud-dextrosa-agar medium enriched with 1% (w/
v) yeast extract (SDAY), and 500 ppm chloram-
phenicol (Lezama et al. 1996) for growing other
fungus species was used. Entomophthorales were
not isolated.
Isolation of Entomopathogenic Fungi and Nematodes
from Soil
In each location of the six surveyed states, a 2
kg combined soil sample was collected. A soil sub-
sample, about 500 g from four different points a
few meters apart, was obtained by digging to a
depth of 10-15 cm with a small shovel. Soil sam-
ples were deposited into double plastic bags,
tagged, stored in a plastic cooler, and taken to the
laboratory where they were kept at 25°C until
processing. The storage time ranged from a few
days to three weeks. Soil was thoroughly mixed
and passed through a 0.4 mm mesh sieve, break-
ing soil lumps and separating any litter.
For isolating entomopathogenic nematodes
and fungi, greater wax moth (GWM) larvae,
Gal-
leria mellonella
L., were used as bait (Bedding &
Akhurst 1975; Tarasco et al. 1997). From the 2 kg
combined soil sample from each location, two
samples were placed in 1000 ml capacity plastic
pots and five GWM last instar larvae were re-
leased into each pot. Pots were incubated at 25°C
in the dark for a 10-day period (Woodring & Kaya

246
Florida Entomologist
86(3) September 2003
T
ABLE
1. G
EOGRAPHIC
LOCATION
,
DATE
,
ALTITIUDE
,
CROP
(*),
SAMPLE
SIZE
(
N
),
AND
TOTAL
PERCENT
FAW
LARVAE
IN-
FECTED
BY
ENTOMOPATHOGENS
AND
PARASITIC
NEMATODES
IN
SIX
M
EXICAN
STATES
(**)
DURING
2000.
Code Date Location Coordinates
Altitude
(m) Crop n
Infected
larvae (%)
C1 08/04 El poblado, 19°13.698’N 422 c 90 0.000
Coquimatlán 103°47.722’W
C2 08/04 Pueblo Juárez, 19°10.752’N 279 c 90 2.222
Coquimatlán 103°54.634’W
C3 08/04 Amachico, 19°10.667’N 328 c 90 0.000
Coquimatlán 103°56.351’W
C4 08/06 Los mezcales, Comala 19°20.811’N 608 c 90 1.111
103°47.639’N
C5 08/06 El remate, Comala 19°24.825’N 817 c 90 0.000
103°47.639’W
C6 08/06 Carrizalillo, Quesería 19°25.389’N 1550 c 90 0.000
103°41.000’W
C7 08/06 Quesería 19°23.362’N 1304 c 90 0.000
103°34.882’W
C8 08/06 Villa de Alvarez 19°17.201’N 515 c 90 0.000
103°47.030’W
C9 08/06 Juluapan, 19°18.890’N 539 c 90 1.111
Villa de Alvarez 103°49.611’W
C10 08/07 Tepames, Colima 19°08.231’N 519 c 90 2.222
103°37.996’W
C11 08/07 Estapilla, Colima 19°59.549’N 304 c 90 1.111
103°31.140’W 1557
J1 08/08 Ciudad Guzmán 19°40.11’N c 90 2.222
103°28.830’W
J2 08/15 Los pinitos, Tonila 19°25.343’N 1326 c 90 11.111
103°32.447’W
J3 08/15 Pialla, Tuxpan 19°27.293’N 1079 c 90 44.444
103°28.514’W
J4 08/15 Atenquique, Tuxpan 19°31.778’N 1338 c 90 11.111
103°27.851’W
J5 08/17 Canoas, Zapotiltic 19°34.073’N 1391 c 90 12.111
103°27.324’W
J6 08/17 Apastepe 19°38.060’N 1709 c 90 3.333
103°30.950’W
J7 08/17 Teocuitatlán 20°07.035’N 1369 c 90 1.111
103°32.704’W
J8 08/17 Zacoalco de Torres 20°11.988’N 1425 c 90 1.111
103°33.806’W
J9 08/17 Acatlán de Juárez 20°25.362’N 1575 c 96 4.166
103°33.406’W
J10 08/17 Tlajomulco de Zúñiga 20°29.396’N 1607 c 92 4.347
103°28.298’W
J11 08/18 Zapopan 20°43.129’N 1670 c 90 10.000
103°29.041’W
J12 08/18 Magdalena 20°53.008’N 1496 c 93 2.150
103°02.509’W
J13 08/23 Crucero de Magdalena 20°56.300’N 1386 c 92 9.782
104°02.509’W
M1 08/09 Totolán 19°58.890’N 1590 c 90 0.000
102°40.183’W
M2 08/09 Santa Inés Tocumbo 19°44.502’N 1630 c 90 0.000
102°34.967’W
*Corn (c), Forage Sorghum (fs), Grain Sorghum (gs), and Sudan Grass (sg).
**Colima (C), Jalisco (J), Michoacan (M), Nayarit (N), Sinaloa (S), and Veracruz (V).

Armyworm Symposium 2002: Molina-Ochoa et al. 247
T
ABLE
1. (C
ONTINUED
) G
EOGRAPHIC
LOCATION
,
DATE
,
ALTITIUDE
,
CROP
(*),
SAMPLE
SIZE
(
N
),
AND
TOTAL
PERCENT
FAW
LARVAE
INFECTED
BY
ENTOMOPATHOGENS
AND
PARASITIC
NEMATODES
IN
SIX
M
EXICAN
STATES
(**)
DURING
2000.
Code Date Location Coordinates
Altitude
(m) Crop n
Infected
larvae (%)
M3 08/09 Peribán 19°33.106’N 1475 c 90 0.000
102°26.586’W
M4 08/10 Cointzio 19°41.609’N 1932 c 90 2.222
101°16.398’W
M5 08/10 Cerro “La Esperanza” 19°41.233’N 1998 c 90 2.222
101°18.890’W
M6 08/11 Tejabán 19°13.342’N 587 c 90 0.000
101°53.714’W
M7 08/11 Carretera a 19°03.290’N 442 c 90 2.222
Nueva Italia 102°02.458’W
M8 08/11 Presa de Zicuirán 18°56.191’N 292 c 63 12.698
101°54.650’W
M9 08/11 El ceñidor, 18°59.651’N 350 c 57 1.754
Nueva Italia 102°11.577’W
M10 08/12 La Guadalupe 19°07.472’N 540 fs 90 2.222
Parácuaro 102°12.519’W
M11 08/12 Las yeguas Parácuaro 18°57.308’N 359 fs 90 2.222
102°16.733’W
M12 08/12 El cirián, Nueva Italia 18°53.661’N 255 c 90 0.000
102°07.483’W
N1 08/18 Santa María del Oro 21°20.121’N 1160 c 90 2.222
104°40.174’W
N2 08/18 El rincón, Tepic 21°32.472’N 849 c 96 2.083
104°56.123’W
N3 08/18 El pichón, Tepic 21°33.479’N 774 c 95 4.210
104°56.937’W
N4 08/19 Xalisco 21°19.601’N 1042 c 107 7.476
104°55.060’W
N5 08/19 El refilión, Xalisco 21°19.407’N 964 c 90 2.222
104°55.323’W
N6 08/19 Compostela 21°17.858’N 920 c 93 21.505
104°54.044’W
N7 08/19 La presa, Compostela 21°13.714’N 928 c 90 2.222
104°52.162’W
N8 08/20 Las lumbres, Acaponeta 22°20.795’N 48 C&gs 60 13.333
105°18.141’W
N9 08/23 Seboruco 21°20.850’N 1134 c 90 2.222
104°40.749’W
N10 08/23 Ahuacatlán 21°06.331’N 1120 c 90 1.111
104°27.427’W
S1 08/21 Bacurimi, Culiacán 24°51.688’N 70 gs 97 0.000
107°29.478’W
S2 08/21 La campana, Culiacán 24°58.415’N 143 gs 100 2.000
107°33.517’W
S3 08/21 Pericos, Mocorito 25°03.574’N 80 gs 95 1.052
107°39.547’W
S4 08/21 Rancho viejo Mocorito 25°06.033’N 89 gs 98 1.020
107°43.165’W
S5 08/22 Aguapepito Mocorito 25°03.861’N 68 sg 95 1.052
107°39.547’W
S6 08/22 Comanito Mocorito 25°09.006’N 91 gs 95 0.000
107°39.645’W
*Corn (c), Forage Sorghum (fs), Grain Sorghum (gs), and Sudan Grass (sg).
**Colima (C), Jalisco (J), Michoacan (M), Nayarit (N), Sinaloa (S), and Veracruz (V).

248
Florida Entomologist
86(3) September 2003
1988, Bidochka et al. 1998). Larval cadavers were
removed and surface-sterilized with 1% Sodium
hypochlorite for a 3 minute-period, then washed
three times with sterile distilled water and placed
on damp filter paper in a 60 mm diameter sealed
Petri dish, and incubated at 25°C for 12 days
(Chandler et al. 1997). Entomopathogenic fungi
from the larvae were isolated using SDAY, with
500 ppm of chloramphenicol (Lezama-Gutierrez
et al. 1996). Fungi were identified by microscopic
inspection of morphological characteristics
in situ
or after isolation in SDAY according to the crite-
ria by Brady (1979) and Samson et al. (1988).
The entomopathogenic nematodes were sepa-
rated to genera by identifying coloration of
Galle-
ria cadavers according to Woodring & Kaya (1988).
Entomopathogenic viruses and bacteria from
FAW larvae and soil, respectively, have not been
yet isolated or identified.
Geographical Coordinates and Collection Data
A Garmin GPS III Plus™ was used for obtaining
the coordinates and altitude data. Location, date,
place, coordinates, altitude, crop, sample size, and
percentage of infected larvae are shown in Table 1.
R
ESULTS
In this survey, out of 5591 FAW larvae col-
lected from 64 locations in six Mexican states, the
entomopathogens and parasitic nematodes killed
197 larvae. Overall larval mortality percentage
due to these organisms was 3.524%. Mortality
percentage per location ranged from 0.000 to
44.444% (Table 1). Considering the total mortal-
ity due to entomopathogens and parasitic nema-
todes, 137 (69.54%) larvae were killed by
entomopathogenic fungi, 26 (13.19%) larvae were
killed by microsporidia, six (3.04%) larvae were
killed by viruses, and 28 (14.21%) larvae were
killed by mermithid nematodes. Two classes of
entomopathogenic fungi were collected. The class
Zygomycetes was represented by Entomophthora
sp. which infected a larva from Colima and a
larva from Veracruz. The class Hyphomycetes
was represented by Nomuraea rileyi and Hirsu-
tella sp. which infected 134 and one larvae, re-
spectively. N. rileyi was responsible for 68.020% of
total mortality due to entomopathogens and par-
asitic nematodes, and was the most abundant and
widely distributed, occurring in all the states.
Hirsutella sp. occurred in Sinaloa, only. Mermith-
S7 08/22 La poma Badiraguato 25°15.749’N 157 c 100 0.000
107°40.739’W c
S8 08/22 La majada Badiraguato 25°14.076’N 145 c 92 0.000
107°39.781’W
V1 09/02 Seis de Enero, Xalapa 19°34.115’N 950 c 91 0.000
96°50.207’W
V2 09/02 Altolucero, Almolonga 19°35.063’N c 33 6.060
96°47.384’W
V3 09/02 Actopan 19°34.623’N c 64 1.562
96°48.589’W
V4 09/02 Los González, Actopan 19°31.894’N 432 c 113 0.884
96°41.294’W
V5 09/02 Bocana, Actopan 19°24.416’N 311 c 119 0.000
96°36.731’W
V6 09/03 El volador, Coatepec 19°21.594’N 709 c 90 3.333
96°51.037’W
V7 09/03 Palmillas 19°12.293’N 702 c 59 0.000
96°46.221’W
V8 09/03 Tierra Colorada 19°13.255’N 46 c 45 4.444
96°21.916’W
V9 09/04 Cerro gordo 19°25.252’N 443 c 45 0.000
96°39.566’W
V10 09/04 Lacumbre 19°23.320’N 366 c 66 0.000
96°38.807’W
TABLE 1. (CONTINUED) GEOGRAPHIC LOCATION, DATE, ALTITIUDE, CROP (*), SAMPLE SIZE (N), AND TOTAL PERCENT
FAW LARVAE INFECTED BY ENTOMOPATHOGENS AND PARASITIC NEMATODES IN SIX MEXICAN STATES (**)
DURING 2000.
Code Date Location Coordinates
Altitude
(m) Crop n
Infected
larvae (%)
*Corn (c), Forage Sorghum (fs), Grain Sorghum (gs), and Sudan Grass (sg).
**Colima (C), Jalisco (J), Michoacan (M), Nayarit (N), Sinaloa (S), and Veracruz (V).

Armyworm Symposium 2002: Molina-Ochoa et al. 249
ids were collected from the states of Nayarit (23),
and Veracruz (5), only, and accounted for approx-
imately 14.21% of total mortality of FAW larvae.
The 26 FAW larvae infected by microsporidia
were collected in five of six states sampled (Mi-
choacán (10), Nayarit (9), Jalisco (5), Colima (1),
Veracruz (1) and Sinaloa (0)). These entomo-
pathogens accounted for 13.19% of the total FAW
larval mortality. The symptoms of larvae infected
with these entomopathogens were similar to the
unidentified microsporidia reported by Lezama-
Gutierrez et al. (2001). They often were dry and
fragile when dead, resembling cigarette ashes.
Few FAW larvae showed symptoms of ascovirosis
infection (Hamm et al. 1986). Two were detected
in Jalisco in one location, two in two separate lo-
cations in the same municipality in Sinaloa, one
in Michoacán, and one in Nayarit. The viruses
were not identified by electron microscopy. The
percentage of fall armyworm larvae infected by
pathogens and parasitic nematodes at each loca-
tion is shown in Table 2.
Entomopathogens Isolated from Soil
Two species of entomopathogenic fungi, Metar-
hizium anisopliae and Beauveria bassiana, were
recovered from 10 of 64 soil samples. M. anisopliae
was recovered from four of eleven locations in
Colima, and in one location in each of Nayarit,
Jalisco, and Michoacán. Beauveria bassiana was
recovered from two locations in Veracruz and one
in Michoacán.
Two genera of entomopathogenic nematodes,
Steinernema sp., and Heterorhabditis sp. were
collected from soil samples. Steinernematid nem-
atodes were recovered from five of 64 locations
(two locations in Colima, two in Jalisco, and in
one in Michoacán). Heterorhabditid nematodes
were recovered in two locations, one in Colima
and one in Michoacán.
D
ISCUSSION
Current research efforts are focused on select-
ing native and exotic entomopathogens, which
are highly virulent to arthropod pests, for devel-
oping efficient and environmentally-sound bio-
insecticides. The high susceptibility of fall army-
worm larvae and other lepidopterous pests to
strains of N. rileyi, B. bassiana and M. anisopliae
has been demonstrated (Bustillo & Posada 1986;
Habib & Patel 1990; Lecuona & Lanteri 1999).
N. rileyi has been reported infecting FAW larvae
naturally in Brasil (Valicente 1989), Venezuela
(Agudelo-Silva 1986), Puerto Rico (Pantoja et al.
1985), Colombia (Vargas & Sánchez 1983), United
States (Fuxa 1982), Mexico (Lezama-Gutiérrez
et al. 2001), and other countries. In this survey,
N. rileyi caused 68.020% of the total FAW larval
mortality due to pathogens and parasitic nema-
todes, and was the most abundant and widely dis-
tributed entomopathogen, occurring in each of the
six states surveyed. Similar results were reported
by Lezama-Gutiérrez et al. (2001) from a survey
conducted in Colima, Jalisco, and Michoacán.
Entomophthora aulicae was reported attack-
ing FAW larvae on grain sorghum in Georgia
(Hamm 1980; Schwehr & Gardner 1982), and Ar-
gentina (Vera et al. 1995). Entomophthora sp.
and Hirsutella sp. were reported attacking larvae
of this pest with parasitism rates that ranged
from 0.6% to 1.1%, respectively (Lezama-Gutiér-
rez et al. 2001). In this study, parasitism rates for
E. sp., and H. sp. were 3.030 and 1.520%, respec-
tively. Total FAW mortality caused by all the
pathogens was 3.524% (197 larvae killed).
An unidentified microsporidian was the third
cause of FAW total larval mortality with 13.19%,
and was similar to that reported by Lezama-Guti-
errez et al. (2001). Most larvae infected with the
microsporidian were collected from Michoacán,
Nayarit, and Jalisco, with 10, 8, and 5 infected
larvae, respectively. The arrangement of the
spores of this microsporidian suggested that this
entomopathogen was neither Nosema nor Vairi-
morpha as previously reported by Gardner &
Fuxa (1980).
A few larvae showed symptoms and signs sim-
ilar to those from ascoviruses, but the identity of
these viruses was not verified by electron micros-
copy. Occurrence of entomopathogenic viruses
has been reported in Latin America, in Puerto
Rico, Argentina, Brasil, and Mexico (Valicente
1989; Pantoja & Fuxa 1992; Vera et al. 1995;
Lezama-Gutierrez et al. 2001).
In this survey, the mermithid nematodes were
important natural enemies of FAW larvae. They
were the second most important mortality factor,
causing 14.21% of total mortality. Nematodes
from the genus Hexamermis have been reported
attacking FAW larvae in Honduras, Brasil, Nica-
ragua, and Argentina (Van Huis 1981; Valicente
1989; Wheeler et al. 1989; Vera et al. 1995).
Mermithids attacking FAW larvae in Mexico were
reported by Alcocer-Gómez & Méndez-Villa (1965).
They found parasitism ranging from 8 to 100%
during a 3-year study. An association between the
pest density and percent of parasitism was deter-
mined. Rainfall also was cited as an important
factor in influencing percent parasitism. In a re-
cent survey conducted in Mexico, mermithid nem-
atodes caused larval mortality ranging from 0.0
to 14.9% in Colima (Lezama-Gutierrez et al.
2001). But in our survey, mermithids were not re-
covered in Colima. However, similar percentages
of mortalities were recorded (0.000 to 15.054%)
from other locations, with the highest rate of par-
asitism from Nayarit and Veracruz. A possible
reason for the difference between the findings of
Lezama-Gutierrez et al. (2001) and those we re-
port is that most of the locations in Colima were

250 Florida Entomologist 86(3) September 2003
TABLE 2. PERCENTAGE OF FAW LARVAE INFECTED BY ENTOMOPATHOGENS AND MERMITHIDS AT EACH LOCATION.
Code* N. rileyi Hirsut. Entomoph. Mermithid Microspo. Viruses
C1 0 0 0000
C2 2.222 0 0 0 0 0
C3 0 0 0000
C4 0 0 1.111 0 0 0
C5 0 0 0000
C6 0 0 0000
C7 0 0 0000
C8 0 0 0000
C9 1.111 0 0 0 0 0
C10 1.111 0 0 0 1.111 0
C11 1.111 0 0 0 0 0
M1 0 0 0000
M2 0 0 0000
M3 0 0 0000
M4 2.222 0 0 0 0 0
M5 2.222 0 0 0 0 0
M6 0 0 0000
M7 2.222 0 0 0 0 0
M8 0 0 0 0 12.698 0
M9 0 0 0 0 1.754 0
M10 1.111 0 0 0 1.111 0
M11 1.111 0 0 0 0 1.111
M12 0 0 0000
J1 2.222 0 0 0 0 0
J2 8.888 0 0 0 0 2.222
J3 44.444 0 0 0 0 0
J4 11.111 0 0 0 0 0
J5 12.222 0 0 0 0 0
J6 2.222 0 0 0 1.111 0
J7 1.111 0 0 0 0 0
J8 0 0 0 0 1.111 0
J9 4.166 0 0 0 0 0
J10 4.347 0 0 0 0 0
J11 7.777 0 0 0 2.222 0
J12 2.150 0 0 0 0 0
J13 8.696 0 0 0 1.086 0
N1 0 0 0 0 2.222 0
N2 1.041 0 0 0 0 1.042
N3 3.157 0 0 0 1.053 0
N4 2.803 0 0 0 4.673 0
N5 2.222 0 0 0 0 0
N6 6.451 0 0 15.054 0 0
N7 1.111 0 0 1.111 0 0
N8 0 0 0 13.333 0 0
N9 2.222 0 0 0 0 0
N10 1.111 0 0 0 0 0
S1 0 0 0000
S2 2.000 0 0 0 0 0
S3 0 0 0 0 0 1.052
S4 0 0 0 0 0 1.020
S5 0 1.052 0 0 0 0
S6 0 0 0000
S7 0 0 0000
S8 0 0 0000
*Locations are described in Table 1.
N. rileyi = Nomuraea rileyi, Hirsut. = Hirsutella sp., Entomoph. = Entomophthora sp., Mermithid = mermithid nematode, Mi-
crospo. = Microsporidia, Viruses = with virosis signs.

Armyworm Symposium 2002: Molina-Ochoa et al. 251
different than those sampled by Lezama-Gutier-
rez et al. (2001) during the summer of 1998.
The entomopathogenic fungi and nematodes
were recovered in 26.5% of the soil samples (17 of
64 locations). M. anisopliae and B. bassiana were
isolated in 15.6% of the samples. They occurred in
Colima, Michoacán, Nayarit, and Veracruz, but
were not found in Jalisco and Sinaloa. M. anisopliae
was recovered from five locations in Colima, and
one location each in Michoacán and Nayarit.
B. bassiana was recovered in two locations of Ve-
racruz and one location in Michoacán. In a study
conducted in Szczecin, Poland using soils col-
lected from forests during the spring and autumn,
the entomopathogenic fungi M. anisopliae and
B. bassiana infected wax moth larvae (Mietkiew-
ski et al. 1998), and M. anispoliae was the domi-
nant species. Recently, Lezama-Gutierrez et al.
(2001) reported three species of entomopatho-
genic fungi recovered from soil samples using the
Galleria technique; M. anisopliae, B. bassiana, and
Paecilomyces fumosoroseus, with M. anisopliae be-
ing the most dominant species. In our soil samples,
M. anisopliae was also the most dominant, occur-
ring in 10.9% of the locations, while B. bassiana
occurred only in 4.7% of the locations (Table 3).
Steinernematid and Heterorhabditid nematodes
were found in seven of 64 locations (10.9%). Stein-
ernematid nematodes were recovered from Colima
in two locations, one in Michoacán, and two in
Jalisco. Heterorhabditids occurred in Colima and
Michoacán, in one location, respectively. Low rates
TABLE 2. (CONTINUED) PERCENTAGE OF FAW LARVAE INFECTED BY ENTOMOPATHOGENS AND MERMITHIDS AT EACH
LOCATION
.
Code* N. rileyi Hirsut. Entomoph. Mermithid Microspo. Viruses
V1 0 0 000 0
V2 3.030 0 3.030 0 0 0
V3 0 0 0 1.562 0 0
V4 0 0 0 0 0.884 0
V5 0 0 000 0
V6 0 0 0 3.333 0 0
V7 0 0 000 0
V8 2.222 0 0 2.222 0 0
V9 0 0 000 0
V10 0 0 0 0 0 0
*Locations are described in Table 1.
N. rileyi = Nomuraea rileyi, Hirsut. = Hirsutella sp., Entomoph. = Entomophthora sp., Mermithid = mermithid nematode, Mi-
crospo. = Microsporidia, Viruses = with virosis signs.
TABLE 3. ENTOMOPATHOGENIC FUNGI (HYPHOMYCETES) AND NEMATODES (RHABDITIDA: STEINERNEMATIDAE AND
HETERORHABDITIDAE) RECOVERED FROM SOIL SAMPLES IN DIFFERENT MEXICAN LOCATIONS.
Code* Location Entomopathogen
C2 Pueblo Juárez, Coquimatlán Heterorhabditis sp.
C5
El remate Metarhizium anisopliae
C5
El remate Steinernema sp.
C6
Carrizalillo Steinernema sp.
C6
Carrizalillo Metarhizium anisopliae
C7
Quesería Metarhizium anisopliae
C10
Tepames Metarhizium anisopliae
J1
Ciudad Guzmán Metarhizium anisopliae
M2
Santa Inés, tocumbo Metarhizium anisopliae
M4
Cointzio Beauveria bassiana
M6
Tejabán Steinernema sp.
M7
Carretera a Nueva Italia Heterorhabditis sp.
J2
Los pinitos, Tonila Steinernema sp.
J3
Pialla, Tuxpan Steinernema sp.
N6
Compostela Metarhizium anisopliae
V8
Tierra Colorada Beauveria bassiana
V9
Cerro Gordo Beauveria bassiana
*Locations are described in Table 1.

252 Florida Entomologist 86(3) September 2003
of entomopathogenic nematode recovery have been
reported in different regions around the world, and
range from 3.9% to 21.4% (Constant et al. 1998;
Tangchitsomkid et al. 1998; Griffin et al. 2000; Rosa
et al. 2000; Lezama-Gutierrez et al. 2001). Soil pH
and type, altitude, habitat, soil temperature, crop-
lands, orchards, pastures, and proximity to coastal
lands were discussed as possible factors affecting
the occurrence of these entomopathogens.
The diversity and distribution of entomopatho-
gens and parasitic nematodes occurring in Mexico
could play an important role in regulating the
FAW larval populations. Additional research is
needed on the identification, biology, and poten-
tial of the microsporidia frequently recovered in
the surveys conducted during 1998 and 2000.
There is also a need to identify the role of
mermithid nematodes as potential biological con-
trol agents. Additional research has already been
conducted at the Universidad de Colima, Mexico,
to identify the steinernematid and heterorhab-
ditid nematodes isolated, and to determine their
potential for biological control of fall armyworm
larvae and other lepidopterous pests.
A
CKNOWLEDGMENTS
This work was supported by funds from Universidad
de Colima, Mexico, we thank Dr. Carlos Salazar Silva,
Rector of this institution. The authors express their
gratitude to Dr. J. J. Hamm, and Dr. R. E. Lynch (USDA-
ARS, Crop Protection and Management Research Unit,
Tifton, GA) for their assistance and identification of the
pathogens. We also thank Dr. J. E. Foster, Dr. E. A. Hein-
richs (University of Nebraska Lincoln, Lincoln, NE),
and Dr. S. R. Skoda (USDA-ARS, Midwest Livestock In-
sects Research Laboratory, Lincoln, NE) for review of
the manuscript. We recognize J. J. Molina-Cárdenas,
O. F. Aguilar-Meza, G. May-Mora, and M. A. Reyes-
Hernández for their technical assistance.
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