TESIS DOCTORAL

Characterization of novel granulovirus strains from against Phthorimaea operculella and solanivora

YANNERY GÓMEZ-BONILLA Pamplona, 2011

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 Francia

Universidad Pública de Navarra E. T. S. Ingenieros Agrónomos Pamplona, 2011 Miembros del Tribunal

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 Dpto. Producción Vegetal: Botánica y Protección Vegetal Universidad Politécnica de Madrid

D. Mª DELIA MUÑOZ LABIANO, 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.

Fdo. Dra. Mª Delia Muñoz Labiano Fdo. Dr. Miguel López-Ferber

Colaboradores

Dr. Primitivo Caballero Universidad Pública de Navarra, Pamplona, Spain

Dr. Xabier Léry Universtité Paris Sud, Orsay, Francia

Dr. Trevor Williams Instituto de Ecología AC (INECOL), Xalapa, México

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. Un largo camino he recorrido para terminar este trabajo, tuve en este largo caminar muchos altibajos, momentos críticos y difíciles, momentos de mucha soledad, sobre todo cuando

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”.

MUCHAS GRACIAS A TODOS Y TODAS Y QUE ESTEN SIEMPRE PURA VIDA

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. 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

LIST OF PUBLICATIONS ...... 99

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 testigos sin tratamiento en los ensayos de invierno y verano. Además, esta reducción no fue

3 Resumen 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 PhopGV-CR3, PhopGV-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 notables características bioinsecticidas en ensayos de laboratorio.

4

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 , no trials have been conducted in 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 untreated controls when applications resulted in complete coverage of potato tuber surfaces.

5 Summary

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 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 outstanding bioinsecticidal characteristics under laboratory conditions.

6

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. 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 ...... 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 6. References ...... 36

7 Chapter I Introduction

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 (: ) 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 , , Perú, Bolivia, and (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 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 consumption.

2.1. The potato tuberworm, Phthorimaea operculella Phthorimaea (Gelechia ) operculella comes from South America. However, the insect was first reported in 1845, in Tasmania, where typical injuries on tubers were observed. However,

8 Chapter I Introduction 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 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 or tubers and can also be laid in non-plant surfaces, like fiber bags used to store tubers, soil

9 Chapter I Introduction 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, , 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 between loss and injury level has been demonstrated: when 100% foliage is destroyed

10 Chapter I Introduction 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 hatching, larvae penetrate into the soil in search for tubers. Cracked ground favours laying

11 Chapter I Introduction 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.

b

a

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 and (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 , Venezuela, Colombia, and Ecuador in South America (Puillandre et al., 2007). In 1999, the insect was found locally in the (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 found in all regions where potato is cultivated except in Abangares, where culture of this crop

12 Chapter I Introduction 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 different categories: cultural, ethological, chemical and biological control. In addition, several

13 Chapter I Introduction 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 the foliage in the field or over the tubers in storage with limited success due to resistance

14 Chapter I Introduction

(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., 1982). These two nematodes can complete their life cycle in the host and kill 50-100% of the

15 Chapter I Introduction 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 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. More than one thousand pathogenic viruses of invertebrates have been described to date,

16 Chapter I Introduction 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 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 GVs, Gammabaculovirus , consisting on NPVs isolated from hymenopteran species, and

17 Chapter I Introduction

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 basic environment. BV penetration into the cells is carried out by pinocytosis when the cell

18 Chapter I Introduction 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 new OBs. This process can be divided into two main stages, primary and secondary

19 Chapter I Introduction 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 early, early, late and very late (or α, β, γ, and δ, respectively) (Rohrmann, 2011).

20 Chapter I Introduction

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 BVs that will start a secondary infection.

21 Chapter I Introduction

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 canopy. P. operculella and T. solanivora infected larvae, for instance, exit their galleries.

22 Chapter I Introduction

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). Primary infection of epithelial cells by occlusion derived virions (ODVs) (B). Secondary infection by budded virions (BVs) and production of new BVs (C) or new OBs in the virogenic stroma (VS) of the cells (D). Cell nuclei disintegrate upon production of GV OBs. From Rohrman, 2011.

23 Chapter I Introduction

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 10 10 progeny genomes, 5×10 7 NPV OBs (Simón et al., 2008) or 4-5×10 9 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 frequent phenomenon. The existence of mobile genetic elements was confirmed in the

24 Chapter I Introduction 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 23 4 5 6 7

16.7

8.9

5.5

3.7 1 2 3 4

17.5 2.8 2.6 1 2 3 2.5 6.8 17.5 a 3.6 6.8

b 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 Bgl II (lanes 1-3) and Pst I (lanes 5-7). In b, different S. exigua nucleopolyhedrovirus isolates from southern Spain were digested Bgl II. In c, a S. exigua nucleopolyhedrovirus isolate (lane 1) was purified and its clone genotypes (lanes 2 and 3) digested with Bgl II. 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 and Hess 1985; Vargas-Osuna et al. 1994), their importance in the generation and

25 Chapter I Introduction 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 species, host plants, and baculovirus-host combinations (Hails et al., 2002).

26 Chapter I Introduction

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 to offspring (Andrealis, 1987; Kukan, 1999).

27 Chapter I Introduction

Baculoviruses are transmitted horizontally through the release of OBs into the environment either by regurgitation and defecation during the latest stages of infection or following death of infected individuals (Vasconcelos, 1996). This type of transmission is very quick in the course of outbreaks, when host populations are very dense and the virus readily meets uninfected hosts. In lower host population densities, horizontal transmission depends on the ability of OBs to be dispersed. Dispersal is important for baculoviruses to remain in contact with a changing host population or with spatially separated host subpopulations. In many instances, dispersal represents the transition between a non-transmissible reservoir population and an ephemeral but transmissible OB subpopulation. Baculovirus OBs are rapidly dispersed by various biotic and abiotic mechanisms. Biotic dispersion may be direct, by the host itself, or indirect, passive dispersal by predators and parasitoids (Dwyer and Elkinton, 1995; Entwistle et al., 1993; Fuxa, 1991; Vasconcelos, 1996). Abiotic dispersing agents include air currents, rainfall, soil type, or agronomic practices (Fuxa and Ritcher, 1999; Murillo et al., 2007).

Vertical transmission takes place in adults, which are focused in reproduction and dispersion. This type of transmission is believed to play an important role in the survival of baculoviruses and gives rise, among other effects, to sublethal infections that may influence the severity of insect pest infestations by affecting insect fecundity or fertility. Indeed, Baculovirus infections may become persistent or latent after a sublethal dose of occlusion bodies (OBs) has been ingested by the host. Latent viral infections have been defined as those in which the virus exhibits minimal gene expression, whereas persistent viral infections are those in which the virus is replicating and a range of viral genes are expressed without signs of disease (Burden et al., 2002, 2003). Both latent and persistent viral infections may be classed as covert in that neither results in obvious signs of disease. The covert infection strategy is likely to be advantageous for pathogen survival when opportunities for horizontal transmission are limited, such as during periods of low host density. Experimental and anecdotal evidence suggest that sublethal infections can switch to patent lethal disease under the appropriate conditions of crowding, physiological stress, or the presence of additional pathogens (Fuxa et al., 1999). Covert infection may also offer opportunities for vertical transmission of the pathogen to the offspring of infected parents (Kukan, 1999).

4.4. Phthorimaea operculella granulovirus against the potato tuber moths

4.4.1. PhopGV isolates

Granulovirus isolates have been collected in most countries where P. operculella is established. The first report of P. operculella granulovirus presented an isolate from Sri

28 Chapter I Introduction

Lanka (Steinhaus and Marsh, 1967), and since then many others were found in Australia (1969), South Africa (1974), New Zealand and India (1979), Peru (1988), Yemen (1990), Kenya (1992), Tunisia (1992), Bolivia (1995), Indonesia (1999) (Zeddam et al. 1999) and Costa Rica (Gómez et al. 2008). In the last decade, several GVs have been isolated from T. solanivora and tested in Venezuela (Niño et al., 2005), Ecuador (Zeddam et al., 2005) and Colombia (Villamizar et al., 2008). All biological and molecular studies performed over these isolates show that these GVs are different strains of the PhopGV species (Espinel-Correal et al., 2010). As occurs with many other GVs, PhopGV shows a narrow host range. It only infects P. operculella , T. solanivora and a few other species of Gelechiidae (Ángeles and Alcázar, 1996; Pokharkar and Kurhade, 1999; Villamizar et al., 2005).

The genome of a Tunisian PhopGV isolate (PhopGV-1346) is one of the 50 baculovirus genomes that have been completely sequenced to date (april 2011). The sequence was deposited in the GenBank data sequence (NC004062) (Croizier et al., 2004). The genome consists of 119217 bp and 130 potential ORFs have been identified.

4.4.2. Efficiency Published studies on the efficiency of PhopGV over P. operculella have shown variably results until recently, mainly because the isolates used were not well characterized. Also, the evaluation protocols were different, including the method to apply the viruses (immersion of tubers in a virus suspension, leave spraying), the larval developmental stage, the method to calculate employed doses, and an accurate description of the virus isolate.

Often, the viral dose was expressed in larval equivalents (leq) (x virus content in larvae dispersed in a y volume of water), or OBs/ml, in which case the concentration is calculated by spectrophotometry. These approaches gave approximate and hardly reproducible results (variability of larval size and of quantity of virus adsorbed by the tuber or leaf surfaces during the soaking process). Spraying contaminated leaves of the Peruvian PhopGV isolate resulted in a median lethal concentration (LC50) of 0.016 Leq/l on neonate P. operculella larvae. Immersion of tubers in a viral suspension resulted in a LD50 of 15 and 18 Leq/100 ml in L2-L3 P. operculella . In this case, three different Indonesian PhopGV isolates were used (Zeddam et al., 1999). Kroschel et al. (1996) obtained a LC50 of 7.3 x10 4 OB/ml after tuber immersion in a suspension containing a Yemen PhopGV isolate. In field evaluations, mortality of P. operculella larvae was between 82.5 and 85.3% (Kroschel et al., 1996). In assays carried out in storage in Indonesia, larval infestation of P. operculella was reduced by 90% compared to controls; with a formulation of 40 Leq/l containing talc (Setiawati et al., 2000). In Peru, PhopGV was used in aqueous suspension to be applied by

29 Chapter I Introduction spraying and as dry powder by dusting. This formulation was used for field treatments. Larval mortality with these formulates ranged between 70 and 100% and the virus persisted up to 60 days after application (CIP, 1992). In Colombia, Pérez et al. (1988) obtained 96% mortality on larvae in the laboratory with a PhopGV applied at a concentration of 4.89 x10 6 OBs/ml. In Bolivia, several formulations and a commercial product developed by the International Potato Center (CIP) with the registered name Matapol, were evaluated over P. operculella . Matapol performed significantly better over P. operculella than over T. solanivora (Crespo et al., 2005).

For the control of the Guatemalan potato tuberworm, different isolates have been obtained from P. operculella and T. solanivora . In Venezuela, an isolate obtained from T. solanivora was evaluated in the field. A mixture of virus, water, and a dispersal agent were applied at a rate of three applications per potato cycle and larval mortalities ranged between 32 and 82% (Niño et al., 2005). Under laboratory conditions, Niño and Notz (2000) assayed a formulation with talc applied over the tubers and obtained between 98 and 100% larval mortality. In Colombia, applications of a bioinsecticide based on a Peruvian PhopGV isolate rendered mortalities over 90% (LC50= 3.4 OBs/mm 2). Not all studies have given successful results. In some instances, PhopGVs isolated from P. operculella showed little effect when evaluated over the alternate host T. solanivora (Pollet et al., 2003; Rebaudo et al., 2006). Further studies indicated that the high efficiency of PhopGV strains over T. solanivora is due to the presence of at least two genotypes, the Peruvian genotype and an additional genotype well adapted to field T. solanivora populations (Espinel et al., 2009, 2010).

In most of these studies, efficiency can not be compared due to the lack of both, a proper identification of the isolates used (the authors only mention the host of origin of each isolate without any further molecular analysis), as well as an accurate method for LC50 determination shared by all authors. On this respect, the development of a procedure to assess the pathogenicity of PhopGV is essential for gaining knowledge on the biology of the virus (virulence, persistence, comparison of different isolates), for the development of effective in vivo mass production systems (Sporleder et al., 2005) and for the search of local isolates, probably better adapted to the local host biotypes (Taha et al., 1998). Carrera et al. (2008) have developed a method consisting in contaminating the tubers by nebulization to achieve a homogeneously covered tuber surface. This method has yielded reliable and reproducible results in different laboratories from France, Ecuador, Costa Rica, and Colombia. For example, the LC50 of the reference isolate, PhopGV-1346 of P. operculella was 16.68 OBs/ml in France, 16.62 OBs/ml in Ecuador and 17.76 OBs/ml in Costa Rica (X. Lery, personal communication). It is thus now possible to systematically analyze the ability of each isolate to control P. operculella and T. solanivora .

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4.4.3. Mass production of PhopGV So far, PhopGV has been produced on P. operculella larvae reared on potato tubers. Production on artificial diet allows more accurate virus dosaging, as occurs with the industrial production of the C. pomonella GV (López-Ferber and Devauchelle, 2005), but none of the artificial diets tested for rearing P. operculella have given satisfactory results. The International Potato Center (CIP) in Peru supports the use of PhopGV-based biopesticides prepared by growers. A simple aqueous formulation can be prepared by homogenizing 20 infected larvae in 1 l of water containing a dispersing agent. A powder formulation can be achieved by adding talc to the aqueous formulation, drying, and grinding (CIP, 1992). However, grower preparations lack quality control procedures and no evaluation on the genetic structure of the virus population or on the amount of microorganisms present, among other things, can be made. The Colombian Corporation of Agricultural Research (CORPOICA) recommends a semi-industrial production of PhopGV by inoculating the virus onto the host egg surface. The eggs are then placed on tubers and infected larvae are extracted and mixed with certain inner ingredients. In addition to the powder formulation, CORPOICA developed two formulations for field application, a granular formulation to obtain water suspensions and an emulsion (Espinel-Correal et al., 2010).

5. BACULOVIRUS-BASED BIOINSECTICIDES The first baculovirus to control insect pests was successfully used in the early 1940´s. A European baculovirus isolate from the hymenopteran Diprion hecyniae (Tenthredinidae) was introduced in the exotic host populations invading Canadian forests (Cameron, 1973). In the 1970´s, the first baculovirus product was marketed for agricultural use (Huber, 1986) and since then a variety of products have been released with several outstanding examples (Cherry and Williams, 2001; Moscardi et al., 1999; Rohrmann, 2011).

5.1. Advantages and limitations Although baculovirus-based pesticides are not and will not be the panacea for all plant-pest systems, their use on several pest-crop systems has shown great benefits. One important advantage is their demonstrated option to reduce or even eliminate our dependence on chemical insecticides (Moscardi et al., 1999; Lasa et al., 2007). For instance, PhopGV provided an alternative to manage field infestations of P. operculella prior to harvest, thus reducing the risk of tuber infestations in storage (Arthurs et al., 2008). Biopesticides are practically non- toxic compared to chemical pesticides. They improve human life quality, particularly that of growers, by offering healthier products to consumers. In addition products labelled as bio are experiencing an increasing acceptance among consumers.

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One further advantage of biopesticides in general resides in their rapid degradation rates, which also reduces pollution risks. This, coupled with their greater specificity, can help maintain biodiversity by conservation of natural enemies (Gröner, 1986). What is more, the use of biopesticides reduces the risk of resistance development in insects, although careful observation of resistance management practices is needed. Finally, given the constraints on the development of baculovirus-based biopesticides, their reasonable use requires an integrated understanding of the biology, ecology and behaviour of the insects to be controlled (Cherry and Williams 2001).

On the other hand, a number of limitations restraint the wider useof baculoviruses. One of the key issues for the development of biological control agents is that they undergo a critical comparison with chemical insecticides, particularly by growers. Since, the mode of action of chemicals and BCAs is different; comparison between the two is not always righteous. However, farmer demands are a clear reflection of their crop protection management and need to be taken into account at some degree. Chemical insecticides act by contact, and/or ingestion and their effect is quick, mostly resulting in action shocks. They are not specific to one species or a limited group of insect species and hence can be applied to a wide range of insect hosts coexisting in the same field. In turn, baculoviruses have a high host specificity making it necessary to apply a different formulate for each of the pests species damaging the crop. This constitutes an extra cost for the growers and also for the industry, which needs to set up as many different facilities as products needed (Guillon, 2005).

Another limitation of baculoviruses is their slow mode of action with respect to chemicals: Baculoviruses need several days to kill their hosts, although feeding ceases sooner, and, unlike chemicals, there is no action shock to ease growers concerns. Low dose applied at multiple time intervals can be applied to kill the earliest most susceptible insect stages though (Cherry and Williams, 2001). Also, the use of baculoviruses requires knowledge of the pest life cycle, and detailed monitoring therein in order to apply the virus before the pest surpasses its economic threshold.

The high sensitivity of baculoviruses to UV light decreases their persistance in the open field very quickly. To overcome this problem, formulations have included photoprotective substances in some instances while virus micro-encapsulation has been performed in other cases (Cherry and Williams 2001). However, this is not always necessary since applications inside greenhouses, over soil insects or in forest agroecosystems have demonstrated to perform successfully over a long period of time (Johnson et al., 1998; Yatsenko, 1984).

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One further drawback of baculoviruses as BCAs is the elevated cost of mass production. So far, these viruses need to be produced in in vivo systems. Generally, insects are reared on artificial diets or natural host plants (potatoes for P. operculella and T. solanivora ) in laboratories. In some cases, field production by widespread infection and subsequent harvest of infected insects was attempted, but the risk of contamination with other pathogens is always possible (Cherry and Williams, 2001). Although several cell lines have been developed for bavuloviruses, large scale production for formulation manufacture is still lagging behind.

Biopesticide registration still requires high costs. This is undoubtedly the biggest obstacle for the development of new products. In addition, the overall structure of the BCA market is significantly different from that of traditional agrochemicals. In the case of BCAs, most businesses are small or medium compared to agrochemicals, which are mostly sold by seven multinational companies (Guillon, 2005). Because of the lack of resources to establish appropriate policies, provide technical information on management applications, and registration of new biopesticides, a common practice in developing countries is the sale of unlicensed biopesticides (Cherry and Williams, 2001).

Resistance is an important factor that may limit the use of baculovirus-based biopesticides. This can be defined as the capacity to tolerate doses of inoculum that normally cause disease or death in the majority of individuals in a normal population of the same species. Resistance cases in virus-host systems reflect a co-evolution strategy with acquisition of resistance from the host and acquisition of genes able to overcome this resistance in the virus (Sosa-Gómez and Moscardi, 2001). There are two well documented cases of resistance: that of Anticarsia gemmatalis (Lepidoptera: Noctuidae) to AgMNPV and that of the codling moth, Cydia pomonella to CpGV). A. gemmatalis populations 2000 times more resistant than susceptible populations can be obtained when subjected to constant exposure to AgMNPV at LC80 doses. This resistance has only been observed in laboratory populations and although there is no evidence of resistance in the field, monitoring of field populations is recommended, particularly in regions with increased risk of appearance (Sosa- Gómez and Moscardi, 2001). So far, the only documented resistance phenomenon taking place in the field is that of the codling moth in Europe. Since the registration of CpGV, its use has been increased in organic orchards or by growers with highly resistant populations to chemicals. In 2003, CpGV resistance was confirmed in organic orchards in Germany and France, later on also in Italy, The Netherlands and Switzerland. Further analysis demonstrated that this resistance is due to the continuous use of a commercial product based on a Mexican CpGV strain with low genotypic heterogeneity (Asser-Kaiser et al.,

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2007). Resistant populations however remain equally susceptible to different CpGV strains (Berling et al., 2009).

5.2. Mass production and field use The use of baculovirus-based biopesticides in IPM requires scaling up virus production and strict quality control of the final active component and formulate (López-Ferber and Devauchelle, 2005). The virus can be amplified in cell cultures in biofermentors, however, no product is currently produced with this methodology because of technical and economical limitations. At the moment, virus formulations depend on in vivo production in larvae reared in specialized insectary facilities or in larvae naturally infesting selected field plots (Claus and Sciocco Cap, 2001). The efficiency of baculovirus-based insecticides does not only depend on the bioinsecticidal properties of the virus but also on other aspects relating its use and application such as virus dose, insect developmental stage, and application timing and frequency. As larvae age, their susceptibility to baculovirus infection decreases, specially in the last larval instar, which may be over 10 5 fold more resistant that the earliest stages (Murillo et al., 2003). In consequence, the dose has to be increased with larval age and it must thus be adjusted to kill all larval instars present. Therefore, as a general rule, the sooner the applications, the most susceptible the insects and the lower the doses needed and the lower the treatment costs. Another important adjustment is application frequency. It is influenced by the persistence of the inocula in the field and must be synchronized with the developmental stages of the pest. For example, Kroschel et al. (1996a) determined a half life of PhopGV in sun-exposed potatoes of 1-3 days. Therefore, frequent low dose rather than less frequent high dose applications are presumed to give better results (Cherry and Williams, 2001).

5.3. Formulation and storage The formulation of the active ingredient is another very important aspect for baculovirus field efficiency. It is essential to place the virus in sites where larvae are likely to ingest it, and ensure their preservation for a sufficient time. On this respect, Arthurs et al. (2008) indicated that water PhopGV suspensions or powder formulations with diluents such as talc, sand, diatom soil, clay and kaolin, were effective for the control of stored tubers when applied over layers of tubers. In the field, Sporleder (2003) investigated the use of colorants, optical brighteners, antioxidants and different types of formulations to protect PhopGV from UV inactivation. The optical brightener Tinopal and several other antioxidants effectively protected the virus from UV decay. However, a better protection was achieved in

34 Chapter I Introduction preparations made with grinded infected larvae in water (Lacey et al., 2010). Also, a formulation must keep the virus stable during the storage period. For example, no significant loss was observed in batches of Orgya pseudotsugata MNPV after 14 years of storage in vacuum packages at -10ºC (Otvos et al., 2006). However, with long-term storage of unpurified suspensions at ambient temperature, loss of insecticidal properties can be high (Jenkins and Grzywacz, 2000). This may be due to the oxidation of unsaturated fats, autocatalysis of insect-derived proteins or the metabolic activity of contaminant bacteria (Jones and Burges, 1998). These factors may result in degradation of OBs through proteolysis, or virus inactivation through the production of free radicals. High temperatures, the presence of oxygen and humidity all contribute to OB degradation. Formulations may need addition of buffers to achieve pH neutrality and freezing can avoid an increase in contaminants. In refrigerated formulations, the use of bacteriostatic additives is often necessary (Shapiro, 1986). Finally, different formulations have been developed to also optimize the application and ingestion of baculoviruses by the pest, increase the insecticidal activity and optimize the persistence in the environment (Williams and Cisneros, 2001) although none of these have been used with PhopGV formulations yet.

5.4. Market issues Currently, the demand for biological insecticides is significantly rising throughout the world. Public increasing concern with environmental issues coupled with regulatory restrictions that aim at reducing chemical pesticide residues, have imposed a particularly favorable scenario for the development of alternative methods (Agronews, 2010). The global market for synthetic chemical pesticides estimated at 26.7 billion U.S. dollars in 2005 decreased to $ 25.3 billion in 2010, with an annual growth rate (AGR) of 1.1%. In turn, the market for biopesticides is growing rapidly, from 672 mill. in 2005 to just over $ 1 bill. in 2010 (9.9% AGR). The largest growth rate (15%) in this period has taken place in Europe (135 mill. dollar sales in 2005 vs. 270 mill. in 2010), followed by Asia (12% AGR) and Latin America (5% AGR, with 70 mill. dollars sales in 2005 vs. 88 mill. in 2010) (Agronews 2010).

Although more than 1000 different products or technologies based on biological control agents (BCAs) are available from more than 350 manufacturers in the world, they represent only 2-4% of the crop protecion market. However, good opportunities for biopesticides are anticipated not only for small organic production but also for large scale extensive crops as a tool in integrated management programs (Guillon, 2005). In Europe, USA and Canada, the use of baculoviruses has been limited to forest ecosystems and a few small niche markets. On the contrary, the situation in developing countries seems to be more promising (Cherry and Williams, 2001).

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Currently, more than twenty species of baculoviruses are commercially available for pest control in agricultural and forest ecosystems. So far, the use of Anticarsia gemmatalis NPV (AgMNPV) in soybean crops in Brazil has been the best documented example of success (Moscardi 1999; Cherry and Williams, 2001). The control program against this insect began in 1980 and by 2005 the area treated was 1 mill. Ha by applying a dose of 1.5 x1011 OB/Ha, and only once per crop cycle (Rohrmann, 2008).

6. REFERENCES Agronews, Biopesticides market to reach 1 billion on 2010. On line: http://news,agropages.com/Feature/FeatureDetail---178.htm , 2010. Andrealis, T. G., 1987. Transmission. In: Fuxa, J. R., Y. Tanada, (Eds.), Epizootiology of Insect Disease. Wiley, New York, pp. 159-176. Angéles Riva, I., Alcázar, J., 1996. Suceptibilidad de la polilla Phthorimaea operculella al virus PoVG. Rev. Peruana de Entomología. 31 , 71-78. Araque, C., García, J., 1999. Manual integrado de la polilla guatemalteca de la papa Tecia solanivora (Povolny). CRECED Provincia de Pamplona, Santander, Colombia, pp. 44. Arthurs, S. P., Lacey, L. A., De la Rosa, F., 2008. Evaluation of a granulovirus (PoGV) and Bacillus thuringiensis subsp. kurstaki for control of the potato tuberworm (Lepidoptera: Gelechiidae) in stored tubers. J. Econ. Entomol. 101 , 1540-1546. Arthurs, S. P., Lacey, L. A., De la Rosa, F., 2008. Evaluation of a granuloviruses (PoGV) and Bacillus thurigiensis subsp. Kurstaki for control of the potato Ttberworm (Lepidoptera: Gelechiidae) in Stored Tubers. J.Econ. Entomol. . 101 , 1540-1546. Asser-Kaiser, S., Fritsch, E., Undorf-Spahn, K., Kienzle, J., Eberle, K., Gund, N. A., Reineke, A., Zebitz, C. P., Heckel, D. G., Huber, J., Jehle, J. A., 2007. Rapid emergence of baculovirus resistance in codling moth due to dominant, sex-linked inheritance. Science 318 , 1916-1917. Benz, G. A., Introduction: Historical perspectives. 1996. In: Granados, R. R., B. A. Federic, Eds., The biology of baculoviruses: Biological properties and molecular biology. CRS-Press, Florida, pp. 1-35. Berling, M., Blachere-López, C., Léry, X., Bonhomme, A., Sauphanor, B., López-Ferber, M., 2009. Cydia pomonella granulovirus (CpGV) genotypes overcome virus resistance in the codling moth and improve virus efficiency by selection against resistant hosts. Applied and Environmental Microbiology. 75 , 925-930. Berlinger, M. J., 1992. Pests of processing tomatoes in Israel and suggested IPM model. Act. Hort. 301 , 185–192. Bosa, C. F., Cotes, A. M., Takehiko, F., Bengtsson, M., Witzgall, P., 2005. Pheromone- mediated communication disruption in Guatemalan potato moth, Tecia solanivora . The Netherlands Entomological Society Entomologia Experimentalis et Applicata. 114 , 137-142. Braunagel, S. C., Summers, M. D., 1994. Autographa californica nuclear polyhedrosis virus, PDV, and ECD viral envelpes and nucleocapsids: structural proteins, antigens, lipid and fatty acid profiles. Virology. 202 , 315-328. Broza, M., Sneh, B., 1994. Bacillus thuringiensis ssp. kurstaki as an effective control agent of Lepidopteran pests in tomato fields in Israel. Journal of Economic Entomology. 87 , 923–928. Burden, J. P., Griffiths, C. M., Cory, J. S., Smith, P., Sait, S. M., 2002. Vertical transmission of sublethal granulovirus infection in the Indian meal moth, Plodia interpunctell . Molecular Ecology 11 , 547-555.

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Burden, J. P., Nixon, C. P., Hodgkinson, E., Possee, R. D., Sait, S. M., King, L. A., Hails, R. S., 2003. Covert infectins as a mechanism for long-term persistence of baculoviruses. Ecol. Lett. 6 , 524-531. Caballero, P., López-Ferber, M., Trevor, W. (Eds.), 2001. Los baculovirus y sus aplicaciones como bioinsecticidas en el control biológico de plagas. Phytoma-España España. Cameron, J. W. M., 1973. Insect pathology. Annu. Rev. Entomol. 18 , 285-306. Carnero, A., Padilla, A., Perera, S., Hernández, E., Trujillo, E., 2008. Pest status of Tecia solanivora (Povolny 1973) (Lepidoptera: Gelechiidae), Guatemalan potato moth, in the Canary Islands. IOBC/WPRS Bull. 31 , 336-339. Carrera, M. V., Zeddam, J. L., Pollet, A., Léry, X., López-Ferber, M., 2008. Evaluation of per os insecticidal activity of baculoviruses by a nebulization method. IOBC/wprs Bulletin. 31 , 40-43. CIP, 1992. Annual Report International Potato Center (CIP). CIP. CIP, Lima, Peru, 1992, pp. 254. CIP, 2007. Ed. Centro Internacional de la Papa, Lima, Perú, 2007. http://www.cipotato.org/potato/ . Claus, J. D., Sciocco de Cap, A., 2001. Producción masiva de baculovirus. In: Caballero, P., et al., Eds.), Los Baculovirus y sus aplicaciones como bioinsecticidas en el control Biológico de Plagas. Phytoma, España, pp. 260-299. Coll, M., Gavish, S., Dori, I., 2000. Population biology of the potato tubermoth, Phthorimaea opercuella (Lepidoptera: Gelechiidae) in two potato cropping systems in Israel. Bulletin of Entomological Research 90 , 309–315. Condreay, J. P., Kost, T. A., 2007. Baculovirus expression vectors for insect and mammal cells. Curr Drug Targets. 8 , 1126-1131. Corredor, D., Flórez, E., 2003. Estudios básicos de biología y comportamiento de la polilla guatemalteca de la papa en un área piloto en el municipio de Villapinzón. Memorias II Taller Nacional, Tecia solanivora . CEVIPAPA. CNP, Bogota, Colombia, pp. 39-45. Cory, J. S., Hails, R. S., Sait, S. M., 1997. Baculovirus ecology. In: Miller, L. K., (Ed.), The Baculoviruses. Plenum Press, New York, 1997, pp. 301-339. Crespo, L., Grzywacs, D., Figueroa, I., Bonea, O. 2005. Una formulación líquida del baculovirus y otros productos naturales para el control de las polillas de la papa Phthorimaea operculella y Symmetrischema tangolias (Lep: Gelechiidae) en Bolivia., Memorias del III Taller internacional sobre la polilla guatemalteca de la papa Tecia solanivora . Centro Internacional de la Papa (CIP), Cartagena de Indias, Colombia, pp. 49-58. Croizier, G., Ribeiro, C. P., 1992. Recombination as a possible major cause of genetic heterogeneity in Anticarsia gemmantalis nuclear polyhedrosis virus populations. Virus Research. 26 , 183-196. Croizier, L., Taha, A., Croizier, G., López-Ferber, M., 2004. Genebank (NC_004062). Online: http://www.ncbi.nlm.nih.gov/sites/entrez?Db=genome&Cmd=ShowDetailView&Term ToSearch=16527&window=7350&begin=108497 . Cherry, A., Williams, T., Control de insectos plaga mediante los baculovirus. In: Caballero, P., et al., Eds.), Los baculovirus y sus aplicaciones como bioinsecticidas en el control biológico de plagas. Phytoma, Valencia, España, 2001, pp. 389- 450. Das, G. P., Raman, K. V., 1994. Alternate hosts of the potato tubermoth, Phthorimaea operculella (Zeller). Crop Protection 13 , 83-86. David, W. A., Gardiner, B. O. C., 1966. Persistence of a granulosis virus of Pieris brassicae on cabbage leaves. J. Invertebr. Pathol. 8 , 180-183. Domingo, E., Baranowski, E., Ruiz-Jarobo, C. M., Martin-Hernández, A. M., Saiz, J. C., Escarmis, C., 1998. Quasispecies structure and persistence of RNA viruses. Emerg. Infect. Dis. 4 , 521–527. Dwyer, G., Elkinton, J. S., 1995. Host dispersal and the spatial spread of insect pathogens. Ecology. 76 , 1262-1275. Eberle, K., Sayed, S., Rezapanah, M., Shojai-Estabragh, S., Jehle, J., 2009. Diversity and evolution of the Cydia pomonella granulovirus. J. Gen. Virol. 90 , 662-671.

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Sporleder, M., Zegarra, O., Kroschel, J., Huber, J., Lagnaqui, A., 2005. Assessment of the inactivation time of Phthorimaea operculella (PoGV) at different intensities of natural irradiation. CIP Program Report. 1999-2000. CIP, 2005, pp. 123-128. Steinhaus, E. A., Marsh, G. A., 1967. Previously unreported accessions for diagnosis and new records. J. Invertebrates Pathology. 9 , 436-438. Steinhaus, M. D., 1963. Insect Pathology, an advanced treatise. Academic Press, New York. Sun, X., Wang, H., Sun, X., Chen, X, Peng, C., Pan, D., Jehle, JA, van der Werf, W., Vlak, J.M. and Hu, Z. H. 2004. Biological activity and field e fficacy of a genetically modified Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus expressing an insect-selective toxin from a chimeric promoter. Biological Control 29, 124–137. Tanada, Y., Kaya, H. K., 1986. Ecology of insect viruses. In: Anderson, J. F., Y. Tanada, (Eds.), Perspective in forest Entomology. Academic Press, New York, pp. 265-283. Torres-Leguizamón, M., Dupas, S., Dardon, D., Gómez, Y., Niño, L., Carnero, A., Padilla, A., Merlin, I., Fossoud, A., Zeddam, J. L., Lery, X., Capdevielle-Dulac, C., Dangles, O., Silvain, J. F., 2011. Inferring native range and invasion scenarios with mitochondrial DNA: the case of T. solanivora successive north–south step-wise introductions across Central and South America. Biol Invasions. DOI 10.1007/s10530-010-9909- 2. Trivedi, T. P., Rajagopal, D., 1992. Distribution, biology, ecology and management of potato tuber moth, Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae): a review. Tropical Pest Management. 38 , 279-285. Van Oers, M., Vlak, J., 2007. Baculovirus genomics. Current Drug Targets. 8 , 1051-1068. Vargas-Osuna, E., Aldebis, H., Caballero, P., Lipa, J., Santiago-Alvarez, C., 1994. A newly described baculovirus (subgroup B) from baetica (Rambur) (Lepidoptera: Actiidae) in sourthen Spain. J. Invertebr. Pathol. 63 , 31-36. Vasconcelos, S. D., 1996. Alternative routes for the horizontal transmission of a nucleopolyhedrovirus. J. Invertebr. Pathol. 68 , 269-274. Villamizar, L., Espinel, C., Grijalba, E., Gómez, J., Cotes, A. M., Torres, L., Barrera, G., Léry, X., Zeddam, J. L., 2008. Isolation, identification and biocontrol activity of colombian isolates of granulovirus from Tecia solanivora . IOBC/WPRS Bulletin. 31 , 85-88. Villamizar, L., Zeddam, J. L., Espinel, C., Cotes, A. M., 2005. Implementacion de técnicas de control de calidad para la producción de un bioplaguicida a base del granulovirus de Phthorimaea operculella PhopGV.(Sección Agrícola). Revista Colombiana de Entomología. 1-16. von Arx, R., Gebhardt, F., 1990. Effects of a granulosis virus, and Bacillus thuringiensis on life-table parameters of the potato tubermoth, Phthorimaea operculella . Entomophaga 35 , 151–159. von Arx, R., Goueder, J., Cheikh, M., Bentemime, A., 1987. Integrated control of potato tubermoth Phthorimaea operculella (Zeller) in Tunisia. Insect Science and Its Application. 8 , 989–994. Wahsburn, J. O., Chan, E. Y., Volkman, L. E., Aumiller, J. J., Jarvis, D. L., 2003. Early synthesis of budded virus envelope fusion protein GP64 enhances Autographa californica multicapsid nucleopolyhedrovirus virulence in orally infected Heliothis virescens . Journal Of Virology. 77 , 280-290. Wang, P., Granados, R., 2001. Molecular structure of the peritrophic membrane (PM): Identification of potencial PM tanget sites for insect control. Arch. Insect Biochem. Physiol. 47 , 110-118. Watmough, R. H., Broodryk, S. W., Annecke, D. P., 1973. The establishment of two imported parasitoids of potato tuber moth ( Phthorimaea operculella) in South Africa: (Copidosoma uruguayyensis , Apanteles subandinus). Entomophaga. 18 , 237-249. Webb, R.E., Thorpe, K.W., Podgwaite, J.D., Reardon, R.C., White, G.B., Talley, S.E. 1999. Efficacy of Gypcheck against the gypsy moth (Lepidoptera: Lymantriidae) and residual effects in the year following treatment. J. Entomol. Sci. 34, 404-414.

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Whiteside, E. F., 1981. The results of competition between two parasites of the potato tuber moth, Phthorimaea operculella (Zeller) Apanteles subandinus , biological control, Copidosoma koehleri South Africa. Journal of Entomological Society Southern Africa. 44 , 359–365. Whiteside, E. F., 1985. An adaptation to overwintering in the potato tuber moth, Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae). Journal of Entomological Society Southern Africa. 48 , 163–167. Williams, T., Cisneros, J., 2001. Formulación y aplicación de los baculovirus bioinsecticidas. In: Caballero, P., et al., Eds.), Los baculovirus y sus aplicaciones como bioinsecticidas en el control biológico de plagas. Phytoma, Valencia, España, pp. 313-372. Williams, G.W. and Faulkner, P. 1997. Cytological changes and viral morphogenesis during baculovirus infection, p. 61-107. In: Miller, L.K. (Ed.), The baculoviruses, Plenum Press, New York. Wouts, W. M., Mracek, Z., Gerdin, S., Bedding, R. A., 1982. Neoaplectana Steiner, 1929 a junior synonym of Steinernema travassos , 1927 (Nematoda: Rhabditida). . Systematic Parasitology 4 , 147-154. Wraight, S. P., Ramos, M. A., 2007. Integrated use of Beauveria bassiana and Bacillus thuringiensis serovar. tenebrionis for microbial biocontrol of colorado potato beetle. Journal of Anhui Agricultural University 34 , 174-184. Xuejuan, F., Maggiorani, A., Gudiño, S., 2000. Uso de nematodos entomopatógenos como una alternativa en el control de la polilla Tecia solanivora , importante plaga de la papa ( Solanum tuberosum) en Mérida, Venezuela. Rev. Forest. Venez. 44 , 115- 118. Yatsenko, V.G. 1984. Primenenie virusnogo preparat Virin-ENSh v borbe s neparnym shelkopryadom v sadakh lesosteprnoi zony Ukrainy, p. 91-96. In: E.V. Orlovskaya (Ed.), Itogi i perspektivy prozvodstva I primeneniya virusnykh prepartov v selskom I lesnom khzyastve. Moskva. Young, S. Y., 1990. Influence of sprinkler irrigation on dispersal of nuclear polyhedrosis virus frum host cadavers on soybean. Environ. Entomol. 19 , 717-720. Young, S. Y., 2001. Persistence of virus in the environment. In: Baur, M. E., J. R. Fuxa, Eds.), Factors affecting the survival of entomopathogens. Southern Cooperative Series Bulletin s-301/s-265, (online: http://www.agctr.Isu.edu/s265/default.htm) . Young, S. Y., Yearian, W. C., 1989. Persistence and movement of nuclearpolyhedrosis virus on soybean plants after death of infected Anticarsia gemmatalis (Lepidoptera: Noctuidae). Environ. Entomol. 18 , 811-815. Zeddam, J. L., Carrera, M., Barragán, A., Pollet, A., López-Ferber, M., Léry, X., 2005. Los virus entomopatógenos para el control de las polillas de la papa: Nuevas perspectivas en el manejo de Tecia solanivora (Lepidoptera:Gelechiidae). Memorias del III Taller internacional sobre la polilla guatemalteca de la papa Tecia solanivora . Cartagena de Indias, Colombia. Centro Internacional de la Papa (CIP), Colombia, pp. 67-78. Zeddam, J. L., Pollet, A., Mangoendiharjo, S., Ramadhan, T. H., Ferber, M. L., 1999. Occurrence and virulence of a granulosis virus in Phthorimaea operculella (Lep., Gelechiidae) populations in Indonesia. Journal of Invertebrate Pathology. 74 , 48-54.

45

CHAPTER II

Characterization of a Costa Rican granulovirus strain highly pathogenic against its indigenous hosts, Phthorimaea operculella and Tecia solanivora

ABSTRACT A granulovirus isolate collected from diseased Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae) larvae in Costa Rican potato [ Solanum tuberosum L. (Solanaceae)] crops was characterized at the molecular and biological level. Restriction endonuclease analysis identified this isolate as a novel Phthorimaea operculella granulovirus (PhopGV) (Baculoviridae: Betabaculovirus ) strain and was designated as PhopGV-CR1. In addition, PCR amplification of four specific variable genomic regions yielded multiple amplicons for two open reading frames, revealing the presence of different genotypic variants within the virus population. Biologically, PhopGV-CR1 was highly pathogenic for its two indigenous hosts, although significant differences of up to four-fold were detected against P. operculella -2 [LD 50 = 17.9 occlusion bodies (OBs) mm ] and Tecia solanivora (Povolny) (Lepidoptera: -2 Gelechiidae) (LD 50 = 69.1 OBs mm ). The two P. operculella colonies, from Costa Rica and France, were equally susceptible to PhopGV-CR1. Serial passage of PhopGV-CR1 over four generations in T. solanivora increased its pathogenicity by five-fold in three generations, suggesting an ongoing adaptation to its alternate host.

This chapter has been accepted for publication in Entomologia Experimentalis et Applicata as: Y. Gómez-Bonilla, X. Lery, M. López-Ferber, P. Caballero, and D. Muñoz. 47 Chapter II Characterization of a Costa Rican granulovirus strain against tuberworms

INTRODUCTION

Two of the most important pests of potatoes [ Solanum tuberosum L. (Solanaceae)] in Central America and the northwestern countries of South America are Phthorimaea operculella (Zeller) and Tecia solanivora (Povolny) (both Lepidoptera: Gelechiidae). Phthorimaea operculella , the potato tuberworm, is a cosmopolitan species (Figure 1) and the most damaging pest of potatoes in tropical and subtropical regions (Rondon, 2010). Tecia solanivora originated from Central America and has rapidly established in various Iberoamerican countries in the last 20 years (Figure 1) and has become a major potato pest in the Andean region (Pollet et al., 2003). In 2000, this pest was reported to have invaded the Canary Islands (CAB International, 2000). The mining larvae of both these species cause severe damage to tubers in the field and in storage, where losses may account for up to 100% under non-refrigerated conditions (Niño, 2004; Raman et al., 1987; von Arx et al., 1987). Tuberworm control with chemical pesticides during the last 50 years has caused the development of multiple resistances to several organophosphorous and synthetic pyrethroids (Shelton et al., 1981; Arévalo & Castro, 2003; Dogramaci & Tingey, 2008; Saour, 2008). All this boosted the development of alternative control methods for these pests that should also protect their natural enemies (Amonkar et al., 1979; Kroschel & Koch, 1996; Kurhade & Pokharkar, 1997; Symington, 2003; Lacey et al., 2010).

Integrated Pest Management (IPM) programs for P. operculella and T. solanivora have been developed and implemented in various countries (von Arx et al., 1987; Chandel & Chandla, 2005; Kroschel & Lacey, 2008; Zeddam et al., 2008). They are based on a handful of similar practices (Das et al., 1992), all with the Phthorimaea operculella granulovirus (PhopGV) (Baculoviridae: Betabaculovirus ) as their main component. Various geographical isolates of PhopGV have proven their efficiency as biopesticides under rustic storage conditions as well as during field outbreaks in several countries, e.g., Peru, Colombia, Bolivia, Ecuador, Venezuela, USA, Tunisia, India, Yemen, and Australia (Reed & Springett, 1971; Raman et al., 1987; Kroschel et al., 1996; Lagnaoui et al., 1996; Setiawati et al., 1999; Lacey et al., 2010). The different PhopGV isolates show distinct activity against alternate hosts (Zeddam et al., 1994, 2003; Pokharkar & Kurhade, 1999; Villamizar et al., 2005).

In spite of the existence of a wide array of efficient PhopGV strains, the search for novel, indigenous isolates is always desirable because they are better adapted than foreign ones to their natural environment (Cory et al., 2005). In addition, novel strains may become excellent tools for managing insect resistance, as has occurred with an Iranian strain of Cydia pomonella granulovirus (CpGV), highly infectious to the codling moth, Cydia pomonella (L.), populations from Europe that had developed resistance against the traditionally commercialized CpGV strain from Mexico (Eberle et al., 2008). Finally, novel strains with

48 Chapter II Characterization of a Costa Rican granulovirus strain against tuberworms efficient insecticidal properties against a certain complex of hosts may offer a better alternative to chemicals than those specialized in a single host (Zeddam et al., 2003; Moura Mascarin, 2010). In Costa Rica, such an isolate would be ideal to simultaneously control P. operculella and T. solanivora , whose populations overlap spatially and temporally in Zarcero and Cartago, the two Costarican regions where potato is mostly cultivated (Anonymous, 2010).

The aim in this work was to characterize novel PhopGV strains from Costa Rica with efficient insecticidal activity against both P. operculella and T. solanivora that will allow their inclusion in IPM programs in the short or medium term.

Fig. 1. Distribution map of Phthorimaea operculella and Tecia solanivora .

MATERIALS AND METHODS Insect rearing

Two P. operculella laboratory colonies (voucher specimens are stored at the Research Center Carlos Durán) were reared. The first, P. operculella -FR, originated from an Egyptian population (kindly provided by Dr. Abol-Ela, Faculty of Agriculture, Cairo, Egypt) and was maintained at the Institut de Recherche pour le Developpement (IRD) laboratory (St Christol- lès-Alès, France). The other colony, P. operculella -CR, originated from Costa Rica and was reared at the Research Center Carlos Durán (Cartago, Costa Rica). Both colonies were maintained under constant environmental conditions: 27 ºC, 60% r.h., and L16:D8 h photoperiod. The colony of T. solanivora was established from a natural population collected in Oreamuno (Cartago, Costa Rica) and reared under similar environmental conditions.

49 Chapter II Characterization of a Costa Rican granulovirus strain against tuberworms

The procedure followed to rear both insect colonies was based on that described by Angeles & Alcázar (1995) with slight modifications. Basically, larvae were fed on potato tubers, which were previously treated with chlorine solution to kill potential contaminating fungi or bacteria. Adults were fed with a 30% (wt/vol) solution of honey or sugar. Females laid their eggs on filter papers which were collected every 24-48 h, incubated in a chamber at 27 ºC until they darkened, and then placed on potato tubers. Under these conditions, the entire life cycle of both insect species varied between 4-5 weeks.

Viruses

The Costa Rican strain (PhopGV-CR1) of the P. operculella granulovirus was isolated from field-collected larval cadavers in Alvarado (09º 56' N, 83º 48' O) (Cartago, Costa Rica) in 2006, amplified on P. operculella -FR, and re-amplified in the same host colony to obtain enough viral inoculum for the whole study. Infected larvae were homogenized in distilled water and this suspension was spread onto potato surfaces at a concentration of approximately 100-500 larval equivalents l -1. Between 15 and 20 neonate larvae were then placed on each potato and these were incubated for 3-4 weeks at 27 ºC, after which time the infected larvae were collected and used as inoculum for another passage in larvae using the same procedure. The other isolates used in this work were (voucher specimens are stored at the IRD): i) PhopGV-1346 from Tunisia (kindly provided by Dr. El Bedewi, International Potato Center, Cairo, Egypt, and multiplied during several years in Egypt), ii) PhopGV-1390.9 from Kayra, Peru (kindly provided by Dr. J. Cory, Oxford, UK) (Vickers et al., 1991), and iii) PhopGV-4.2, a clone of the PhopGV-1346 isolate (Vickers et al., 1991; Lery et al., 1998), whose genome has been completely sequenced (GeneBank NC004062) (INRA/CNRS/Université de Montpellier II, Saint Christol-lès-Alès, France). All three reference isolates were amplified in 100 P. operculella -FR neonates as described above.

To purify occlusion bodies (OBs), ca. 50 larval cadavers were collected and homogenized in 10 ml 0.01 M Tris-HCl pH 7.5 using a Potter-Elvehjem homogenizer (Bellco Glass, Vineland, NJ) and centrifuged at 664 g for 5 min at 4 ºC. Supernatants were centrifuged at 20 000 g for 20 min, pellets resuspended in 1 ml bidistilled H 2O, and then placed on a continuous 30-70% (wt/vol) sucrose gradient and centrifuged al 20 000 g for 20 min. Occlusion bodies were collected with a Pasteur pipette, resuspended in 1 ml 1× TE buffer (0.1 M Tris-HCl, pH 7.5, and 10 mM EDTA, pH 8.0), centrifuged at 20 000 g for 20 min, and stored at -20 ºC. Occlusion body concentration was determined with a 8 spectrophotometer and calculated using the following formula: 6.8 × 10 × OD 450 × dilution = number of granules ml -1. A linear regression between absorbance and OB concentration was 8 -1 determined at λ = 450 nm with 1 OD 450 for 6.8 × 10 OBs ml (Zeddam et al., 2003).

50 Chapter II Characterization of a Costa Rican granulovirus strain against tuberworms

DNA extraction and restriction endonuclease (REN) analysis

Purified OB suspensions were incubated with 25 l of 2M Na 2CO 3 for 5 min, and DNA was extracted using a phenol/chloroform/isoamyl alcohol protocol and then precipitated with ethanol, as described in previous works (Muñoz et al., 1998). Between 0.25-0.5 g of viral DNA was incubated with 10 units of each of these restriction enzymes: Sma I, Bam HI, Hin dIII, Nru I, Mlu I, Hpa I, Nsi I, Nde I, Dra III, Bst EII, and Bst ApI (Promega, Charbonnières-les-Bains, France) at the conditions specified by the supplier, according to the results obtained previously (Vickers et al., 1991; Lery et al., 1998). After addition of loading buffer (0.25% bromophenol blue, 40% wt/vol sucrose in water), samples were loaded in 1% (wt/vol) agarose gels with TAE buffer (40 mM Tris-acetate, 1mM EDTA, pH 8.0) and subjected to electrophoresis at 80 V. Ethidium bromide stained gels were then photographed on a UV transilluminator. The restriction endonuclease fragment molecular weights were determined by comparison with the corresponding sequenced PhopGV-4.2 fragments and the lambda DNA marker fragments. Marker fragments for the PhopGV-CR1 isolate were labeled using the same letter as the closest larger fragment of PhopGV-1346 in lower case, and with a sub index in cases in which when marker fragments of different sizes shared the same letter.

Analysis of DNA by PCR

Previous restriction fragment length polymorphism (RFLP) analysis on the genetic diversity of PhopGV isolates originating from various countries allowed to determine four regions of high genomic sequence variability (variable regions), affecting open reading frames (ORFs) 46, 84, 109, and 129 (Léry et al., 2005). A fifth region, affecting ORFs 90 and 91, was recently found in isolates coming from Colombia (Léry et al., 2008). Primers encompassing these variable regions were designed using the complete PhopGV sequence (NC004062). In addition, primers for lef-4 and granulin genes were included as controls.

PCR reactions were carried out in a total volume of 25 µl, containing 10-100 ng of

DNA, 1 pmol of each primer, 3.5 mM MgCl 2 and 12.5 µl of a mixture prepared by the supplier containing dNTP´s and Taq polymerase (Promega, Charbonnières-les-Bains, France). Amplifications were carried out in a thermocycler under the following conditions: a first cycle of 94 ºC per 4 min, continued by 30 cycles of 94 ºC min -1, 50 ºC min -1, and 72 ºC min -1, and a final cycle of 72 ºC per 5 min. The PCR products (amplicons) were electrophoresed in 2% wt/vol agarose gels at 150 V. The PhopGV-1346 strain was used in all PCR reactions as a reference.

51 Chapter II Characterization of a Costa Rican granulovirus strain against tuberworms

Table 1. Forward and reverse primers used for PCR amplification of the different PhopGV isolates.

Primers 1 PhopGV CpGV AT (°C) Name Sequence ORF 2 ORF Forward: 83-1 ATGTAGACGCGTCGTTAACCTGGGTGTA 3 63 83 ODV 25 Reverse: 83-2 ATGAACTGTTAAACGGCTTGAGTGAGCG Forward: 84-1bis CCGCGCCGATTACCAACAGCAGCACTAT 63 84 ORF 92 Reverse: 84-2 CCTTGTAGCGTAACACTGTTTGTGTCTC Forward: 109-1 CGGTAGACGTGTAGATAATGTGCGCGTT 4 64 109 Lef 9 Reverse: 109-2 TCATCAATGATACCATAAGGCCCGGGCG Forward: 129-1 GCGATGATGAGAATGGGAATGTGAAGAC 5 64 129 EGT Reverse: 129-2 TGCCTGCTGTGCTCGACAACAATAGACC Forward: Po 1 GAGATTAGACGAGTTCATCCAGAC 70 1 Granuline Reverse: Po2 TTGTTGTCGCTTTGGAGCTAGTAC Forward: Po 5 CTGTCAGGACGTTCTTTGATTACT 68 ORF 87 Lef 4 Reverse: Po6 CTGCTATACGCGTACATGTCACCA 1. AT: annealing temperature; 2. ORF: open reading frame; 3. ODV: occlusion derived virion; 4. Lef: late expression factor; 5. EGT: ecdysteroid UDP-glucosyltransferase

Bioassays

The bioassays to determine the mean lethal concentration at which 50% of the test organisms die (LC 50 ), an expression of pathogenicity, were carried out with neonate larvae (L1) as described by Espinel-Correal et al. (2010).

To assess the LC 50 of PhopGV-CR1 in P. operculella -FR, P. operculella -CR, and in T. solanivora , the viral inoculum was previously amplified in each host colony. Six viral concentrations were prepared (10 5-10 10 OBs ml -1 in 2 ml of water) and applied homogeneously on the surface of the potato tuber using a nebulizer (Carrera et al., 2008). Two tubers of ca. 5 cm diameter were used for each concentration and 10 neonate larvae were placed on each. For each concentration, 3-5 replicates were arranged. The final concentrations applied on the potato surface were 0.1, 1, 10, 100, 1 000, and 1 000 OBs mm 2. As most larvae leave the tubers before dying, the numbers of dead and infected larvae were recorded daily for 3 weeks to allow pupation. At the end of the experiment, tubers were opened to register dead and infected larvae remaining inside the galleries as well as the infected pupae. Corpses were dissected and microscopy preparations were performed to confirm the presence of OBs. Mortality results were subjected to probit analysis (Finney, 1971) using the POLO-PC program (LeOra Software, 1987).

Successive passages of OBs in vivo

Occlusion bodies obtained from all P. operculella larvae in the first bioassay were pooled, amplified separately in P. operculella -CR and T. solanivora , and designated passage zero. These OBs were used as inocula to infect five groups of 60 larvae. Dead larvae were pooled to obtain enough inoculum (designated passage I) for the subsequent passage (II), and the same procedure was followed for two further passages (III and IV).

52 Chapter II Characterization of a Costa Rican granulovirus strain against tuberworms

To assess the pathogenicity of PhopGV-CR1 throughout four generations of larvae, a bioassay identical to that described above was performed with five viral concentrations: 5 × 10 5, 5 × 10 6, 5 × 10 7, 5 × 10 8, and 5 × 10 9 OBs ml -1. PhopGV-1346 was used as a reference. This bioassay was replicated three times.

RESULTS

Molecular characterization of PhopGV-CR1

PhopGV-CR1 genomic profiles obtained with 10 restriction endonucleases and by PCR with four sets of primers were compared to those of PhopGV-1346, PhopGV-4.2, and PhopGV- 1390.9. Restriction endonuclease profiles produced with Nde I were unique for the three isolates compared with five RFLPs, allowing profile discrimination (Table 2). With Bam HI (Figure 2) and Bst EII, PhopGV-CR1 showed a novel RFLP that distinguished this isolate from PhopGV-4.2 and PhopGV-1390.9, which were identical. Hpa I, Mlu I, and Nsi I did not differentiate PhopGV-CR1 from PhopGV-4.2 but they were useful enzymes to tell apart PhopGV-4.2 from PhopGV-1390.9. With Nru I, PhopGV-CR1 and PhopGV-1390.9 showed identical profiles but they differed by one fragment from that of PhopGV-4.2 (Table 2). Finally, no polymorphisms were observed between restriction endonuclease profiles produced by Sma I, Hin dIII, or Dra III. Submolar fragments were observed in both field isolates, PhopGV- CR1 and PhopGV-1390.9, with several enzymes (Figure 2 and Table 2).

Table 2. Polymorphic fragments present in the restriction endonuclease (REN) profiles of PhopGV- CR1 and PhopGV-1390.9 with the RENs Bam HI, Nru I, Mlu I, Hpa I, Nsi I, Nde I, and Bst EII with respect to the cloned genotype PhopGV-4.2. Their approximate molecular weight (in bp) is indicated in brackets.

REN PhopGV-4.2 PhopGV-CR1 PhopGV-1390.9 I (6114) +* + Bam HI - i1 (ca. 6000) - - - g1* (ca. 5000) Hpa I - - m1* (ca. 3400) - c1 (ca. 14500) - G (6746) - - Nde I - g1 (ca. 6500) - I (5994) + - - j1 (ca. 5800)* - Bst EII - g1 (6400) - Mlu I - - l1 (3500) I (6104) - - Nru I - + h1 (6300) Nsi I - - i1 (2600) a +/-: presence/absence of fragment; *: submolar fragment

53 Chapter II Characterization of a Costa Rican granulovirus strain against tuberworms

By PCR, only the set of primers encompassing ORF-84 generated a PhopGV-CR1 profile which differed from those of the reference strains, PhopGV-1346 and PhopGV-1390.9 (Table 3). Using the set of primers for ORF129, PhopGV-CR1 appears similar to PhopGV- 1390.9, both differing from PhopGV-1346 by the presence of amplicons of 1023 and 869 bp, and the absence of one amplicon of 723 bp (Table 3). A single amplicon was amplified with the primer sets ORF1, ORF83, ORF87, and ORF109, with identical size for the three isolates analyzed. These amplicons were not further analysed.

Fig. 2. Bam HI restriction endonuclease profiles of the genomic DNA extracted from PhopGV-4.2 (4.2), PhopGV-CR1 (CR1), and PhopGV-1390.9 (1390.9). Restriction fragment polymorphic fragments Bam HI-I (I) and Bam HI-i1 (i1) are indicated to the right of the picture. The asterisk points out the submolar fragment I in CR1. Lambda DNA digested with Hin dIII was used as molecular weight marker (M) and its fragment sizes are included to the left of the picture.

Table 3. PCR amplicon sizes (bp) of genomic regions ORF129 and ORF84 amplified for PhopGV- 1346, PhopGV-1390.9, and PhopGV-CR1.

ORF of region amplified (gene PhopGV-1346 PhopGV- PhopGV-CR1 function) 1390.9 - 1023 + 937 + + ORF129 ( egt ) - 869 + 723 - - ORF84 - - 330 241 + + a +/-: presence/absence of amplicon

54 Chapter II Characterization of a Costa Rican granulovirus strain against tuberworms

Pathogenicity of PhopGV-CR1

The pathogenicity of PhopGV-CR1 was assayed on indigenous populations of its two natural host, P. operculella and T. solanivora . In addition, two P. operculella populations were tested in order to detect potential differences in susceptibility to the virus by geographically distant host biotypes. The dose-mortality responses of PhopGV-CR1 for the two P. operculella populations and for T. solanivora were fitted with a common slope; the interaction between host populations and log [virus dose] was not significant ( χ2 = 3.82, d.f. = 2, P= 0.15).

The pathogenicity (expressed as LC 50 ) of PhopGV-CR1 for the two P. operculella biotypes is not significantly different, indicating that this PhopGV strain is equally pathogenic against host populations as distant as those from Costa Rica and Egypt (Table 4). However, the pathogenicity of PhopGV-CR1 for T. solanivora was four-fold lower than for P. operculella from France, as indicated by the 95% confidence intervals of the relative potency, which did not include 1 (Robertson & Preisler, 1992). The relative potency is the ratio between the

LC 50 s of the reference treatment (PhopGV-CR1 for P. operculella -FR in this particular assay) and each of the other LC 50 s.

Table 4. LC 50 values of PhopGV-CR1 obtained from two colonies of Phthorimaea operculella and the Costa Rican population of Tecia solanivora .

Confidence LC Relative limits 95% Insect population Regression line 50 (OBs/mm 2) potency lower upper

P. operculella-FR 0.48x + 4.54 17.0 1 - - P. operculella-CR 0.48x + 4.53 17.9 0.95 0.42 2.15 T. solanivora 0.48x + 4.24 69.1 0.25 0.10 0.59

Pathogenicity of PhopGV-CR1 upon serial passage in P. operculella and T. solanivora

The pathogenicity of PhopGV-CR1 was significantly enhanced at the second and third passages in P. operculella (by six-fold) and T. solanivora , (by five-fold), respectively, as indicated by the potency 95% confidence limits, which were above 1 (Robertson & Preisler,

1992). LD 50 values did not vary thereafter upon serial passage (Table 5), indicating that the virus got quickly adapted to its host. Moreover, the final pathogenicity of the viruses, once adapted to one or the other host, was not statistically different for any of the virus isolates tested (Table 5). The pathogenicity of PhopGV-1346 increased, by 7.5- and 13.1-fold, at the second passage in P. operculella and T. solanivora , respectively (Table 5).

55 Chapter II Characterization of a Costa Rican granulovirus strain against tuberworms

Table 5. LC 50 values of PhopGV-1346 and PhopGV-CR1 obtained from Phthorimaea operculella and Tecia solanivora upon serial passage.

Confidence limits 95% LC 50 Virus Host Passage Regression line 2 Potency (OBs/mm ) lower upper I 0.56x+4.26 21.1 1 - - Po II 0.56x+4.35 2.8 7.54 2.54 22.58 III 0.56x+4.58 3.0 7.09 2.39 21.58 1346 I 0.48x+4.18 47.7 1 - - Ts II 0.48x+4.73 3.6 13.09 4.75 38.20 III 0.48x+5.85 2.0 23.38 5.32 110.69 I 0.45x+4.42 18.8 1 - - II 0.45x+4.78 3.0 6.28 2.43 16.94 Po III 0.45x+4.75 3.6 5.19 1.86 15.15 IV 0.45x+4.74 3.7 5.13 1.55 17.87 CR1 I 0.50x+4.27 28.9 1 - - II 0.50x+4.41 14.6 1.97 0.79 4.98 Ts III 0.50x+4.63 5.4 5.35 2.00 14.71 IV 0.50x+4.65 4.9 5.91 2.22 16.29

DISCUSSION

The present study describes the molecular and biological characterization of a granulovirus (GV) isolated from P. operculella in Costa Rica and named PhopGV-CR1. Restriction endonuclease analysis showed a high similarity of PhopGV-CR1 to reference strains, revealing that the novel Costa Rican isolate is a geographical strain of this viral species, namely PhopGV. Several studies have shown that variation among restriction patterns of PhopGV isolates collected in very distant locations (Australia, Peru, Yemen, Indonesia) is limited, although at least several of these virus populations have probably been isolated for quite long periods. The reason for such a strong conservation of restriction sites in viral sequences is unclear but this fact is not unique among GVs (Zeddam et al., 1999). The presence of submolar fragments in several restriction endonuclease profiles and the existence of multiple fragment amplification with the same set of primers strongly suggest the presence of different genotypes within PhopGV-CR1. Genotypic heterogeneity within the same virus strain has also been observed for other PhopGV isolates (Lery et al., 1998), and is very common among other GV and nucleopolyhedrovirus (NPV) populations (Smith & Crook, 1988; Caballero et al., 1992; Burden et al., 2006; Figueiredo et al., 2009). This denotes the existence of functional diversity among PhopGV genotypes. Indeed, purified genotypes with significantly different pathogenicity, virulence, or OB yield to that of the wild- type mixture have been described (Muñoz et al., 2000; Hodgson et al., 2001; Simón et al., 2008). This constitutes evidence of the importance of genetic diversity in the capacity of a pathogen to adapt to its environment, in particular to its hosts.

Adaptation of a virus isolate to different host colonies was tested using the same bioassay methodology. The LC 50 of PhopGV-CR1 was determined in two populations of P.

56 Chapter II Characterization of a Costa Rican granulovirus strain against tuberworms operculella from France and Costa Rica, and also in a Costa Rican colony of the alternate host, T. solanivora . The pathogenicity of PhopGV-CR1 in its homologous host is similar to that obtained with the reference strain, PhopGV-1346, its purified clone PhopGV-4.2, and two isolates from Colombia and Peru, and lower than that of two other Colombian isolates (Espinel-Correal et al., 2010). Comparison of these values with those of strains from Yemen (Kroschel et al., 1996) or Indonesia (Zeddam et al., 1999) is difficult because of the different methodologies employed for the bioassays.

The pathogenicity of PhopGV-CR1 was four times lower for its heterologous host, T. solanivora . Similarly, for Colombian T. solanivora , a non-adapted virus strain is less efficient (Espinel-Correal et al., 2010). Larvae of T. solanivora have been found infected by baculoviruses naturally (Zeddam et al., 2003; Niño, 2004; Villamizar et al., 2005). The viruses they contain appeared to be related to PhopGV. However, the process of adaptation was not explored, and the efficacy of these isolates in the control of potato tuber moths was not indicated.

Granulovirus strains with good insecticidal performance are desirable for an efficient control of both pests. This may be achieved, for example, by thoroughly screening isolates from various geographical regions. Indeed, three Colombian PhopGV strains, recently isolated by Espinel-Correal et al. (2010), have been found to be up to 50-fold more pathogenic than the Costa Rican or the Peruvian PhopGV strains, showing LC 50 values as low as 1.16 OBs mm -2. These highly pathogenic strains were isolated from T. solanivora in regions where both hosts coexist, whereas the Peruvian strain had not had previous contact with this host. Tecia solanivora ’s origin has been traced back to Central America. It is thus likely that virus populations infecting potato tuber moths in this region, like PhopGV-CR1, had previously been in contact with both host species, and retain the ability for quick adaptation to their hosts. Increasing pathogenicity of the virus adaptation to its host has been observed to occur upon serial passage of genotypically heterogeneous virus populations (Kolodny- Hirsch & Van Beek, 1997; Berling et al., 2009). Both the known genome plasticity of the baculoviruses and the genotypic heterogeneity of baculovirus populations play a role in this adaptation. As the PhopGV-CR1 strain was composed of an array of distinct genotypes, quick adaptation to its natural hosts was suspected. To demonstrate this, PhopGV-CR1 was passaged serially for four host generations, and indeed its pathogenicity significantly -2 -2 increased, reaching LC 50s as low as 3.0 OBs mm for P. operculella and 5.4 OBs mm for T. solanivora . These values are strikingly similar to some of the field-adapted Colombian PhopGV strains (Espinal-Correal et al., 2010). Likewise, 15-fold increased pathogenicity was registered for AcMNPV against Plutella xylostella (L.) when passaged 20 times in this host (Kolodny-Hirsch & Van Beek, 1997). More recently, a similar observation was made on the

57 Chapter II Characterization of a Costa Rican granulovirus strain against tuberworms

CpGV NPP-R1 strain that was passaged four times in a colony of C. pomonella resistant to a commercialized CpGV strain from Mexico (Berling et al., 2009). In this case, genotype selection accounted, at least partially, for host adaptation, as the proportion of a genotype similar to the one dominant in the Mexican CpGV strain was sharply reduced in this strain after only four serial passages (Berling et al., 2009). It is likely that adaptation of PhopGV- CR1 to T. solanivora can also be explained by genotype selection, but molecular characterization of the genotypes composing the PhopGV-CR1 strain from the first and fourth serial passage in P. operculella and T. solanivora are needed to confirm this hypothesis. Surprisingly, the reference strain, PhopGV-1346 also got quickly adapted upon contact with the Costa Rican populations of both the original and the heterologous hosts. This constitutes further evidence of the ability of this virus to adapt to its hosts. In addition, the fact that

PhopGV-1346 shows LC 50 values similar to those of the T. solanivora field-adapted Colombian strains seems to invalidate a link between pathogenicity and a 86 bp genomic insertion in the egt gene (observed as a 1 023 bp fragment by PCR). This insertion occurs in the Colombian isolates (Espinel-Correal et al., 2010) and also in PhopGV-CR1, but is absent in the PhopGV-1346 strain.

Preliminary results on the efficiency of PhopGV-CR1 have been obtained under both field and storage conditions (Gómez-Bonilla et al., 2011a,b). In the field, the virus reduced damage between 50 and 80% compared with the untreated controls, whereas over 70% damage reduction was obtained in stored potatoes. These data favor the inclusion of PhopGV-CR1 formulations in IPM programs.

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60 Chapter II Characterization of a Costa Rican granulovirus strain against tuberworms

Shelton AM, Wyman JA & Mayor AJ (1981) Effects of commonly used insecticides on the potato tuberworm and its associated parasites and predators in potatoes. Journal of Economic Entomology 74: 161-184. Simón O, Williams T, López-Ferber M, Taulemesse JM & Caballero P (2008) Population genetic structure determines the speed of kill and occlusion body production in Spodoptera frugiperda multiple nucleopolyhedrovirus. Biological Control 44: 321- 330. Smith IR & Crook NE (1988) In vivo isolation of baculovirus genotypes. Virology 166: 240- 244. Symington CA (2003) Lethal and sublethal effects of pesticides on the potato tuber moth, Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae) and its parasitoid Orgilus lepidus Muesebeck (Hymenoptera: Braconidae). Crop Protection 22: 513- 519. Vickers JM, Cory JS & Entwistle PF (1991) DNA characterization of eight geographic isolates of granulosis virus from the potato tuber moth ( Phthorimaea operculella ) (Lepidoptera, Gelechiidae). Journal of Invertebrate Pathology 57: 334-342. Villamizar L, Zeddam JL, Espinel C & Cotes AM (2005) Implementación de técnicas de control de calidad para la producción de un bioplaguicida a base del granulovirus de Phthorimaea operculella PhopGV. Revista Colombiana de Entomología 31: 127- 132. von Arx R, Goueder J, Cheikh M & Termine AB (1987) Integrated control of potato tubermoth Phthorimaea operculella (Zeller) in Tunisia. Insect Science and its Application 8: 889-994. Zeddam JL, Léry X, Giannotti J, Niño de Gualdrón L, Angeles I & Alcazar L (1994) Susceptibility of different potato moth species to a same granulosis virus. Proceedings of the XXVIIth Annual Meeting of the Society for Invertebrate Pathology, 28 August-2 September, Montpellier, France (ed. by the Society for Invertebrate Pathology), pp. 239-240. Société Viganaise d´Imprimerie, Ganges, France. Zeddam JL, Orbe K, Léry X, Dangles O, Dupas S & Silvain JF (2008) An isometric virus of the potato tuber moth Tecia solanivora (Lepidoptera : Gelechiidae) has a tri- segmented RNA genome. Journal of Invertebrate Pathology 99: 204-211. Zeddam JL, Pollet A, Mangoendiharjo S, Haris Ramadhan T & López-Ferber M (1999) Occurrence and virulence of a granolosis virus in Phthorimaea operculella (Lepidoptera, Gelechiidae) populations in Indonesia. Journal of Invertebrate Pathology 74: 48-54. Zeddam JL, Vásquez M, Vargas Z & Lagnaoui A (2003) Producción viral y tasas de aplicación del granulovirus usado para el control biológico de las polillas de la papa Phthorimaea operculella y Tecia solanivora (Lepidoptera: Gelechiidae). Plagas 29: 659-667.

61

CHAPTER III

Potato crops in Costa Rica are efficiently protected from Phthorimaea operculella and Tecia solanivora by an indigenous granulovirus strain

ABSTRACT The control efficiency of a Phthorimaea operculella granulovirus isolate from Costa Rica (PhopGV-CR1) against the insect pests P. operculella and Tecia solanivora under field conditions was evaluated. The virus reduced damage between 50 and 80% in fields compared with the untreated controls. These data favor the inclusion of PhopGV-CR1 formulations in IPM programs.

This chapter has been accepted for publication in the 2011 Proceedings of the IOBC/wprs 13th European Meeting of the IOBC/WPRS Working Group "Insect Pathogens and Entomoparasitic Nematodes", Innsbruck, Austria as: Y. Gómez-Bonilla, M. López-Ferber, P. Caballero, and D. Muñoz. 63 Chapter III Field efficiency of a Costa Rican PhopGV strain

INTRODUCTION The tuber moths, Phthorimaea operculella (Lepidoptera: Gelechiidae) (Zeller) and Tecia solanivora, (Lepidoptera: Gelechiidae) (Povolny), are the most devastating potato pests in Latin American field crops (Arthurs et al., 2008). P. operculella attacks the aerial parts of the tomato plant causing major damage to tuber yield. Infestation of tubers occurs mainly in storage conditions, where tuber quality is severely affected. T. solanivora only infests the tubers, both in the field, before harvest, and in storage. Protection of tubers entering warehouses is thus not sufficient to avoid T. solanivora derived losses. Pre-harvest intervals limit the use of broad spectrum pesticides, when the most economically significant damage typically occurs (Arthurs et al., 2008). Therefore, alternative options for late-season and postharvest control that also provide tools for insecticide-resistance management and organic production are needed. Field applications of biopesticide formulations have demonstrated not only to protect the pest natural enemies but also maintain the ecological balance in the environment. Several baculovirus-based biopesticides have been successfully used against several pests (Berling et al., 2009; Moscardi et al., 1999; Lasa et al., 2007), most which can be included in Integrated Pest Management (IPM) programs.

Successful IPM programs against the tuber moths have included granulovirus-based formulations for field use (Chaparro et al., 2010). However, in Costa Rica, IPM programs for these pests have not yet included them given the lack of indigenous strains, which have not been isolated and characterized until only very recently (Gómez-Bonilla et al., submitted). The novel Costa Rican P. operculella GV (PhopGV) strains have proved their efficiency under laboratory conditions against the two pests. Their inclusion in IPM programs in this country only depends on their field efficiency. This paper describes the performance of a highly pathogenic strain from Costa Rica (PhopGV-CR1) under field conditions.

MATERIAL AND METHODS Insect rearing The insect populations of P. operculella and T. solanivora originally came from Costa Rica and were maintained at 25ºC and a 16:8 light:dark photoperiod at the Research Center Carlos Duran (Cartago, Costa Rica). Larvae were fed on potato tubers previously treated with 0.5% chlorine solution. Adults were fed honey or 30% (w/v) sugar.

Viral formulations Viral formulations were based on the Costa Rican PhopGV-CR1 strain, previously characterized (Gómez-Bonilla et al., 2011). A solid formulation was manufactured with 20 P. operculella infected larvae (2.13 x 10 10 OBs) which were homogenized and added: 0.2% Tween 20, 1 l water and 1 kg talc. The resulting mixture was air dried at room temperature

64 Chapter III Field efficiency of a Costa Rican PhopGV strain for at least 15 days and then grinded. Finally, it was packed in 2 plastic bags containing 500 g each. The liquid formulation was prepared with 20 P. operculella infected larvae which were homogenized and added, 0.2% Tween 20 and 1 l water (Zeddam et al., 2003).

Field assays Field assays were carried out in Alvarado (Cartago, Costa Rica) throughout the two seasons in 2009: summer assays from February to May and winter assays from April to July. Both fields were divided into 25 m 2 plots, each consisting of five 5 m long rows with 20 potatoes seeded 25 cm apart in each row. A row was kept empty between plots to avoid treatment drift. Plots were subjected with one of the following three treatments: i) baculovirus formulations alone (Bac); ii) baculovirus formulations together with insecticide and fungicide applications (Bac+I+F); and iii) insecticide and fungicide applications (I+F). Three additional plots were kept untreated as controls. The treatments were applied as scheduled in Fig. 1 and arranged in a complete randomize block design. The whole assay was replicated six times. The weight of the potatoes produced by the four plants placed along 1 m of two central rows in each plot was registered at the end of the season and damage (those potatoes with at least one tunnel) calculated as a percentage between the weight of damaged potatoes and the total weight. Pheromone traps for both moths were placed in the four field corners from sowing to harvest and monitored each week.

A modified Shapiro-Wilks test was applied to check for the normality of the data andthe analysis of the variance (ANOVA), and the Duncan test (SAS software package, SAS Institute Inc., USA) was applied to determine statistical differences.

RESULTS AND DISCUSSION Field applications of a PhopGV-CR1-based powder and liquid formulations, alone or together with an insecticide, protected field potatoes as effectively as chemical insecticides (Table 1). Damage in the treated plots was significantly reduced: between 56.1 and 78.6% in the summer field and between 61.1 and 82.5% in the winter field compared with untreated control plots, where damage reached 19.1 and 14.4% in summer and winter, respectively. No significant differences were obtained between treatments in any of the two experimental fields, indicating that PhopGV-CR1 can be used against these pests alone, as an alternative to chemicals or, rather, included in IPM programs to diminish the chances of chemical resistance development.

Damage inflicted in the untreated plots was moderately low because of the temperate climate of this region of Costa Rica, with frequent rains. Although rain leads to low moth densities, this pest has gradually adapted to higher moisture conditions. As occurred in the

65 Chapter III Field efficiency of a Costa Rican PhopGV strain warehouses, incidence of P. operculella was lower than T. solanivora during both seasons (Fig. 1). Both populations suffered drastic population decreases upon chemical insecticide applications against moths (Fig. 1).

Fig. 1. Population fluctuations of T. solanivora and P. operculella along the potato crop cycle during the summer and winter assays. Arrows indicate timing of control applications with baculovirus formulations in powder (dark green) or liquid (bright green) and/or chemicals in plots where a combination of baculovirus and chemicals were applied (black arrows) or in plots where only chemicals were applied (red arrows).

Frequency of virus application may seem too numerous in comparison with other baculovirus-host systems. However, weekly PhopGV applications are a common practice against potato tuber moths in Southern America given the overlapping host generations commonly found in tropical regions. Up to 10 T. solanivora generations per year can occur under optimal conditions (Notz, 1996), with a length cycle of 43 days at 25°C (Niño, 2004). In Venezuela, Niño captured adults from January to October (Niño, 2004).

Table 1. Percent damage by P. operculella and T. solanivora in summer and winter assays.

Damage (%) ± S.E. Treatment Summer Winter Control untreated 19.05 ± 1.62 a 14.38 ± 2.23 a Bac 8.37 ± 2.27 b 5.59 ± 0.90 b Bac+I+F 4.07 ± 1.43 b 2.64 ± 0.95 b I+F 4.27 ± 0.97 b 2.52 ± 0.71 b

66 Chapter III Field efficiency of a Costa Rican PhopGV strain

In conclusion, the results of this study indicate that PhopGV-CR1 based formulations can be incorporated into IPM practices for field use against P. operculella and T. solanivora to protect potatoes in Costa Rica and that they may be as efficient as chemical insecticides.

REFERENCES Arthurs, S. P., Lacey, L. A. & De la Rosa, F., 2008: Evaluation of a granulovirus (PoGV) and Bacillus thuringiensis subsp. kurstaki for control of the potato tuberworm (Lepidoptera: Gelechiidae) in stored tubers. J. Econ. Entomol. 101, 1540-1546. Berling, M., Blachere-Lopez, C., Soubabere, O., Lery, X., Bonhomme, A., Sauphanor, B. & Lopez-Ferber, M., 2009: Cydia pomonella granulovirus genotypes overcome virus resistance in the codling moth and improve virus efficiency by selection against resistant hosts. Appl. Environ. Microbiol. 925–930. Chaparro, M., Espinel, C., Cotes, A.M. & Villamizar, L. 2010: Photostability and insecticidal activity of two formulations of granulovirus against Tecia solanivora larvae. Rev. Col. Entomol. 36: 25-30. Gómez-Bonilla, Y., Lopez-Ferber, M., Caballero, P., Lery, X. & Muñoz, D. Characterization of a Costa Rican granulovirus strain highly pathogenic against its indigenous hosts, Phthorimaea operculella and Tecia solanivora . Entom. Exp. Appl., submitted. Lasa, R., Caballero, P. & Williams, T., 2007: Juvenile hormone analogs greatly increase the production of a nucleopolyhedrovirus. Biol. Con. 41, 389–396. Moscardi, F., 1999: Assessment of the application of baculoviruses for control of Lepidoptera. Ann. Rev. Entomol. 44, 257-263. Niño, L., 2004 : Revisión sobre la polilla de la papa Tecia solanivora en Centro y Suramérica. Suplemento Revista latinoaméricana de la papa. 4 : 1-18. Notz A. 1996 : Influencia de la temperatura sobre la biología de Tecia solanivora (Povolny) (Lepidoptera: Gelechiidae) criadas en tubérculos de papa Solanum tuberosum L. Boletín Entomología Venezolana. 11 (1): 49-54 Zeddam, J. L., Vasquez Soberon, R. M., Vargas Ramos, Z. & Lagnaoui, A. 2003: Producción viral y tasas de aplicación del granulovirus usado para el control biológico de las polillas de la papa Phthorimaea operculella y Tecia solanivora (Lepidoptera: Gelechiidae). Rev. Chil. Entomol. 29, 29-36.

67

CHAPTER IV

Stored potatoes in Costa Rica are efficiently protected from Phthorimaea operculella and Tecia solanivora with an indigenous granulovirus strain

ABSTRACT The control efficiency of a Phthorimaea operculella granulovirus isolate from Costa Rica (PhopGV-CR1) against the insect pests P. operculella and Tecia solanivora under storage and field conditions was evaluated. The virus reduced damage by over 70% compared with the untreated controls. These data favour the inclusion of PhopGV-CR1 formulations in IPM programs.

This chapter has been accepted for publication in the 2011 Proceedings of the IOBC/wprs 13th European Meeting of the IOBC/WPRS Working Group "Insect Pathogens and Entomoparasitic Nematodes", Innsbruck, Austria as: Y. Gómez-Bonilla, M. López-Ferber, P. Caballero, and D. Muñoz. 69 Chapter IV Efficiency of a Costa Rican PhopGV strain in warehouses

INTRODUCTION Potato tubers constitute one of the key resources of human diet in the world, especially in Central and South America, the geographical origin of this commodity. The number of production cycles greatly varies between regions, from a single per year up to four cycles under optimal conditions. The tubers can be stored without major processing in warehouses, which need only to be dry and dark, without needs of temperature or humidity control. This traditional simple storage allows tuber conservation until required for consumption or processing.

Harvest storage constitutes an attraction point for pests, mainly rodents and insects. In potatoes, important postharvest losses are related to the potato tuber moth, Phthorimaea operculella (Lepidoptera: Gelechiidae) (Zeller), infestations. P. operculella is widely distributed all around the world in temperate to warm regions. It attacks the aerial parts of the potato plant in the field, and may cause damage on the quantity or the quality of tuber production. However, in warehouses it feeds on tubers, rendering them unsuitable for consumption. In recent years, a second insect, Tecia solanivora (Lepidoptera: Gelechiidae) (Povolny), originating from Guatemala, started to colonize new areas from Costa Rica, towards southern countries. Since 1999 it has been observed in the Canary Islands (Torres- Leguizamón et al., 2011). T. solanivora larvae feed only on the potato tubers. It can infest tubers in the field which, upon arriving to the warehouses constitute the inoculum for a wide scale infestation that can result in a complete loss (Rondon 2010). Therefore, alternative options for postharvest control that also provide tools for insecticide-resistance management and organic production are needed.

Successful IPM programs against the tuber moths have lately included granulovirus- based formulations for storage use (Arthurs et al.; 2008; Zeddam et al., 2003). However, in Costa Rica, IPM programs for these pests have not yet included granuloviruses (GVs) given the lack of indigenous strains, which have not been isolated and characterized until only very recently (Gómez-Bonilla et al., submitted). The novel Costa Rican P. operculella GV (PhopGV) strains have proved their efficiency under laboratory conditions against the two pests. Their inclusion in IPM programs in this country only depends on determining their efficiency. This paper describes the performance of a highly pathogenic strain from Costa Rica (PhopGV-CR1) under storage conditions.

MATERIAL AND METHODS Insect rearing The insect populations of P. operculella and T. solanivora originally came from Costa Rica and were maintained at 25ºC and a 16:8 light:dark photoperiod at the Research Center

70 Chapter IV Efficiency of a Costa Rican PhopGV strain in warehouses

Carlos Duran (Cartago, Costa Rica). Larvae were fed on potato tubers previously treated with 0.5% chlorine solution. Adults were fed honey or 30% (p/v) sugar.

Viral formulations Viral formulations were based on the Costa Rican PhopGV-CR1 strain, previously characterized (Gómez-Bonilla et al., submitted). Twenty P. operculella infected larvae (2.13 x 10 10 OBs) were homogenized and added: 0.2% Tween 20, 1 l water and 1 kg talc. The resulting mixture was air dried at room temperature for at least 15 days, grinded and packed in two plastic bags containing 500 g each.

Field assays Warehouses were located 23 km apart in the province of Cartago (Costa Rica) at 2,100 m (warehouse 1) and 2340 m (warehouse 2) of altitude. The former was made of plastic, and storage boxes rested on the soil whereas the latter was a concrete building. Twenty kg of potatoes (var. Floresta) were treated with 100 g of the baculovirus powder formulation. Two types of applications were performed: a traditional dusting application (DA) over potato layers placed in storage boxes, or a bag application (BA) consisting in thoroughly mixing the potatoes with the virus formulation in bags before storing them in similar boxes. BA applications were performed with the baculovirus formulation alone (BacB treatment) whereas different treatments were performed with the DA application: i) the baculovirus alone (BacD treatment); ii) the baculovirus and 20 g of the fungicide Vitavax 40 WP (BacD+F treatment); and iii) 100 g of the insecticide Vydate 24 SL and 20 g Vitavax 40 WP (F+I). Groups of five boxes were piled up, stored in diffuse light for four months and rotated every month. Three replicates were carried out and non-treated boxes were used as controls. A randomized complete blocks design was used. Results were expressed as percentage of damaged potatoes (those with at least one tunnel) over the total number of potatoes. Pheromone traps for both moths were placed and monitored each week. Assays were carried out with the 2008 potato harvest.

A modified Shapiro-Wilks test was applied to check for the normality of the data and the analysis of the variance (ANOVA), and the Duncan test (SAS software package, SAS Institute Inc., USA) was applied to determine statistical differences.

RESULTS AND DISCUSSION In warehouse 1, the BacB treatment resulted in significantly lowest damage than any treatment applied as DA or the non-treated control (Table 1). In warehouse 2, percent damage in seed potatoes was significantly lower in all four treatments compared to the

71 Chapter IV Efficiency of a Costa Rican PhopGV strain in warehouses control (Table 1). The best control in both warehouses was achieved when BA was applied, indicating that the extra step taken to apply the virus inside a bag results in better potato coverage as has occurred in other instances (Alcázar et al., 1993; Niño de Guadrón and Notz, 2000). The added operational step might be worth the extra cost.

Table 1. Percent damage by P. operculella and T. solanivora in warehouses 1 and 2. Damage (%) ± S.E. Treatment Warehouse 1 Warehouse 2 Control untreated 38.00 ± 4.51 a 29.00 ± 4.04 a BacB 6.33 ± 1.76 b 8.33 ± 1.20 c BacD 29.00 ± 4.51 a 10.67 ± 2.03 b c BacD+F 21.00 ± 9.07 a b 19.33 ± 3.53 b F+I 26.67 ± 1.20 a 8.33 ± 0.88 b c

Different results were obtained with the other three treatments in the two warehouses, probably because of their different structure. The concrete-made warehouse 2 represents a greater barrier for moth entry than warehouse 1, as demonstrated by moth catches (Fig. 1). In warehouse 2, T. solanivora populations increased during the last month most probably because of the emergence of adults from the second generation. In any case, the level of protection after 2-3 months storage has been reported to decrease dramatically in other instances (Das et al., 1992; Arthurs et al., 2008). T. solanivora was the dominant species throughout the whole season in this study (Fig. 1) and in previous recordings in this region (Gómez-Bonilla & Guzmán, 1997), indicating that T. solanivora can effectively be controlled by PhopGV-CR1 as occurs with other PhopGV isolates elsewhere (Espinel-Correal et al., 2010; Zeddam et al., 2003). In conclusion, it seems that PhopGV-CR1 offers an efficient alternative to control T. solanivora and P . operculella in the warehouses of this region.

72 Chapter IV Efficiency of a Costa Rican PhopGV strain in warehouses

Fig. 1. Number of adults of T. solanivora and P. operculella captured in pheromone traps in warehouses 1 (a) and 2 (b) during the 2008-2009 period.

REFERENCES Alcázar, J., Cervantes, M., Raman, K. V., 1993: Efectividad de un virus granulosis formulado en polvo para controlar Phthorimaea operculella en papa almacenada. Rev. Per. Entomol. 35, 113-116. Arthurs, S. P., Lacey, L. A., De la Rosa, F., 2008: Evaluation of a granulovirus (PoGV) and Bacillus thuringiensis subsp. kurstaki for control of the potato tuberworm (Lepidoptera: Gelechiidae) in stored tubers. J. Econ. Entomol. 101, 1540-1546. Das, G. P., E., Magallona, D., Raman, K. V., C.B., A., 1992: Effects of different components of IPM in the management of the potato tuber moth, in storage. Agric. Ecosyst. Environ. 41, 321-325 Espinel-Correal, C., Léry, X., Villamizar, L., Gómez, J., Zeddam, J. L., Cotes, A. M. & López- Ferber, M. 2010: Genetic and biological analysis of Colombian Phthorimaea operculella granulovirus isolated from Tecia solanivora (Lepidoptera: Gelechiidae). Appl. Environ. Microbiol. 76, 7617–7625. Gómez-Bonilla, Y., Guzmán M., J., 1997: Manejo integrado del cultivo de papa. Archivos Técnicos. Ministerio de Agricultura y Ganadería, San José, pp. 47 Gómez-Bonilla, Y., Lopez-Ferber, M., Caballero, P., Lery, X. & Muñoz, D. Characterization of a Costa Rican granulovirus strain highly pathogenic against its indigenous hosts, Phthorimaea operculella and Tecia solanivora . Entom. Exp. Appl., submitted. Niño de Gualdrón, L. & Notz, J. 2000: Patogenicidad de un virus granulosis de la polilla de la papa Tecia solanivora (Povolny) 1973 (Lepidoptera: Gelechiidae) en el estado Mérida, Venezuela. Bol. Entomol. Ven. 15, 39-48. Rondon, S. L. 2010: The potato tuberworm: a literature review of its biology, ecology, and control. Am. J. Pot. Res 87, 149-166. Torres-Leguizamón, M., Dupas, S., Dardon, D., Gómez, Y., Niño, L., Carnero, A., Padilla, A., Merlin, I., Fossoud, A., Zeddam, J-L., Léry, X., Capdevielle-Dulac, C., Dangles, O., Silvain, J-F. 2011: Inferring native range and invasion scenarios with mitochondrial DNA: the case of T. solanivora successive north–south step-wise introductions across Central and South America. Biol. Invasions. DOI 10.1007/s10530-010-9909-2. Published on line 26 january 2011. Zeddam, J. L., Vasquez Soberon, R. M., Vargas Ramos, Z. & Lagnaoui, A. 2003: Producción viral y tasas de aplicación del granulovirus usado para el control biológico de las polillas de la papa Phthorimaea operculella y Tecia solanivora (Lepidoptera: Gelechiidae). Rev. Chil. Entomol. 29, 29-36.

73

CHAPTER V

Costa Rican soils contain highly insecticidal granulovirus strains against Phthorimaea operculella and Tecia solanivora

ABSTRACT The aim of this work was to isolate and characterize novel Phthorimaea operculella granulovirus (PhopGV) strains from Costa Rican soils. Three novel strains, named PhopGV- CR3, PhopGV-CR4, and PhopGV-CR5, were isolated from three locations in Costa Rica, Alvarado, Zarcero, and Abangares, respectively, by means of soaking potato tubers with diluted soil samples. An additional strain, PhopGV-CR2, was identified from diseased larvae from a T. solanivora laboratory culture. Restriction fragment length polymorphisms obtained for each isolate with six restriction endonucleases (RENs), allowed their identification as four distinct PhopGV strains. Both REN and PCR analyses indicated the existence of an array of genotypic variants present in all isolates. Bioassays in P. operculella and T. solanivora showed that PhopGV-CR3, was well adapted to the two coexisting hosts with high levels of pathogenicity against both pest species. The mean lethal dose values of this strain were 2.8 OBs/mm 2 for P. operculella and less than 0.5 OBs/mm 2 for T. solanivora . We conclude that PhopGV-CR3 shows great promise for soil application against these pests in Costa Rican potato crops.

This chapter has been accepted for publication to the Journal of Applied Entomology as: Y. Gómez- Bonilla, M. López-Ferber, P. Caballero, and D. Muñoz 75 Chapter V Costa Rican granulovirus soil isolates against tuberworms

INTRODUCTION The potato tuberworms, Phthorimaea operculella Zeller and Tecia solanivora Povolny (Lepidoptera: Gelechiidae), are the most devastating pests of potato in Central America (Rondon., 2010; Vargas et al., 2004) and particularly in Costa Rica (Hilje, 1994). In Costa Rica, T. solanivora is more abundant than P. operculella . Larvae of both these species cause serious damage in the field and in storage, where losses may be as high as 100% (Rondon et al., 2010; Vargas et al., 2004). In the field, P. operculella females lay their eggs on the plant, usually on foliage throughout the growing season. Hatching larvae mine leaves, stems and petioles causing irregular galleries. Larvae attack tubers by excavating tunnels mainly under storage conditions (Rondon, 2010). T. solanivora attacks potato tubers exclusively. In the field, females usually lay their eggs at the base of plant stems and emerging larvae reach the tubers where they dig galleries that result in partial or complete tuber destruction (Niño, 2004). Management of P. operculella and T. solanivora has been traditionally achieved using broad-spectrum chemical insecticides applied at weekly intervals (Bekheit et al., 1997; Niño, 2004; Rondon, 2010), which has prompted highly resistant biotypes of both tuber moth species and adverse effects on beneficials and the environment (Arévalo and Castro, 2003; Dogramaci and Tingey, 2008; Shelton et al., 1981).

Baculoviruses (Baculoviridae) are environmentaly friendly biocontrol agents (BCA) that are compatible with beneficial organisms and which represent a useful alternative to chemicals for pest control in an increasing number of plant-pest systems (Berling et al., 2009; Moscardi, 1999; Lasa et al., 2007). This is due, among other characteristics, to their high persistence in the environment when protected from UV light (Christian et al., 2006; Fuxa and Richter, 2001). In this respect, baculoviruses stand out as promising control agents in the context of potato crop protection. They can be applied onto the ground, which shields viral occlusion bodies from UV light and where direct damage occurs on developing tubers. In addition, most baculovirus species have a high degree of intraspecific diversity, which renders them adaptable to unstable habitats like agroecosystems (Cory et al., 1997, 2005; Hogdson et al., 2004; Muñoz and Caballero, 2001). Such heterogeneity is manifested, for instance, by the existence of geographical strains and genotypic variants with differing levels of pathogenicity against their hosts (Cory et al., 2005; Léry et al., 1998; Murillo et al., 2006, 2007; Hogdson et al., 2004). Several baculovirus isolates from different regions of the world have been isolated from P. operculella or T. solanivora (Espinel-Correal et al., 2010; Gómez- Bonilla et al., 2008; Niño and Notz, 2000; Setiawati et al., 1999; Zeddam et al., 2003). However, only a handful of these have proven highly pathogenic against both P. operculella and T. solanivora (Espinel-Correal et al., 2010; Gómez-Bonilla et al., 2008, 2011). All of them belong to the Betabaculovirus genus (formerly Granulovirus genus or GVs) and all have been isolated from infected larvae. So far, no soil GV isolates of P. operculella and T. solanivora

76 Chapter V Costa Rican granulovirus soil isolates against tuberworms have been studied. Soil-derived isolates will probably contain higher numbers of soil-adapted genotypes than field-collected isolates from dead insects and may constitute ideal strains for pest control in potato crops.

Baculovirus soil isolates can be obtained by incorporating soil samples into insect rearing diets (Richards and Christian, 1999; Murillo et al., 2006). In this study, we have slightly modified these techniques for isolation of P. operculella granuloviruses (PhopGVs) from Costa Rican soil samples with the aim of selecting those with high pathogenicity and virulence against both T. solanivora and P. operculella.

MATERIAL AND METHODS

Insect rearing The populations of Phthorimaea operculella and Tecia solanivora originally came from Costa Rica and were maintained under constant environmental conditions: 27 ºC temperature, 60% relative humidity and 16:8 (light:dark) photoperiod at the Research Center Carlos Duran (Cartago, Costa Rica). Larvae were fed on potato tubers, which had been previously treated with hypochlorite solution. Adults were fed honey or 30% (wt/vol) sugar.

Collection of virus samples and amplification Single soil samples taken from depths of 0-30 cm were obtained from four different sites in Costa Rica: Alvarado and Oreamuno (Cartago), Zarcero (Alajuela), and Abangares (Guanacaste), located at 2320, 1870, 2140, and 1100 m altitude, respectively. Alvarado, Oreamuno, and Zarcero recorded potato damage by both P. operculella and T. solanivora during the last decade. However, given its low altitude, Abangares has not traditionally been a site for the production of potato crops and no pest outbreaks have been recorded to date for either of the two insect species in this region, where new potato varieties better adapted to higher temperatures are now being cultivated. The sample from Alvarado was 1 kg of vermicompost prepared two years before with virgin soil taken from close to a nearby volcano (Irazú). The sample from Oreamuno was 1 kg of soil taken from the edge of a potato field. In this location of intensive agriculture, tuber moths and other pests are controlled with repeated applications of chemical pesticides and potato crops are rotated with onion, brassica and carrot crops every year. The low soil pH resulting from the use of certain chemical pesticides in this region is corrected by application of quicklime. Samples from Zarcero and Abangares consisted of 1 kg soil from fields that had remained uncultivated for several years. For each soil sample, 100 g of soil were mixed with 100 ml bidistilled water. Five potatoes were then soaked in these suspensions for 30 min., air

77 Chapter V Costa Rican granulovirus soil isolates against tuberworms dried, placed in individual plastic boxes, infested with 20 neonate T. solanivora each, and incubated at 27 °C for 25 days. At least one larva w ith symptoms of viral infection was obtained from all samples. An additional virus sample was collected from more than 200 T. solanivora larvae with symptoms of granulovirus infection at the insect rearing facilities of the Research Station Carlos Durán (2440 m altitude) (Cartago) in 2007. As soon as the virus was detected, the chamber was cleaned and quarantined before rearing was resumed.

For amplification of viral isolates, infected larvae from both species were individually homogenized in 1 ml bidistilled water and used to contaminate four tubers at a concentration of 2 x 10 9 OBs/ml. The OB concentrations were determined with a spectrophotometer and calculated using the method described by Zeddam et al. (2003), based on the equation: OBs/ml= 6.8 x 10 9 x OD450 x dilution factor. Each tuber was then infested with 25 neonate larvae and incubated for 25 days. The resulting infected larvae were collected and used again for one further amplification round. Finally, infected larvae were stored at -20 °C.

PhopGV-CR1, a Costa Rican isolate that had been characterized previously (Gómez-Bonilla et al., 2008) was used as a reference. PhopGV-1346 is an isolate from Tunisia kindly provided by Dr. El Bedewi, IPC Egypt, and was multiplied in Egypt for several generations. It is considered the type strain and was used as a reference for biological analyses. PhopGV-4.2 is a purified genotypic variant obtained by plaque purification from the PhopGV-1346 isolate (Lery et al., 1998; Vickers et al., 1991). The entire genome of PhopGV- 4.2, of 119217 bp, has been sequenced (GeneBank NC004062) (INRA/CNRS/Université de Montpellier II, Saint Christol les Ales, France) and used as a reference for molecular analysis.

Purification of occlusion bodies (OBs) and titration of OB suspensions To purify OBs, ca. 50 larval cadavers were collected and homogenized in 0.01 M Tris-HCl pH 7.5 using a Potter-Elvehjem homogenizer (USA) and centrifuged at 664 g for 5 min. at 4 ºC. The pellet was discarded and supernatant-containing OBs were centrifuged at 15,000 g for 20 min., and pellets resuspended in 1 ml bidistilled water, and then placed on a discontinuous 30%-70% (w/v) sucrose gradient and centrifuged at 30,000 g for 120 min. Granules were resuspended in 1x TE buffer (0.1 M Tris-HCl, pH 7.5, and 10mM EDTA, pH 8.0) and stored at -20 ºC. OB concentration was estimated using the spectrophotometer- based method described by Zeddam et al. (2003).

78 Chapter V Costa Rican granulovirus soil isolates against tuberworms

DNA extraction and restriction endonuclease (REN) analysis

Purified OB suspensions were incubated with 1/10 vol. 2M Na 2CO 3. DNA was extracted with phenol/chloroform and then precipitated with ethanol, as described by Muñoz et al. (1997). Between 0.5-1 g of viral DNA were incubated with 10 units of restriction enzymes Sma I, Bam HI, Hin dIII, Nru I, Mlu I, Nde I, Dra III, BstEI I, at the conditions specified by the supplier. Reactions were stopped by addition of loading buffer (0.25% bromophenol blue, 40% w/v sucrose in water), loaded in 1% agarose gels with TAE buffer (40 mm Tris-acetate, 1mM EDTA, pH 8.0) and electrophoresed at 100V for 2 hrs. Ethidium bromide stained gels were then photographed on a UV transilluminator. REN analysis was performed using PhopGV- CR1 as a reference. In silico restriction digestion was performed on the PhohpGV-4.2 genotype sequence using the Clone Manager 5 Software (Sci-Ed Central). REN fragments that were not present in all isolates or in PhopGV-4.2 were considered restriction fragment length polymorphisms (RFLPs). Also, RFLPs absent in PhopGV-4.2 were considered novel and the nomenclature used to designate them was the same as that used previously for another Costa Rican isolate (Gómez-Bonilla et al., 2008). Specifically, the same RFLP nomenclature of PhopGV-4.2 was employed for the novel restriction fragments migrating immediately below those of PhopGV-4.2, but lower case letters followed by a number were used instead of the upper case lettering used for the sequenced genotype REN fragments.

Polymerase chain reaction (PCR) Amplification of four different PhopGV variable genomic regions (within ORFs 46, 109 and 129 and a region encompassing ORFs 83 and 84), was carried out using different primers (Gómez-Bonilla et al., 2008). PCR reactions were carried out in a total volume of 25 µl, containing 0.75-2.5 ng of DNA, 1 pmol of each primer, 3.5 mM MgCl 2 and 0.5 vol. of a mixture prepared by the supplier containing dNTP´s and Taq polimerase (Promega, Charbonnieres, France). A sample containing all PCR components except virus DNA was used as a negative control. Amplifications were carried out in a thermocycler under the following conditions: a first cycle of 94ºC/4 min., followed by 30 cycles of 94ºC/1 min., 50ºC/1 min. and 72ºC/1 min., and a final cycle of 72ºC/5 min. The PCR products (amplicons) were subjected to electrophoresis in 2% agarose gel at 150 v. for 105 min.

Bioassays

Bioassays to determine the median lethal concentration (LC 50 ) values of all viral isolates on Costa Rican P. operculella and T. solanivora populations were carried out with neonate larvae (L1). Five different virus concentrations (final concentrations of 0.5, 5, 50, 500, and 5000 OBs/mm 2 on the surface of the potato tuber) were prepared by means of a nebulizer apparatus from purified OB solutions as previously described (Carrera et al. 2008). Two

79 Chapter V Costa Rican granulovirus soil isolates against tuberworms tubers were used for each concentration, twelve larvae were placed on each tuber, and tubers were incubated at 27°C for 25 days. The numb er of dead and infected (white) larvae, outside the tubers, was recorded daily. At day 25, tubers were opened and dead or infected larvae remaining inside the galleries were also registered. Each bioassay was replicated four times. Regression lines (relating log-dose and mortality) and LC 50 values were calculated by Probit analysis (Finney, 1971) using Polo PC (LeOra Software, 1987). To compare the LC50 values, the broadly used analysis described by Robertson and Preisler (1992) was followed. When parallelism between regression lines could be assumed to occur, then the relative potencies (LC 50 ratios) and their corresponding 95% fiducial limits were estimated by constraining the slopes of all response lines to be the same. If the lines could not be considered parallel, the LC50 ratio was calculated and then their 95% confidence limits compared.

RESULTS

Isolation of Costa Rican granuloviruses from soil GVs were isolated in three out of the four soil samples collected. In the samples from Alvarado, Zarcero, and Abangares, 47, 4, and 1% of T. solanivora larvae present on potatoes contaminated with the diluted soil samples died, respectively. No larvae died on potatoes soaked with the soil sample from Oreamuno. Mortality derived from other entomopathogenic organisms such as fungal spores, nematode larvae or Bacillus thuringiensis was not observed in larvae that developed on soil-contaminated diet.

Characterization of Costa Rican granulovirus isolates The three soil isolates and a fourth one obtained from diseased T. solanivora larvae could be differentiated at the molecular level by size differences in genomic restriction fragments (RFLPs) obtained with Bam HI, Nde I, Sma I, Nru I, Mlu I, and Bst EII. This allowed their identification as four distinct PhopGV strains (Table 1), that were named PhopGV-CR2 for the isolate collected from dead larvae in the Carlos Durán Research Centre, and PhopGV- CR3, PhopGV-CR4 and PhopGV-CR5 for the soil isolates from Alvarado, Zarcero and Abangares, respectively.

The novel PhopGV strains could be differentiated between them and from the sequenced genotype (PhopGV-4.2) and the reference strain (PhopGV-CR1) using Bst II, except PhopGV-CR2 and PhopGV-CR3, which displayed identical profiles with this enzyme (Table 1). Only two fragments allowed their identification as distinct strains: Nde I-c1 (Fig. 1) and Sma I-d1. All four strains shared the following six RFLPs: Mlu I-L (3620 bp); Mlu I-l1 (3410 bp); Bst EII-I (4614 bp); Bst EII-i1 (4450 bp), Bst EII-J (4267 bp); and Bst EII-j1 (4325 bp)

80 Chapter V Costa Rican granulovirus soil isolates against tuberworms

(Table 1). Submolar concentrations were observed in digestions with several endonucleases for all Costa Rican isolates (Fig. 1 and Table 1). REN profiles were identical for all isolates following treatment with Hin dIII or Dra III,.

Table 1. Restriction fragment length polymorphic (RFLP) fragments observed between the reference genotype PhopGV-4.2 (4.2) and the Costa Rican isolates PhopGV-CR1 (CR1), PhopGV-CR2 (CR2), PhopGV-CR3 (CR3), PhopGV-CR4 (CR4), and PhopGV-CR5 (CR5) with Bam HI, Nde I, Sma I, Nru I, Mlu I, and Bst EII. RFLPs (mol. weight) 4.2 CR1 CR2 CR3 CR4 CR5 Bam HI-I (6114) + +* +* +* +* + Bam HI-i1 1 (ca. 6000) - + + + + - Nde I-c1 (ca. 14500) - + - +* + - Nde I-G (6746) + - - - - - Nde I-g1 (ca. 6500) - + + + + + Nde I-I (5994) + + +* +* +* + Nde I-j1 (ca. 5300) - +* - - +* - Sma I-D (7469) + +* +* +* - +* Sma I-d1 (ca. 5500) - - - +* - - Nru I-h1 (ca. 6000) - + - - +* + Nru I-I (6104) + - + + +* - Mlu I-b1 (ca. 5500) - +* +* +* +* +* Mlu I-L (3620) + + - - - - Mlu I-l1 (ca. 3500) - - + + + + Bst EII-g1 (ca. 6400) - + - - +* + Bst EII-H (6218) + - + + +* - Bst EII-I (4614) + + - - - - Bst EII-i1 (ca. 4500) - - + + + + Bst EII-J (4267) + + - - - - Bst EII-j1 (ca. 4300) - - +* + + + 1 In bold, RFLPs obtained from the Costa Rican isolates that are absent in the reference PhopGV-4.2. These were named using the restriction endonuclease fragment name of PhopGV-4.2 which migrated closest above it in lower case letters followed by a number. Their molecular weight is approximate (ca.). Asterisks indicate fragments present in submolar concentrations.

CR1 CR2 CR3 CR4 CR5

23130

* Nde I-c1 9416

6557 Nde I-g1 *** Nde I-I ** Nde I-j1 4361

2322 2027

Fig. 1. Nde I restriction endonuclease profiles of the genomic DNA of PhopGV-CR1 (CR1), PhopGV- CR2 (CR2), PhopGV-CR3 (CR3), PhopGV-CR4 (CR4), and PhopGV-CR5 (CR5). Nde I RFLPs are shown at the right of the panel. Asterisks indicate submolar fragments. Reference molecular size fragments are indicated to the left of the panel.

81 Chapter V Costa Rican granulovirus soil isolates against tuberworms

Primers within ORFs 46, 109, and within a region encompassing ORFs 83 and 84 yielded a single amplicon of the same size (1200, 1000, and 250 bp, respectively) for all strains tested, including the reference PhopGV-CR1. Primers within ORF 129 produced a total of six different amplicons in the Costa Rican strains (Fig. 2, Table 2). According to Espinel-Correal et al. (2010), each ORF 129 amplicon denotes the presence of a genotypic variant and they designated genome types 1 to 5 to amplicons of 937, 723, 1023, 569, and 869 bp, respectively. Each PhopGV strain comprised between 2 and 4 of these genome types except for PhopGV-CR5, which had only a type 1 amplicon plus an additional amplicon of 783 bp that had not been described so far and which was designated here as type 6 (Table 2).

Table 2. PCR amplicons (bp) of ORF 129 yielded by PhopGV-CR1 (CR1), PhopGV-CR2 (CR2), PhopGV-CR3 (CR3), PhopGV-CR4 (CR4), and PhopGV-CR5 (CR5). Amplicon size (bp) CR1 CR2 CR3 CR4 CR5 1023 + - + + - 937 - + - - + 869 + - + + - 783 - - - - + 723 - + + + - 569 - + + + - +/- presence/absence of amplicon

MMCR1 CR2 CR3 CR4 CR5

1500

1000 900 800

700 600 500 400

Fig. 2. PCR analysis of ORF 129 from PhopGV-CR1 (CR1), PhopGV-CR2 (CR2), PhopGV-CR3 (CR3), PhopGV-CR4 (CR4), and PhopV-CR5 (CR5). A DNA ladder ranging from 1.5 to 0.1 kb was used as a molecular size marker (M) and its fragment sizes are indicated to the right of the panel.

Insecticidal activity of the Costa Rican granulovirus strains Bioassays in P. operculella indicated that the newly isolated Costa Rican PhopGVs, as well as the laboratory isolated strain, PhopGV-CR2, were all as pathogenic as the reference strains PhopGV-1346 and PhopGV-CR1, with LC 50 values statistically similar to those of these strains (Table 3). In T. solanivora , isolate PhopGV-CR3 and PhopGV-CR2 killed more than 50% of the larvae at the lowest (0.5 OBs/mm 2) and second lowest (5 OBs/mm 2)

82 Chapter V Costa Rican granulovirus soil isolates against tuberworms concentrations, respectively, indicating a high pathogenicity of both these strains against T. solanivora . The LC 50 values of the rest of the soil isolates, PhopGV-CR4 and PhopGV-CR5, were statistically similar to those of PhopGV-1346 and PhopGV-CR1 (Table 3). For all virus treatments in both species, mortality increased with increasing OB concentration. A common slope for concentration-mortality could be fitted for T. solanivora but not for P. operculella . No virus mortality was registered in mock-infected control insects.

Table 3. LC 50 values of viral strains PhopGV-1346 (1346), PhopGVCR-1 (CR1), PhopGVCR-2 (CR2), PhopGVCR-3 (CR3), PhopGVCR-4 (CR4), PhopGV-CR5 (CR5) for Phthorimaea operculella (PO) and Tecia solanivora (TS).

Fiducial limits LC50 Relative Host Virus strain Regression line 2 (OBs/mm ) potency Lower upper

1346 0.50x + 4.50 7.0 1 - - CR1 0.61x +4.24 14.0 0.50 0.07 3.32 CR2 0.28x + 4.60 32.2 0.22 0.04 1.15 PO CR3 0.26x + 4.89 9.5 0.73 0.12 4.52 CR4 0.56x + 4.37 10.5 0.67 0.13 2.58 CR5 0.35x + 4.58 18.8 0.37 0.07 1.97 1346 0.44x +4.41 23.1 1 - - CR1 0.44x + 4.21 65.8 0.35 0.11 1.08 TS CR4 0.44x + 4.22 60.6 0.38 0.12 1.15 CR5 0.44x + 4.62 7.5 3.07 0.98 9.71

DISCUSSION Efficient protection of potato tubers against P. operculella and T. solanivora in the field may be achieved by soil application of baculovirus-based bioinsecticides (Gómez-Bonilla et al., 2011). Given that high levels of diversity occur within baculovirus populations, the existence of viral strains or genotypes well adapted to soil habitats and, at the same time, with elevated levels of pathogenicity for both pest species was hypothesized. In this study, three soil PhopGV strains were identified at the molecular level and their performance as BCAs evaluated in terms of OB pathogenicity.

Several baculovirus strains of Helicoverpa armigera single nucleopolyhedrovirus (HaSNPV) or Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) have been previously isolated from soil samples by incorporating soil samples into the artificial diets used to rear insect hosts (Richard and Christian, 1999; Murillo et al., 2006). Since we lacked artificial diets for P. operculella or T. solanivora , we had to modify this technique slightly by soaking potato tubers in aqueous soil suspensions. With this technique, we were able to detect PhopGVs in three out of the four Costa Rican soil samples analyzed, from Alvarado, Zarcero, and Abangares.

83 Chapter V Costa Rican granulovirus soil isolates against tuberworms

Overall, 47% of larvae treated with Alvarado vermicompost soil died, indicating a high prevalence of virus in this sample. Considering this soil preparation had not been used for cultivation before, the presence of active virus may seem surprising. However, it was kept next to potato crops and warehouses with stored potatoes, and therefore next to pest populations from which the vermicompost may have acquired the virus naturally. Dispersal of OBs by abiotic agents such as air currents, agronomic practices, rain, etc. is crucial for the transition between non-transmissible reservoir populations and ephemeral but transmissible subpopulations (Murillo et al., 2007). The presence of virus in the other two soil samples is more noteworthy. Although GV prevalence in the sample from Zarcero was much lower (4%) than in Alvarado, this sample came from a pasture ground that had remained uncultivated for ten years, and the closest solanaceum crops were located at a distance of ca. 1 km. The amount of GV OBs in the sample from Abangares was even lower (1%). The lower altitude and higher temperatures of this location are unfavourable conditions for T. solanivora or P. operculella outbreaks and potato cultivation was initiated in Abangares only in the last decade using low altitude varieties. The presence of active GVs in Abangares and Zarcero, although tiny, constitutes further evidence of the long persistence of OBs in the soil in the absence of the insect host, as has been observed previously for other baculoviruses (De Moraes et al., 1999; Murillo et al., 2007). In any case, high persistence in the uppermost soil layers is favoured by the affinity of OBs to soil particles, especially to the clay components, to which most OBs remain strongly bound (Fuxa and Richter, 2001; Hukuhara and Namura, 1972; Jaques, 1985).

The lack of PhopGV strains from Oreamuno, where the incidence of tuberworms is continuous, was unexpected. However, the presence of entomopathogens is sometimes higher in uncultivated than in cultivated soils (Martin and Travers, 1989). The presence/absence of 20 RFLP fragments, obtained from six different restriction endonucleases (Table 1), allowed differentiation of these strains. Genomic changes such as point mutations, deletions, transpositions, or recombinations usually account for the observed polymorphisms (Jehle, 1996; Muñoz et al., 1997; Simón et al., 2004). On the basis of these analyses, each soil isolate was identified as a novel strain (PhopGV-CR3, PhopGV-CR4, and PhopGV-CR5). These three constitute the very first PhopGV strains obtained and identified from soils. An additional strain, PhopGV-CR2, was identified from diseased larvae from a T. solanivora colony maintained at the Research Station Carlos Durán.

The high OB pathogenicity values of PhopGV-CR3 against T. solanivora suggested that this strain may have originated from a viral population that is well adapted to T. solanivora . However, PhopGV-CR3 also showed high pathogenicity against P. operculella , indicating that it is adaptated to both coexisting hosts. Other PhopGV strains have been

84 Chapter V Costa Rican granulovirus soil isolates against tuberworms reported to be well adapted to T. solanivora , P. operculella or to both host species apparently after having had previous contact with one, or both hosts (Espinel-Correal et al., 2010; Gómez-Bonilla et al., 2008). This phenomenon has also been observed in NPVs (Hogdson et al., 2004; Ribeiro et al., 1997), and is probably explained by viral intraspecific heterogeneity, which renders viral populations more adaptable to varying environmental conditions (Muñoz & Caballero, 2001), including different hosts or different haplotypes of the same host. In this respect, a recent study revealed a high diversity of T. solanivora haplotypes in Central America (Torres-Leguizamón et al., 2011). Indeed, the presence of multiple genotypic variants in a single population may include genotypes more specialized in infecting particular hosts (species and haplotypes), or even coexisting hosts, as occurs with Pannolis flammea multiple NPV (Hogdson et al., 2004). Accordingly, all Costa Rican isolates contained mixtures of genotypes, as indicated by the presence of submolar bands in the REN profiles and multiple amplicons in the ORF129 PCR profiles.

A survey of PhopGV isolates recently performed in Colombia collected diseased T. solanivora larvae along the invasion pathway of this insect (Espinel-Correal et al., 2010). These isolates contained mixtures of genotypic variants well adapted for replication in both T. solanivora and P. operculella . All Costa Rican strains shared many of the RFLPs and genome types identified in the Colombian isolates, which strongly suggests that PhopGV- CR2, PhopGV-CR4, and PhopGV-CR5 have the potential to adapt to coexisting hosts when exposed to both of them. In fact, previous studies with the reference Costa Rican strain, PhopGV-CR1, resulted in a five-fold increase in OB pathogenicity against T. solanivora after only three passages in this host (Gómez-Bonilla et al., 2008). Finally, these isolates may also be useful for the control of Tuta absoluta Meyrick (Lepidoptera: Gelechiidae), another pest of many solanaceous crops, as reported recently for a Brazilian PhopGV strain (Mascarin et al., 2010). In summary, all novel Costa Rican PhopGV strains seem likely to be good candidates for soil application against P. operculella and T. solanivora , in particular PhopGV-CR3, as suggested by their soil origin, persistence, potential for host adaptability and high OB pathogenicity.

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Mascarin G, Alves S, Rampelotti-Ferreira F, Urbano M, Demétrio C, Delalibera I. 2010. Potential of a granulovirus isolate to control Phthorimaea operculella (Lepidoptera: Gelechiidae). BioControl, 55, 657-671. Moscardi F, 1999. Assessment of the application of baculoviruses for control of Lepidoptera. Ann. Rev. Entomol. 44, 257-263. Muñoz D, Caballero P, 2001. Diversidad natural de los baculovirus. In: Los baculovirus y sus aplicaciones como bioinsecticidas en el control biológico de plagas. Ed. by Caballero P, López-Ferber M, Williams T, Phytoma-España, Valencia, Spain 95- 118. Muñoz D, Vlak JM, Caballero P, 1997. In vivo recombination between two strains of the genus nucleopolyhedrovirus in its natural host, Spodoptera exigua . Appl. Environ. Microbiol. 63, 3025-3031. Murillo R, Elvira S, Muñoz D, Williams T, Caballero P, 2006. Genectic and phenotypic variability in Spodoptera exigua nucleopolyhedrovirus isolates from greenhouse soils in southern Spain. Biol. Control, 38, 157-165. Murillo R, Muñoz D, Ruíz-Portero MC, Alcázar MD, Belda JE, Williams T, Caballero P, 2007. Abundance and genetic structure of nucleopolyhedrovirus populations in greenhouse substrate reservoirs. Biol. Control 42, 216-225. Niño L. 2004. Revisión sobre la Polilla de la Papa Tecia solanivora en Centro y Suramérica. Rev. Latinoam. Papa - Supl. 4-21. Niño L, Notz J, 2000. Patogenicidad de un virus granulosis de la polilla de la papa Tecia solanivora (Povolny) 1973 (Lepidoptera: Gelechiidae) en el estado Mérida, Venezuela. Boletín de Entomología Venezolana. 15, 39-48. Ribeiro HCT, Pavan OH, Muotri AR, 1997. Comparative susceptibility of two different hosts to genotypic variants of the Anticarsia gemmatalis nuclear polyhedrosis virus. Entomol. Exp. Appl. 83, 233-237. Richards AR, Christian PD, 1999. A rapid bioassay screen for quantifying nucleopolyhedorvirus (Baculoviridae) in the environment. J. Virol. Methods 82, 63- 75. Robertson JL, Preisler HK, 1992. Pesticide bioassays with arthropods. CRC Press, Boca Raton, USA. Rondon SL, 2010. The potato tuberworm: a literature review of its biology, ecology, and control. Am. J. Pot. Res. 87, 149-166. Setiawati W, Soeriaatmadja RE, Rubiati T, Chujoy E, 1999. Control of potato tubermoth (Phthorimaea operculella ) using an indigenous granulosis virus in Indonesia. Indian J. Crop Sci. 14, 10-16. Shelton AM, Wyman JA, Mayor AJ, 1981. Effects of commonly used insecticides on the potato tuberworm and its associated parasites and predators in potatoes. J. Econ. Entomol. 74, 161-184. Simón, O., Williams, T., López-Ferber, M., Caballero, P., 2004. Genetic structure of a Spodoptera frugiperda nucleopolyhedrovirus population: high prevalence of deletion genotypes. Appl. Environ. Microbiol. 70 , 5579-5588. Torres-Leguizamón M, Dupas S, Dardon D, Gómez Y, Niño L, Carnero A, Padilla A, Merlin I, Fossoud A, Zeddam JL, Lery X, Capdevielle-Dulac C, Dangles O, Silvain JF, 2011. Inferring native range and invasion scenarios with mitochondrial DNA: the case of T. solanivora successive north–south step-wise introductions across Central and South America. Biol. 13 (7), 1505-1519. Vargas B, Rubio S, López-Ávila A, 2004. Estudios de hábitos y comportamiento de la polilla guatemalteca Tecia solanivora (Lepidoptera: Gelechiidae) en papa almacenada. Rev. Colomb. Entomol. 30, 211-217 Vickers JM, Cory JS & Entwistle PF (1991) DNA characterization of eight geographic isolates of granulosis virus from the potato tuber moth ( Phthorimaea operculella ) (Lepidoptera, Gelechiidae). J. Invert. Pathol. 57, 334-342. Zeddam JL, Vásquez-Soberon R, Vargas-Ramos Z, Lagnaoui A, 2003. Cuantificación de la producción viral y tasas de aplicación de un virus de granulosis usado para el

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88

CHAPTER VI

Resultados y Discusión General

El excesivo uso que se les ha dado a los insecticidas químicos desde su aparición en la década de los 40 para el control de plagas en la agricultura ha hecho que muchos de ellos sean hoy en día inútiles contra una serie de especies de plagas clave debido a resistencias y problemas de salud ambiental y humana. Tales problemas impulsaron en la década de los 70 una nueva filosofía en el control de plagas que rechaza los tratamientos de calendario, tiene en cuenta criterios de sostenibilidad económica y ambiental para decidir la necesidad de un tratamiento, e integra todos los métodos posibles de control para cada caso. Es el denominado Manejo Integrado de Plagas (MIP). El desarrollo y la puesta en marcha de los programas MIP han demostrado ser eficaces en el mantenimiento de las poblaciones de plagas por debajo de los umbrales económicos. Además han conseguido en muchos casos reducir o incluso eliminar el uso de productos químicos y han demostrado ser sostenibles.

Para el control de Phthorimaea operculella en patata de campo y almacenada se han puesto en práctica con éxito varios programas MIP en diversos países del mundo. En dichos programas se incluyen métodos de control cultural, etológico y microbianos (Kroshchel y Lacey, 2008). Además, un virus de la familia Baculoviridae, el granulovirus de P. operculella (PhopGV) ha formado parte de muchos de ellos desde los primeros desarrollos. Las propiedades bioinsecticidas, el alto nivel de especificidad, la ausencia de toxicidad para los seres humanos o benéficos y la compatibilidad con la mayoría de otros métodos de control, han hecho que muchos baculovirus se hayan utilizado con éxito el control en programas MIP contra diferentes plagas de lepidópteros e himenópteros de los ecosistemas agrícolas y forestales (Cherry y Williams, 2001; Rohrmann, 2011). Sin embargo, no se han desarrollado

89 Chapter VI Resultados y Discusión General programas MIP contra T. solanivora hasta hace muy poco. Esta especie se convirtió en una plaga clave para los productores de papa de América Latina sólo a partir de finales de 1970, después de su rápida expansión desde Guatemala hacia el sur por el resto de América Central y el norte de América del Sur. Dado que P. operculella y T. solanivora tienen una biología similar y co-existen en muchas regiones de América Latina, se pueden desarrollar métodos de control comunes para las dos plagas. El PhopGV es uno de ellos. Aunque los primeros aislamientos de PhopGV estadounidenses provenían de larvas enfermas de P. operculella , su espectro de acción incluye a T. solanivora (Villamizar et al., 2005). Además, se han obtenido recientemente otros aislados de PhopGV procedentes de T. solanivora con mejores propiedades bioinsecticidas contra este hospedero (Niño et al, 2005;.. Espinel- Correal et al, 2010). Sin embargo, aunque son varias las formulaciones de PhopGV que han demostrado una gran eficacia en la protección de los tubérculos de papa en varios países de América del Sur, especialmente en condiciones de almacenamiento (CIP, 1992; Crespo et al, 2005; Zeddam et al., 2003), no se han realizado ensayos en América Central por falta de aislamientos autóctonos. Biológicamente, no todos los granulovirus (GV) funcionan igual contra sus hospederos naturales y, a su vez, no todas las especies plagas o incluso los distintos biotipos de una especie concreta son igualmente susceptibles a un virus. Como regla general, los GV autóctonos suelen ser más eficaces que los GV procedentes de otros lugares. La co-evolución de los virus con sus biotipos hospedadores en sus ambientes naturales les ha permitido contar con las herramientas necesarias para adaptarse a las cambiantes condiciones bióticas y abióticas.

El objetivo de esta tesis consistió en aislar cepas costarricenses de PhopGV, seleccionar aquellas con mayores propiedades insecticidas en bioensayos de laboratorio, y evaluar su eficacia bioinsecticida en la protección de los tubérculos de patata en condiciones de campo y almacén.

Inicialmente se recolectó un aislado de GV a partir de larvas enfermas de P. operculella de la Estación Carlos Durán (Cartago, Costa Rica). Su identificación molecular confirmó que se trataba de una nueva cepa de PhopGV, denominado PhopGV-CR1, y que su composición genotípica era heterogénea. Mediante bioensayos de laboratorio se determinó su alta patogenicidad, no sólo para una colonia de P. operculella de Costa Rica, sino también para una colonia de P. operculella procedente de Egipto y criada en Francia. Sin embargo, la patogenicidad de PhopGV-CR1 fue cuatro veces menor contra las larvas del hospedador alternativo, T. solanivora . Otras cepas de PhopGV aisladas de P. operculella también mostraron una patogenicidad disminuída sobre T. solanivora y similares resultados se obtienen con cepas de PhopGV aisladas T. solanivora y utilizadas contra P. operculella (Villamizar et al, 2005; Espinel-Correal et al, 2010), sugiriendo una adaptación del virus a

90 Chapter VI Resultados y Discusión General sus especies hospedadoras. En este sentido, se ha observado un aumento de la patogenicidad en aislados virales que se multiplican durante varios pases sucesivos en una especie de hospedador determinada (Berling et al, 2009;. Kolodny-Hirsch y Van Beek, 1997). Esto, unido a la gran plasticidad de los genomas de baculovirus y a la heterogeneidad genotípica de PhopGV-CR1, hizo prever una alta capacidad de esta cepa para adaptarse a sus especies hospedadoras. En efecto, el pase seriado de PhopGV-CR1 a lo largo de tres generaciones de T. solanivora aumentó su patogenicidad cinco veces, lo que indica una adaptación continua del virus a su huésped alternativo. Sorprendentemente, la cepa de referencia de PhopGV utilizada en nuestro estudio, y que también fue obtenida a partir de larvas enfermas de P. operculella , también se adaptó rápidamente al entrar contacto con las poblaciones costarricenses de ambos hospedadores, lo que constituye una prueba más de la capacidad de adaptación del virus a sus hospederos. En otros sistemas virus- hospedadores se han realizado observaciones similares. Por ejemplo, en AcMNPV se detectó un aumento de patogenicidad de hasta quince veces para Plutella xylostella después de pases sucesivos en este hospedador (Kolodny-Hirsch y Van Beek, 1997). Más recientemente, un aislado nuevo de Cydia pomonella GV (CpGV) mostró un incremento de su patogenicidad después de cuatro pases en una colonia de C. pomonella resistente al formulado comercial de CpGV (Berling et al., 2009). En este caso, la selección de genotipos fue responsable, al menos parcialmente, de la adaptación al biotipo hospedador (Berling et al., 2009). Es probable que la adaptación de PhopGV-CR1 a T. solanivora también pueda explicarse por la selección de genotipos, pero para confirmar esta hipótesis se hace necesaria la caracterización molecular de los genotipos que componen la cepa PhopGV- CR1 a lo largo de la serie de pases en P. operculella y T. solanivora . En cualquier caso, todos estos análisis mostraron que PhopGV-CR1 tenía un gran potencial para el control de P. operculella y T. solanivora , en particular después del pase seriado en la especie a controlar. Con estas credenciales, los estudios de campo con esta cepa no necesitaron mayores justificaciones.

Se evaluó la eficiencia de control de PhopGV-CR1 para P. operculella y T. solanivora en los cultivos de Costa Rica y los almacenes de papa. En el campo, el virus produjo una reducción de daños de entre el 50 y el 80% en comparación con los controles no tratados en los ensayos de invierno y verano. Por otra parte, esta reducción no fue significativamente diferente de la que dio por sustancias químicas o por una combinación de PhopGV y productos químicos, que fueron utilizados como referencia. Este resultado sugiere la posibilidad de reducir e incluso eliminar los productos químicos utilizados para el control de las polillas de la patata en el campo. Además, el virus fue eficaz contra T. solanivora , que fue la especie dominante a lo largo de las dos estaciones del año, según lo indicado por capturas de las trampas de feromonas. Por último, se llevaron a cabo aplicaciones de virus

91 Chapter VI Resultados y Discusión General al suelo en siembra y aporque. Esta es probablemente la aplicación que rinde mejores resultados que las aplicaciones a la parte aérea de la planta dada la protección del virus ofrecida por el suelo frente a la luz ultravioleta. Sin embargo, este punto necesita confirmación con pruebas de campo pertinentes.

En condiciones de almacenamiento, el virus redujo los daños en un 70% en comparación con los controles no tratados cuando las aplicaciones cubrieron por completo la superficie del tubérculo. Esto se logró mediante la mezcla de los tubérculos con el virus en las bolsas, un paso adicional en el proceso de almacenamiento que requiere una evaluación de su rentabilidad desde una perspectiva económica. Mediante una aplicación más sencilla consistente en espolvorear el virus sobre las distintas capas de patatas almacenadas también se produjo una importante protección de los tubérculos respecto a los no tratados en el almacén de hormigón. Sin embargo, en el almacén rústico de plástico, las aplicaciones simples de PhopGV, de PhopGV con insecticidas o de los insecticidas químicos no protegieron las patatas. En general, el uso de PhopGV ha sido muy eficaz en la mayoría de las experiencias en condiciones de almacenamiento (Lacey et al., 2010). El virus está bien protegido de la dañina luz ultravioleta y mantiene una alta actividad durante largos periodos de tiempo sin la necesidad de nuevas aplicaciones. Por último , T. solanivora también fue la especie dominante durante toda la temporada en almacén, lo que indica que esta especie también puede ser controlada efectivamente por PhopGV-CR1 en condiciones de almacenamiento. El alto nivel de protección que ofrece PhopGV-CR1 tanto en campo como en almacén es una clara indicación de su potencial para el control eficaz de T. solanivora y P. operculella en Costa Rica.

A pesar de las excelentes propiedades del PhopGV-CR1, se continuó la búsqueda de nuevos aislamientos de Costa Rica. La amplia diversidad genotípica de los baculovirus, que da lugar a fenotipos diferentes y especializados, alentaron la exploración. Hasta el momento se han descrito muchos genotipos de baculovirus especializados en determinadas funciones. Por ejemplo, se han observado genotipos especializados en infectar una especie huésped concreto (Ribeiro et al., 1997), en generar progenies virales altas (Muñoz et al., 2000), en incrementar la patogenicidad de la población viral en su conjunto (Simon et al., 2005), e incluso en la transmisión vertical u horizontal (Cabodevilla et al., 2011). Esta heterogeneidad representa un valioso recurso para el desarrollo de bioinsecticidas (Cory y Myers, 2003), ya que permite la selección de las mejores cepas para un determinado contexto de plagas. Por esta razón, la búsqueda de nuevas de PhopGV cepas se centró no sólo en insectos enfermos, sino que se amplió a los hábitats del suelo, donde los genotipos presentes podrían funcionar mejor como bioinsecticidas de suelo. Se obtuvieron tres aislados de GV a partir de tres localidades de Costa Rica (Alvarado, Zarzero y Abangares),

92 Chapter VI Resultados y Discusión General cubriendo las patatas con muestras diluidas de suelo. Sorprendentemente, dos de ellas fueron aisladas de suelos no utilizados para el cultivo de patata. La presencia de virus en la muestra de suelo de Alvarado se puede explicar por la existencia de cultivos de papa y almacenes cercanos, ya que los baculovirus se dispersan fácilmente por diferentes factores abióticos y bióticos (Fuxa y Richter, 2001). Sin embargo, la presencia de virus en las muestras de suelo de Zarcero y Abangares, alejadas de los cultivos de patata, puede constituir una prueba más de la alta persistencia de OBs en el suelo en ausencia de su huésped de insectos y plantas hospederas. La alta persistencia de los baculovirus en suelo ha sido demostrada previamente para otras especies (De Moraes et al., 1999; Murillo et al., 2007). Tiene lugar principalmente en las capas superiores, favorecida por la afinidad de los OBs a las partículas del suelo, en especial a los componentes de arcilla, a la que las que se unen fuertemente los cuerpos de oclusión (Fuxa y Richter, 2001). Sorprendentemente, no se aislaron virus en la muestra de suelo correspondiente a un cultivo de patata donde las poblaciones originan plagas constantemente. Probablemente, el uso de cal para aumentar el pH del suelo después de la aplicación intensiva de insecticidas químicos inactivó el virus. Aunque inesperada, la presencia de los entomopatógenos es a veces más alta en lugares no cultivados que en los suelos cultivados (Martin y Travers, 1989).

La presencia o ausencia de 20 fragmentos polimórficos obtenidos con seis enzimas de restricción permitió diferenciar los aislados de suelo de PhopGV. Cambios genómicos tales como mutaciones puntuales, deleciones, transposiciones o recombinaciones normalmente justifican los fragmentos polimórficos (Jehle, 1996; Muñoz et al., 1997; Simón et al., 2004). En base a estos análisis, cada aislado de suelo se identificó como una nueva cepa (PhopGV-CR3, PhopGV-CR4 y PhopGV-CR5). Las tres constituyen por tanto las primeras cepas de PhopGV obtenidas e identificadas de suelo. Se identificó una cepa adicional, PhopGV-CR2, de larvas enfermas de una colonia de T. solanivora criada en el Centro de Investigación Carlos Durán (Cartago, Costa Rica).

Los extraordinarios valores de patogenicidad de PhopGV-CR3 para T. solanivora sugirieron que esta cepa pudo haberse originado de una población viral bien adaptada T. solanivora . Sin embargo, PhopGV-CR3 también mostró ser altamente patogénica para P. operculella , lo cual indicó su adaptación a ambos hospedadores. En otras cepas de PhopGV también se ha observado una buena adaptación a T. solanivora , P. operculella o a ambas especies, aparentemente después de su previa exposición a uno, otro o ambos hospederos (Espinel-Correal et al., 2010; Gómez-Bonilla et al., 2008). Este fenómeno se ha comprobado también en otros baculovirus (Hogdson et al., 2004; Ribeiro et al., 1997), y tiene probablemente su explicación en la heterogeneidad viral intraespecífica, que hace más adaptables las poblaciones a condiciones ambientales cambiantes (Muñoz & Caballero,

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2001), entre las que se incluyen diferentes especies hospederas o diferentes haplotipos de la misma especie. Recientemente se hizo un análisis de diferentes cepas colombianas de PhopGV a partir de larvas de T. solanivora enfermas recolectadas a lo largo de la ruta de invasión de este insecto (Espinel-Correal et al., 2010). Dichas cepas contenían mezclas de variantes genotípicas bien adaptada a las dos especies coexistentes, P. operculella y T. solanivora . Todas las cepas de Costa Rica compartían muchos de los marcadores moleculares y los tipos genómicos identificados en el genoma de los aislados colombianos, lo que representa una sólida evidencia molecular de que también PhopGV-CR2, PhopGV- CR4 y PhopGV-CR5 tienen potencial para conseguir adaptarse a ambas especies hospederas. Como ocurrió con PhopGV-CR1, este fenómeno también podría explicarse por la heterogeneidad intraespecífica viral, ya que todos los aislamientos de Costa Rica contienen mezclas de genotipos.

En resumen, todas las nuevas cepas de Costa Rica PhopGV parecen buenas candidatas para su utilización en el control de P. operculella y T. solanivora , en particular PhopGV-CR1, cuya eficacia ha sido demostrada tanto en el campo como en almacenamiento. La alta patogenicidad, el origen del suelo, la persistencia y la alta adaptabilidad al hospedero de PhopGV-CR3 también hacen de esta cepa una alternativa prometedora para el control de campo. Sin embargo, todavía se requieren estudios de campo de esta cepa. En cualquier caso, la existencia de una colección de cepas de PhopGV costarricenses permitiría el uso de cepas alternativas en caso de resistencias, como ha ocurrido con otros virus (Berling et al., 2009).

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CONCLUSIONES

1. Los cinco nuevos aislados de granulovirus recolectedos a partir de larvas enfermas (PhopGV-CR1, PhopGV-CR2) o muestras de suelo (PhopGV-CR3, PhopGV-CR4, y PhopGV-CR5) en Costa Rica constituyen distintas cepas del granulovirus de Phthorimaea operculella (PhopGV) y están formadas por mezclas heterogéneas de genotipos, como lo revelan los análisis moleculares con endonucleasas de restricción y la reacción en cadena de la polimerasa.

2. La cepa PhopGV-CR1, aislada de P. operculella , se adapta a T. solanivora , como queda demuestrado por sus valores de patogenicidad, que se incrementan significativament después de tres multiplicaciones sucesivas del virus en T. solanivora cuando la patogenicidad de esta cepa para T. solanivora después de una multiplicación en P. operculella es cinco veces menor.

3. La cepa PhopGV-CR1 puede utilizarse en campo como bioinsecticida en sustitución y/o en combinación con insecticidas químicos de síntesis; así lo justifican los valores de reducción de daños de patata en campo en las temporadas de invierno y verano a niveles similares a los obtenidos con insecticidas químicos de síntesis o a una combinación de virus y químicos.

4. En almacenamiento, la cepa PhopGV-CR1 puede utilizarse como bioinsecticida en sustitución y/o combinación con insecticidas químicos de síntesis y tanto en almacenes rústicos como en almacenes robustos siempre y cuando se cubra totalmente la superficie del tubérculo, tal y como lo señalan los valores de reducción de daño, por encima del 70%.

5. El aislado de suelo PhopGV-CR3 está bien adaptado a ambos hospederos, P. operculella y T. solanivora , como queda revelado por los extraordinarios valores de patogenicidad de esta cepa para las dos especies obtenidos en ensayos de laboratorio.

6. Los suelos costarricenses, especialmente los no cultivados, son un rico reservorio de cepas de PhopGV, así lo demuestran el aislamiento de tres cepas diferentes a partir de tres de las cuatro muestras de suelo recolectadas en distintos puntos de Costa Rica.

7. La dispersión y la persistencia son importantes fenómenos que contribuyen a la transmisión de los baculovirus en general y en particular del PhopGV, como lo revela el

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aislamiento de cepas de PhopGV, como PhopGV-CR4 and Phop-CR5, de suelos pertenecientes a zonas distantes de donde ocurren las plagas de polilla de patata.

REFERENCIAS

Berling M, Blachere-Lopez C, Soubabere O, Lery X, Bonhomme A, Sauphanor B, Lopez- Ferber M, 2009. Cydia pomonella granulovirus genotypes overcome virus resistance in the codling moth and improve virus efficiency by selection against resistant hosts. Appl. Environ. Microbiol. 75, 925–930. Cabodevilla, O., Ibañez, I., Simón, O., Murillo, R., Caballero, P., Williams, T. 2011. Occlusion body pathogenicity, virulence and productivity traits vary with transmission strategy in a nucleopolyhedrovirus. Biological Control, 56, 184–192. CIP, 1992. Annual Report International Potato Center (CIP). CIP. CIP, Lima, Peru, 1992, pp. 254. Cherry, A., Williams, T., Control de insectos plaga mediante los baculovirus. In: Caballero, P., et al., Eds.), Los baculovirus y sus aplicaciones como bioinsecticidas en el control biológico de plagas. Phytoma, Valencia, España, 2001, pp. 389- 450. Cory, J.S. and Myers, J.H. 2003. The ecology and evolution of insect baculoviruses. Annu. Rev. Ecol. Evol. Syst. 34, 239-372. Crespo, L., Grzywacs, D., Figueroa, I., Bonea, O. 2005. Una formulación líquida del baculovirus y otros productos naturales para el control de las polillas de la papa Phthorimaea operculella y Symmetrischema tangolias (Lep: Gelechiidae) en Bolivia., Memorias del III Taller internacional sobre la polilla guatemalteca de la papa Tecia solanivora . Centro Internacional de la Papa (CIP), Cartagena de Indias, Colombia, pp. 49-58. De Moraes RR, Maruniak JE, Funderburk JE, 1999. Methods for detection of Anticarsia gemmatalis nucleopolyhedrovirus DNA in soil. Appl. Environ. Microbiol. 65, 2307- 2311. Espinel-Correal C, Léry X, Villamizar L, Gómez J, Zeddam JL, Cotes AM, López-Ferber M, 2010. Genetic and biological analysis of Colombian Phthorimaea operculella granulovirus isolated from Tecia solanivora (Lepidoptera: Gelechiidae). Appl. Environ. Microbiol. 76, 7617–7625. Fuxa JR, Richter AR, 2001. Quantification of soil-to-plant transport of recombinant nucleopolyhedrovirus: effects of soil type and moisture, air currents, and precipitation. Appl. Environ. Microbiol. 67, 5166-70. Jehle JA, 1996. Transmission of insect transposons into baculovirus genomes: an unusual host-pathogen interaction, pp. 81-97. In : Tomiuk L, Wohrmann K, Sentker A (eds.), Transgenic organisms-biological and social implications. Birkhauser Verlag, Basel, Switzerland. Kolodny-Hirsch DM &Van Beek NAM (1997). Selection of a morphological variant of Autographa californica nuclear polyhedrosis virus with increased virulence following serial passage in Plutella xylostella . Journal of Invertebrate Pathology 69: 205-211. Kroschel, J., Lacey, LA (eds.). 2008. Integrated Pest Management for the Potato Tuber Moth, Phthorimaea operculella (Zeller) - a Potato Pest of Global Importance. Tropical Agriculture 20, Advances in Crop Research 10. Margraf Publishers, Weikersheim, Germany. 147 pp. Lacey, L. A., Headrick, H. L., Horton, D. R., Schreiber, A., 2010. Effect of a granulovirus on mortality and dispersal of potato tuber worm (Lepidoptera: Gelechiidae) in refrigerated storage warehouse conditions. Biocontrol Science and Technology, 20, 437-447. Martin P, Travers R, 1989. Worldwide abundance and distribution of Bacillus thuringiensis isolates. Appl. Environ. Microbiol. 55(10), 2437-2442.

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Muñoz, D., Ruiz de Escudero, I., and Caballero, P. 2000. Phenotypic characteristics and relative proportions of three genotypic variants isolated from a nucleopolyhedrovirus of Spodoptera exigua. Entomol. Exp. Appl. 97, 275-282. Murillo, R., Muñoz, D., Ruíz-Portero, M. C., Alcázar, M. D., Belda, J. E., Williams, T., Caballero, P., 2007. Abundance and genetic structure of nucleopolyhedrovirus populations in greenhouse substrate reservoirs. Biological Control. 42 , 216-225. Niño, L., Acevedo, E., Becerra, F., 2005. Evaluación de un virus de la granulosis nativo e insecticidas químicos para el control en campo de la polilla guatemalteca Tecia solanivora (Lepidoptera:Gelechiidae) en el estado Mérida, Venezuela. Memorias del III Taller internacional sobre la polilla guatemalteca de la papa Tecia solanivora . Cartagena de Indias, Colombia. Centro Internacional de la Papa (CIP), Venezuela, pp. 59-66. Ribeiro, H.C.T., Pavan, O.H., and Muotri, A.R. 1997. Comparative susceptibility of two different hosts to genotypic variants of the Anticarsia gemmatalis nuclear polyhedrosis virus. Entomol. Exp. Appl. 83, 233-237. Rohrmann, G.F., 2011.Baculovirus Molecular Biology.2nd Ed. Bethesda (MD): National Library of Medicine (US), NCBI; http://www.ncbi.nlm.nih.gov/books/NBK49500/ Simón, O., Williams, T., López-Ferber, M., Caballero, P., 2004. Genetic structure of a Spodoptera frugiperda nucleopolyhedrovirus population: high prevalence of deletion genotypes. Applied and Environmental Microbiology. 70, 5579-5588. Simón, O., Williams, T., López-Ferber, M., Caballero, P., 2005. Functional importance of deletion mutant genotypes in an insect nucleopolyhedrovirus population. Applied and Environmental Microbiology. 71 , 4254-4262. Villamizar, L., Zeddam, J. L., Espinel, C., Cotes, A. M., 2005. Implementacion de técnicas de control de calidad para la producción de un bioplaguicida a base del granulovirus de Phthorimaea operculella PhopGV.(Sección Agrícola). Revista Colombiana de Entomología. 1-16. Zeddam JL, Vásquez-Soberon R, Vargas-Ramos Z, Lagnaoui A, 2003. Cuantificación de la producción viral y tasas de aplicación de un virus de granulosis usado para el control biológico de las polillas de la papa, Phthorimaea operculella y Tecia solanivora (Lepidoptera: Gelechiidae). Rev. Chil. Entomol. 29, 29-36.

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LIST OF PUBLICATIONS

Gómez-Bonilla, Y., Léry, X., Muñoz, D., López-Ferber, M., Caballero, P. 2008. Molecular and biological characterization of a novel granulovirus isolate from Phthorimaea operculella found in Costa Rica. IOBC/WPRS Bulletin, 31, pp. 89.

Gómez-Bonilla, Y., López-Ferber, M., Caballero, P., Léry, X., Muñoz, D. 2011. Characterization of a Costa Rican granulovirus strain highly pathogenic against its indigenous hosts, Phthorimaea operculella and Tecia solanivora . Entomologia Experimentalis et Applicata, 140 (3): 238-246.

Gómez-Bonilla, Y., López-Ferber, M., Caballero, P., Léry, X., Muñoz, D. Costa Rican soils contain highly insecticidal granulovirus strains against Phthorimaea operculella and Tecia solanivora . Journal of Applied Entomology. In press.

Gómez-Bonilla, Y., López-Ferber, M., Caballero, P., Muñoz, D., 2011. Potato crops in Costa Rica are efficiency protected from Phthorimaea operculella and Tecia solanivora by an indigenous granulovirus strain. IOBC/WPRS Bulletin. In press.

Gómez-Bonilla, Y., López-Ferber, M., Caballero, P., Muñoz, D., 2011. Stored potatoes in Costa Rica are efficiently protected from Phthorimaea operculella and Tecia solanivora with an indigenous granulovirus strain. IOBC/WPRS Bulletin. In press.

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Para la realización de esta Tesis, Yannery Gómez-Bonilla obtuvo una beca predoctoral del programa de cooperación Iberoamericana del Instituto Nacional de Investigaciones Agrarias (INIA) de España.