TESIS DOCTORAL
Characterization of novel granulovirus strains
from Costa Rica against Phthorimaea
operculella and Tecia solanivora
YANNERY GÓMEZ-BONILLA
Memoria presentada por
YANNERY GÓMEZ-BONILLA
Para optar el grado de Doctora por la Universidad Pública de Navarra
Characterization of novel granulovirus strains from Costa Rica
against
Phthorimaea operculella
and
Tecia solanivora
Directores: Dra. Delia Muñoz
Profesora Titular de Universidad Departamento de Producción Agraria Universidad Pública de Navarra España
Dr. Miguel López-Ferber
Directeur du centre LGEI École des Mines d´Alès FranciaUniversidad Pública de Navarra E. T. S. Ingenieros Agrónomos
Presidente
Dr. Pedro del Estal Padillo
Dpto. Producción Vegetal: Botánica y Protección Vegetal Universidad Politécnica de Madrid
Secretario
Dr. Rosa Murillo Pérez
Dpto. Producción Agraria Universidad Pública de Navarra
Vocal
Dr. Enrique Vargas Osuna
Dpto. Ciencias y Recursos Agrícolas y Forestales Universidad de Córdoba
Suplentes
Dr. Oihane Simón de Goñi
Instituto de Agrobiotecnología
Centro Superior de Investigaciones Científicas-Universidad Pública de Navarra
Dr. Elisa Viñuela Sandoval
Profesora Titular del área de Producción Vegetal del Departamento de Producción Agraria de la Universidad Pública de Navarra, en España,
y
Dr. MIGUEL LÓPEZ-FERBER,
Professeur des Ecoles des Mines, Directeur du centre LGEI, École des Mines d´Alès, Francia,
INFORMAN:
que la presente memoria de Tesis Doctoral titulada “Characterization of novel granulovirus strains from Costa Rica against Phthorimaea operculella and Tecia solanivora” elaborada por DÑA. YANNERY GÓMEZ BONILLA ha sido realizada bajo nuestra dirección y que cumple las condiciones exigidas por la legislación
vigente para optar al grado de Doctor.
Y para que así conste, firman la presente en Pamplona a 30 de junio de 2011.
Dr. Primitivo Caballero
Universidad Pública de Navarra, Pamplona, Spain
Dr. Xabier Léry
Universtité Paris Sud, Orsay, Francia
Dr. Trevor Williams
AGRADECIMIENTOS
Quiero agradecer el apoyo que he recibido de todas aquellas instituciones que me ayudaron a la realización de la tesis doctoral. Al Departamento de Producción Agraria de la Universidad Pública de Navarra y al Centre de Pharmacologie et Biotetechnologie pour la Santé d` Centre Nacional de la Recherche Scientifique (CNRS), St Chrystol Les Ales, Francia.
Mi más sincero agradecimiento al INTA por haberme dado la oportunidad de poder realizar este doctorado que fue de mucho enriquecimiento científico. Al personal de la Estación Carlos Durán por su colaboración en este trabajo y compañeros del Laboratorio de Fitoprotección por su gran apoyo moral.
INIA-España quienes dieron el dinero para que pudiera realizar los estudios.
MICIT-CONICIT, por toda la ayuda económica complementaria, porque sin esta no hubiera sido posible terminar los estudios.
A mi directora de Tesis Delia Muñoz por su gran ayuda en la corrección escrita de esta tesis, por su amistad, siempre me dio mucho animo y esperanza, gracias Delia. Al Dr. Primitivo Caballero, por darme la oportunidad de entrar a su grupo de investigación en el mundo de los Baculovirus.
Al Dr. Xavier Léry, por toda su enseñanza, paciencia y sobre todo el auxilio prestado en el desarrollo de las investigaciones.
A los amigos y compañeros de la Universidad Pública de Navarra (Sonia, Emi, Amaia, Gabriel, Mariangeles, Iñigo, Sarhay, Rosa, Oihane, Rodrigo, Laura, Carlos, Pili, Noelia, Oihana, Maite, Alex por su apoyo y lo más valioso su amistad. Y a todas aquellas personas que tuve la dicha de conocer, no solo en Pamplona, sino también en St Chrystol Les Ales, Francia, ha sido la experiencia más enriquecedora de toda mi vida, porque en un poco tiempo conocí gente de muchos países de Europa y Latinoamérica que me brindaron cariño, amistad y ayuda.
A todo mi familia y en particular a mis hermanos por su apoyo incondicional, porque siempre me dieron la fuerza y su admiración para seguir adelante a pesar de todas la dificultades. Dedico esta tesis a mis hijas Diana, Cathy, Silvia y mi nieto Aidan que me dan la alegría de todos los días, su energía positiva, su admiración y su gran amor. A mi querida madre Vilma, quien siempre me tiene en su corazón, en sus oraciones, quien me brinda todo su amor, comprensión, amistad y ayuda, si no hubiera sido por ella, no gozara de todos los éxitos que he tenido.
estuve trabajando a lo que yo llame el “monasterio” en Francia, pero al mismo tiempo esto te ayuda a valorar, lo importante de la familia y las amistades. Entre como investigadora en un nuevo mundo con los Baculovirus y la biología molecular, y entre más avanzaba más ignorante me sentía. Termino tomando las palabras de un gran científico e investigador Dr. Louis Pasteur que dijo “UN POCO DE CIENCIA NOS APARTA DE DIOS. MUCHA, NOS APROXIMA”.
TABLE OF CONTENTS
RESUMEN ...3
SUMMARY ...5
CHAPTER I. Introduction ...7
CHAPTER II. Characterization of a Costa Rican granulovirus strain highly pathogenic against
its indigenous hosts, Phthorimaea operculella and Tecia solanivora...47
CHAPTER III. Potato crops in Costa Rica are efficiently protected from Phthorimaea
operculella and Tecia solanivora by an indigenous granulovirus strain ...63
CHAPTER IV. Stored potatoes in Costa Rica are efficiently protected from Phthorimaea
operculella and Tecia solanivora with an indigenous granulovirus strain...69
CHAPTER V. Costa Rican soils contain highly insecticidal granulovirus strains against
Phthorimaea operculella and Tecia solanivora ...75
CHAPTER VI. Resultados y Discusión General ...89
RESUMEN
En la búsqueda de alternativas a los insecticidas químicos utilizados contra el complejo de
especies de la polilla de la papa, formado por los lepidópteros Phthorimaea operculella y
Tecia solanivora, destaca un granulovirus (GV) con prometedoras propiedades como agente
de control. El virus, originalmente aislado de larvas de P. operculella y designado por ello
granulovirus de P. operculella (PhopGV), es capaz de infectar también a T. solanivora. Se
han aislado cepas de PhopGV en la mayoría de los países donde se encuentra P.
operculella. Ensayos de laboratorio y campo han demostrado la eficacia de algunos de estos
aislados geográficos como biopesticidas de P. operculella, aunque su actividad
bioinsecticida es menor para T. solanivora. Hasta hace poco, todas las cepas de PhopGV se
habían obtenido de larvas de P. operculella. Sin embargo, varias cepas colombianas de
PhopGV aisladas recientemente de T. solanivora muestran mejores propiedades insecticidas
contra este hospedador.
A pesar de que son varias las formulaciones de PhopGV que ya han demostrado su
eficacia en la protección de los tubérculos de patata en condiciones de almacén y en
infestaciones de campo en Asia, Norteamérica y Sudamérica, todavía no ha sido posible
realizar ensayos en Centroamérica por falta de aislados autóctonos. El primer objetivo de
esta tesis fue por tanto recolectar cepas autóctonas de PhopGV de Costa Rica, donde las
dos especies de polilla de la papa constituyen las principales plagas en campo y almacén.
En un primer intento se aisló un granulovirus de larvas enfermas de P. operculella recogidas
en Alvarado (Cartago, Costa Rica). El análisis molecular lo identificó como una nueva cepa
de PhopGV, que recibió el nombre de PhopGV-CR1, y reveló su heterogénea composición
genotípica. Biológicamente, esta cepa demostró una alta patogenicidad no sólo para una
colonia costarricense de P. operculella sino también para una colonia criada en Francia. Sin
embargo, la patogenicidad de PhopGV-CR1 contra T. solanivora fue cuatro veces menor. En
un intento por mejorar la patogenicidad de PhopGV-CR1 contra su hospedador alternativo,
se realizó un pase seriado del virus en tres generaciones de T. solanivora, incrementándose
por cinco su patogenicidad. Este resultado indicó que la adaptación de esta cepa a su
hospedador alternativo está en marcha.
El elevado potencial insecticida demostrado por PhopGV-CR1 en condiciones de
laboratorio justificó el siguiente objetivo de la tesis: la evaluación de la eficiencia de control
en campo y almacén. En campo, el virus redujo el daño entre 50 y 80% comparado con los
significativamente diferente de la producida por un insecticida químico o por una
combinación del virus con un insecticida químico. En almacén, el virus redujo el daño en
más de un 70% comparado con el testigo sin tratamiento cuando las aplicaciones cubrieron
por completo la superficie de los tubérculos. Esto se consiguió mezclando exhaustivamente
los tubérculos con el virus en sacos. T. solanivora fue la especie dominante a lo largo de
toda la temporada en campo y almacén, permitiendo deducir que PhopGV-CR1 puede
controlar a T. solanivora de manera efectiva.
Para incrementar la colección costarricense de PhopGV se realizaron nuevas
prospecciones, no sólo de insectos enfermos, sino también de tierra, donde podrían
encontrarse genotipos con mejor potencial como bioinsecticidas de suelo. Se obtuvieron tres
nuevas cepas costarricenses, denominadas CR3, CR4, and
PhopGV-CR5, procedentes de suelos no empleados para el cultivo de patata, lo que sugiere la
importancia de la dispersión y persistencia en la transmisión de estos virus. Una última cepa,
PhopGV-CR2, se aisló de larvas enfermas de T. solanivora. Las cuatro nuevas cepas
costarricenses compartían muchos marcadores moleculares y tipos genómicos identificados
en los aislados colombianos de PhopGV bien adaptados a ambos huéspedes, indicio del
potencial de las cepas costarricenses para adaptarse a los dos huéspedes coexistentes al
exponerse a ambos. Los extraordinarios valores de patogenicidad de PhopGV-CR3 contra
las dos especies dieron mayor apoyo a la hipótesis y destacaron su potencial como
bioinsecticida. Además, su origen de suelo, su persistencia y su adaptabilidad al huésped
hacen de este aislado una alternativa prometedora para su control en campo.
En resumen, todas las cepas costarricenses de PhopGV muestran ser buenas
candidatas para su aplicación contra P. operculella y T. solanivora, en especial
PhopGV-CR1, cuya eficiencia ha sido demostrada en campo y almacén, y PhopGV-CR3 por sus
SUMMARY
In the search for alternatives to chemical insecticides against the potato tuberworm complex,
formed by the lepidopterans Phthorimaea operculella and Tecia solanivora, a granulovirus
(GV) stands out as a very promising control agent. The virus, originally isolated from P.
operculella larvae and thus designated P. operculella granulovirus (PhopGV), can also infect
T. solanivora. PhopGV isolates have been collected in most countries where P. operculella is
established. Laboratory bioassays and field performance studies have proved the efficiency
of several of these geographical isolates as biopesticides against P. operculella, although,
their bioinsecticidal activity against T. solanivora is lower. Until very recently, all PhopGV
strains had been obtained from P. operculella larvae. However, the novel Colombian
PhopGV strains isolated from T. solanivora display better bioinsecticidal properties against
this host.
While several PhopGV formulations have demonstrated their efficiency for
protecting potato tubers in rustic storage conditions as well as during field outbreaks in Asia,
and North and South America, no trials have been conducted in Central America for lack of
indigenous isolates. The first aim of this thesis was to isolate indigenous PhopGV isolates in
Costa Rica, where potato tuberworms are the primary pests in the field and storage. During
the first attempt, a GV was isolated from diseased P. operculella larvae in Alvarado (Cartago,
Costa Rica). Molecular analysis identified it as a novel PhopGV strain, which was designated
PhopGV-CR1, and indicated a heterogeneous composition of genotypes. Biologically, this
strain was highly pathogenic not only for a Costa Rican P. operculella colony, but also for a
P. operculella colony reared in France. However, the pathogenicity of PhopGV-CR1 was four
times lower against T. solanivora. In an attempt to improve the pathogenicity of PhopGV-CR1
against its alternate host, the virus was serially passaged over three generations of T.
solanivora, which increased its pathogenicity by five fold. This indicated an ongoing
adaptation of the virus to its alternate host.
Given the bioinsecticidal potential of PhopGV-CR1 in the laboratory, the next aim of
the thesis was to evaluate its control efficiency in Costa Rican potato crops and warehouses.
In the field, the virus reduced damage between 50 and 80% compared with the untreated
controls in winter and summer assays. What is more, this reduction was not significantly
different from that yielded by chemicals or by a combination of PhopGV and chemicals.
Under storage conditions, the virus reduced damage by over 70% compared with the
This was achieved by thoroughly mixing the tubers with the virus in bags. T. solanivora was
the dominant species throughout the whole season both in the fields and in the warehouses,
indicating that T. solanivora can be effectively controlled by PhopGV-CR1.
To increase the Costa Rican PhopGV strain collection, further efforts were made in
this thesis to isolate PhopGV strains not only from insects but also from soil habitats. Such
isolates were hypotheized to perform better as soil bioinsecticides. Three novel strains,
named PhopGV-CR3, PhopGV-CR4, and PhopGV-CR5, were isolated from three Costa
Rican locations from soils not employed for potato cultivation, suggesting the importance of
dispersal and persistence in granulovirus transmission. An additional strain, PhopGV-CR2,
was identified from T. solanivora diseased larvae. All four novel Costa Rican strains shared
many of the molecular markers and genome types identified in Colombian PhopGV isolates
well adapted to both hosts, which represents robust molecular indication of the potential of
Costa Rican PhopGV strains to get adapted to coexisting hosts upon exposure to both of
them. The extraordinary pathogenicity values of PhopGV-CR3 against the two species gave
further support to this hypothesis and highlighted its potential as a bioinsecticide. In addition,
its soil origin, persistence and host adaptability also make this isolate a promising alternative
for field control.
In summary, all novel Costa Rican PhopGV strains seem good candidates for
application against P. operculella and T. solanivora, in particular PhopGV-CR1, whose
efficiency has been demonstrated in the field and in storage and PhopGV-CR3 for its
CHAPTER I
Introduction
Contents
1. General introduction and scope of research...8
2. The potato tuberworms ...8
2.1. The potato tuberworm, Phthorimaea operculella ...8
2.2. The guatemalan potato tuberworm, Tecia solanivora ...11
3. Control methods against the potato tuberworms ...13
3.1. Cultural control...14
3.2. Ethological control...14
3.3. Chemical control ...15
3.4. Biological control...15
4. Biological control with baculoviruses ...16
4.1. Taxonomy and structure...17
4.2. Pathology...19
4.2.1. Primary and secondary infections ...19
4.2.2. Phases of gene expression...20
4.2.3. Types of infection...23
4.3. Ecology ...24
4.3.1. Diversity...24
4.3.2. Soil reservoirs and persistence ...26
4.3.3. Environmental spreading ...27
4.4. Phthorimaea operculella granulovirus against the potato tuber moths ...28
4.4.1. PhopGV isolates ...28
4.4.2. Efficiency ...29
4.4.3. Mass-production of PhopGV ...31
5. Baculovirus-based bioinsecticides ...31
5.1. Advantages and limitations ...31
5.2. Mass production and field use...34
5.3. Formulation and storage ...34
5.4. Market issues...35
1. GENERAL INTRODUCTION AND SCOPE OF RESEARCH
Potatoes (Solanum tuberosum) originate from South America highlands, where it has been a
basic source of food for more than eight thousand years (Graves, 2000). At present, it is the
most important food crop in the world, with an annual production of nearly 300 million tons.
More than a third of the global production comes from developing countries
(http://www.cipotato.org/potato/ 2007). In Costa Rica, potatoes are cultivated at altitudes
between 1000 and 3000 m, where temperatures range between 7 and 25 ºC and
precipitations between 1400 and 2600 mm. The major threat of potato yields in general in
Central America and particularly in Costa Rica, is posed by two caterpillar species:
Phthorimaea operculella Zeller (Lepidoptera: Gelechiidae) and Tecia solanivora Povolny
(Lepidoptera: Gelechiidae), which form the so called potato tuberworm complex. Traditional
control with chemical insecticides is no longer efficient and alternative control methods based
on more specific and environment-friendly methods are greatly needed.
Some entomopathogenic viruses, particularly baculoviruses (family Baculoviridae)
comprising the nucleopolyhedroviruses (NPVs) and granuloviruses (GVs) have demonstrated
to be effective control agents of certain agricultural and forest pests (Hunter-Fujita et al.,
1998). A granulovirus of P. operculella (PhopGV) is a particularly effective biopesticide of this
pest in several countries (Fig. 1), including Colombia, Ecuador, Perú, Bolivia, and Venezuela
(Lacey et al., 2010). In addition, several isolates of this virus are also efficient against the
altenate host, T. solanivora (Espinel-Correal et al., 2010). However, no programs are
currently available in Costa Rica for neither of the two insect pests given the lack of
indigenous GV isolates.
The aim of this thesis is to isolate Costa Rican PhopGV strains and evaluate their
bioinsecticidal properties against P. operculella and T. solanivora in the laboratory. Those
showing promising bioinsecticidal properties will be selected for tuber protection assays in
the field and under storage conditions.
2. THE POTATO TUBERWORMS
The potato tuberworm complex causes the most important damage in tubers both in the field
and in storage in Central America. Injuries drastically reduce their commercial value since
tubers can no longer be utilized as seeds or food for human or animal consumption.
2.1. The potato tuberworm, Phthorimaea operculella
Phthorimaea (Gelechia) operculella comes from South America. However, the insect was
it was not until 1873 when it was first described by Zeller from a specimen found in Texas.
Afterwards, Ortega and Fernandez (2000) reported its presence in California. The first record
of P. operculella in Costa Rica was in the 1970´s (Murillo, 1980).
Fig. 1. Distribution of Phthorimaea operculella and Tecia solanivora in the world and countries where
integrated pest management (IPM) programs including granuloviruses have been successfully implemented against P. operculella.
The adult moth is a microlepidopteran of 12-15 mm wing span, 10 mm body length
and narrow wings, being the forewings yellowish grey sprinkled with little black spots and the
hindwings grey with long bristles (Fig. 2). Eggs are whitish and their diameter is ca. 0.45 mm.
Neonate larvae are 1.3 mm long whereas fully developed larvae (Fig. 2) range between 10 to
12 mm. Their body is rosy white, with brown black head and prothorax, a few black spots,
and a small number of bristles on each segment. Pupae are light brown and between 7-10
mm long.
As a poikilotherm organism, P. operculella developmental time largely depends on
environmental conditions, being temperature and humidity the most important factors.
Therefore, the life cycle from egg to adult may well vary between 22 and 55 days. P.
operculella developmental threshold is 14.6 ºC. In Central America, environmental conditions
allow between 8-10 generations per year. At night, adult moths are inactive, hidden in the
crop foliage, but at dusk they start flying actively to find prospective mating. Female fecundity
is around 200 eggs throughout their short adult life span. Eggs may be laid on leaves, stems
mounds, or different debri. Upon hatching, larvae penetrate the plant tissues mining leaves or
stems or boring tubers (López-Avila, 1996).
Fig. 2. Morphology of Phthorimaea operculella adult and fully developed larva.
P. operculella has established in most of the tropical and subtropical regions of the
world (Fig. 1), where it has become one of the most important insect pests of potatoes. It has
been found from north to southern latitudes in Europe, Australia and New Zeland and from
east to west in Japan and the United States. Populations are found in the fields at
temperatures around 16ºC and at altitudes of up to 2000 m. Cold highland regions in
Colombia, Peru, Venezuela, Kenya and Nepal (Ortega and Fernandez 2000) have also
reported damage on tubers by this insect species.
P. operculella larvae mine leaves stems, petioles and tubers, but the most important
damage is caused on stored potatoes (Sporleder et al. 2005) (Fig. 3). In the field, larvae
perform the first attack during the germination of the plantlet. Larvae feed on the bud
terminals and petioles and live in the drilling shaft. Heavy infestations are observed in less
than 90 days. The second attack is produced between hilling and flowering, with no negative
consequences on crop yield. The third attack takes place just before harvest on exposed
tubers, resulting in infestation of stored tubers (Ortega and Fernández, 2000).
When the infestation occurs on foliage, injuries weaken plants, and may cause plant
death in extreme cases. In any case, these attacks cause plant decline. A positive correlation
during the flowering stage, yield loss is reduced between 50 and 80%. Injuries exceeding
25% affect tuber quality (Ortega and Fernandez, 2000). The most severe injuries are caused
on stored tubers, whose quality and weight are affected directly and indirectly by increased
sweating and secondary infection by microorganisms. Studies on potential injuries in storage
indicate that low P. operculella infestations (eg. 60 larvae per 20 kg of potato) can attack
100% of tubers in only 110 days (Ortega and Fernández, 2000). P. operculella can feed on
60 other wild and cultivated plant species, most of them belonging to the Solanaceae family
(Das and Raman, 1994).
2.2. The Guatemalan potato tuberworm, Tecia solanivora
T. solanivora was first described by Povolny in 1973 as Scrobipalpopsis solanivora, from
larvae collected in Costa Rica. It has become the main pest of potatoes in Central and South
America. In addition, the low temperature requirements for its development make this insect
species a threatening potential pest for European potato crops, where it has been included in
the EPPO Quarantine list (http://www.eppo.org/QUARANTINE/listA1.htm).
T. solanivora adult moths present a clear sexual dimorphism. Female are 12 mm
long with grey head and thorax and light brown forewings. Males are smaller (10 mm long),
with brown head and thorax and dark brown forewings. In both genders, forewings bear three
bright white longitudinal spots, which are stronger in females and hindwings are light grey
with grey to black margins (Fig. 4).
Eggs are white and oval, between 0.46 and 0.6 mm wide and 0.39 and 0.43 mm
long (López-Ávila and Espitia, 2000). Larvae undergo three molts from neonates (1.2-1.4 mm
long) to fourth stage (12.4-14.2 mm long). First instars are light colored with brown head and
prothorax, while later instars (Fig. 4) become bright red dorsally and pink laterally. Upon
pupation, coloration turns from green to brown-red. Pupal length ranges between 8.5
(female) and 7.8 (male) mm.
The life cycle of T. solanivora varies between 55 and 93 days, depending mainly on
temperature conditions. Adults are difficult to observe due to their coloration and daylight
inactivity. They rest hiding on the foliage or the ground and start flying only at night and
dawn, when they copulate and females lay eggs. Female fecundity ranges between 180 and
235 eggs, of which 80% are laid during the first 10 days after emergence. In the field, they
are laid in clusters over the base of the stems, or, if possible, closer to the tubers. In
warehouses, eggs are laid directly onto the tubers (Araque and García, 1999). Upon
eggs closer to tubers, facilitating neonate food source search and, hence, tuber infestations.
Unlike P. operculella, T. solanivora larvae do not feed on leaves or stems. Prepupae leave
the tubers and build up a cocoon surrounding the pupae.
a
b
c
d
Fig. 3. Injuries inflicted by Phthorimaea operculella in the leaves (a) and stems (b) of potato plants
and by P. operculella (c) and Tecia solanivora (d) in tubers.
T. solanivora originates from the region of Tehuantepec in Mexico, the North of
Honduras and Guatemala (Puillandre et al., 2007). However, it was first described from a
Costa Rican population, which was introduced through Guatemalan infested potato seeds in
1970 (Niño, 2004). Since then, this highly invasive species has established gradually in
Panama, Venezuela, Colombia, and Ecuador in South America (Puillandre et al., 2007). In
1999, the insect was found locally in the Canary Islands (Carnero et al., 2008) (Fig. 1).
T. solanivora has become adapted to different agricultural and ecological conditions
in the different potato producing regions. In Costa Rica, T. solanivora populations have been
was initiated only 10 years ago with low altitude varieties. Nevertheless, moth population
densities vary depending on climatic conditions, being rain and altitude the most important
limiting factors.
Molecular studies have allowed to delimit the area of origin of T. solanivora, and to
analyze the genetic structure of the species in its native region (Torres-Leguizamón et al.,
2011). According to these authors, the number of haplotypes has decreased over the
colonization process, ending with the observation of a single haplotype in Colombia, Ecuador
and the Canary Islands. Consequently, the invasion of South American countries by T.
solanivora is likely to have had a front-like step-wise progression, where the most recently
invaded country becomes the source of subsequent invasion.
Fig. 4. Morphology of Tecia solanivora female adult (a) and fully developed larva (b). For
comparison, larvae of Phthorimaea operculella (top) and T. solanivora, were photographed together.
Larvae originate serious injuries in potato tubers both in the field and in storage (Fig.
3) leading to important losses. No alternative plant hosts are known. After emergence, larvae
search tubers to feed on. As they eat, they form galleries inside tubers where they leave their
excrements and exuvia. Injuries increase with larval size and end up promoting secondary
infections by microorganisms. In the field, infestations start during the initial phase of
tuberization and become bigger as the tubers develop. Usually, injuriers are more important
along the crop edges but, as the crop develops, the infestation progresses further and
injuries can also be present at the center of the plot (Galindo and Español, 2003). The most
important injuries are mostly inflicted on stored potatoes in rustic warehouses where losses
can be total (Sotelo, 1997).
3. CONTROL METHODS AGAINST THE POTATO TUBERWORMS
A wide variety of methods are available for the control of the potato tuber moths belonging to
approaches for the development of integrated pest management systems are also available
(Kroschel and Lacey, 2008).
3.1. Cultural control
Considering that most of the economic damage posed by P. operculella and T. solanivora
occurs on tubers, preventive attempts to control larvae are desirable. Cultural control aims at
destroying infestation sources and at creating unfavorable conditions for population growth.
Deep seedling and hilling establish a large distance between eggs and tubers and
hatching larvae die of starvation while searching food. In these cases, it is very important to
keep fields well watered in order to avoid cracks in the ground that may constitute shelters for
adults or egg laying sites (Corredor and Flórez, 2003).
Harvesting in time and sanitation procedures such as destruction of injured tubers
or plant residues are important cultural methods to avoid or diminish infestation sources
(Ortega and Fernández, 2000). Also, a thorough cleaning and disinfestation of warehouses
and potato containers is encouraged previous to storage. During storage, surveillance is
strongly recommended to avoid population development. Adult flights in the warehouse warn
their presence and pheromone traps can be of great use in such cases.
Crop rotation interrupts food source and hence limits the building up of moth
populations. In Costa Rica, potatoes are rotated with carrots, onions and brassica twice a
year. This is theoretically more effective for narrow host range species, like T. solanivora,
and not that much against species with a wider host range like P. operculella.
3.2. Ethological control
Traps containing synthetic sexual female pheromones are used to capture large amounts of
adult males and hence cause a gender disproportion. They are placed on the edges and also
inside field crops at densities of 16 traps/Ha. Lower trap densities (4 traps/Ha) are used by
many growers routinely to survey moth population densities and make decisions on whether
control measures need to be taken (Niño, 2004).
Pheromones have proven promising when used as mating disruptors in field and
storage (Bosa et al., 2005). Dispensers flood the environment and avoid the encounter of
males and females.
3.3. Chemical control
Carbamates, organophosphates and pyretroid based formulations have been pulverized over
(King and Saunders, 1984; Raman and Alcazar, 1988; Trivedi and Rajagopal, 1992). In
addition, use of these insecticides may cause environmental problems on water and soil and
acute or chronic toxicity on beneficial organisms and humans. In robust warehouses
fumigation has proved effective but many Costa Rican growers store their tubers in rustic
warehouses where fumigation is not advisable. Insect growth regulators have been used
successfully in the field when applied to the lower part of the canopy (Berlinger, 1992).
3.4. Biological control
A lot of information on the biology and the potential of natural enemies including parasitoids,
predators, and diseases can be found in the literature but their impact on P. operculella and
T. solanivora populations is unknown given current chemical-based pest management
practices. The advantage of using biological control agents is that they have no pre-harvest
intervals, and are safer for application personnel, food supply and non-target organisms.
Several parasitoid and predator species have been described for P. operculella
(Kroschel and Lacey, 2008). Copidosoma koehleri Blanchard (Hymenoptera: Encyrtidae) and
Apanteles subandinus Blanchard (Hymenoptera: Braconidae) are believed to be excellent
parasitoids worldwide of P. operculella eggs and larvae, respectively (Watmough et al. 1973;
Whiteside 1981, 1985) along with Trichogramma spp. (Hymenoptera: Trichogrammatidae)
(Kfir, 1981, 1983). Other parasitoids of P. operculella have also been described from the
orders Diptera (fam. Tachinidae) and Hymenoptera (fam. Braconidae, Encyrtidae,
Eulophidae, Ichneumonidae, Mymaridae, Perilampidae, Pteromalidae, Scelionidae, and
Trichogrammatidae) (Rondon, 2010). Among the predators species of P. operculella are
Coccinella septempunctata Linnaeus (Coleoptera: Coccinellidae); Chrysoperla carnea
Stephens (Neuroptera: Chrysopidae), and Orius albidipennis Reuter (Heteroptera:
Anthocoridae) (Coll et al., 2000).
Regarding T. solanivora, Rubio et al. (2004) describe the parasitoid species
Thrichogramma lopezandinensis Sarmiento (Hymenoptera: Trichogrammatidae) as a
relatively successful agent under storage conditions.
A number of insect pathogens have been described as natural mortality factors of
potato tuberworm populations and several of them have already been developed as microbial
insecticides (Lacey et al. 2010). Among the entomopathogenic nematodes are Steinernema
feltiae (Rhabditida: Steinernematidae) and Heterorhabditis spp. (Rhabditida: Heterorhabditidae) and their obligate bacterial symbionts Xenorhabdus spp.
(Enterobacteriales: Enterobacteriaceae) and Photorhabdus spp. (Enterobacteriales:
Enterobacteriaceae) which are directly responsible of killing the insect hosts (Wouts et al.,
insects exposed after only 24 h. (Xuejuan et al., 2000; Saenz, 2003). Several nematodes are
currently formulated for the control of different insect pests (Grewal et al., 2005). Recent
results of nematodes isolated from Perú and Ecuador highlands revealed promising results
over L4 P. operculella larvae. Soil applications or treatment over discarded potato piles are
being considered (Lacey et al., 2010).
Bacillus thuringiensis (Bt) is a spore forming bacteria very effective against several
insect pests in agriculture (Garczynski and Siegel, 2007). A wide range of Bt subspecies
have been characterized so far and Bt subspecies kurstaki is the most widely employed
against lepidopteran pests. Commercial formulations have proved efficient against P.
operculella in the field and in storage alone and in combination with chemical insecticides
(von Arx and Gebhardt, 1990; Hamilton and MacDonald, 1990, Arthurs et al., 2008; Salama
et al., 1995; Broza and Sneh, 1994; Kroschel, 1995). Although their cost and low persistence
are two important limitations for large-scale use in the field (Ortega and Fernández, 2000),
treatments over stored potatoes in integrated management program have reported promising
results (Farrag, 1998; Raman et al., 1987; von Arx et al., 1987).
Numerous entomopathogenic fungi are effective against insect pests (Goettel et al.,
2005; Ekesi and Maniania, 2007), including several potato pests (Lacey et al., 1999, 2009;
Wraight et al., 2007). However, research against P. operculella and T. solanivora remains
scarce. Although few, studies with Metarhizium anisopliae Metschnikoff (Hypocreales:
Clavicipitaceae), Beauveria bassiana Balsamo (Hypocreales: Clavicipitaceae) and Muscodor
albus Stroble (Xylariales: Xylariaceae) have shown promising results for the control of P. operculella (Hafez et al., 1997; Lacey et al., 2008; Sewify et al., 2000)
Finally, baculoviruses are arthropod specific viruses with a narrow host range. Their
safety for the environment along with outstanding properties as bioinsecticides have made
them successful biocontrol agents (BCAs) of several field crop pests (Berling et al., 2009;
Moscardi et al., 1999; Lasa et al., 2007) storage pests (Cowan et al., 1986), and forest pests
(Martignoni, 1999; Olofsson, 1988; Webb et al., 1999), and very promising BCAs for many
other agricultural and forest pests (Cherry and Williams, 2001; Sun et al., 2004). Several
baculovirus isolates have been isolated from P. operculella and T. solanivora worldwide and
their field performance has encouraged both the Food and Agriculture Organization (FAO)
and the World Health Organization (WHO) to recommend them as alternatives to chemicals.
4. BIOLOGICAL CONTROL WITH BACULOVIRUSES
Viruses are an important group of insect pathogens both in number and variety of families.
being baculoviruses the most extensively studied family of arthropod specific viruses for two
main reasons. Their agronomic interest derives from their lethality to both, insect pests and
beneficials, like the silkworm, Bombyx mori (Lepidoptera: Bombycidae). In addition, their
genomic characteristics have made baculoviruses an important tool in biotechnology for
mass-production of recombinant proteins of veterinary and medical interest (Kost et al., 2005;
Condreay and Kost, 2007).
While most viruses are studied due to their harmful effects on humans or human
activities, baculoviruses are mainly beneficial. The first description of illness by baculoviruses
comes from silk breeders in Italy, back in the sixteenth century (Benz, 1986), but it was not
until the 1950´s when their development as BCAs was set on (Steinhaus, 1963). In 1975, the
first baculovirus was registered as a pesticide in the United States (Ignoffo, 1981). For a
variety of reasons, this product was a commercial failure and the first notable success of a
baculovirus as a BCA did not come until three years later, when the US Forest Service used
a baculovirus-based product specific for Orgya pseudotsugata McDunnough (Lepidoptera:
Lymantriidae) in pine-tree forest extensions (Martignoni, 1984).
Baculoviruses are practically ubiquitous. They infect arthropods of terrestrial and
marine ecosystems and, on land, they have been identified in hundreds of species inhabiting
forests, fields, rivers, and households (Martignoni and Iwai, 1986). Structurally, they have
been designed to survive outside their host and may remain active for years in soil, plant
crevices, and other shelters (Jaques, 1975).
4.1. Taxonomy and structure
The taxonomy of baculoviruses is changing rapidly. Here, we will follow the last proposal of
the International Committee for the Taxonomy of Viruses (ICTV). Historically, the major
criterion for establishing the classification of the Baculoviridae family was the morphology of
occlusion bodies (OBs). All viruses grouped in the family Baculoviridae have OBs, a resistant
structure allowing virus horizontal transmission between host individuals while outside the
host. Until recently, the Baculoviridae family was divided into two genera according to the two
described morphological types: the nucleopolyhedroviruses (NPVs) with polyhedral shaped
OBs called polyhedron, 1-5 microns in diameter, and occluding multiple virions; and the
granuloviruses (GVs), with ovoid OBs called granulum, 250-450 nm in diameter, and
generally occluding a single virion (Caballero et al., 2001) (Fig. 5). This classification was
changed in 2009 to base it on phylogenetics (ICTV, 2009). The new classification includes
four genera: Alphabaculovirus, including lepidopteran NPVs, Betabaculovirus, the former
Deltabaculovirus, which includes the only baculovirus isolated from dipteran species, Culex
nigripalpus (Jehle et al., 2006; van Oers and Vlak, 2007).
Polyhedra contain multiple virions, called occlussion body derived virions (ODVs). In
turn, Alphabaculovirus ODVs may contain one or several nucleocapsids. This characteristic
is not considered a taxonomic criterion, but is reflected in the virus name as single (SNPV) or
multiple (MNPVs) NPVs. The Betabaculovirus ODVs contain only one virion, which in turn
contains a single nucleocapsid (Fig. 5). Baculoviruses are circular double-stranded DNA
viruses with a genome size varying between 90 and 180 Kb. The viral genome is condensed
by the P6.9 protein, a basic arginine-rich protein. The packed genome is enclosed in a
nucleocapsid, whose main component is formed by a viral encoded protein, VP39. The
nucleocapsid has a helical symmetry that confers baculoviruses a characteristic rod shape
with a width between 30 and 60 nm and a length between 250 and 300 nm, which varies
depending on the genome size of each viral species (Caballero et al. 2001; López-Ferber
and Devauchelle 2005). The cylindrical nucleocapsid is wrapped inside a lipid-protein bilayer
membrane to form the virion.
A peculiarity of baculoviruses is the existence of two types of virions during the viral
infection cycle, which play different roles along the infection: ODVs and budded virions (BVs).
ODVs are protected in OBs and can be released in the alkaline conditions of the insect
midgut allowing host to host transmission. BVs are responsible for viral colonization of cells
inside the individual. These virion types are produced by budding from the plasma membrane
in the infected cell, hence their name (Caballero et al. 2001; López-Ferber and Devauchelle
2005). The two viral forms are produced in all infected cells sequentially. Firstly BVs, which
invade the different insect host cells, and then ODVs, which ensure transmission between
individuals.
BVs and ODVs contain the same genome but differ in their structure and chemical
composition of lipid, fatty acids and proteins (Braunagel and Summers, 1994). For instance,
BVs contain a single nucleocapsid per virion, which is not always the case for ODVs. Also,
the origin of the envelope is different for each virion type. That of ODVs originates from the
host nuclear membrane whereas that of BVs comes from the citoplasm membrane and
undergoes modifications by the attachment of certain viral encoded proteins responsible for
host cell penetration. The ODV envelope contains unique proteins, in particular some
responsible for entry into midgut epithelial cells. These proteins have been designated
collectively as per os infectivity factors (PIFs) (Kuzio et al., 1989; Slack et al., 2010). The
entry of ODVs into midgut epithelial cells is accomplished by direct fusion between the ODV
envelope and the cytoplasm membrane located in the cell brush border and takes place in a
engulfs nonspecific molecules in tiny vesicles mediated by the envelope fusion protein (F
protein). This protein is encoded by a well conserved gene in all baculoviruses. In certain
Alphabaculovirus species, a second gene codes for a far more active protein in the BV
envelope, gp64. Presence or absence of this gene allows grouping Alphabaculovirus species
into group I and II, respectively (Herniou et al., 2003). Concentration of fusion proteins in the
apical part of the BV originates distinct structures, the peplomers, which are clearly visible
under light microscopy (Gutiérrez and López-Ferber, 2004).
Alphabaculovirus
Betabaculovirus
Fig. 5. Three major occlusion body (OB) phenotypes. Alphabaculovirus OBs are larger than
Betabaculovirus OBs because of their larger content of occlusion-derived virions (ODVs). Betabaculovirus OBs are capsule sheped and contain only single virions. The NPVs are grouped into the multiple NPVs (MNPVs) and single NPVs (SNPVs) depending on the number of nucleocapsids that are found in each virion. The ODVs are illustrated in dissected views and the GV ODV is illustrated as partially encapsulated. From Slack and Arif, 2007.
4.2. Pathology
4.2.1. Primary and secondary infections
The infection cycle starts when larvae ingest the OBs present in their food substrate,
continues by BV generation and dispersion inside the host and finishes with the release of
infections, depending on the cell type infected and the infecting viral form, ODV and BV,
respectively.
In the primary infection, once OBs have been ingested, they are rapidly dissolved in
the midgut epithelium by the high pH present (pH>10) releasing the ODVs along with the
proteins forming the OBs (Sciocco de Cap, 2001) (Fig. 6). These particles have then to
surpass the perithropic membrane, made of chitin and mucines (Wang and Granados, 2001).
This structure protects cells from abrasion of ingested particles and forms a barrier against
pathogens (López-Ferber and Devauchelle, 2005). A mucine-degrading enzyme, a
metalloprotease called enhancine, has been identified in most granuloviruses and also in
certain NPVs (Li et al., 2003). After surpassing the perithrophic membrane, the ODVs attach
to the microvilli of midgut epithelial cells and nucleocapsids enter the cytoplasm by direct
membrane fusion (Horton and Burand, 1993) mediated by PIF proteins. Shortly afterwards,
the nucleocapsids travel towards the cell nucleus through actin wires fixed by the action of
two nucleocapsid proteins, P39 and P78/83 (Rohrmann, 2011). Once in the nucleus, viral
replication takes place. Washburn et al. (2003) showed groups of nucleocapsids migrating
directly to the basal cell membrane and do not entering the nucleus. Among the first viral
protein products are fusion glycoproteins responsible of BV infectivity. These proteins travel
towards the basal cell membrane to allow the formation of novel BVs containing the
nucleocapsids that had travelled directly through the cell without entering the nucleus. This
mechanism permits a rapid set off of secondary infections, even before primary infection is
completed, and confers a selective advantage to overcome the shedding of midgut cells,
which takes place at a frequent pace. Therefore, this can only take place when a midgut cell
receives more than a single nucleocapsid. In MNPVs, the presence of multiple nucleocapsids
inside an ODV guarantees this process.
In the secondary infection, several questions pertaining the main dissemination
route in the larval haemocoel remain unanswered. BV propagation is believed to take place
mainly through the tracheal system (Engelhard et al., 1994). Virtually all larval tissues are
infected being the most important infection levels taking place in the fat body cells.
4.2.2. Phases of gene expression
Viral replication takes place in the host cell nuclei. Upon arrival of nucleocapsids to the
nucleus membrane pores, the viral DNA is released and gene expression starts. Four
different phases (Fig. 7) can be distinguished with regard to gene expression timing: very
Fig. 6. Infection by baculoviruses. A baculovirus occlusion body (OB) enters by the per os route by
way of contaminated food. OBs pass through the foregut and enter the midgut where they dissolve in the alkaline midgut lumen and release occlusion derived virions (ODVs). The inset figure depicts the translocation of released ODVs past the peritrophic membrane (PM) to midgut columnar epithelial cells. From Slack and Arif, 2007.
In the very early phase (0 to 15 min. after infection), viral genes involved in the
control activities of host cells are expressed, namely, viral transactivators which bear
cell-type promoters and are thus recognized by cell RNA polymerases. Their role is to derive the
recognition of cell promoters towards those of the virus.
In the early phase (15 min to 8 hours post infection or hpi) viral proteins needed for
BV formation are synthesized (eg. EFP, gp64, and F protein) along with most proteins
involved in inhibition of reactions at the cellular and organismal level (eg. apoptosis inhibitors,
molting inhibitors). Other proteins being synthesized at this stage are those needed for
regulation of both the viral cycle and late genes expression. In fact, late and very late genes
are under the control of promoters that are not recognized by host cell RNA polymerases but
by a viral encoded polymerase that is produced at this stage. At the end of the early phase
the replication of the viral genome takes place.
In the late phase (8 to 20 hpi), nucleocapsid and BV structural proteins are
expressed. In the virogenic stroma, nucleocapsid assembling takes place and newly formed
nucleocapsids are exported outside the nucleus towards the cell basal membrane to form the
Fig. 7. Phases of baculovirus infection cycle. Several phases of virus replication are illustrated
beginning with the rounding of newly infected cells and finishing with the lytic release of occlusion bodies. Indicated times are relative to the infection cycle of AcMNPV. The purpose of the figure is to illustrate the progression of phases from budded virus (BV) production to occlusion-derived virus production (ODV). Nucleocapsids are initially translocated to the cell membrane for BV production and later become retained in the nuclear ring zone for ODV production. INM is in reference to the inner nuclear membrane which provides the ODV envelope. From Slack and Arif, 2007.
In the very late phase, approximately 20 hpi, the virogenic stroma is condensed and
the infection evolves towards OB production. Migration of nucleocapsids towards the
cytoplasm membrane stops and nucleocapsids remain in the nucleus. Occlusion of virions in
a granuline or polyhedrine protein matrix starts at around 24 hpi (Slack and Arif, 2007) (Fig.
7). At the end of the infection (48 to 72 hpi) cells disintegrate and OBs are released into the
environment and tissue liquefaction takes place (Fig. 7). Granulovirus infection cause larval
symptoms such as whitening of the whole body (Sciocco de Cap, 2001). Larval integuments
are destroyed by the mediation of viral encoded chitinases and cathepsinases, and OBs are
released into the environment to initiate a new infection cycle in healthy larvae (Williams and
Faulkner, 1997).
During the infection process, several metabolic changes occur in the infected
larvae: delay in larval development, inhibition of molting, increase of weight, lengthening of
larval stages, and inhibition of ecdysis (Sciocco de Cap, 2001). What is more, larval
behaviour is also modified. For example, at the last stages of infection, foraging insects leave
their shelters or climb up to the tree top (instead of crawling down to the soil for pupation) to
facilitate OB dispersion in the most exposed uppermost and adjacent branches of the tree
4.2.3. Types of infection
NPVs and GVs differ in some parts of the infection process. In NPVs, replication takes place
in the nucleus, the virogenic stroma is formed in its center, then BVs are formed and
eventually ODVs. The nucleus membrane remains intact throughout these stages. In GVs,
replication starts in the nucleus, then the stroma is concentrated at the nucleus edges, and
immediately afterwards, the nuclear membrane disintegrates and the contents of cytoplasm
and nucleus are mixed (Fig. 8). Viral replication takes place there (Sciocco de Cap, 2001).
Three different types of GVs can be distinguished regarding the viral infection tropism
(Federici, 1997). Type I is represented by Trichoplusia ni GV (TnGV) and other noctuid
lepidopteran GVs. They invade the midgut transiently and then remain in the fat body. There
is no breakdown of larval skin and the virus kills its host in a period that varies between 10
and 35 days. Type II is represented by Cydia pomonella GV (CpGV) and is characterized by
a generalized infection and the pathology has similar characteristics with that of NPVs in
lepidopteran hosts. The most affected tissues are the fat body, the trachea and the
epidermis. They take between 5 and 6 days to kill larvae and liquefy and rupture their host
integuments. Type III has only one member, Harrisinia brillians GV (HbGV), which infects
lepidopterans of the Zygaenidae family and produces infections limited to the midgut in the
larvae and also in the adult host stage in a period of 4-7 days.
Fig. 8. Granulovirus infection cycle. Ingestion of occlusion body (OB) particles along with food (A).
4.3. Ecology
The behaviour of baculoviruses in nature is key to our understanding of the effective use of
baculoviruses as BCAs and the assessment of any perceived risks attached to their release
into the environment. Molecular techniques have aided in the identification of individual
isolates and the monitoring of the structure and changes in viral populations. In addition,
DNA sequencing information on the baculovirus genome is beginning to highlight the variety
of mechanisms employed by a baculovirus to manipulate the host for its own survival.
4.3.1. Diversity
NPVs and GVs have been isolated from hundreds of insect species. The morphology and
structure, initially observed by electron microscopy and through serological assays, indicated
that the different isolates were not identical. Molecular techniques, in particular restriction
endonuclease (REN) profiles, allowed both to define these differences on a molecular level
and to coin the terms interspecific and intraspecific heterogeneity for different baculovirus
species and different baculovirus strains of the same species, respectively (Fig. 9). In
addition, these techniques have allowed analysis of heterogeneity of single baculovirus
populations. For example, the presence of submolar fragments in the REN profiles (Fig. 9)
indicate the heterogeneity of baculovirus isolates, which was first confirmed in NPVs (Lee
and Miller., 1978) and then in GVs (Smith and Crook, 1988). More recent and abundant
studies verify the existence of a high genotypic variability within viral populations of a single
baculovirus isolate (Léry et al., 1998; Rezapanah et al., 2008; Eberle et al., 2009; Erlandson,
2009; Muñoz et al., 1998; Simón et al., 2004).
This variability is expected for the viral populations because of the short time
elapsing between generations and the large progeny being produced in each replication
cycle. A single genome infecting a larva may produce an average of 1010 progeny genomes,
5×107 NPV OBs (Simón et al., 2008) or 4-5×109 GV OBs (Zeddam et al., 1999).With such elevated progenies at the end of an infection cycle, the likelihood of mutations is high,
although the baculovirus genome is as stable as that of host cells. Another source of
variation is recombination, which renders deletions, inversions, duplications and acquisition
of DNA from the host or even other organisms (Muñoz and Caballero, 2001). Baculoviruses
have demonstrated highly recombinable in both culture cells (Croizier and Ribeiro, 1992;
Erlandson, 2009) or in vivo by injection of DNA genomes from two distinct species (Kamita et
al., 2003) or natural ingestion of OBs from different populations of the same virus species
(Muñoz et al., 1997). Exchange of genetic material between the host and the virus, or even
between the host plant and the virus or the parasites present in the same host is also a
baculovirus genomes by Jehle et al. (1998). These mobile elements may transport genetic
information between the two genomes, between the host cell and the virus, between two
viruses or inside the same genome conducting to insertions or duplications.
1 2 3 4 5 6 7
16.7
8.9
5.5
3.7
2.6 2.8
2.5
a
17.5
6.8
3.6
1 2 3 4
b
1 2 3
17.5
6.8
c
Fig. 9. Restriction endonuclease (REN) profiles of baculovirus genomes depicting interspecific
diversity (a) and intraspecific diversity of geographical isolates (b) or purified genotypes present in the same isolate (c). In a, the genomes of nucleopolyhedroviruses of Spodoptera littoralis (lanes 1 and 5), S. exigua (lanes 2 and 6) and S. frugiperda (lanes 3 and 7) were digested with
BglII (lanes 1-3) and PstI (lanes 5-7). In b, different S. exigua nucleopolyhedrovirus isolates from
southern Spain were digested BglII. In c, a S. exigua nucleopolyhedrovirus isolate (lane 1) was purified and its clone genotypes (lanes 2 and 3) digested with BglII. Arrow points out submolar fragments. Molecular weight marker fragments are indicated in the center (a) or the left (b and c) of the pictures. From Muñoz and Caballero, 2001.
The different level of genetic heterogeneity between NPVs and GVs may be related
to the transmission between individuals. Initiation of infection requires a limited number of
OBs, even a single OB. Presence of multiple virions in the same OB and multiple
encapsulation occurring in MNPVs allows infection of each single larva with an array of
genotypes present in the original population. This strategy facilitates the variability inside the
larvae and, in consequence, the exchange of genetic material between different genomes
through recombination. In GVs, low multiplicity of infection presumably limits genetic
variability since they are structured as a single virion per OB and a single nucleocapsid per
virion. Although some exceptions to this structural GV model have been described (Falcon
maintaining of diversity still needs to be evaluated. In any case, the variability would most
likely be lower, and the possible exchanges between genomes would be reduced, especially
outside outbreak periods or when OB densities in the environment are low and hence the
probability for a high multiplicity of infection is reduced.
Intrapopulation heterogeneity is important for viral adaptation to environmental
conditions (Domingo et al., 1998). A naturally occurring population of Spodoptera frugiperda
multiple nucleopolyhedrovirus (SfMNPV) has been extensively studied (Simon et al., 2004).
Nine genotypes, some were defective, were isolated and identified. Combinations of two or
more genotypes were analyzed in terms of both bioinsecticidal performance and proportion
of each genotype in each infection cycle. These experiments proved that the population
genetic structure was maintained over time since different genotypic proportions tended to a
single equilibrium population structure similar to that occurring in the wild-type population of
origin (Simon et al., 2006). In addition, cooperation between genotypes was observed: all
mixed infections were more pathogenic than each genotype separately (Lopez-Ferber et al.,
2003). In this example, the maintenance of at least some of the genetic diversity is important
for keeping maximum pathogenicity in this population. In other cases, other genetic traits also
leading to higher transmissibility may be favored. In S. exigua nucleopolyhedrovirus,
phyllogeneticaly close to SfMNPV, genotypic variants did not contribute to enhanced
pathogenicity (Muñoz et al., 1998) but to increased OB production (Amaya Serrano, pers.
comm.).
Co-evolution between baculoviruses and their hosts is probably one or the main
phenomena driving baculovirus heterogeneity. A handful of studies have shown that
baculoviruses get adapted to their host biotypes (Espinel-Correal et al., 2010). This leads to
a continuous search for indigenous strains against the host biotypes that need to be
controlled in each world region.
4.3.2. Soil reservoirs and persistence
Baculoviruses can remain active outside their host thanks to their occluded nature. However,
OBs can be quickly inactivated by certain environmental factors, UV light particularly (Evans,
1986). Therefore, viral location directly influences their survival possibilities and transmission
to uninfected hosts and hence, their probabilities to generate epizootics (Tanada and Kaya,
1986). Inocula are unevenly distributed in space, more highly concentrated in places were
infected insects have died, but OB spatial distribution may vary among different insect
The eventual reservoir for most baculoviruses is the soil after transportation by
gravity, rain or insect hosts that find shelter or food in the soil at some stage in their life cycle
(Jaques, 1967, 1970). In the soil, OB activity may remain intact for several years, serving as
inoculum for a number of host generations (Jaques, 1967). In some instances, OBs do not
have to be translocated elsewhere to meet their host insects. It is the case, among others, of
P. operculella, T. solanivora, A. segetum, and many lymantriid species, which keep in contact
with the soil by feeding on tubers, roots, and plant undergrounds (Richards et al., 1999). In
other virus-host systems, OBs are transported upwards to the plant leaves by rainfall splash,
wind, or tillage (Fuxa and Richter, 2001; Olofsson, 1988). Field studies on the release of OBs
in soil, stress the importance of soil viral populations in the prevalence of disease (Fuxa et
al., 2001).
OBs persist in the soil despite unstable conditions of seasonal crop habitats (Fuxa
and Richter, 1999). The main factors responsible for OB stability in the soil seem to be clay
content and pH (Evans, 1996). Laboratory and field studies have shown that OBs bind very
strongly to soil particles close to the surface (Jaques, 1969, 1975), especially to the clay
components of the soil (Hukuhara and Namura, 1972), allowing them to persist even after
rain leaching (Hukahara and Namura, 1971; Jaques, 1985). However, OB survival in soil is
believed to decline with increasing pH as the integrity of the OB matrix is compromised by
alkaline conditions (Jaques, 1974; McLeod et al., 1982). Values of pH ranging from 5.1 to 6.0
have been generally considered suitable for good OB persistence (Jaques, 1985). High
temperatures and the presence of microbial agents can also reduce the persistence of OBs
in the soil (Peng et al., 1999; Young, 2001).
Baculovirus OBs persist shortly on host plants being solar radiation the principal
factor limiting OB persistence in exposed surfaces (Young, 2001). Between 50% and 100%
of viral depositions are usually inactivated in 2-3 days to two weeks, respectively (Jaques,
1985). Viral populations may persist longer if protected from solar radiation (Cory et al., 1997;
Young and Yearian, 1989). Other abiotic factors such as rainfall, brushing, irrigation, high
temperatures or even host plan physiology can also decrease viral activity on the plant
surfaces (David and Gardiner, 1966; Young, 1990; Evans, 1986; Ritcher et al., 1987).
4.3.3. Environmental spreading
Two general transmission pathways are defined for baculoviruses: horizontal transmission,
between individuals of the same generation, and vertical transmission, directly from parents
