BR112016030501B1 - OCCLUSION BODY COMPRISING OCCLUSION-DERIVED VIRIONS AND THEIR PRODUCTION PROCESS, METHOD FOR IDENTIFYING IN A SAMPLE THE PRESENCE OF A SIMPLE NUCLEOPOLYEDROVIRUS OF H. ARMÍGERA, COMPOSITIONS AND THEIR USES - Google Patents

Abstract

SIMPLE NUCLEOPOLYEDROVIRUS OF HELICOVERPA ARMIGERA (HEARSNPV), OCCLUSION BODY AND ITS PRODUCTION PROCESS, COMPOSITION AND ITS USE, AND METHOD FOR IDENTIFYING IN A SAMPLE THE PRESENCE OF A SIMPLE NUCLEOPOLYEDROVIRUS OF H. ARMIGERA. Two new genotypes of the simple nucleopolyhedrovirus of Helicoverpa armígera are described, HearSNPV, HearSNPV-SP1 B and HearSNPV-LB6, each coming from mixtures of genotypes obtained from different locations and cultivations. Each of them has a specific insecticidal activity against H. armígera larvae comparable to that of the usual commercial insecticides, in addition, the mixture of the two genotypes, particularly in a 1:1 ratio with co-occluded virions of mixed genotypes, is capable of controlling H. armígera pests in tomato cultivation, being as effective as commonly used chemical or biological insecticides. Its use as a bioinsecticide represents a safe technology for vertebrates, as it is specific to arthropods. Furthermore, they can be produced easily and in good yield by oral inoculation of H. armígera larvae with HearSNPV occlusion bodies.

Description

Field of invention

[0001] The invention is aimed at the technical sector of biological pesticides for the control of insect pests. Specifically, the invention relates to two new genotypes of a nucleopolyhedrovirus that is capable of infecting larvae of the lepidopteran Helicoverpa armigera (Hlibner, 1809), to compositions comprising one or more of the new genotypes, to a process for their production and use for pest control of the aforementioned insect.

Background of the invention

[0002] In Spain, tomato cultivation occupies 59,300 hectares, with a production of more than 4.3 million tons per year, it is the fourth tomato producing country, behind only the United States (California), China and Italy (http: //wvvw.magrama.gob.es/estadistica/pags/ anuario/2011/AEm201 1_13_06_27_01.pdf). Most tomatoes are grown in Extremadura (73%), Andalusia (13%) and the Ebro Valley (10%) (http://www.navarraagraria.com/n184/artoma11.pdf). In Portugal, tomato cultivation also occupies an important place, with 15,300 hectares and a production of more than 1.1 million tons (http://www.ine.pt/ine__novidades/Estatisticas__Agricolas_2011/index. html#). Among the pests that affect tomato cultivation, the most important is the tomato borer, Helicoverpa armigera (Lepidoptera: Noctuidae) (Torres-Vila et al., 2003). On a global scale, few pests cause as many economic losses as the noctuid H. armigera (Cunningham et al., 1999; Reed and Pawar, 1982). In Spain, H. armigera was one of the key pests in extensive crops such as cotton and corn, but for just over a decade it has been gaining an equal level of importance in horticultural greenhouses in the Spanish Levant, from where it spread to the rest of Spanish regions. and for Portugal (Torres-Vila et al., 2003). It is currently considered the most problematic phytophagous species in most outdoor tomato crops in the Mediterranean region (Torres-Vila et al., 2003). Larvae can attack crops in any phenological state, however, the preferred period for females to lay eggs is flowering. Its preference for parts of the plant with a high concentration of nitrogen, such as reproductive structures (flower and fruit) and growth points, means that its action has a very direct influence on the harvest. Furthermore, it is a very polyphagous species, and has great mobility, high fecundity and multivolitism, and as a result, its population levels can vary quickly in space and time. Quality controls at tomato canning factories set the damage limit at between 2 and 5% of harvested tomatoes. If larvae are present, this limit is reduced to 0-2% (Torres-Vila et al., 2003). These reduced quality limits highlight the need for an effective control method against the pest H. armigera.

[0003] Control of H. armigera is usually done by applying chemical insecticides (Torres-Vila et al., 2003). However, the indiscriminate use of synthetic insecticides has caused several problems, such as increased production costs, the emergence of resistance to different active substances, a reduction in useful fauna, a decrease in quality due to an increase in chemical residues in fruits and derivatives ( Torres-Vila et al., 2000). These facts encouraged the search for other control methods, including viruses and other entomopathogenic microorganisms (Moscardi, 1999).

[0004] The Baculoviridae (baculovirus) family is the most widely studied of all those that affect insects due to their usefulness to man, since they present very desirable characteristics as bioinsecticides: high pathogenicity, compatibility with natural enemies of pests, high specificity (they specifically infect arthropods) (Groner, 1986), long-lasting persistence in places protected from ultraviolet light, high horizontal transmission and, therefore, the capacity to originate epizootics (Caballero et al., 1992; Gelernter and Federici, 1986). Furthermore, they can be formulated in the same way as synthetic chemical insecticides, are perfectly compatible with them and can be applied with conventional equipment (Cherry and Williams, 2001). Baculovirus isolates have already been collected in different places around the world, which have been characterized at a biological and biochemical level (Gelernter and Federici, 1986; Caballero et al., 1992; Hara et al., 1995). Furthermore, some of them are currently registered as insecticides in various parts of the world and are used to control pests (Moscardi, 1999).

[0005] Previously, baculoviruses were classified based on the morphology of the viral occlusion bodies (OBs for their acronyms in English), comprising two genera: Nucleopolyhedrovirus, in which the occlusion bodies are formed by polyhedrin, in the form of an irregular polyhedron, and Granulovirus (GV), in which the occlusion bodies are formed by granulin, in the form of a granule (Theilmanm et al., 2005). However, a more current classification, based on phylogeny (homology at the genomic level), divides the Baculoviridae family into four genera: Alphabaculovirus (lepidopteran-specific nucleopolyhedroviruses [NPV], Betabaculovirus (lepidopteran-specific GV), Deltabaculovirus (NPV specific from dipterans) and Gammabacliovirus (hymenoptera-specific NPV) (Jehle et al., 2006).

[0006] Baculoviruses have a circular genome of double-stranded DNA surrounded by a capsid of a proteinaceous nature, forming the nucleocapsid, which in turn is surrounded by a trilaminar envelope composed of a layer of proteins between two layers of lipids, which it is acquired during virus replication, constituting the virion (Caballero et al., 2001). Said lipoprotein membrane can be acquired in two different ways, constituting two types of virions. If the nucleocapsids remain in the same cell in which they were formed, they acquire a newly synthesized membrane, giving way to virions derived from occlusion bodies (occlusion derived viruses, ODV), which are subsequently enveloped in a matrix formed by a single protein. giving way to the occlusion body (OB). However, other nucleocapsids, once synthesized, move and leave the host cell, acquiring the membrane from the host cell's cytoplasmic membrane when they cross it through specific points where a glycoprotein encoded by the virus (GP84 or F protein) is inserted. , depending on the virus). These virions are called budded virions (BV) and are found free in the host's hemocoelic cavity, being responsible for spreading infection to cells in different tissues. At this stage, all baculoviruses synthesize large quantities of polyhedrin (in the case of nucleopolyhedroviruses, NPVs) or granulin (in the case of granuloviruses, GVs), which crystallize forming a matrix or occlusion body (OB) in the form of an irregular polyhedron (polyhedrin). or granule (granulin). For this reason, polyhedrin OBs are also known as polyhedra and granulin OBs are also known as granules. At the end of the infectious process, which ends between three and six days, the larva dies containing large quantities of occlusion bodies in its hemocoelic cavity that are easily observed under an optical microscope. As a consequence of the infectious process, the larval integument is degraded, releasing millions of occlusion bodies that contaminate the plant foliage, which constitute the inoculum that serves to give rise to a new infectious process in other susceptible hosts (Caballero et al., 2001).

[0007] Therefore, baculoviruses present two types of infectious virions or viral particles that are morphologically and functionally different. ODVs are present in all known baculoviruses, and are the infectious particles responsible for the primary infection in the epithelial cells of the mesentery (digestive tube) and therefore, responsible for the horizontal transmission of the virus between susceptible individuals. BVs, in turn, always contain only one nucleocapsid and are, in all cases, morphologically the same (Fig. 1A). These BVs are the infectious particles responsible for disseminating the infection among the organs and tissues of the host's hemocoeic cavity that are susceptible, giving rise to secondary infection, as in in vitro cell cultures (Caballero et al., 2001). In the occlusion bodies of the NPVs, several ODVs are included, whereas in the granule or GVs only one. Morphologically, nucleopolyhedrovirus ODVs can be of two distinct types: the so-called simple ones (giving rise to simple nucleopolyhedroviruses or single nucleopolyhedroviruses, SNPV) which contain a single nucleocapsid per virion, or the multiple type (multiple nucleopolyhedroviruses or multiple nucleopolyhedroviruses). , MNPV) that contain one to several nucleocapsids per virion (Fig. 1 B).

[0008] Occlusion bodies, whether polyhedra or granules, provide protection to virions, preserving the infectious capacity of these viruses outside the host, since they are capable of persisting in the environment for long periods in places protected from ultraviolet light, they are insoluble in water, resistant to putrefaction and disintegration by chemical agents and also to physical treatments such as freezing, desiccation or freeze-drying. On the contrary, occlusion bodies are soluble in alkaline solutions, such as those produced in the digestive tract of some insects (pH 9-11), which allows the release of ODVs for infection to begin (Caballero et al., 2001 ).

[0009] Baculoviruses have been isolated from more than 500 species of insects, mainly from the order Lepidoptera, among which are many of the most important agricultural pests. In addition to an important interspecific diversity, baculoviruses present a great intraspecific diversity, which has already been demonstrated both in the characterization of different geographic isolates of the same virus and within the same isolate, as wild isolates often comprise distinct genotypic variants. To differentiate and characterize both isolates and genotypes present within the same isolate, viral DNA analysis with restriction enzymes is generally used, as it provides characteristic profiles for each isolate or genotype (Erlandsom et al., 2007; Figueiredo et al. , 1999; Harrison and Bonning, 1999).

[00010] These genomic differences between different isolates and genotypes of the same virus can give rise to significant differences in their insecticidal characteristics, such as pathogenicity, which is defined as the amount of inoculum necessary to kill a percentage of the population, virulence, or speed at which the insect is killed, and viral productivity. Other phenotypic characteristics that may be affected are host spectrum, occlusion body size, and larval liquefaction (Cory et al., 2005; Harrison et al., 2012). Knowing the intrapopulation diversity of baculoviruses is, therefore, especially important when designing bioinsecticides, whose active substances must include strains or genotypes with the greatest insecticidal potential. On the other hand, it is known that local insect populations are more susceptible to native isolates of the virus (Barrera et al., 2011; Bernal et al., 2013a), and therefore it would be convenient to select a virus isolate with the same geographic origin than the populations you want to combat.

[00011] Naturally, H. armigera larvae are infected by a nucleopolyhedrovirus known in short as HearSNPV (Helicoverpa armigera single nucleopolyhedrovirus, genus Alphabaculovirus). It is a simple nucleopolyhedrovirus (SNPV) that also infects larvae of other members of the genus Helicoverpa spp. and Heliothis spp., such as, for example, larvae of Helicoverpa zea. HearSNPV isolates have already been characterized in different regions of the world, such as China or Kenya (Chen et al., 2001 ; Ogembo et al., 2005). Isolates of this virus have also been obtained in Spain and Portugal (Figueiredo et al., 1999, 2009), where it causes natural epizootics in populations of H. armigera. To date, several isolates of this virus have been characterized, with the following being the most studied: - Two pure genotypes from China, whose genomes have been completely sequenced, HearSNPV-G4 (Chen et al., 2001) and HearSNPV-C1 (Zhang et al., 2005), which will be referred to throughout the descriptive report in abbreviated form as HearG4 and HearC1. Guo and colleagues (2006) compared the biological activity of these two genotypes. In terms of the concentration/dose-mortality relationship, HearC1 was 2.8 times more pathogenic than HearG4 for third-stage larvae of a population of H. armigera from China. Furthermore, larvae infected with HearC1 died 9 hours before those infected with HearG4. The article by Zhang and collaborators, from 2005, compares the genomes of these two genotypes, observing an identity in the nucleotide sequence of 98.1%. Comparison of these two genomes shows four variable regions between these two genotypes, the homologous regions 1, 4 and 5 (hr1, hr4 and hr5) and the bro-b region. Homologous regions (hr) are intergenic zones that are present in many baculoviruses, and are located multiple times throughout the genome. They are characterized by the presence of multiple and imperfect repeated sequences.

[00012] Five homologous regions appear in the HearSNPV genome. In figure 1 of the article by Chen and collaborators (2000) the restriction profiles with the restriction endonucleases BamI, BglII, EcoRl, Hindlll, Kpnl, Pstl, Sacl and Xhol appear (Fig. 2 of the present application). Table 1 of the aforementioned article shows the estimated sizes of the restriction fragments generated by each of the aforementioned restriction endonucleases (Table 1). The complete genomes of HearG4 and HearC1 are available in the GenBank database under accession numbers AF271059 and AF303045, respectively. The HearG4 genotype is currently commercialized for the control of H. armigera in cotton cultivation in China (Zhang, 1994). Table 1: Estimated sizes of HearG4 fragments obtained by digestion with BamHI, BglII, EcoRI, HindlII, Kpnl, PstI, SacI and XhoI and estimated total genome size (Chen et al., 2000). - An isolate from Kenya, HearSNPV-NNg1, which will be mentioned throughout the descriptive report as HearNNg1, whose genome has also been completely sequenced (Ogembo et al, 2009). HearNNg1 was selected by Ogembo and collaborators (2007) as the isolate with the best characteristics to be developed as a bioinsecticide for H. armigera larvae in Japan. HearNNg1 was shown to be between 3.2 and 82.6 times more pathogenic than the rest of the isolates studied, and 311.5 times more pathogenic than the Chinese isolate HearG4, for third stage larvae. Furthermore, the NNg1 isolate killed H. armigera third-stage larvae between 0.4 and 1.8 days before the rest of the isolates, and 4.3 days before the HearG4 genotype. Figure 1 of the aforementioned article shows the restriction profiles with the endonucleases BglII and XbaI of the characterized isolates (Fig. 3 of the present application). Table 2 of the same article shows the estimated sizes of the restriction fragments of the different isolates digested with the endonucleases BglII, XbaI and HindII (Table 2). Table 2: Estimated fragment sizes of HearNNg1 (NNg1) and other isolates from South Africa (NS2), Kenya (Nma1), Zimbabwe (NZ3), Thailand (NT1) and China (G4) obtained by digestion with BglII, XbaI and HindIII and estimated total genome size (Ogembo et al., 2007).

[00013] On the other hand, the article by Ogembo and collaborators (2009) compares the HearNNg1 genome with the genomes of the Chinese genotypes HearC1 and HearG4, as well as with the genome of the Helicoverpa zea simple nucleopolyhedrovirus (HzSNPV). The NNg1 genotype shows the greatest differences with the HearC1, HearG4 and HzSNPV genomes in the homologous regions (hr) and in the bro genes, as occurs when comparing the HearC1 and HearG4 genomes. The complete HearNNg1 genome is available in the GenBank database under accession number AP0109Q7. - An isolate from Australia, HearSNPV-Aus, which will be referred to throughout the descriptive report in abbreviated form as HearAus, whose genome has been completely sequenced and is available in the GenBank database under accession number JN584482. - Seven isolates from the Iberian Peninsula, five Spanish, HearSP1, HearSP2, HearSP4, HearSP7 and HearSP8, and two Portuguese, HearPT1 and HearPT2 (Arrizubieta et al., 2014; Figueiredo et al., 1999, 2009). Figueiredo and collaborators (1999) selected the HearSP1 isolate as the isolate with the best insecticidal characteristics, as it was twice as pathogenic as the HearSP2 isolate for second-stage larvae of a population of H. armigera from Portugal. Subsequently, in a new study carried out by Figueiredo and collaborators (2009) it was determined that the HearSP7, HearPT1 and HearPT2 isolates presented the most favorable characteristics as bioinsecticides, but this work did not include the HearSP1 isolate. In a study recently carried out in our laboratory in which all these isolates from the Iberian Peninsula were compared, the HearSP1 isolate was selected for its best insecticidal characteristics against H. armigera, since although it presents the same pathogenicity as the rest of the isolates studied, it is the most virulent and additionally among the most productive in terms of the quantity of occlusion bodies produced in each infected insect (Arrizubieta et al., 2014). Figure 1 B of the article by Figueiredo and collaborators (2009) shows the restriction profiles with the BglII endonuclease of the Spanish isolates HearSP1, HearSP2, HearSP3, HearSP4, HearSP7 and HearSP8, and of the Portuguese isolates HearPT1 and HearPT2 (Fig. 4A of the present order). Figure 1 of the article by Arrizubieta and colleagues (2014) shows the profiles of isolates HearSP1, HearSP2, HearSP4, HearSP7, HearSP8, HearPT1, HearPT2 and HearG4 with the EcoRI endonuclease (Fig. 4B of the present application), and in Table 1 of In this article, the sizes of the restriction fragments are indicated (Table 3). Table 3. Estimated sizes of the HearSP1, HearSP2, HearSP4, HearSP7, HearSP8, HearPT1, HearPT2 and HearG4 fragments, and actual size of the HearG4 fragments generated in silico (G4*) from the sequence (AF271059) obtained by digestion with EcoRI and estimated total genome size (Arrizubieta et al., 2014).

[00014] The difference in the number of fragments of the different genotypes with those of the HearG4 genotype generated in silico is due to the fact that its genome is completely sequenced, which is why small fragments are detected that are impossible to detect by analysis of banding patterns as they are not visible in REN profiles. In the case of HearSP1, small-sized fragments were detected by PCR amplification and sequencing of the amplified fragment using primers designed at the ends of the cloned fragments (Arrizubieta et al., 2014).

[00015] After selecting the appropriate active substance, before marketing a bioinsecticide, it is necessary to carry out field tests to prove its efficiency in the conditions in which it will be applied, since its effectiveness in the field may vary in relation to that obtained under conditions laboratory controlled. However, to carry out the aforementioned tests and thus be able to treat extensive cultivation surfaces, it is necessary to obtain large quantities of occlusion bodies, making it necessary to develop a system for mass production of the virus. Currently, the methodology used for the mass production of most baculoviruses is in vivo production in permissive hosts (Kalia et al., 2001; Lasa et al., 2007). This technique consists of feeding susceptible larvae with a superficially contaminated artificial diet. with a suspension of occlusion bodies. Some of the essential aspects of this methodology, such as artificial insect diet or mass rearing methods, must be developed specifically for each host-pathogen system (Lasa et al., 2007). On the other hand, in the United States a HearSNPV production system was developed that involves both in vivo and in vitro production (US Patent No. US 7521219 B2). This technique consists of first multiplying the virus in H. armigera larvae, and then carrying out a limited number of serial passes in cells to obtain a large number of occlusion bodies.

[00016] As a consequence of the increasingly frequent resistance developed by H. armigera larvae to synthetic chemical insecticides, the applied quantity necessary for this type of substances to achieve the desired effect has been progressively increased. In the Iberian Peninsula, due to the large area cultivated with tomatoes, among others, this fact has become a problem whose consequences are tremendously negative for farmers, consumers and the environment.

[00017] The contamination of soils, aquifers and other natural spaces, the effect on other living organisms, as well as the increase in the production costs of agricultural products and the decrease in their quality pose a serious threat to several strategic sectors in the Iberian Peninsula . Due to the resistance developed by H. armigera larvae to synthetic chemical insecticides, it would be interesting to have an alternative with good insecticidal capacity and which, in addition, had a very reduced host spectrum, so as not to harm natural enemies or other beneficial organisms, such as a biological control agent. Among them, it would be especially desirable for it to be an efficient control method for the Iberian Peninsula, and for it to be powerful enough to solve the threats and problems presented by H. armigera pests in the Iberian Peninsula. In addition to being highly effective against pests from the Iberian Peninsula, it would be interesting if its production method were also highly effective, so that the cost of its production and the amount of insecticide to be applied did not imply an increase in costs that would not make it competitive.

[00018] The invention provides an effective solution to this problem.

Summary of the Invention

[00019] The present invention is based on obtaining new genotypes of the simple nucleopolyhedrovirus of Helicoverpa armigera, which were isolated by in vitro purification. Two of these genotypes were purified from the isolate HearSNPV-SP1 (HearSP1) (Figueiredo et al., 1999), named HearSNPV-SP1A and HearSNPV-SP1B (or HearSP1A and HearSP1B for short), while another six genotypes were isolated from from larvae killed during an epizootic produced in the laboratory in the second generation of a population of H. armigera from a cotton crop in Lebrija (Sevilla), called HearSNPV-LB1, HearSNPV-LB2, HearSNPV-LB3, HearSNPV-LB4, HearSNPV -LB5 and HearSNPV-LB6 (or for short HearLB1, HearLB2, HearLBS, HearLB4, HearLB5 and HearLB8). These genotypes are distinct from all isolates and genotypes characterized to date.

[00020] Surprisingly, the tests carried out with these genotypes demonstrate that two of the new isolated genotypes HearSNPV-SP1B (CNC I-4806) and HearSNPV-LB8 (CNCM I-4807), and in particular, the mixture of the same HearSNPV-SP1B: LB6 in a 1 : 1 ratio, are among the most active nucleopolyhedroviruses that have been developed as bioinsecticides to date.

[00021] This product represents a clean and safe technology as it does not leave toxic residues in the soil or on crops and is not toxic to humans or other animals, including natural enemies of pests, such as predators and parasitoids. Furthermore , these nucleopolyhedroviruses have the additional advantage of ease and good yield in their production.

[00022] Therefore, in a first aspect, the objective of the present invention refers to a simple nucleopolyhedrovirus of H. armigera (HearSNPV) that belongs to a genotype selected from the group of: i) the HearSNPV genotypes deposited in the Collection Nationale of Cultures of Microorganismes (CNCM) with deposit numbers CNCM 1-4808 (HearSNPV- SP1B), CNCM I-4807 (HearSNPV- LB6), or ii) the genotypes whose genome is represented by SEQ ID NO: 13 (HearSNPV- SP1B), or SEQ ID NO: 14 (HearSNPV-LB6).

[00023] Said nucleopolyhedrovirus can be in different forms, either in the form of a viral particle or virion, or in the form of occlusion bodies, which is the form in which nucleopolyhedroviruses are found in nature, and, therefore, the form in which the larvae ingest. An occlusion body may contain virions of only one of the HearSNPV- SP1B (CNCM I-4808) or HearSNPV-LB6 (CNCM I-4807) genotypes, or virions of more than one of said genotypes co-occluded within the same occlusion body. . Virions can be occlusion body-derived virions (ODV) (the propagating form that is included in the occlusion bodies and which is released into the intestine of the larvae after dissolution of the polyhedrin), or budded virions (BV) (the form by means of which the infection is propagated to the different tissues of an infected insect, and which can also be found in cell cultures).

[00024] An aspect of the present invention also constitutes an occlusion body containing several virions, at least one of which belongs to a genotype of the H. armigera simple nucleopolyhedrovirus selected from the group of HearSNPV-SP1B (CNCM I-4806) and HearSNPV-LB6 (CNCM I-4807). The occlusion body may contain several virions of the same genotype or virions of different genotypes co-occluded in the same occlusion body. When the virions are of the same genotype, this may be any of the HearSNPV-SP1B or HearSNPV-LB6 genotypes. While when it comes to co-occluded virions, the co-occluded genotypes can be any of HearSNPV-SP1B and/or HearSNPV-LB6, in different proportions. Furthermore, virions of other genotypes of the H. armigera simple nucleopolyhedrovirus may also be included in the mixture or it may be that all virions belong to one of the genotypes of the HearSNPV-SP1B and HearSNPV-LB6 group. In either case, the virions contained in the occlusion bodies will be occlusion body-derived virions (ODVs).

[00025] The HearSNPV-SP1B and HearSNPV-LB8 genotypes can be distinguished by the specific sequence they present in certain areas of their genomes, of great variability, such as the regions of the genome known as homologous regions (hr) 1 and 5 (hr1 and hr5), as described in the examples of the present application. Therefore, occlusion bodies containing at least one virion (ODV) whose genome comprises a DNA fragment whose sequence is represented by: i) SEQ ID NO:5 or SEQ ID NO:6 are also possible embodiments of this aspect of the invention. (the specific sequences of the homologous region 1 (hr1) amplified by PCR using the primers F-hr1 and R-hr1 in examples of the present application, belonging, respectively, to the genotypes HearSNPV-SP1B (CNCM I-4806) and HearSNPV-LB6 ( CNCM I-4807)). ii) SEQ ID NO:7 or SEQ ID NO:8 (the specific sequences of the homologous region 5 (hr5) amplified by PCR using the primers F-hr5 and R-hr5 in examples of the present application, belonging, respectively, to the HearSNPV genotypes - SP1B (CNCM I-4806) and HearSNPV-LB6 (CNCM I-4807)). iii) SEQ ID NO:9 or SEQ ID NO: 10 (the complete sequences of homologous region 1 (hr1), belonging, respectively, to the genotypes HearSNPV-SP1B (CNCM I-4806) and HearSNPV-LB6 (CNCM I-4807) ). iv) SEQ ID NO: 11 or SEQ ID NO: 12 (the complete sequences of the homologous region 5 (hr5), belonging, respectively, to the genotypes HearSNPV-SP1B (CNCM I-4806) and HearSNPV-LB6 (CNCM I-4807) ).

[00026] Another aspect of the invention is a composition that contains nucleopolyhedroviruses of at least one of the genotypes HearSNPV-SP1B (CNCM I-4806) and HearSNPV-LB6 (CNCM I-4807), or combinations thereof. As in the previous case, nucleopolyhedroviruses can be in different forms, such as free virions or, preferably, in the form of occlusion bodies, which can have a variable number of co-occluded virions (virions that, as mentioned previously, will be virions derived from occlusion bodies (ODVs)). In this case, the virions contained in the occlusion body may be of a single genotype or of several, as long as at least one of the genotypes is HearSNPV-SP1B (CNCM I-4806) or HearSNPV-LB6 (CNCM I-4807). Therefore, this aspect of the invention relates to a composition comprising a nucleopolyhedrovirus of the invention or an occluding body of the invention. In particular, possible embodiments of the invention are those that comprise mixtures of virions of different genotypes with which the assays described later in the examples of the present invention are carried out, with preference for compositions that comprise a mixture of virions of the HearSNPV-SP1B genotypes ( CNCM I-4806) and HearSNPV-LB8 (CNC I-4807).

[00027] The different genotypes can be in any relative proportion, preferably in the proportion that presented the best results in the examples described below, that is, that in which the genotypes HearSNPV-SP1B (CNCM I-4808) and HearSNPV-LB6 (CNCM I-4807) are in the HearSNPV-SP B:HearSNPV-LB6 1:1 ratio.

[00028] Additionally, the compositions of the invention may comprise any excipient or vehicle appropriate in the agricultural sector, with preference for those that make it suitable for application according to any of the usual methods in agriculture: spraying, whether at ground level or aerial , application in suspension or powder form, or by any type of irrigation system. The composition may be in any form, whether in aqueous form or in solid form. The composition may contain any other component, preferably those of particular agricultural interest; therefore, H. armigera simple nucleopolyhedroviruses can be mixed, for example, with a fertilizer, a fertilizer or a pesticide, or mixtures thereof. A concrete case may be one in which the composition of the invention additionally comprises an insecticide based on the bacteria Bacillus thuringiensis selected from endosporas of said bacteria, crystals of Cry proteins or mixtures thereof.

[00029] On the other hand, possible embodiments of said invention are also compositions that may comprise agents that enhance the pathogenic effect of the nucleopolyhedrovirus on the lepidoptera.

[00030] An additional aspect of the invention is the use as an insecticide of at least one of the nucleopolyhedroviruses of the present invention, or of a composition containing at least one of them. The insect you want to control is preferably H. armigera, specifically when it is in the form of larvae or caterpillars. It is preferable for nucleopolyhedroviruses to be in the form of occlusion bodies, which is the form commonly ingested by larvae. It is also preferred that the composition contains a mixture of the genotypes HearSNPV-SP1B (CNCM I-4806) and HearSNPV-LB6 (CNCM I-4807), preferably mixtures in which said genotypes are in the ratio HearSNPV-SP1B: HearSNPV-LB6 1 : 1.

[00031] Another aspect of the invention is a process for producing occlusion bodies comprising a step in which H. armigera larvae are fed an artificial diet containing occlusion bodies of the H. armigera nucleopolyhedrovirus with virions of any one of the genotypes HearSNPV-SP1B (CNCM I-4808) or HearSNPV-LB6 (CNCM I-4807) or mixtures thereof.

[00032] An aspect of the invention also constitutes a method for identifying the presence in a sample of a genotype of the H. armigera simple nucleopolyhedrovirus selected from HearSNPV-SP1B (CNCM I-4806) and HearSNPV-LB6 (CNCM I-4807) that comprises the steps of: i) amplifying by PCR the DNA extracted from said sample using a pair of primers, which are amplified in the homologous region (hr) 1 or 5, which are selected among those formed by: a. SEQ ID NO: 1 (F-hr1) and SEQ ID NO:2 (R-hr1), or b. SEQ ID NO:3 (F-hr5) and SEQ ID NO:4 (R-hr5); ii) analyze the amplified fragment to determine its size or sequence; iii) digest the amplified fragment with the NdeI endonuclease: iv) analyze the fragments generated after digestion to determine the number of fragments and the size of each of them; v) conclude that one of the HearSNPV-SP1B (CNCM 1-4808) or HearSNPV-LB6 (CNCM 1-4807) genotypes is present if: a. the fragment amplified by the pair of SEQ ID NO: 1 and SEQ ID NO:2 has: i. a size of 2,177 (HearSNPV-SP1B) or 2,117 (HearSNPV-LB6) nucleotides; ii. digestion of said fragment with the Ndel endonuclease generates 8 fragments of 857, 508, 381, 306, 78 and 47 nucleotides (HearSNPV-SP1B) or 5 fragments of 1,210, 475, 307, 78 and 47 nucleotides (HearSNPV-LB6); iii. the sequence represented by SEQ ID NO:5 (HearSNPV-SP B) or SEQ ID NO:8 (HearSNPV-LB6); or, alternatively, b) the fragment amplified by the pair of SEQ ID NO:3 and SEQ ID NO:4 has: i. a size of 2,326 (HearSNPV-SP1B) or 2,330 (HearSNPV-LBo) nucleotides; ii. digestion of said fragment with the Ndel endonudease generates 4 fragments of 1,120, 917, 211 and 78 nucleotides (HearSNPV-SP1B) or 3 fragments of 1,120, 998 and 212 nucleotides (HearSNPV-LB6); iii. the sequence represented by SEQ ID NO:7 (HearSNPV-SP1B) or SEQ ID NO:8 (HearSNPV-LB6).

[00033] The invention will be explained in more detail through the following Figures and Examples.

Description of Figures

[00034] Figure 1. (A) Photos in the transmission microscope and schematic representation of virions derived from occlusion bodies (ODVs) and budded virions (BVs), and (B) of multiple-type nucleopolyhedroviruses (NPV) with virions with a variable number of nucleocapsids, and the simple type (SNPV) with virions with a single nucleocapsid.

[00035] Figure 2. Restriction profiles of the HearSNPV-G4 isolate obtained after digestion of genomic DNA with the endonucleases BamHI, BglII, EcoRl, Hindlll, KpnI, Pstl, SacI and Xhol. On the left of the figure is shown the molecular weight marker Lambda (X) digested with BamHl-EcoRl-HindIIl, and their sizes are indicated in kilobases (Chen et al., 2000).

[00036] Figure 3. Restriction profiles of different HearSNPV isolates: NNg1 (from Kenya), NS2 (South Africa), NMa1 (Kenya), NZ3 (Zimbabwe) and NT1 (Thailand) obtained after digestion of genomic DNA with the endonucleases Bglll (A) and XbaI (B). To the left of the figures is shown the molecular weight marker Lambda (X) digested with Hindlll (MI) and with EcoRl-Hindlll (MIl), and their sizes are indicated in kilobases (Ogembo et al., 2007).

[00037] Figure 4. (A) Restriction profiles of isolates HearSP1, HearSP2, HearSP3, HearSP4, HearSPS, HearSP6, HearSP7, HearSPS, HearPT1 and HearPT2 after digestion of genomic DNA with the endonuclease Bglll, shown on the left of the figure the molecular weight marker Lambda (X) digested with Hindlll, and their sizes are indicated in base pairs (Figueiredo et al., 2009). (B) Restriction profiles of isolates HearSP1, HearSP2, HearSP4, HearSP7, HearSPS, HearPT HearPT2 and HearG4 with the EcoRl endonuclease, on the left of the figure the molecular weight marker HyperLadder I (Bioline) is shown, and their sizes are indicated in kilobases (Arrizubieta et al., 2014).

[00038] Figure 5. Banding pattern obtained in a continuous sucrose gradient after centrifugation of the ODVs obtained from (A) HearSNPV and (B) Ac NPV. White asterisks indicate bands representing ODVs. As can be seen in panel A, only one band is visible, therefore all virions have the same morphology, containing a single nucleocapsid. However, in panel B several bands are observed, each of which represents ODVs with a specific number of nucleocapsids, and depending on the number of nucleocapsids they will have greater or lesser weight, appearing at a smaller or greater height.

[00039] Figure 6. Schematic representation of a mixture of occlusion bodies of different genotypes, where each occlusion body is formed by ODVs of the same genotype, and of a mixture of genotypes co-occluded in the same occlusion body, where each occlusion body is formed by ODVs of different genotypes.

[00040] Figure 7. (A) Electrophoresis of the restriction fragments obtained by treating the viral DNA of the HearSP1 isolate and the HearSP1A and HearSP1B genotypes with the restriction endonucleases BglII and EcoRI. (B) Electrophoresis of restriction fragments obtained by treatment of viral DNA from isolates HearSP1, HearSP2, HearSP4, HearSP7, HearSP8, HearPT1 and HearPT2 and genotypes HearG4, HearSP1A, HearSP1B, HearLB1, HearLB2, HearLB3, HearLB4, HearLBS and HearLB6 with the EcoRi restriction endonuclease. (C) Electrophoresis of restriction fragments obtained by treatment of viral DNA from isolates HearSP1, HearSP2, HearSP4, HearSP7, HearSP8, HearPT1 and HearPT2 and genotypes HearG4, HearSP1A, HearSP1B, HearLB1, HearLB2, HearLB3, HearLB4, HearLBS and HearLB6 with the BglII restriction endonuclease. To the left of the figures is shown the 1 kb molecular weight marker (NI PPON Genetics, Europe GmbH), and the sizes of its fragments are indicated in kilobases.

[00041] Figure 8. (A) Fragments obtained by PCR amplification of the variability zones of the homologous region hr1 (primers identified by SEQ ID NO: 1 and SEQ ID NO:2) and hr5 (primers identified by SEQ ID NO:3 and SEQ ID NO:4) of the HearSP1B and HearLB6 genotypes, the HearSP1 isolate and the Chinese HearG4 genotype, with c-: negative control without viral DNA. The 1 kb molecular weight marker (NIPPON) is shown on the left of the figure, and the sizes of its fragments are indicated in kilobases. (B) Fragments obtained after digestion with NdeI endonuclease of fragments obtained by PCR from the hr1 and hr5 variability zones of the HearSP1B and HearLB6 genotypes, the HearSP1 isolate and the Chinese HearG4 genotype. The 100 bp molecular weight marker (NIPPON Genetics, Europe GmbH) is shown on the left of the figure, and the sizes of its fragments are indicated in base pairs.

[00042] Figure 9. (A) Alignment of the nucleotide sequences of the fragments amplified by PCR of the homologous region 1 (hr1) corresponding to the HearSP1B and HearLB6 genotypes and the HearG4, HearC1, HearNNg1 and HearAus isolates. (B) Alignment of the nucleotide sequences of the PCR-amplified fragments of the homologous region 5 (hr5) corresponding to the HearSP1B and HearLB6 genotypes and the HearG4, HearC1, HearNNg1 and HearAus isolates.

[00043] Figure 10. Average production of occlusion bodies (x107 occlusion bodies/larva) in second-stage larvae of H. armigera after being infected with the individual genotypes HearSP1A and HearSP1B and with the HearSP1 isolate. Vertical bars indicate standard deviation. The letters with the same meaning that accompany the values indicate no significant differences between treatments (P>0.05).

[00044] Figure 11. Average production of occlusion bodies (x108 occlusion bodies/larva) in H. armigera second-stage larvae after being infected with the individual genotypes HearLB1, HearLB2, HearLB3, HearLB4, HearLBS and HearLB6 and with the HearSP1 isolate. Vertical bars indicate standard deviation. The letters with different meanings that accompany the values indicate that there are significant differences between the treatments (P<0.05).

[00045] Figure 12. Average production of occlusion bodies (x107 occlusion bodies/larva) in H. armigera second-stage larvae after being infected with the individual genotypes HearSP1A, HearSP1B, HearLB1, HearLBS and HearLB6 and with the mixtures co-occluded HearSP1A:SP1B (1 : 1), HearSP1A:SP1B (1:2), HearLB1:LB3, HearLB3:LB6, HearLB1:LB3:LB6, HearLBmix, HearSP1B:LB1 and HearSP1B:LB6. Vertical bars indicate standard deviation. The letters with different meanings that accompany the values indicate that there are significant differences between the treatments (P<0.05).

[00046] Figure 13. Percentage of mortality due to infection, survival (or reached the pupa state) and cannibalism in third, fourth and fifth stage larvae (L3, L4 and L5), healthy and infected with the lethal concentration of 90% (LC90) of the HearSP1B:LB6 co-occluded mixture at different larval densities (1, 5, 10 and 20 larvae per box). The letters with different meanings that accompany the values indicate that there are significant differences between the treatments (P<0.05).

[00047] Figure 14. Percentage of larval mortality after inoculating newly molted H. armigera larvae to the third, fourth and fifth stages (L3> L4 and L5) and one day after molting (L3+1, L4+1 and L5+I) with a lethal concentration of 95% (LC95), 90% (LC90) or 80% (LC80) of the HearSP1B:LB6 co-occluded mixture. Vertical bars indicate standard deviation. The letters with different meanings that accompany the values indicate that there are significant differences between the treatments (P<0.05).

[00048] Figure 15. (A) Average production of occlusion bodies (x 108 occlusion bodies/larva) in H. armigera larvae recently moulted to the third, fourth and fifth stages (L3, L4 and L5) and a day after molting for the aforementioned stages (L3+1, L4+1 and L5+1) inoculated with a lethal concentration of 95% (LC95), 90% (LC90) or 80% (LC80) of the HearSP1B co-occluded mixture :LB6. (B) Average production of occlusion bodies (x 1010 occlusion bodies/100 larvae) in H. armigera larvae recently moulted to L3, L4 and L5 and one day after moulting to the aforementioned stages (L3+1, L4 +1 and L5+1) inoculated with CL95, CL90 and CL80 from the HearSP1B:LB6 co-occluded mixture. Vertical bars indicate standard deviation. The letters with different meanings that accompany the values indicate that there are significant differences between the treatments (P<0.05).

[00049] Figure 16. Average production of occlusion bodies (x 109 occlusion bodies/larva) in fifth stage larvae (L5) of H. armigera inoculated with the 95% lethal concentration (LC95) of the HearSP1B co-occluded mixture :LB6 and incubated at 23, 26 and 30°C. Vertical bars indicate standard deviation. The letters with the same meaning that accompany the values indicate that there are no significant differences between the treatments (P>0.05).

[00050] Figure 17. Percentage of mortality obtained in second-stage larvae of H. armigera collected from tomato plants treated under laboratory conditions. Larvae were collected 1, 3 and 5 days after application of HearSNPV at three concentrations (106, 107 and 108 occlusion bodies/mi) of the HearSP1B:LB6 co-occluded mixture, and reared individually in the laboratory in pots with a semi-synthetic diet until death or pupation.

[00051] Figure 18. Percentage of damaged fruits in tomato cultivation in a greenhouse 10 days after the application of Turex, Spintor and HearSNPV. The letters with different meanings that accompany the values indicate that there are significant differences between the treatments (P<0.05).

[00052] Figure 19. Percentage of larval mortality observed in tomato cultivation in a greenhouse 10 days after the application of Turex, Spintor and HearSNPV. The letters with different meanings that accompany the values indicate that there are significant differences between the treatments (P<0.05).

[00053] Figure 20. Percentage of residual insecticidal activity (Turex, Spintor and HearSNPV) in greenhouse tomato leaves over time, in relation to the amount of insecticide present in tomato leaves one hour after applying the treatments. Vertical bars indicate standard deviation.

[00054] Figure 21. Amount of residual insecticidal activity per gram of tomato leaves in a greenhouse 1, 72, 144 and 216 hours (0, 3, 8 and 9 days) after application of the treatments: A) Turex (mg), B) Spintor (μl) and C) HearSNPV (occlusion bodies). Vertical bars indicate standard deviation.

[00055] Figure 22. Percentage of damaged fruits, whether scarred or fresh, in outdoor tomato cultivation after application of HearSP1B: LB6, HearSP1, Spintor, Turex and Dursbam during (A) the first, (B) second , (C) third, and (D) fourth fortnight. The letters with different meanings that appear in the columns in each group indicate that there are significant differences within each group between the different treatments (P<0.05).

[00056] Figure 23. Percentage of damaged fruits harvested, whether rotten red, scarred red or chopped green, in outdoor tomato cultivation, after the application of HearSP1B: LB6, HearSP Spintor, Turex and Dursban. The letters with different meanings that appear in the columns in each group indicate that there are significant differences within each group between the different treatments (P<0.05).

[00057] Figure 24. Tons of A) green tomatoes, both chopped and healthy and B) healthy red tomatoes, scarred or rotten per hectare in outdoor cultivation after application of HearSP1B: LB6, HearSP1, Spintor, Turex and Dursban. The letters with different meanings that appear in the columns in each group indicate that there are significant differences within each group between the different treatments (P<0.05).

[00058] Figure 25. Percentage of residual insecticidal activity (HearSP1B: LB6, HearSP1, Spintor, Turex and Dursban) present in tomato leaves in outdoor cultivation over time, in relation to the amount of insecticide present in tomato leaves one hour after applying treatments. Vertical bars indicate standard deviation.

[00059] Figure 26. Amount of residual insecticidal activity per gram of tomato leaf in outdoor cultivation 1, 72, 168 and 240 hours (0, 3, 7 and 10 days) after application of the treatments: A) HearSP1B: LB6 (occlusion bodies), B) HearSP1 (occlusion bodies), C) Spintor (μ?), D) Turex (mg) and E) Dursbam (mg). Vertical bars indicate standard deviation.

Detailed description of the invention

[00060] The objective of the present invention refers to obtaining new genotypes of the simple nucleopolyhedrovirus of Helicoverpa armigera (Fig. 5). The aforementioned genotypes were isolated in two different ways: i) from the isolate HearSNPV-SP1 (or, in short, HearSP1), using a plate assay with in vitro cell culture. The genotypes present in that isolate, different from all isolates and genotypes characterized to date, were named HearSNPV-SP1A and HearSNPV-SP1B (or, in short, HearSP1A and HearSP1B). ii) from larvae killed during an epizootic in the laboratory in the second generation of a population of H. armigera from a cotton plantation in Lebrija (Sevilla). The genotypes obtained from these larvae, different from all isolates and genotypes characterized to date, were named HearSNPV-LB1, HearSNPV-LB2, HearSNPV-LB3, HearSNPV-LB4, HearSNPV-LB5 and HearSNPV-LB6 (or, more precisely, abbreviated, HearLB1, HearLB2, HearLB3, HearLB4, HearLBS and HearLB6). Each of these genotypes comes from an individual larva killed by the aforementioned epizootic. The absence of submolar bands in the restriction profiles suggests that these genotypes are pure genotypes since the aforementioned submolar bands are produced by the presence of several genotypes in different proportions. However, in order to be sure of their purity, in vitro cloning of the different isolates was carried out using an end point dilution (EPD) test. Said cloning and subsequent analysis by restriction enzymes confirmed their purity, demonstrating that each of the larvae had died due to the infection of a single genotype.

[00061] On the other hand, the restriction profiles obtained after digesting the genome of each of these genotypes with different restriction enzymes (endonucleases) confirmed that they were different genotypes (HearSP1A, HearSP1B, HearLB1, HearLB2, HearLBS, HearLB4, HearLBS and HearLB8) (Fig. 7) and were also different from other isolates and genotypes characterized to date (Table 4), such as the Chinese genotypes HearC1 and HearG4 (Fig. 2), the isolate from Kenya HearNNg1 (Fig. 3) and the Iberian Peninsula isolates HearSP1, HearSP2, HearSP4, HearSPT, HearSPS, HearPT1 and HearPT2 (Fig. 4).

[00062] Of the different genotypes found, two of them were selected, called HearSP1B and HearLB6, which can be easily distinguished from each other and differentiated from other HearSNPV isolates and genotypes by the profiles obtained by treating their genomes with restriction enzymes, which can be EcoRI and Bglll. Figures 2, 3, 4 show the restriction profiles obtained for the previously characterized HearSNPV isolates, while Figure 7 shows the restriction profiles of the genotypes HearSP1 A, HearSP1B, HearLB1, HearLB2, HearLB3, HearLB4, HearLB5 and HearLB6, as well as those corresponding to the Spanish isolates HearSP1, HearSP2, HearSP4, HearSP7 and HearSPS, the Portuguese isolates HearPT1 and HearPT2, and the Chinese genotype HearG4. Differentiation is based on the presence of characteristic polymorphic fragments in the restriction profiles of each genotype or isolate. The submolar bands (bands that contain a smaller amount of molecules than the rest of the bands of the same DNA profile) indicate the presence of a mixture of isolates as observed in the case of HearSP1 in Figure 7A. Another example is the HearSP1B genotype, which when digested with the Ndel enzyme shows a 9.73 kb band that is not observed in the profile of the HearSP1 isolate (Figure 8B). Furthermore, the HearSP1 isolate shows several submolar bands around 6.5-7 kb, which are not observed in the profile of the pure HearSP1B genotype (Figure 8B). On the other hand, in the profiles obtained with the BglII endonuclease, the HearSP1 isolate shows a submolar band of 18.8 kb that is not observed in the HearSP1B genotype profile. The presence of the aforementioned submolar bands proves that the wild isolate HearSP1 is composed of a heterogeneous mixture of genotypes.

[00063] In order to more clearly differentiate the HearSP1B and HearLB6 genotypes, and also differentiate them from other HearSNPV isolates whose genomes have been completely sequenced (HearG4, HearC1, HearNNg1 and HearAus), the numerical values of the sizes of restriction fragments generated after digestion of said isolates and genotypes with the EcoRI endonuclease. Table 4: Estimated sizes (kb) of DNA fragments generated after digestion of genomic DNA from different isolates and genotypes with the EcoRI endonuclease and estimated total genome size. The DNA fragments are named in alphabetical order, with fragment A being the largest.

[00064] When comparing the data reflected in Table 4, it is observed that there are differences in the number and size of fragments, which indicates that the HearSP1B and HearLB6 genotypes are different from those already known and, therefore, new. For example, the EcoRI-B fragment of the HearLB6 genotype (10.50 kb) is larger than that of the HearSP1B genotype (10.18 kb). The EcoRI-F fragment (6.45 kb) of the HearLB6 genotype is not found in the HearSP1B profiles or in those of the sequenced genotypes. However, the EcoRI-U (1.74) fragment of the HearSP1B genotype does not appear in the HearLB6 genotype profile, but appears in the sequenced genotypes. This can be seen in figure 7.

[00065] The HearSP1B and HearLB6 genotypes also differ from each other and from other HearSNPV isolates and genotypes described in the literature, due to the specific nucleotide sequences that each one presents in specific regions of the genome. One can use, for example, the region of the genome known as homologous region 1 (hr1), taking as reference the corresponding sequence of the genomes of two isolates from China, HearG4 (Chen et al., 2001; with the accession number in GenBank AF271059) and HearC1 (Zhang et al., 2005; GenBank AF303045), from a Kenyan isolate, HearNNg1 (Ogembo et al., 2007; GenBank AP010907), and from an Australian isolate, HearAus (GenBank JN584482). It is also possible to use the region where hr5 is located (homologous region 5).

[00066] Therefore, a rapid and accurate differentiation of each of these two genotypes can be obtained using the technique of PCR amplification and subsequent digestion with the NdeI restriction endonuclease of the amplified fragments using specific primers that are amplified, for example, in one of the following alternative regions: i) The homologous region 1, hr1. In this region of the HearSNPV genome, it was proven that the specific primers F-hr1 (5'- CGAAATCGACAACACCATGCA-3') and R-hr1 (S'-ACTTTTGTACGCCA GAGACGA-S') amplify a fragment of 2.177 and 2.1 17 nucleotides for the HearSP1B and HearLB6 genotypes, respectively. Digestion of these amplified fragments with the restriction endonuclease NdeI produces unique profiles for each genotype: for HearSP1B, six fragments of 857, 508, 381, 306, 78 and 47 nucleotides or five fragments of 1,210, 475, 307, 78 and 47 nucleotides in the case of HearLB8. In turn, these profiles are different from those obtained for the sequenced genotypes (Table 6, Fig. 8B). Specifically and as an example, in Figure 8B it is observed how the bands of 508 and 381 nucleotides do not coincide with any other band on the gel, the bands of the HearSP1 isolate being slightly higher, indicating a larger size than those of HearSP1B. ii) In homologous region 5, hr5. In this region of the HearSNPV genome, it was proven that the specific primers F-hr5 (5!- CTAGCCGGTCCGTTTCTGTT-3:) and R-hr5 (5'-GCCCCACCCAAAAC ATAACG-3') amplify a fragment of 2,326 and 2,330 nucleotides for the genotypes HearSP1B and HearLB8, respectively. In this case, the digestion of these amplified fragments with the NdeI restriction endonuclease also produces unique profiles for each genotype: five fragments of 1,120, 917, 211 and 78 nucleotides for HearSP1B or three fragments of 1,120, 998 and 212 nucleotides for HearLB8 . In turn, these profiles are different from those obtained for the sequenced genotypes (Table 6, Fig. 8B).

[00067] Figure 8, in panel A, shows the photograph obtained after subjecting the fragments amplified by PCR to electrophoresis using the specific primers for the hr1 and hr5 regions. Panel B shows the photograph after the fragments obtained by digestion with the Ndel endonuclease, the fragments amplified by PCR for hr1 and hr5, from the previous item, have been electrophoresed. In the aforementioned photograph, it can be seen that the fragments obtained for each genotype are different and distinguishable from each other. It can also be observed that the fragments obtained for each genotype are different and distinguishable from each other. For example, in the case of hr1, the 1.20 bp fragment is characteristic of the HearLB6 genotype, while the HearSP1B genotype presents a characteristic fragment of 857 bp. In the case of hr5, the HearSP1B genotype has a fragment of 917 bp, while the HearLB6 genotype has a fragment of 998 bp.

[00068] In this way, with a single PCR reaction followed by digestion with the Ndel endonuclease, the different genotypes can be differentiated from each other and from any other genotype of the virus described in the literature (see Table 6 further in detail, in Example 2).

[00069] In the case of natural isolates or artificial mixtures that may contain mixtures of genotypes, the proportion of the two genotypes HearSP1B and HearLB6 in the mixture can be determined by a quantitative PCR, using specific primers for each of the genotypes, as mentioned later in Example materials and methods section.

[00070] On the other hand, sequencing the aforementioned fragments generated by PCR would also make it possible to prove the identity of the different genotypes in the mixture. Therefore, the sequences represented by SEQ ID NO:5 and SEQ ID NO:6 correspond to the sequences of the fragments amplified using the primers F-hr1 and R-hr1 that are amplified in hr1 of the HearSP1B and HearLB6 genotypes, respectively, while the SEQ ID NO:7 and SEQ ID NO:8 correspond to the sequences of the fragments amplified for hr5, for the same genotypes.

[00071] As commented later, the complete genome sequence of each of these two genotypes HearSP1B and HearLB6 was obtained, sequences that are shown, respectively, as SEQ ID NO: 13 and SEQ ID NO: 14 and which can be used to differentiate some genotypes from others. Specifically, due to their variability, the complete sequences of the variability zones corresponding to homologous region 1 (hr1) are shown individually (SEQ ID NO: 9 and SEQ ID NO: 10, corresponding, respectively, to the genotypes HearSP1B and HearLB6) and the homologous region 5 (hr5) (SEQ ID NO: 1 1 and SEQ ID NO: 12, corresponding, respectively, to the HearSP1B and HearLB8 genotypes). Both in the case of homologous region 1 (hr1) and in the case of homologous region 5 (hr5), these sequences are indicated in the sense in which they appear in the complete genome sequence. As they are intergenic zones, located between two open reading phases, there is no coding direction as occurs in the reading phases. The latter can be transcribed in sense (coding sequence) or antisense (sequence complementary to the coding sequence).

[00072] In the present invention, the complete genome sequences of each of these two genotypes HearSP1B (SEQ ID NO: 13) and HearLB6 (SEQ ID NO: 14) were obtained, a characteristic and defining trait of each of them. Therefore, said genotypes are described herein in such a way that the person skilled in the art can reproduce the invention. Furthermore, the complete sequences of each of the genomes are complemented with the data provided in the present application that the Helicoverpa armigera nudeopolyhedrovirus is a simple-type nudeopolyhedrovirus (SNPV), that is, each complete viral particle or virion contains a single nucleocapsid. , and therefore a single copy of the nudeopolyhedrovirus genome. Additional data are also provided to identify each of the genotypes according to the profile obtained after digestion of their genome with different restriction endonucleases, as well as the size and sequence of the fragments obtained after PCR amplification of the variability zones of the homologous regions 1 and 5 (hr1 and hr5), using the primers SEQ ID NO: 1 and SEQ ID NO:2 or SEQ ID NO:3 and SEQ ID NO:4, respectively, as per the pattern of the bands obtained after digestion of these fragments of PCR with the NdeI enzyme. The Examples also show data on the insecticidal activity of each genotype and of mixtures of occlusion bodies that contain co-occluded virions of different genotypes within the same occlusion body, as well as how to obtain the different mixtures. The differences in pathogenicity, virulence and productivity are significant between genotypes, and between mixtures of the genotypes of the invention, with the mixture of the two genotypes HearSP1B and HearLB6, in a 1:1 ratio, being more pathogenic than the rest of the genotypes and mixtures and of the same virulence as the faster genotypes.

[00073] The large number of possible genotype combinations and the differences between them in terms of their relative potency mean that under no circumstances would it be possible to predict in advance that the combination of the present invention could give better results than the rest.

[00074] Of all the genotype mixtures constructed, the HearSP1B: LB6 mixture in a 1: 1 ratio was the one that presented the most desirable effects with synergistic activity from a bioinsecticide point of view. However, this synergistic activity is not observed in many other mixtures of genotypes (one in which an additive or even antagonistic effect may simply occur) and there is no way to predict in which mixtures this activity will be produced. This is a result that is not obvious or predictable, specifically taking into account that the genotypes come from different geographical areas (Badajoz and Lebrija) and that both genotypes (HearSP1B and HearLB6) had not yet been obtained in pure form, until now, from complex wild mixtures such as field isolates.

[00075] Furthermore, data on the deposit of the two genotypes according to the Budapest Treaty are provided, enabling reference to said genotypes by their deposit number: CNC 1-4806 (HearSP1B) and CNCM 1-4807 (HearLB8).

[00076] With regard to the possible applications of the nucleopolyhedroviruses of the present invention, it has been proven that each of the new genotypes HearSP1B and HearLB8 has a specific insecticidal activity towards H. armigera larvae, which can be considered comparable to that of chemical insecticides, such as Dursbam and Spintor, or the biological type based on Bacillus thuringiensis, such as Turex, commonly used for H. armigera. But it was additionally found that the mixture of the two HearSP1B: LB6 genotypes in a concrete proportion (1 : 1), as occlusion bodies that include ODVs that became co-occluded so that the same occlusion body can contain different HearSNPV genotypes, has better insecticidal activity than that of each of the individual genotypes or any of the currently known wild HearSNPV isolates, as it presents greater pathogenicity than the rest of the genotypes and mixtures, and the same mean mortality time (MMR) as the genotypes faster. This implies a significant advantage, since the biggest drawbacks when developing baculoviruses as an active substance in bioinsecticides are their pathogenicity and speed of action. On the other hand, this virus can be produced in a short time since by inoculating 100 larvae recently moved to the fifth stage (L5) and incubating them with a diet at 30°C, around 5 x 1011 occlusion bodies are obtained in a period of time of 5 or 6 days.

[00077] Furthermore, tests on tomato plants, both in the laboratory, in a greenhouse or outdoors, revealed that concentrations of the order of 1013 occlusion bodies/hectare of the nucleopolyhedrovirus of the present invention are capable of effectively controlling pests produced by the larvae of this caterpillar, with the same efficiency as the insecticides commonly used to combat this pest in tomato cultivation (Spintor, based on two spinosyn toxins (spinosad); Turex, based on Bacillus thuringiensis var. Aizawai; and Dursban , based on the chemical insecticide chlorpyrifos). These applications achieve a significant reduction in the number of damaged fruits harvested, both green and ripe, in relation to the control treatment, and do not present differences with the rest of commonly used insecticides.

[00078] The fact that the host spectrum of baculoviruses is restricted to invertebrates, and the great specificity of HearSNPV in particular (which appears to only affect the larvae of a few species of moths of the genus Helicoverpa, all of which are very phylogenetically related) makes that the product represents a clean and safe technology, as it does not leave toxic residues in the soil or on crops and is not toxic to humans and other animals, including natural enemies such as parasitoids or predators.

[00079] Surprisingly, the results presented in the present patent application demonstrate that the co-occluded mixture of these two cloned genotypes (HearSP1B and HearLB6) in a 1:1 ratio is among the most active nucleopolyhedroviruses among those that were developed as bioinsecticides to date.

[00080] With regard to HearNPV isolates native to the Iberian Peninsula, HearSP1B and HearLB6 are better adapted to the environmental conditions prevailing in southern Europe than isolates from other geographic origins. This fact is especially significant taking into account the negative effect exerted by UV radiation on the deposits of a bioinsecticide application, which requires that the NPV remain active until the moment of ingestion by H. armigera where they can exert their insecticidal effect. . Furthermore, it was observed that there is a certain predisposition for natural isolates from a given geographical area to be more pathogenic and virulent for larvae native to the same area.

[00081] An additional advantage of these nucleopolyhedroviruses is that they can be mass produced. Their occlusion bodies, in which the insecticidal activity resides, can be mass-produced in vivo by inoculating H. armigera larvae with occlusion bodies previously obtained after oral infection of larvae with 1 : 1 mixtures of pure HearSP1B and HearLB6. The occlusion bodies can contain virions of any of the HearSP1B or HearLB6 genotypes, if one wants to obtain occlusion bodies with virions of a single genotype, or mixtures thereof, if one wants to obtain virions of different genotypes co-occluded within the same genotype. occlusion body. The concrete process used for the production of the new occlusion bodies can be any of those known to those skilled in the art or that which was used in the Examples of the present application described below. In said Examples, an example of the composition of the artificial diet is also described, which is compatible with the occlusion body production process of the present invention. Therefore, the process of producing occlusion bodies may comprise the steps of: i) feeding fifth-stage larvae of H. armigera with an artificial diet comprising occlusion bodies of the H. armigera nucleopolyhedrovirus that contain virions of any of the genotypes HearSP1B (CNC I-4806) and HearLB6 (CNCM I-4807) or mixtures thereof; ii) keep individual larvae at 30°C until death; iii) purify the occlusion bodies generated in the larvae by grinding the larvae corpses in water, filtering the resulting suspension, precipitating the occlusion bodies, washing the precipitate and re-precipitating them; iv) resuspend the final precipitate in water at neutral pH; v) optionally, store the suspension obtained in one of the following conditions: a) at room temperature b) under refrigeration c) freeze-dry the suspension and store it at room temperature.

[00082] As used in the present application, refrigerated conditions are understood to be those in which the product is maintained between 0°C and 8°C, and freezing conditions would correspond to maintenance below 0°C. For the purposes of the present invention, it is preferable that refrigeration conditions are maintained between 0°C and 6°C and freezing conditions between -20°C and -80°C.

[00083] It is also possible to produce said occlusion bodies by feeding the fifth stage larvae with an aqueous solution, which contains 10% sucrose together with the selected co-occluded mixture at a lethal concentration of 95% (LC95). This method was described by Hughes and Wood in 1986, and consists of administering in the form of drops a suspension containing the occlusion bodies suspended at the desired concentration, as well as a dye that indicates whether the larvae ingested the drop, such as food coloring. blue color Fluorella blue (Hilton-Davis, Cincinnati, Ohio). This method is less complicated than the previous one, since in the artificial diet the diet must be well impregnated with the viral suspension, and the preparation of the virus-impregnated portions of the diet takes longer.

[00084] The artificial diet through which the larvae are fed or infected is administered in solid form, such as tablets which, in addition to the occlusion bodies of the nucleopolyhedrovirus of Helicoverpa armigera (when the objective is to infect said larvae), contain 7.2 % wheat germ, 2.5% soy protein, 1.4% brewer's yeast, 1.9% agar, 2.9% sugar, 1% mixed salts, 0.1% cholesterol , 0.4% ascorbic acid, 0.2% sorbic acid, 0.02% streptomycin, 0.04% chlortetracycline hydrochloride, 0.1% nipagin, 0.1% nipasol, 0.2 % benzoic acid, 0.1% choline chloride, 0.01% vitamins, 15% agar and 80% distilled water. Larvae can be infected by administering the occlusion bodies either as an aqueous suspension in the form of drops or as an artificial diet in solid form. Typically a volume of several liters of diet is prepared by mixing the previously mentioned ingredients, which are then autoclaved to sterilize and allow the agar to dissolve, and before it cools completely (at a temperature of 50°C) antibiotics are included, and After being well mixed, aliquots are obtained in square Petri dishes measuring 120x120 mm. Then the diet in the Petri dishes is cut into 5x5 mm cubes.

[00085] In Example 4 of the present application, the mass production method designed for the host-pathogen system described in this application, H. armigera-HearSNPV, is shown. There are many factors that can influence the final production of occlusion bodies, such as the larval stage, the concentration of the initial inoculum, or even the temperature. These parameters can be modified so that different final productions are obtained. In the tests carried out in our laboratory, it was proven that some specific conditions are preferred, considering that they obtain the best results and, therefore, greater final production of occlusion bodies. Below are shown the different parameters that can be modified indicating their preference: i) H. armigera larvae from the third (L3), fourth (L4) and fifth (L5) stages, although larvae from the fifth stage are preferred; ii) different concentrations of occlusion bodies provided in the artificial diet, as demonstrated by trials with different concentrations in the range of 5.5 x 106 to 1.5 x 108 occlusion bodies/mi, although L5 larvae are preferred at a concentration of 1.5 x 108 occlusion bodies/ml; iii) individual larvae in 12-well plates, to avoid cannibalism, iv) larvae incubated at 30°C until death.

[00086] The study of different larval ages and different viral doses demonstrated that the ideal production of occlusion bodies is obtained when larvae recently moved to the fifth stage are treated, inoculating the larvae with a concentration of about the concentration that promotes the death of 95% of the larvae of the aforementioned stage (CL95), which in this case is a concentration of 1.5 x 108 occlusion bodies/ml, and maintaining them individually, due to the high degree of cannibalism presented by the larvae of this species, with a diet at 30°C until death. These conditions allow approximately 5 x 109 occlusion bodies/larva to be obtained in a period of 5 to 6 days. Therefore, by infecting 100 larvae, the order of 5 x 1011 occlusion bodies would be obtained.

[00087] The occlusion bodies produced in H. armigera larvae can be purified, formulated in solid or liquid form and sprayed as aqueous suspensions, which very effectively protect tomato crops, both in greenhouses and outdoors, from pests caused by H. armigera larvae.

[00088] The nucleopolyhedrovirus may also be applied by other methods, such as ground or aerial spraying, or application in suspension, in the form of powder, or irrigation. Furthermore, as mentioned previously, occlusion bodies can be mixed with excipients, and used with appropriate vehicles in the agricultural sector, especially those that facilitate preparation in the form suitable for the desired application method. The same composition may also contain, for example, a fertilizer, a fertilizer or a pesticide. Furthermore, it may also contain an agent that enhances the pathogenic effect of the nucleopolyhedrovirus on H. armigera.

[00089] It is convenient to include agricultural humectants in products that include these occlusion bodies, as is the case with the commercial product Agral® (Syngenta), which is used in the Examples of the present application. The humectant compound present in the product is ethoxylated isotridecyl alcohol, which increases the biological action of insecticides, herbicides, fungicides and pesticides in general, since better coverage and penetration of the product in the crop to be treated is obtained. The web page where its characteristics are described (http://www. syngenta.com/country/es/sp/produtos/proteccion__cultivos/mojantes/P aginas/agr al.aspx) indicates that it is a surfactant humectant and dispersant non-ionic, especially suitable for mixing with any class of insecticides, fungicides and agrochemicals.

[00090] Another case of special interest is one in which the composition contains another pesticide, in order to increase the spectrum of action for other possible pests that affect the same crops, without limiting it exclusively to H. armigera. The pesticide can be, for example, another biological insecticide, such as those based on Bacillus thuringiensis (Bt) previously mentioned, such as the Turex® product used later in Example 6 of the present application, which is used for crops attacked by H. armigera. The mixture with Bt-based insecticides is interesting, since cases of synergistic interactions of insecticidal activity between these products and baculoviruses for noctuids have been described (Granados et al., 2001).

[00091] In the tests described in the Examples of the present application described below, it is shown that each of these two genotypes has a characteristic insecticidal activity for H. armigera larvae, determined by pathogenicity, the mean mortality time (MMR) and the capacity to produce occlusion bodies in H. armigera larvae.

[00092] In previous work carried out in the research group of the present inventors, it was observed that mixtures of occlusion bodies or mixtures of virions co-occluded in the same occlusion body on certain occasions present better insecticidal qualities than individual genotypes (Bernal et al. al., 2013b; López-Ferber et al., 2003; Simóm et al., 2005) or even better than the wild isolate (Munoz et al., 1998). On the other hand, mixtures of virions from different genotypes co-occluded in the same occlusion body may have a different activity than mixtures of occlusion bodies where the virions of each occlusion body belong to a single genotype (López-Ferber et al. , 2003), given that there may be synergism or antagonism between some genotypes. Therefore, in the present invention, a study was carried out on the insecticidal activity of different mixtures of virions co-occluded in the same occlusion body, to check whether said mixtures had different insecticidal characteristics than the occlusion bodies of a single genotype, if the genotypes presented antagonistic or synergistic activity, and determine the variations that could occur between different combinations and different types of mixtures.

[00093] The mixture of the two HearSP1B:LB6 genotypes, co-occluded in the same occlusion body in a 1:1 ratio (i.e., each of these occlusion bodies contains the two genotypes in that proportion), contrary to expectations, proved to have a insecticidal activity in terms of pathogenicity, greater than that of individual genotypes. However, on the other hand, its virulence (TMM) is similar to that of the fastest larvae-killing genotypes. For this reason, these genotypes were selected for application in their co-occluded form in the same occlusion body, compared to the remaining individual genotypes isolated from HearSNPV.

[00094] In Example 3 of the present invention, tests of the insecticidal activity of different genotypes and mixtures are described, which surprisingly demonstrate that the new isolated nucleopolyhedroviruses, and in particular their combination, are among the biological insecticides with the greatest activity against insect pests. Therefore, its use as an insecticide is proposed, particularly for the control of insects of the genera on which it is known to act, Helicoverpa or Heliothis, with special preference for use for the control of H. armigera.

[00095] Among the combinations chosen, there is no type of experience and/or advance prediction that suggests that the combination of both genotypes will present results in terms of relative potency that are notably superior to the rest of the isolates. The synergistic activity that occurs in the case of HearSP1B:LB6 is not observed in many other mixtures of genotypes, including in cases in which a clearly antagonistic effect is produced. This activity is surprising since the synergistic activity of two different genotypes that are naturally found in distant geographical areas was not expected.

[00096] The plants to which this formulation can be applied can be any plants that are damaged by this species of lepidoptera and where it is desired to control the damage caused by this insect, whether they grow or are cultivated both in a greenhouse and outdoors, highlighting tomato cultivation, especially in the Iberian Peninsula, where its effectiveness has been proven, in growing tomatoes both in greenhouses and outdoors.

[00097] Taking all these data into account, it can be said that: i) each of the new isolated genotypes, HearSP1B and HearLB6 is new, as each of them is different from the other genotypes and different from the H. armigera nudeopolyhedroviruses so far known, from which they can be distinguished both by differences in the sequences of their genomes (particularly, in the areas of homologous regions 1 and 5, hr1 and hr5) and by differences in the profiles generated by digestion with restriction enzymes of said genomes, in particular EcoRl and/or BglII. ii) the two new isolated genotypes share, among others, the technical characteristics of: a) their insecticidal activity and productivity, individually, is greater than or equal to that of any of the previously known natural isolates; b) their mixtures, in particular the co-occluded mixture of the two, HearSP1B: LB6, in a 1 : 1 ratio, presents a pathogenicity and virulence for H. armigera larvae better or equal to those of wild isolates of this virus and comparable to those of insecticides (but without the drawbacks) that are commonly used against this pest, such as insecticides sold under the names Dursban®, Spintor® or the Bt-based biological insecticide, Turex®; c) given that the two genotypes were isolated in relatively close geographical areas, it is expected that they will be especially active for possible variants of H. armigera from that geographical area, the south of the Iberian Peninsula or Andalusia and Extremadura specifically.

Examples

[00098] To carry out the tests described in this application, the following materials and methods were used:

- Insects

[00099] There are no officially recognized strains or strains of H. armigera. The H. armigera larvae used for the amplification of the different viruses, for carrying out bioassays in the laboratory and for the greenhouse tests, were obtained from a laboratory culture at the Public University of Navarra (UPNA) established from pupae received from the Center for Ecology and Hydrology (NERC-CEH) in Oxford (United Kingdom). The population was maintained in the UPNA insectary at 25±1°C, with a relative humidity of 70±5% and a photoperiod of 16:8 (light:dark). Larvae were fed an artificial diet previously described by Greene et al. (1976) and adults ad libitum with honey diluted to 30% (weight/volume).

[000100] The H. armigera larvae used to carry out the outdoor field trials came from a natural infestation of tomato cultivation in Guadajira (Badajoz), - Isolation and amplification of occlusion bodies

[000101] Occlusion bodies (OBs) or occlusion bodies were extracted from dead larvae by crushing the corpses in sterile double-distilled water with sodium dodecyl sulfate (SDS) at 0.1% (weight/volume) and filtering the resulting suspension through museline. The occlusion bodies were pelleted by centrifugation at 6,000 x g for 10 minutes. Subsequently, 2 washes were carried out with water and the occlusion bodies were sedimented under the same conditions. Finally, the purified occlusion bodies were resuspended in sterile double-distilled water and their concentration was determined by recounting the samples in triplicate using an enhanced Neubauer hemocytometer (Hawksley, Laucing, United Kingdom) by phase contrast microscopy at 400x.

[000102] The occlusion bodies of the different isolates were multiplied by a single pass in fourth-stage larvae of H. armigera. Groups of 24 larvae from the laboratory colony were individualized and kept without food for approximately 12 hours. After this time, they were infected by the drop method (Hughes and Wood, 1981) with a concentration of 106 occlusion bodies/ml, 10% sucrose (weight/volume) and 0.001% (weight/volume) of the food coloring Fluorella Blue. (Hilton-Davis, Cincinnati, Ohio). The food coloring makes it possible to distinguish the larvae that ingested the occlusion body suspension from those that did not. The larvae with blue intestines, and therefore those that drank the suspension, were reared individually on an artificial diet until death. The advantage of this droplet method is that the dose or viral concentration is ingested in a short period of time, which is especially important when calculating some parameters such as the mean time to mortality (MMR).

[000103] The purified occlusion bodies were stored at - 20°C until their molecular and biological characterization. - Purification of genotypes by plaque assay

[000104] To purify the different genotypes present in the HearSP isolate, a plaque assay was carried out (Munoz et al., 2001). To this end, 25 fourth-stage larvae of H. armigera were infected orally at the concentration that produced 90% mortality (LC90) of 106 occlusion bodies/ml. 48 hours after infection, a small incision was made in the last pair of pseudopods of the larvae in order to extract the hemolymph. At this time the hemolymph is full of BVs (budded virions) that contain a single nucleocapsid and, therefore, a single genotype. Hemolymph was filtered through a 0.45 filter.

[000105] To eliminate possible contaminants such as bacteria, it was subsequently serially diluted with a dilution factor of 5 with EX-CELL 420 medium (Sigma). Subsequently, 2 x 106 HzAM1 cells were incubated in six-well plates (35 mm in diameter) at 27°C for three hours to allow cell deposition. After this time, the medium was replaced by 100 μl of hemolymph dilutions. After one hour, the viral inoculum contained in the hemolymph dilutions was replaced with new EX-CELL 420 medium with 1% antibiotics (penicillin-streptomycin) (Lonza) and 2% agarose to prevent excessive spread of the infection. After 5 days, the cells were stained neutral red in order to differentiate healthy cells from infected ones, as healthy cells acquire a reddish color, while infected cells give rise to a non-colored area called a plaque or bald spot, which corresponds to a set of dead cells due to infection by a single BV, and therefore, by a single genotype. These single infection zones (plaques) were extracted with a sterile Pasteur pipette and were individually diluted in 50 μl of EX-CELL 420 medium. Each suspension was subsequently injected into H. armigera fourth-stage larvae for in vivo multiplication of the same to thus obtaining large quantities of occlusion bodies, which were analyzed at the molecular DNA level in order to determine the number of different genotypes. - Purification of genotypes by cutoff point dilution

[000106] To purify the genotypes obtained from larvae killed during an epizootic in the laboratory in the second generation of a population of H. armigera from a cotton crop in Lebrija, 25 fourth-stage larvae of H. armigera were infected orally with 106 occlusion bodies/ml. 48 hours after infection, hemolymph was extracted, filtered through a 0.45 μm filter, and subsequently serially diluted with a dilution factor of 5 with EX-CELL 420 medium (Sigma) with 1% antibiotics (penicillin - streptomycin) (Lonza). To a volume of 100 μl of each dilution was mixed 900 μl of a HzAMI cell suspension at a concentration of 2 x 105 cells/ml. 100 μl of the virus-cell suspension was added to the first 10 wells of a row of a 96-well plate, the last two being left with a suspension that contained only cells (no virus) as a negative control. A total of four repetitions were performed. These plates were incubated at 28°C for 7 days. After this time, all wells were observed under a microscope to determine the presence of infected cells. The nuclei of infected cells were full of occlusion bodies. In dilutions in which less than 10% of infected wells were found, indicating that the infection of the well was caused by a single budded virion (and, therefore, a single genotype), the supernatant from said wells was extracted with a sterile Pasteur pipette. This supernatant contains budded virions (BVs) that were injected into fourth-stage larvae of H. armigera for multiplication, so that sufficient occlusion bodies were obtained to be able to carry out their molecular characterization, and to be able to determine the purity of each genotype or the number of different genotypes. - Determination of the number of nucleocapsids per virion

[000107] To determine whether the virions derived from two occlusion bodies (ODVs) of the Spanish isolates of the H. armigera nucleopolyhedrovirus were of the single or multiple type, the ODVs found within the occlusion bodies were released by incubating a suspension of 109 occlusion bodies with an alkaline solution (1 volume of 0.1 M Na2CO3) for 30 minutes at 28°C. Polyhedrin and other residues were pelleted by low speed centrifugation (2,500 x g) for 5 minutes. To separate the different bands (if multiple) or the single band (single type), the supernatant containing the virions was centrifuged at density equilibrium (90,000 x g) for 1 hour in a continuous sucrose gradient at 30-60% (wt. /volume). After that, a visual inspection was carried out and they were photographed, so that it was possible to determine their nature. - DNA extraction and analysis with restriction endonucleases

[000108] To extract DNA, 100 μl of an occlusion body suspension at a concentration of 109 occlusion bodies/ml was incubated with 100 μl of 0.5 M sodium carbonate (Na2CO3), 50 μl of 10% SDS (weight/volume) and 250 μl of H2O at 60°C for 10 minutes, to dissolve the polyhedrin and release the virions. Undissolved occlusion bodies and other residues were removed by centrifugation at low speed (3,800 x g) for 5 minutes. The supernatant containing the virions was incubated with 500 μg of proteinase K at 50°C for 1 hour. Viral DNA was extracted twice with one volume of saturated phenol, followed by one with chloroform, and was precipitated with 1/10 volume of 3 M sodium acetate (pH 5.2) and 2.5 volumes of cold absolute ethanol at 12,000 x g for 10 minutes. Subsequently, it was washed with cold 70% ethanol and centrifuged for 5 minutes. Finally, the DNA was resuspended in 100 μl of 0.1x TE buffer (Tris-EDTA, pH 8) at 80°C for 10 minutes. The concentration was estimated by reading the optical absorption at 260 nm on a spectrophotometer (Biophotometer Plus, Eppendorf, Freiberg, Germany).

[000109] For restriction endonuclease analysis, 2 μg of viral DNA, or fragments amplified by PCR, were incubated with 10 U of one of the following enzymes: EcoRI and BglII (Takara Bio Inc., Japan) for 4 to 12 hours at 37°C. In the case of PGR fragments, the NdeI enzyme, from the same supplier, was used. Reactions were stopped by adding 4 μl of loading buffer (0.25% (w/v) bromophenol blue, 40% (w/v) sucrose). Electrophoresis was performed using 1% (w/v) horizontal agarose gels in TAE buffer (0.04 M Tris-acetate, 0.001 M EDTA, pH 8.0) at 20 V for 12 to 16 hours. The DNA fragments were colored with ethidium bromide and visualized under an ultraviolet transilluminator (Cheni-Doc, BioRad, California, USA). - Complete genome sequencing

[000110] For the complete sequencing of the two genotypes HearSP1B and HearLB6, DNA purification was carried out in cesium chloride (CICs) (King and Possee, 1997). Initially, ODVs were released and purified as indicated in the item determining the number of nucleocapsids per virion. To this end, 500 μl of occlusion bodies (109 occlusion bodies/ml) were mixed with 500 μl of 0.1 M sodium carbonate (Na2CO3), and after centrifugation in a continuous sucrose gradient, the single band was obtained. for each of the three genotypes. Using a 1 ml needle and syringe, the centrifuge tube where the band was located was punctured and aspirated, so that the entire band was collected, that is, the simple ODVs. These virions were diluted 1:3 in TE buffer (Tris-EDTA, pH 8), and to concentrate they were sedimented at 24,000 rpm for 1 hour, and resuspended in 400 μl of SURFACTANT. For DNA extraction, these 400 μl of purified virions were mixed with 100 μl of a suspension containing 20% (w/v) sarkosyl (sodium lauroyl sarcosine or N-laurylsarcosine sodium salt, Sigma) and incubated at 60 °C for 30 minutes. This allowed the lysis of the virions and the rupture of the nucleocapsids, releasing the DNA into the medium. Immediately, this lysate was transferred to 5 ml of a suspension of cesium chloride (50% in TE (w/w)) which in turn contained 12.5 μl of ethidium bromide (10 mg/ml), which allowed staining of the DNA and, therefore, its visualization, and was subsequently centrifuged at 35,000 rpm for at least 18 hours at 20°C. After centrifugation, the DNA was visible as two orange bands (due to ethidium bromide). The two bands corresponded to supercoiled DNA (the bottom band) and circular open DNA (the top band). Using a 1 ml needle and syringe, the tube was punctured and both bands were extracted. Once extracted, the ethidium bromide was removed with several passes of butanol. To this end, the same volume of butanol was added, mixed and centrifuged, and the upper phase containing butanol and ethidium bromide was eliminated. This procedure was repeated several times until the solution became clear. Finally, the sample was dialyzed in a precipitate vessel containing 500 ml of TE buffer under stirring at 4°C, performing 2-3 TE changes every 8 hours. After dialysis, the DNA was transferred to a tube, quantified on a spectrophotometer, and stored at 4°C until use. A restriction analysis was also performed with the endonucleases EcoRl and BglII to confirm the identity and quality of the DNA.

[000111] The DNA sequencing of the two genotypes was carried out by the company Lifesequencing S.L. (Paterna, Valencia), using PacBio technology. Between 5 and 10 g of DNA purified by CICs were used. Basically, a genomic library was made in a sequencing vector with the DNA of each of the genotypes, with 0kb insertions. 24,627 and 3,731 reads were made for the HearSP1B and HearLB6 genomes, respectively. Finally, all information was gathered, requiring the use of the HGAP v2.0.2 program. The complete sequences obtained for each of the genotypes were compared with those already existing from other HearSNPV isolates (HearSNPV-G4, HearSNPV-C1, HearSNPV-NNg1 and HearSNPV-Aus) and with each other, using the Clone Manager program (Scientific & Educational Software, 1994-2007). - Construction of genotype mixtures

[000112] In order to find the mixture of genotypes that presented the best insecticidal properties for the control of H. armigera, mixtures were constructed with different genotypes.

[000113] To this end, five genotypes were selected based on their insecticidal characteristics in order to optimize biological activity and thus obtain a mixture with greater pathogenicity, virulence and/or viral productivity. On the one hand, the only two genotypes obtained from the HearSP1 isolate, HearSP1A and HearSP1B, were selected, as HearSP1 is the isolate with the best insecticidal characteristics against H. armigera larvae in Spain (Arrizubieta et al., 2014). On the other hand, the following three genotypes were selected from infected larvae collected in Lebrija: HearLB1, because it is one of the most virulent and one of the most productive in terms of the quantity of occlusion bodies produced in infected insects: HearLB3 because it is one of the most fast; and HearLB6 for being the most virulent. In total, eight genotype mixtures were made.

[000114] Among those that exclusively include the HearSP1 genotypes we find HearSP1 A: HearSP1B in the ratio 1 : 1, which throughout this report will be called HearSP1A:SP1B (1 : 1) and HearSP1A:HearSP1B in the ratio 1 : 2, and also called HearSP1 A:SP1B (1 :2). On the other hand, four mixtures were also made that exclusively contained the Lebrija genotypes as HearLB1 :HearLB3 in the ratio 1 : 1, and also called HearLB1 :LB3, HearLB3:HearLB6 in the ratio 1 : 1, and also called HearLB3:LB6, HearLB1 :HearLB3:HearLB8 in the ratio 1 : 1 : 1 and also called HearLB1 :LB3:LB6 and finally a mixture was also constructed with the six Lebrija genotypes in the proportions found in the population, which was called HearLBmix. Finally, two mixtures were made that included a genotype from the HearSP1 isolate and another from Lebrija, thus the HearSP1B:HearLB1 mixture contained the HearSP1B and HearLB1 genotype in a 1:1 ratio, also called HearSP1B:LB1, and the HearSP1B:HearLB6 mixture that contains the genotype HearSP1B and HaerLB6 also in a 1 : 1 ratio, and also called HearSP1B:LB6.

[000115] On the other hand, it is known that in co-occluded mixtures, with the genotypes in a proportion within the same occlusion body, the proportion is maintained when entering the host (Bernal et al., 2013b; Clavijo et al., 2010). But when occlusion bodies are simply mixed with the same genotype, the proportion does not need to be maintained when entering the epithelial cells of the mesentery. Furthermore, in work recently carried out in our laboratory it was possible to prove that co-occluded mixtures are faster in killing the host than mixtures of occlusion bodies (Bernal et al., 2013b). Therefore, to construct the co-occluded mixtures, the concentrations of the different genotypes were initially homogenized, diluting them to the same concentration of 109 occlusion bodies/ml and subsequently mixing the same volume of each of them, and therefore, the proportions in all cases were 1 : 1, except in the case of the HearSP1 A:SP1B (1 : 2) mixture, in which twice the volume of HearSP1B was mixed as HearSP1 A. In these mixtures the occlusion bodies contain virions of the same genotype. Subsequently, to co-oclude the different genotypes within the same occlusion body (co-occluded mixtures), fourth stage H. armigera larvae were infected orally with different mixtures of occlusion bodies at a concentration of 106 bodies. of occlusion/ml (the occlusion body mixtures produced above were diluted by a factor of one thousand (103) before infecting the larvae). In this way, the mixture of occlusion bodies with virions (ODVs) of the same genotype enters the digestive tract, and after the release of the virions, a mixture of virions of different genotypes (coming from different occlusion bodies) is produced. After entering and replicating in the same cell, they are co-occluded in the same occlusion body, forming co-occluded mixtures where virions of different genotypes are co-occluded in the same occlusion body and, moreover, in the same proportion as were inoculated (Bernal et al., 2013b; López-Ferber et al., 2003) (Fig. 6).

[000116] In short, in total eight co-occluded mixtures were constructed: HearSP A:SP1B (1:1), HearSP1A:SP1B (1:2), HearLB1:LB3 (1:1), HearLB3:LB6 (1:1 ), HearLB1 :LB3:LB6 (1 : 1 : 1), HearLBmix (six genotypes in their natural ratio, HearLB1-6), HearSP1B:LB1 (1 : 1) and HearSP1B:LB6 (1 : 1). - Identification of genotypes in mixtures by PCR and restriction analysis of the PCR product

[000117] In order to determine the nature of the different pure genotypes, in addition to complete genome restriction analysis, the viral DNA obtained from them was subjected to PCR amplification using the primer pairs F-hr1/R-br1 and F-hr5-/R-hr5. For PCR, 20.5 μl of H2O, 2.5 μl of polymerase buffer (10x), 0.75 μl of magnesium chloride (50 mM MgCI2), 0.25 μl of dNTPs (phosphate nucleotides), 0, 25 μl of the respective primers (R-hr1/F-hr1 or F-hr5/R-hr5), 0.25 μl of Taq polymerase and 0.25 μl of extracted DNA. The reaction conditions were a period of denaturalization at 94°C for 2 minutes, followed by 35 cycles that include denaturalization at 94°C for 1 minute, alignment which is produced at 60°C for 1 minute and elongation at 72°C for 3 minutes, followed finally by 10 minutes at 72°C to complete elongation.

[000118] Subsequently, the fragments amplified by PCR for hr1 and hr5 were digested with the endonuclease NdeI in the manner described previously. - Bioassays on insects

[000119] The insecticidal activity of the HearSNPV genotypes purified from the HearSP1 isolate (HearSP1A and HearSP1B) and those from Lebrija (Sevilla) (HearLB1, HearLB2, HearLBS, HearLB4, HearLBS and HearLB6), as well as the co-occluded mixtures HearSP1A:SP1B (1 : 1), HearSP1A:SP1B (1 :2), HearLB1 :LB3, HearLBS: LB6, HearLB1 :LB3:LB6, HearLBmix, HearSP1B:LB1 and HearSP1B:LB8, was compared with that of the wild isolate HearSP1 , previously selected as the isolate from the Iberian Peninsula with the best insecticidal characteristics (Arrizubieta et al., 2014). Concentration-mortality curves (lethal concentration 50, LC50), mean mortality time (MMR) and viral productivity (number of occlusion bodies produced by a single larva, occlusion bodies/larva) were determined by per os assays. (orally), carried out using the drop feeding method, described previously.

[000120] To determine the LC50 of the different genotypes, mixtures of ests and the HearSP1 isolate, five viral concentrations were used: 5.7 x 105, 1.9 x 105, 6.3 x 104, 2.1 x 104 and 7 .0 x 103 occlusion bodies/ml for second stage larvae, which had previously been determined to kill between approximately 95% and 5% of experimental insects. Larvae that ingested the suspension within the next 10 minutes were transferred to individual wells of a 24-well culture plate that contained the artificial diet in cube form as previously described. Bioassays with 24 larvae per viral concentration and 24 larvae as negative controls were carried out on three occasions. Larvae were reared at 25°C and mortality data were collected every 24 hours until the insects died or pupated. Virus-induced mortality results were subjected to logit analysis using the POLO-PC program (Le Ora Software, 1987).

[000121] The mean mortality time (MMR) of individual genotypes, different mixtures of genotypes and the HearSP1 isolate was determined by a bioassay on second-stage H. armigera larvae. Larvae were inoculated by ingestion with the LC90 (concentration that kills approximately 90% of inoculated larvae) of each virus calculated in the pathogenicity assays described previously (2.0 x 105, 1.8 x 105, 9.9 x 104, 1 .5x105, 1.5x105, 2.5x105, 3.5x105, 1.5x105, 9.8x104, 1.0x105, 1.5x105, 1.2 x 105, 1.8 x 106, 9.3 x 104, 1.2 x 105, 5.8 x 104 and 5, 1 x 104 occlusion bodies/ml for the wild isolate HearSP1 and the pure genotypes HearSP1 A, HearSP1B , HearLB1, HearLB2, HearLB3, HearLB4, HearLB5, HearLB6, and for the co-occluded mixtures HearSP1 A:SP1B (1 : 1), HearSP1A:SP1B (1 :2), HearLB1 :LB3, HearLB3:LB8, HearLB1 :LB3 :LB6, HearLBmix, HearSP1B:LB1 and HearSP1B:LB6, respectively). A group of larvae treated with the same solution without occlusion bodies was included as a control. Larvae were maintained individually on a diet at 25°C and mortality was recorded every 8 hours until all larvae died or pupated. 24 larvae were infected per treatment and three independent replicates were performed. Mortality data over time were subjected to a Weibull survival analysis using the “Generalized Linear Interactive Modeling” (GLIM) program (Crawley, 1993). The distribution of mortality over time of the different isolates was analyzed graphically By microscopic observation of the dead larvae, it was possible to identify those that had died due to disease caused by nucleopolyhedroviruses, and these were included in the analysis.

[000122] The production of occlusion bodies of pure genotypes, mixtures of genotypes and the HearSP1 isolate was determined in second-stage larvae of H. armigera infected by the drop method and with concentrations of occlusion bodies that produce 90% of mortality (the same concentrations used in the study of mean mortality time). All larvae that died from nucleopolyhedrovirus disease were collected and stored at − 20°C until used for occlusion body recounts. To this end, each larva was homogenized in 100 μl of distilled water and the total yield of occlusion bodies per larva was estimated by recounting the samples in triplicate using an improved Neubauer hemocytometer. Data were normalized by a log transformation and analyzed by analysis of variance (ANOVA) using the SPSS 15.0 program. Example 1: Isolation of new genotypes of the H. armigera nucleopolyhedrovirus 1.1. From the HearSNPV-SP1 isolate

[000123] The isolate HearSNPV-SP1, or HearSP1 for short, was selected in previous studies as the isolate from the Iberian Peninsula with the best insecticidal characteristics against H. armigera (Figueiredo et al., 1999; Arrizubieta et al., 2014 ). Furthermore, the restriction profiles performed with different endonucleases in the aforementioned studies showed submolar bands, which indicates the presence of different genotypic variants within the wild isolate (Fig, 4, 7, 8).

[000124] To isolate the possible genotypes within the HearSP1 isolate, an in vitro plaque assay described in the material and methods section was performed. In this way, 145 clones were obtained, each consisting of a single genotype. Using molecular techniques based on the use of restriction endonucleases, two different genotypes were identified among the different clones isolated, which were named HearSNPV-SP A and HearSNPV-SP1B, or in short, HearSP1A and HearSP1B (Fig. 7A). The HearSP1A genotype was found in 69% of clones, whereas the HearSP1B genotype was found in 31% (Fig. 7A). 1.2. From insect corpses killed during an epizootic in the laboratory

[000125] During an epizootic that occurred in the second generation of a population of H. armigera established in the laboratory from larvae collected in cotton cultivation in Lebrija (Sevilla) in August 2009, 17 corpses were individually collected with typical signs of death due to disease caused by nucleopolyhedroviruses. The occlusion bodies from each individual cadaver were purified as mentioned in the materials and methods section described previously. Sometimes the number of occlusion bodies obtained from a single larva is not sufficient to carry out characterization and it is necessary to amplify these isolates by inoculating healthy larvae from a laboratory colony using the droplet method. Therefore, amplification of samples was carried out in larvae under laboratory conditions, as previously mentioned in the section on isolation and amplification of occlusion bodies. Among the 17 multiplied isolates, it was only possible to identify 6 different profiles, which were named HearSNPV-LB1, HearSNPV-LB2, HearSNPV-LB3, HearSNPV-LB4, HearSNPV-LB5 and HearSNPV-LB6 or, in short, HearLB1, HearLB2, HearLBS , HearLB4, HearLBS and HearLB6 (Fig. 7B and 7C). The 6 genotypes were found in different proportions, with the HearLBS genotype being the most abundant, since it was isolated in 6 different larvae (representing 35.3% of the total genotypes), followed by HearLB1 and HearLB2, which were isolated in 4 larvae (23.5%), and finally the genotypes HearLB4, HearLBS and HearLB6, which were isolated in one larval sole each (5.9%).

[000126] Subsequently, in order to determine the purity of the six identified isolates, an end point dilution (EPD) test was carried out as described in the material and methods section. After oral infection of H. armigera larvae with the different isolates, the hemolymph was extracted, which was serially diluted and used for cell infection. Subsequently, 20 wells were selected with the presence of occlusion bodies in the dilution that produced less than 10% viral infection (around 1/500 for all isolates). The BVs obtained were multiplied into larvae by intrahemocoelic injection and the viral DNA from the occlusion bodies obtained with the endonucleases BglII and EcoRI was analyzed as mentioned in the material and methods section. All clones/wells obtained from a single isolate presented the same restriction profile as that of the original isolate, from which the clones were obtained, thus deducing that each of the 8 isolates is composed of a single genotype. Example 2: Molecular characterization of new HearSNPV 2.1 genotypes. Determination of the number of nucleocapsids per virion

[000127] To determine whether the different genotypes were of the simple or multiple type, the ODVs were released and centrifuged in a continuous sucrose gradient. All genotypes showed a single band, which indicates that all virions contain a single nucleocapsid (Fig. 5A). If these were multiple-type isolates, several bands would be observed, and each of them would contain ODVs with a different number of nucleocapsids, since the weight of the virions varies depending on the number of nucleocapsids they contain (Fig. 5B). From this observation it was concluded that all HearNPV isolates were of the simple type with a single nucleocapsid per virion (ODV). 2.2. Constraint Profiles

[000128] Digestion of the viral DNA of the different genotypes with the EcoRI restriction endonuclease produces a characteristic and unique profile for each of them (Fig. 7A, 7B and 7C; Table 5), some of the restriction fragments generated by this enzyme may be used as markers to differentiate them. For example, the EcoRI-B fragment of the HearLB4 genotype (11.0 kb) is larger than that in the HearLB2, HearLB3 and HearLB6 genotypes (10.5 kb), the HearSP1A and HearSP1B genotypes (10, 18 kb) and the HearLB1 genotype ( 10.15 kb), whereas it is not found in the HearLB5 genotype. The genotypes HearLB1 (EcoRID), HearSP1A (EcoRI-D) and HearSP1B (EcoRI-E) present a single fragment common to the three genotypes (9.20 kb), while the genotypes HearLB2 (EcoRI-D), HearLB3 (EcoRI -D), HearLB4 (EcoRI-D), HearLB5 (EcoRI-C) and HearLB6 (EcoRI-D) said fragment is 9.38 kb. The EcoRI-E fragment (9.01 kb) of the HearLB1 genotype is only present in this genotype, as is the EcoRI-E fragment (8.70 kb) of the HearLB4 genotype, which is only found in the HearLB5 genotype (EcoRID), on the other hand. , the EcoRI-F fragment (7.16 kb) of the HearSP1A genotype is only located in the profile of the HearLB2 genotype (EcoRI-F), although its size is smaller (7.10 kb), while the EcoRI-M fragment of HearSP1A genotype (5.26 kb) is not present in HearLB2 or HearLBS genotypes. The HearLB5 genotype presents a single 3.10 kb fragment (EcoRI-S), whereas it does not present a 2.83 kb fragment, present in the rest of the genotypes. No submolar bands were observed in the restriction profiles of these genotypes after one pass in larvae and the profiles were maintained in all passes, which indicates the stability and purity of the genotypes.

[000129] The restriction profiles of these genotypes can also be differentiated by the use of other restriction endonucleases, such as BglII (Fig. 7A and 7C).

[000130] In the profiles obtained with both enzymes, the presence of submolar bands in the wild isolate HearSP1 is clearly observed, showing that the wild isolate is composed of a heterogeneous mixture of genotypes. Therefore, in the profile generated with the EcoRI enzyme, the HearSP1 isolate shows several submolar bands around 6.5-7 kb, which are not observed in the profile of the pure HearSP1B genotype. Likewise, in the profile obtained with the BglII endonuclease, the HearSP1 isolate shows a submolar band of 18.8 kb, which does not appear in the HearSP1B genotype profile. on the contrary, the absence of these bands in the pure genotypes demonstrates their purity, thus the HearSP1B genotype shows a band of 9.73 kb that was not observed in the profile of the HearSP1 isolate.

[000131] Table 5 shows the estimated sizes of the restriction fragments generated after digestion of the viral DNA of the different genotypes with the EcoRI enzyme. The difference in the number of fragments of the HearSP1A, HearSP1B, HearLB1, HearLB3, HearLB6, HearG4, HearC1, HearNNg1 and HearAus genotypes with the HearLB2, HearLB4 and HearLB5 genotypes is due to the fact that their genomes are completely sequenced, and therefore they are Small-sized fragments were detected that cannot be detected by band pattern analysis as they are not visible in the restriction profiles (marked with an asterisk [*] in Table 5). Table 5: Estimated fragment sizes of the genotypes HearSP1A, HearSP1B, HearLB1, HearLB2, HearLBS, HearLB4, HearLB5 and HearLB6, and of the isolates HearG4, HearC1, HearNNg1 and HearAus obtained by digestion with EcoRI, and estimated total genome size. * Small-sized fragments detected by sequencing that are not visible in restriction profiles. Continued Table 5: * Small-sized fragments detected by sequencing that are not visible in restriction profiles. 2.2. Differentiation by PCR amplification and digestion of the amplified fragment

[000132] A more precise differentiation of each genotype is obtained by amplification of regions of the genome characteristic for each of the genotypes by the PCR technique (polymerase chain reaction), using specific primers designed in the areas of variability, followed by a digestion of the fragments amplified by PCR with restriction endonucleases.

[000133] Comparisons of the complete genomes sequenced to date by HearSNPV have shown that the zones of variability correspond mainly to the homologous regions (hr1, hr2, hr3, hr4 and hr5) and the bro genes (Zhang et al., 2005; Ogembo et al., 2009). In this case, specific primers were designed to amplify the hr1 and hr5 homologous regions.

[000134] Therefore, the following primers were designed: - For hr1: - forward F-hr1: 5'- CGAAATCGACAACACCATGCA-3 (SEQ ID NO: 1), - reverse R-hr1: 5'- ACTTTTGTACGCCAGAGACGA-3' (SEQ ID NO:2). - And for hr5: - forward: F-hr5: 5'- CTAGCCGGTCCGTTTCTGTT-3' (SEQ ID NO:3), - reverse: R-hr5: 5'- GCCCCACCCAAAACATAACG-3' (SEQ ID NO:4).

[000135] Its usefulness for the amplification of homologous regions 1 and 5 (hr1 and hr5) was proven, respectively, by PCR as indicated in the item on the techniques used. The result obtained after the amplified fragments were subjected to electrophoresis is shown in figure 8A. For hr1, amplified fragments of 2,177 and 2,117 nucleotides were obtained for HearSP1B and HearLB6, respectively, and for hr5, fragments of 2,326 and 2,330 nucleotides were obtained for HearSP1B and HearLB8.

[000136] In order to clearly differentiate the genotypes, the fragments amplified by PCR for hr1 and hr5 were digested with the endonuclease Ndel. Said fragments, once digested, were subjected to electrophoresis, as previously described. The result obtained after the digested fragments were subjected to electrophoresis is shown in Figure 8B and Table 6. In the case of hr1, digestion with NdeI generated 6 fragments of 857, 508, 381, 306, 78 and 47 nucleotides for HearSP1B, and 5 fragments of 1,210, 475, 307, 78 and 47 nucleotides for HearLB8, in the case of hr5, digestion with NdeI generated four fragments of 1,120, 917, 211 and 78 nucleotides for HearSP1B, and 3 fragments of 1,120, 998 and 212 nucleotides for HearLB6.

[000137] The complete sequences of the homologous region 1 (hr1) corresponding to each of the two genotypes HearSP1B and HearLB6 are represented by SEQ ID NO: 9 and SEQ ID NO: 10, respectively. On the other hand, the complete sequences of the homologous region 5 (hr5) corresponding to each of the two genotypes HearSP1B and HearLBo are represented by SEQ ID NO: 11 and SEQ ID NO: 12, respectively. The alignments of the aforementioned sequences with those of the analogous zones of the HearG4, HearC1, HearNNg1 and HearAus genomes are shown in figure 9. Table 8: Specific primers designed in hr1 and hr5, nucleotide sequence, size of the amplified fragments for each genotype, number of fragments obtained after digestion of the fragment amplified by PCR with the endonuclease NdeI, size of said fragments and reference number of the sequence of the fragment amplified by PCR. Example 3: Insecticidal activity of individual genotypes and mixtures of co-occluded genotypes

[000138] To construct the mixtures, different combinations of genotypes were used in various proportions as described in the previous item of techniques called "Construction of genotype mixtures". Briefly, to obtain the co-occluded mixtures L4 larvae of H. armigera were inoculated orally (per os) with the occlusion body mixtures, which were obtained by preparing occlusion body mixtures of different genotypes in the desired proportions, and after the infection process, occlusion bodies were obtained with virions of different genotypes co-occluded in the same occlusion body in the desired proportions. 3.1. Insecticidal activity of the wild isolate HearSPI and the pure genotypes HearSPI A and HearSPIB

[000139] In order to determine the biological activity of the individual genotypes purified from the HearSP1 isolate, the biological activity of the two genotypes individually and the wild HearSP1 isolate was determined (Figueiredo et al., 1999; Arrizubieta et al., 2014). Table 7 shows the LC50 values and relative potency of the individual genotypes HearSP1 A and HearSP1B compared to that of the wild isolate HearSP1. Relative potencies refer to the relationship between the LC50 of the different genotypes in relation to the wild isolate HearSP1

[000140] Pathogenicity bioassays showed that the pathogenicity of the HearSP1B genotype is 2.8 times higher than that of the wild HearSP1 isolate. However, the HearSP1 A genotype presents an intermediate pathogenicity, being similar to both the wild isolate HearSP1 and the HearSP1B genotype (Table 7). Table 7. Relative insecticidal activity of occlusion bodies of the wild isolate HearSP1 and the individual genotypes HearSP1 A and HearSP1B. *The same letters that accompany the values indicate that there are no significant differences between the treatments (t test, P>0.0; 5).

[000141] No significant differences were observed in the mean mortality times (MMR) between the pure genotypes and the wild isolate, with HearSP1A and HearSP1B being statistically equal to the wild isolate in terms of speed in killing the second stage larvae of H. Armígera ( Table 7).

[000142] Furthermore, the genotypes HearSP1A (5.2 x 107 occlusion bodies/larva) and HearSP1B (5.3 x 107 occlusion bodies/larva) are the same as the HearSP1 isolate (7.3 x 107 occlusion bodies/ larva) in terms of productivity, in larvae inoculated in the second stage of H. armigera (Fig. 10).

[000143] Therefore, we can conclude that the pure genotype HearSP1B presents better insecticidal qualities since its pathogenicity is greater than that of the wild isolate or the pure genotype HearSP1A, while its virulence (TMM) and production of occlusion bodies are not inferior to those of other isolates/genotypes. 3.2. Insecticidal activity of individual Lebrija genotypes (HearLB)

[000144] In order to carry out the biological characterization of the individual genotypes coming from Lebrija, the biological activity of the different genotypes was determined individually and compared with the HearSP1 isolate (Figueiredo et al., 1999; Arrizubieta et al., 2014) in terms of pathogenicity, virulence and productivity as described in item 3.1.

[000145] Table 8 shows the LC50 values and relative potency of the HearSP1 isolate and the individual genotypes HearLB1, HearLB2, HearLB3, HearLB4, HearLB5 and HearLB6. These values allow us to observe how the fiducial limits at 95% of the relative potencies calculated for the LC50 largely overlap in all treatments, which indicates that the pathogenicity is similar for the pure genotypes and the HearSP1 isolate. Table 8. Relative insecticidal activity of occlusion bodies of the wild isolate HearSP1 and the individual genotypes HearLB1, HearLB2, HearLBS, HearLB4, HearLBS and HearLB8. The different letters that accompany the values indicate that there are significant differences between the treatments (t test, P<0.05).

[000146] On the other hand, the HearLB1, HearLB2, HearLBS and HearLB6 genotypes were significantly faster to kill the second stage larvae of H. armigera than the rest of the genotypes and the HearSP isolate (Table 8),

[000147] The occlusion body production data were analyzed by ANOVA and Tukey's Test analysis with the SPSS 15.0 statistical program (Fig. 11). The HearLB1 genotype is the most productive (5.3 x 108 occlusion bodies/larva), although it does not present significant differences with the HearLB4 genotype (4.2 x 108 occlusion bodies/larva). Furthermore, the HearLB1, HearLB4 and HearLBS genotypes are more productive than the HearSP1 isolate in second stage larvae of H. armigera. 3.3. Insecticidal activity of the co-occluded mixtures obtained with the five genotypes selected in the previous paragraphs (item 3.2 and 3.3), and of the HearLBmix mixture

[000148] Based on the minimal differences in insecticidal activity observed between the different genotypes, five of them were selected in the previous paragraphs (item 3.2 and 3.3) and several mixtures were made with the purpose of optimizing the biological activity and thus obtaining a mixture with insecticidal qualities best. To this end, eight co-occluded mixtures were made which included: - HearSP1 A:SP1B in a 1 : 1 ratio. The objective of this mixture is to increase pathogenicity since the HearSP1B genotype is more pathogenic than HearSP1, and in this mixture it is found in a greater proportion than in the wild isolate HearSP1 (natural ratio 2: 1). - HearSP1 A:SP1B in a 1 :2 ratio. The objective of this mixture is also to increase pathogenicity since the HearSP1B genotype is more pathogenic than HearSP1, and in this mixture it is even found in a greater proportion than in the previous mixture. - HearLB1 :LB3 in a 1 : 1 ratio. The HearLB1 genotype is one of the fastest and is also among the most productive. On the other hand, the HearLBS genotype is among the most productive, because it is the slowest. The objective of this mixture is to maintain the virulence of the HearLB1 genotype and the productivity of both. - HearLB3:LB6 in a 1:1 ratio. The HearLB6 genotype is one of the fastest and least productive genotypes, while HearLB3 is among the most productive. In this case, the aim is to maintain the virulence of HearLB6 and the productivity of HearLB3. - HearLB1:LB3:LB6 in a 1:1:1 ratio. This mixture could maintain the virulence of the HearLB1 and HearLB6 genotypes, and the productivity of HearLB1 and HearLB6. - HearLBmix (HearLB1-6) in 4:4:6:1:1:1 ratio. This mixture includes the six Lebrija genotypes in the proportion in which they were isolated. The fact that each of the genotypes was isolated in a proportion after an epizootic may have some significance at a biological level. - HearSP1B:LB1 in a 1:1 ratio. This mixture could maintain the pathogenicity of the HearSP1B genotype and the virulence of HearLB1, and increase productivity, as HearLB1 is one of the most productive genotypes. - HearSP1B: LB8 in a 1:1 ratio. In this case, the aim is to maintain the pathogenicity of HearSP1B and the virulence of HearLB8.

[000149] The insecticidal activity of the different co-occurrence mixtures was compared in terms of pathogenicity, virulence and productivity as described in item 3.1. The individual genotypes HearSP1A, HearSP1B, HearLB1, HearLB3 and HearLB8 were included as references. Table 9: Relative insecticidal activity of occlusion body mixtures HearSP1A:SP1B (1:1), HearSP1A:SP1B (1:2), HearLB1 :LB3, HearLB3:LB6, HearLB1 :LB3:LB6, HearLBmix, HearSP1B:LB1 and HearSP1B: LB6, and the individual genotypes HearSP1A, HearSP1B, HearLB1, HearLB3 and HearLB6. *The different letters that accompany the values indicate that there are significant differences between the treatments (t test, P<0.05).

[000150] Table 9 shows the LC50 values, the relative potency of the co-occluded mixtures and individual genotypes (in relation to that of HearSP A), as well as the average mortality time. Surprisingly, the HearSP1B:HearLB6 genotype mixture (5.7 x 103 occlusion bodies/ml) proves to be the most pathogenic, between 1.7 and 3.7 times more pathogenic than the individual genotypes and the rest of the mixtures. Furthermore, this mixture, with a MRT of 108.8 hours, is equal in terms of virulence to the fastest larval killing genotypes, such as HearSP1A, HearSP1B and HearLB8. Analyzing the data presented in Table 9, it was concluded that one or another mixture is not expected to be more or less pathogenic, as there is no standard or norm that predicts a priori which of all the mixtures is the most potent.

[000151] Productivity bioassays showed that the genotypes HearLB1 and HearLBS and the co-occluded mixtures HearLB1 :LB3 and HearLB1 :LB3:LB6 were the most productive (4.9 x 08, 5.7 x 08, 5.7 x 108 and 4.0 x 108 occlusion bodies/larva, respectively) (Tukey, P<0.05), followed by the HearLB6 genotype and the co-occluded mixtures HearSP1A:SP1B (1 :2), HearLB3:LB6, HearLBmix, HearSP1B:LB1 and HearSP1B:LB6 (3.4 x 108, 2.5 x 108, 3.7 x 108, 2.2 x 08, 2.5 x 108 and 1.6 x 108 occlusion bodies/larva, respectively). Lastly, the HearSP1A and HearSP1B genotypes and the HearSP1A:SP1B (1 : 1) mixture were the least productive, with a viral productivity of 6.3 x 107, 1.4 x 108 and 9.3 x 107 occlusion bodies/ larva, respectively (Tukey, P<0.05) (Fig. 12).

[000152] The mixture of co-occluded genotypes HearSP1B: LB6 is more pathogenic than the rest of pure genotypes and mixtures and, in addition, is of the same virulence as the faster genotypes. It is expected that these characteristics would allow the rapid suppression of pest populations in the field using a minimum amount of product, reducing crop production costs. For these reasons, we selected the HearSP1B:LB6 mixture as the active substance of a new bioinsecticide for the control of H. armigera. Thus, the mass production and effectiveness trials described below were carried out with the aforementioned. Example 4: Mass production of HearSNPV 4.1. Study of H. armigera cannibalism

[000153] To determine the ideal conditions for mass production of HearSNPV, the number of occlusion bodies that produce lethally infected larvae was used as a criterion. Mass production of the HearSP1B:LB6 co-occluded mixture in H. armigera larvae can be done with individual larvae in 12-well plates or in larger volume containers with a greater number of larvae. However, this last method may present problems depending on the degree of cannibalism presented by the species. Cannibalism normally depends, among other things, on larval density, although there is no limitation on food (Polis, 1981). Typically, cannibalism also increases with larval age (Chapmam et al., 1999).

[000154] In this case, the cannibalism of three larval stages of H. armigera, L3, L4 and L5, was studied, both in healthy larvae and those infected with CL90, which was 8.1 x 105, 2.4 x 106 and 2 .5 x 107 occlusion bodies/ml for stages L3, L4 and L5, respectively. These concentrations were estimated in preliminary bioassays, at three different densities: 5, 10 and 20 larvae per plastic box with a capacity of 0.5 liters. As a control, five individual larvae from each stage were included, both healthy and infected. The test was repeated three times.

[000155] The percentages of cannibalism, mortality due to nucleopolyhedroviruses and larvae that reached the pupal stage were analyzed by ANOVA analysis and Tukey's test with the SPSS 15.0 statistical program. The cannibalism observed in H. armigera was similar between the L3 and L4 stages, and between healthy and infected larvae (around 30% of cannibalism being observed) (Tukey, P>0.05), However, at the L5 stage it was A significantly higher percentage of cannibalism was observed in infected larvae (between 77 and 87%) than in healthy larvae (20-55%) (Tukey, P<0.05) (Fig. 12). On the other hand, cannibalism increased significantly with larval density (Tukey, P<0.05), being approximately 40% at a density of 5 larvae per box, increasing to 50-60% at a density of 10 larvae per box and reaching finally 80% in boxes with 20 larvae. However, in infected L5 larvae the percentage of cannibalism was similar for all densities, varying between 77 and 87% (Tukey, P>0.05) (Fig. 13).

[000156] The percentage of mortality due to nucleopolyhedroviruses obtained in individual larvae was greater than 90%; however, in higher density containers, 50% mortality was not achieved, due to the fact that the sick larvae were cannibalized before their death (Fig, 13).

[000157] Due to the high percentage of cannibalism observed in H. armigera larvae, which leads to a reduction in mortality and, therefore, the production of occlusion bodies, mass production of HearSNPV is much more efficient if done with larvae individualized. 4.2. Effect of larval stage, timing of inoculation and viral concentration on HearSNPV production

[000158] To achieve a higher production of occlusion bodies per larva it is necessary to select the larval age, the time of inoculation and the viral concentration that allow greater growth of the larva, and, therefore, a higher viral production (Shieh, 1989; Gupta et al., 2007).

[000159] To select the stage and time of inoculation, the study was carried out with the last three larval stages, L3, L4 and L5, infected at two different times; after moulting (recently moulted) and one day after moulting (1 day after moulting). On the other hand, it is known that when concentrations that produce high percentages of mortality are applied, the larvae develop more slowly and thus produce fewer occlusion bodies. Therefore, it is advisable to optimize the viral concentration that produces a high percentage of mortality and with the highest possible production of occlusion bodies/larvae. For this, each stage was infected with three different concentrations of the virus, corresponding to CL80 (1.5 x 105, 4.8 x 105 and 5.5 x 106 occlusion bodies/ml, for stages L3, L and L5, respectively), CL90 (6.1 x 105, 2.4 x 106 and 2.5 x 107 occlusion bodies/ml, for stages L3, L4 and L5, respectively) and CL95 (1.9 x 106, 9, 1 x 106 and 1.5 x 108 occlusion bodies/ml, for stages L3, L4 and L5, respectively); these concentrations were previously determined in preliminary bioassays. The larvae were inoculated individually according to the drop method described by Hughes and Wood (1981) and were deposited in individual pots to avoid cannibalism with an artificial diet until they died from viruses or until they reached the pupal stage. The occlusion bodies produced by each dead larva were extracted, purified and titrated as previously indicated. 24 larvae were inoculated per treatment and three replications were performed. The data obtained were analyzed by ANOVA analysis and Tukey's test with the SPSS 15.0 statistical program.

[000160] The percentages of mortality obtained in larvae infected after molting were as expected (between 80 and 100%), however, larvae inoculated one day after molting showed a significantly lower percentage of mortality (F17,36 = 16, 30, P<0.05), reaching between 31 and 47% mortality in the case of fourth and fifth stage larvae (Fig. 14). This may be due to the fact that these larvae are more resistant to infection since their size is larger one day after they have moulted and the characteristics of the midgut change according to interstage developmental status (Washburm et al., 1998). . Within each larval age, the three doses used produced statistically similar mortality percentages, although a slight increase in mortality was observed as the viral dose increased (Tukey, P>0.05) (Fig. 14).

[000161] The larvae produced significantly greater quantities of occlusion bodies with increasing age when inoculated (F17.36=14.25; P<0.05) (Fig. 15A). Therefore, the larvae inoculated one day after moving to L4 and L5 and the newly moved L5 produced significantly more occlusion bodies than the rest of the larvae (between 5.6 and 9.1 x 109 occlusion bodies/ larva) (Tukey, P<0.05). However, as mentioned previously, larvae inoculated the day after moulting to L and Ls showed a much lower percentage of mortality than those inoculated recently moulted to L5, and thus the final production of occlusion bodies was lower (Fig. 15B). Newly moulted L5 larvae inoculated with CL95 produced 6.9 x 1011 occlusion bodies/100 inoculated larvae, versus 1.6 x 1011 - 4.2 x 1011 occlusion bodies/100 inoculated larvae one day after moulting to L5 .

[000162] Therefore, the ideal stage for producing the HearSP1B:LB6 genotype mixture in H. armigera larvae is L5 being freshly moulted inoculated with CL95 (1.5 x 108 occlusion bodies/ml). This treatment produces a mortality rate of around 100% and is the treatment that achieves the highest productivity (6.9 x 1011 occlusion bodies/100 inoculated larvae). 4.3. Effect of incubation temperature on HearSNPV production

[000163] The incubation temperature can influence larval development and, therefore, viral productivity (Subramaniam et al., 2006). Therefore, a study was carried out to determine the ideal temperature for the production of HearSNPV.

[000164] Newly moulted L5 larvae were inoculated with CL95 (conditions selected in item 4.2.) and were incubated at 23, 26 and 30°C. Mortality was recorded every 8 hours to determine larvae mortality time as a function of temperature and cadavers were collected individually to determine the production of occlusion bodies. 24 larvae were infected per treatment and five replications were performed.

[000165] The production of occlusion bodies/larvae and the TMM were calculated in the manner described previously. There were no significant differences in productivity between larvae incubated at different temperatures (F2,12=0.30; P>0.05) (Fig. 16). However, at 30°C the larvae die between 13 and 34 hours faster than at 28°C and 23°C, respectively (Table 10). Therefore, the ideal temperature for HearSNPV production is 30°C, as it allows the same quantity of occlusion bodies to be obtained more quickly than other incubation temperatures. Table 10: Mean mortality time (MMR), expressed in hours after infection, of H. armigera L5 larvae infected with CL95 and incubated at 23, 28 and 30°C. *The different letters that accompany the values indicate that there are significant differences between the treatments (Mest, P<0.05). Example 5: Efficacy trials of HearSNPV for the control of H. armigera in tomato plants 5.1. Tests on tomato cultivation under laboratory conditions

[000166] Initially, to determine the effectiveness of the HearSP1B:LB6 co-occluded mixture for controlling H. armigera, a test was carried out on tomato plants treated and maintained under laboratory conditions. Tomato plants were treated by spraying with an aqueous suspension containing the HearSP1B:LB6 co-occluded mixture at different concentrations (109, 1010 and 1011 occlusion bodies/liter) together with an agricultural humectant (Agral®, Syngenta) at 0 .2% (vol/vol). As a control, plants were used treated with a solution containing water and Agral® (0.2%), but without occlusion bodies. Once treated, the plants were allowed to dry and were placed in 50 ml pots with Hoagland nutrient solution inside 10 liter crystal containers and infested with 150 second stage H. armigera larvae (L2). The plants were maintained at 25±1°C, 70±5% relative humidity and a photoperiod of 16:8 hours light:dark.

[000167] Assessment of treatment effectiveness was determined by quantifying the percentage of mortality. For this, 15 larvae were collected from each of the treatments on days 1, 3 and 5 after treatment. These larvae were deposited individually in pots with an artificial diet and mortality 7 days after they were collected from the plants was recorded.

[000168] The results obtained are shown in figure 16. No mortality was observed in the larvae collected in the control treatment, which indicates the absence of viral contamination in the plants used. In plants treated with 109 occlusion bodies/liter, a mortality percentage of 88.9, 96.7 and 88% was obtained in larvae collected on days 1, 3 and 5, respectively. However, in plants treated with 1010 and 1011 occlusion bodies/liter, 100% mortality was obtained for all larvae on all collection days (Fig. 17).

[000169] The concentration of 1x1010 occlusion bodies/liter is the minimum concentration that produces 100% mortalities of all collection days. Therefore, said concentration is selected as the optimum for the control of H. armigera larvae in tomato crops under laboratory conditions. 5.2. Trials on tomato cultivation in a greenhouse in Lisbon (Portugal)

[000170] To determine the effectiveness of HearSP1B:LB6 to protect tomato cultivation under greenhouse conditions against H. armigera, trials were carried out in an experimental greenhouse at the Instituto Superior de Agronomia (Technical University of Lisbon). Based on the results obtained in laboratory tests, the effectiveness of the HearSP1B:LB6 co-occluded mixture was evaluated at a concentration of 1x1013 occlusion bodies/Ha (equivalent to 1010 occlusion bodies/liter, using 1,000 liters/ha) . In the present study, the efficacy of HearSP1B:LB6 was compared with that of: - a biological insecticide based on the entomopathogenic bacterium Bacillus thuringiensis aizawai (Turex®, from Certis, Elche, Spain, which contains 50% B. thuringiensis in the form of wettable powder). This bioinsecticide is usually used at a concentration of 1-2 kg/ha, in this case 1.5 kg/ha was used (using 1,000 liters/ha), - a biological insecticide based on spinosad, a product of two spinosyn toxins , which are obtained naturally by fermentation of the bacterium Saccharopoiyspora spinosa (Spintor 480SC®, Dow AgroSciences, Madrid, Spain, which contains Spinosad at 48% by weight/volume). Said insecticide is usually used at a concentration of 250 ml/ha (using 1,000 liters/ha).

[000171] As a control, a water treatment was carried out. The application method was by spraying with an aqueous suspension containing the different treatments.

[000172] The experimental design consisted of two blocks with four experimental plots each, totaling 8 replications. In each treatment, a total of 28 tomato plants were included, of which the 6 central plants were observed to determine the percentage of larval mortality, the percentage of damaged fruits and the persistence of the different treatments.

[000173] To carry out the tests, insects were artificially released, 4 larvae of H. armigera in the L2 stage being placed in the fruits (selected at random) of each of the tomato plants. The following day the different treatments were applied.

[000174] Firstly, the percentage of damaged fruits was determined 10 days after applying the treatment. The percentage of larval survival due to treatment was also determined. For this, the number of larvae that remained alive on each plant 10 days after applying the treatment was counted. The data obtained were analyzed by ANOVA analysis and Tukey's test with the SPSS 15.0 statistical program.

[000175] The three insecticides significantly reduced the percentage of damaged fruits in relation to the control (F3,20=9.79; P<0.05). However, there were no significant differences between the different insecticides (Tukey, P>0.05) (Fig. 18). Treatments with HearSP1B: LB8, Turex and Spintor significantly increased larval mortality in relation to the control treatment (F3 2o=37.70; P<0.05). Furthermore, HearSP1B:LB6 and Spintor produced significantly higher larval mortalities than Turex (Tukey, P<0.05) (Fig. 19).

[000176] Finally, the persistence of the different treatments on tomato leaves was determined.

[000177] For this, 15 leaves were collected individually per treatment and repetition from the medium-high part of the plants 1 hour after treatment and after 3, 6 and 9 days, and these were immediately frozen. These leaves were crushed individually and mixed with an artificial diet (in a 1:4 weight:weight ratio). The mixture was distributed into five plastic pots, with one L2 larva placed in each of the five pots to avoid cannibalism.

[000178] At 7 days the percentage of mortality was determined. The relationship between mortality and the amount of viable insecticide was obtained by calibrating the bioassay. The calibration curves of the three insecticides were obtained by mixing leaves collected before treatment and, therefore, not infected, with an artificial diet, and with five known and different concentrations of the insecticides. 50 larvae were used per concentration. The amount of persistent insecticide on the leaves was estimated by comparing the percentage of mortality obtained in the different treatments with the calibration curves. The insecticide quantity data obtained were analyzed by ANOVA and Tukey's test with the SPSS 15.0 statistical program. In order to compare the persistence of the different treatments on tomato leaves in the greenhouse, the percentage of residual insecticidal activity of each of the treatments was calculated in relation to that obtained one hour after application, at which time it was considered that 100% were present in the plant. of the insecticidal activity applied.

[000179] When comparing the residual insecticidal activity of the different treatments at different leaf collection times, significant differences were found between the persistence of HearSNPV and Turex 6 and 9 days after application, with the persistence of Turex being lower (Tukey, P <0.05) (Fig. 20). On the rest of the days, a similar degree of residual insecticidal activity was observed in all treatments.

[000180] Residual insecticidal activity decreased significantly over time (F15,48 = 88.25; P<0.05) in all cases (Fig. 20 and 21). The persistence of HearSNPV and Spintor was maintained up to 6 days after application of the treatments, decreasing significantly on day 9 (Tukey, P<0.05), although 59% and 49% insecticidal activity still persisted, respectively (Fig. 21B and 21C). In the case of Turex, the residual insecticidal activity after 6 days of applying the treatments was significantly lower than the insecticidal activity that was present in the leaves one hour after treatment (Tukey, P<0.05), and at 9 days they were maintained only 32% insecticide (Tukey, P<0.05) (Fig. 21A). 5.3. Trials on outdoor tomato cultivation in Badajoz (Spain)

[000181] To determine the effectiveness of the HearSP1B:LB6 co-occluded mixture in outdoor tomato cultivation, trials were carried out in a part of the La Ordem facility (Guadajira, Badajoz). In these tests, the same dose of HearSP1B:LB8 was used as that used in the greenhouse tests, 1013 occlusion bodies/ha (an application volume of 1,000 liters/ha was used) and its effectiveness was compared with that of: - the wild isolate HearSP1 from Badajoz (Figueiredo et al., 1999), where the tests were carried out, using the same dose as for HearSP1B:LB6, 1010 occlusion bodies/liter (equivalent to 1013 occlusion bodies/ha, treatment being applied in a volume of 1,000 liters/ha). - a biological insecticide based on the entomopathogenic bacteria Bacillus thuringiensis aizawai (Turex®, from Certis, Elche, Spain, which contains 50% B. thuringiensis in the form of a wettable powder). Usually it is used at a concentration of 1-2 kg/ha, in this case 1.5 kg/ha was used (applied in a volume of 1000 liters/ha). - a biological insecticide based on two spinosyn toxins, which are obtained naturally by fermentation of a soil organism, the bacterium Saccharopolyspora spinosa (Spintor 480SC®, Dow AgroSciences, Madrid, Spain, which contains Spinosad at 48% weight/volume ). It is usually used at a concentration of 250 ml/ha (by diluting 250 ml in 1,000 liters, using 1,000 liters/ha). - a chemical insecticide based on chlorpyrifos (Dursbam 75WG®, Dow AgroSciences, Madrid, Spain, which contains chlorpyrifos at 75% weight/weight). Usually it is used at a concentration of 1-1.25 kg/ha, in this case 1.25 kg/ha was used (which was diluted again in 1,000 liters to use the same volume in one ha).

[000182] As a control, a treatment with water and 0.2% agral was carried out. The application method was by spraying with an aqueous suspension containing the different treatments.

[000183] The test consisted of 48 lots (1.5 m x 4 m), each of which consisted of approximately 30 plants. The design was made of random blocks. Each of the blocks consisted of two rows with 6 elementary lots and to half of the lots in each block the different treatments were applied three times while to the other half five times, carrying out a total of 4 repetitions for 3 and 5 applications. All applications were carried out 15 days apart. Core plants were observed to determine the percentage of damaged fruit, the persistence of the different treatments, and the yield of each batch.

[000184] Firstly, the percentage of damaged fruits, both with fresh and healed damage, was determined every 3 or 4 days, throughout the test period. The data obtained were grouped by fortnightly averages and were analyzed by ANOVA analysis and Tukey's test with the statistical program SYSTAT (1990).

[000185] No significant differences were observed in the percentage of damaged fruits between the lots treated 3 and 5 times for the different treatments (F1,174= 0.22; P>0.05), and therefore the data from all lots treated with each insecticide were grouped having a total of 8 repetitions.

[000186] Figure 22 shows the percentage of fresh and healed damaged fruits in each of the fortnights for each treatment. In the first fortnight there were no differences in the percentage of damaged fruits obtained in the lots treated with the different insecticides, being similar to that obtained with the control treatment (F5,15=0.55; P>0.05) (Fig. 22A). However, in the second and third fortnight in the control lots there was a higher percentage of damaged fruits, both fresh and healed, than in the lots treated with the different insecticides (Tukey, P<0.05) (Fig. 22B and 22C). In the fourth fortnight, a period of cultivation that is not usually attacked by H. armigera larvae, the percentage of damaged fruits healed was also higher in the control lots (Tukey, P<0.05), but there were no differences in the percentage of fruits with fresh damage (P>0.05) (Fig. 22D).

[000187] These results demonstrate that HearSNPV significantly reduces the number of damaged fruits, both with fresh and healed damage, in relation to the control treatment and, in addition, with equal effectiveness as the rest of the insecticides usually used to control the pests caused by H. armigera.

[000188] Subsequently, the yield of each batch was determined. For this, the fruits from the central meter of each lot were harvested and separated into green and red. The green fruits were separated into healthy and chopped ones, and the red ones into healthy ones, healed chopped ones and rotten chopped ones. Subsequently, each group was weighed. The data obtained were analyzed by ANOVA and Tukey's test with the SYSTAT statistical program. Quality controls at canning companies reject batches of tomatoes in which less than 80% of the fruits are ripe, and in which more than 5% of the ripe tomatoes are damaged. Unripe fruits are rejected before they arrive at the company.

[000189] In this case, there were also no differences between the batches treated 3 or 5 times, that is, in the number of applications, which is why the data from all batches treated with each insecticide were grouped. The percentage of damaged fruits harvested in each of the treatments is shown in figure 23. The percentage of damaged fruits, whether chopped green, scarred red or rotten red, was higher in the control lots than in those treated with the different insecticides (Tukey, P< 0.05). Furthermore, in lots treated with Dursbam and Spintor a significantly lower percentage of scarred red fruits was obtained than in those treated with Turex and HearSP1B:LB6 (Tukey, P<0.05), in lots treated with HearSP1 and Turex a percentage of rotted fruits greater than in those treated with Dursbam (Tukey, P<0.05) (Fig. 23).

[000190] Tons of healthy green fruits per hectare (T/ha) were similar in all treatments (F5,39 = 0.68; P>0.05) (Fig. 24A). However, the tons of chopped green fruits per hectare were significantly higher in the control plots than in those treated with the different insecticides (F5,39=4.95; P<0.05) (Fig. 24A). Tons of healthy red fruits per hectare were significantly lower in control lots than in lots treated with insecticides, except with Turex P<0.05), although it did not show significant differences with the rest of the insecticides (Tukey, P> 0.05) (Fig. 24B). As for damaged red fruits, both scarred and rotten, a greater number of tons per hectare was obtained in the control plots than in those treated with insecticides (Tukey, P<0.05). Furthermore, there were no significant differences between the tons of scarred red fruits obtained in lots treated with HearSP1B:LB6 and HearSP1 in relation to lots treated with the rest of the insecticides (Tukey, P>0.05), although in lots treated with Dursbam and Spintor fewer tons of scarred red fruits were obtained than in those treated with Turex (Tukey, P<0.05) (Fig. 24B). Furthermore, in the lots treated with Dursbam, fewer tons per hectare of rotten red fruits were obtained than in those treated with HearSP1 and Turex (Tukey, P<0.05) (Fig. 24B), but they did not show significant differences with HearSP1B:LB8 ( Tukey, P>0.05),

[000191] In lots treated with HearSP1B:LB6 or HearSP1 it is possible to obtain a harvest similar to that in lots treated with other insecticides, since the tons of healthy red fruits, that is, those that are marketable, are similar in all treatments, except in the control treatment. Furthermore, the percentage of damaged fruits is very low, as is the case with the rest of the lots treated with insecticides. This information is very important when marketing tomatoes, as Spanish canning companies do not accept batches with more than 5% of the fruits damaged.

[000192] Finally, the persistence of the different treatments on tomato leaves was determined. To this end, leaves close to the fruit were harvested one hour after the first treatment and after 3, 7 and 10 days. 25 leaves from each batch were harvested and immediately frozen. These leaves were crushed in groups of five, mixed with an artificial diet (in a 1:4 ratio, weight:weight) and distributed into 10 individual pots with one L2 larva in each to avoid cannibalism. At 7 days the percentage of mortality was determined. The relationship between mortality and the amount of viable insecticide was obtained by calibrating the bioassay. The calibration curves of the five insecticides were obtained by mixing leaves harvested before treatment with an artificial diet and five known and different concentrations of the insecticides. 50 larvae/concentration were used. The amount of persistent insecticide on the leaves was estimated by comparing the percentage of mortality obtained in the different treatments with the calibration curves. The insecticide quantity data obtained were analyzed by ANOVA and Tukey's test with the SPSS 15.0 statistical program. To be able to compare the persistence of the different treatments on tomato leaves outdoors, the percentage of residual insecticidal activity of each of the treatments was calculated after one hour of application.

[000193] If we compare the percentage of residual insecticidal activity of the different treatments at different leaf harvest times, we only found significant differences between the amount of HearSP1 and Spintor at 7 days after application, with the persistence of HearSP1 being lower (Tukey, P< 0.05) (Fig. 25), and after 10 days between amounts of HearSP1B:LB8 and HearSP1 with Spintor and Dursban, the persistence of baculoviruses was lower (Tukey, P<0.05) (Fig. 25).

[000194] Residual insecticidal activity in outdoor tomato plants decreases significantly over time (F19,140=34.24; P<0.05) in all cases (Fig. 25 and 28). The amount of HearSNPV (both HearSP1B:LB6 and HearSP1) remains the same from the first day to 3 days after application of the treatment, and from that moment it decreases significantly (Tukey, P<0.05). 7 days after application, 66% and 52% of the insecticidal activity of the occlusion bodies of HearSP1B:LB6 and HearSP1, respectively, still persist in the plant, while after 10 days only 9 and 2% of the activity of the occlusion bodies persist, and although no significant differences are observed between the two, it appears that the selected mixture is able to persist for longer (Figs. 26A and 26B). The activity of Dursbam and Spintor remains in the plant up to 3 days after application (Tukey, P>0.05), decreasing significantly in 7 days (Tukey, P<0.05), presenting the same insecticidal activity as in 10 days (Tukey, P>0.05), at which time 59% of Spintor and 46% of Dursbam activity still persist in the plant (Figs. 26C and 26E). In the case of Turex, the insecticidal activity decreases significantly after 3 days (Tukey, P<0.05) and remains up to 7 days (Tukey, P>0.05), decreasing significantly again after 10 days (Tukey, P< 0.05) persisting 27% (Fig. 26D).

[000195] In the case of HearSNPV isolates, which are harmless to humans and other vertebrates, the fact that more than 50% of the insecticidal activity persists 7 days after application of the treatment is positive, since the larvae that ingest the contaminated leaves can contract the disease. In the case of Dursban, which is toxic to humans, a negative point is the fact that approximately 50% still remains after 10 days, which increases the safety period for harvesting tomato fruits, in addition to the consequent environmental contamination.

[000196] In view of the results, it is concluded that the application of treatments with HearSNPV at a dose of 1013 occlusion bodies/ha allows to protect tomato crops, both in the greenhouse and outdoors, in a satisfactory way with the same effectiveness as the chemical and biological treatments that are currently used in this cultivation and avoiding the inconveniences that they present.

Biological material deposit

[000197] The new genotypes HearSP1B and HearLB8 were deposited at the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, France, in accordance with the rules of the Budapest Treaty. The deposit numbers and their dates were as follows: Genotype Abbreviation Deposit number Deposit date HearSNPV- HearSP1B CNCM I-4806 October 15, SP1B 2013 HearSNPV-LB6 HearLB6 CNCM I-4807 October 15, 2013

[000198] The two genotypes were deposited by one of the inventors, Prof. Dr. Primitivo Caballero (Instituto de Agrobiotecnología y Recursos Naturales, Universidad Pública de Navarra, Campus de Arrosadía, Mutilva Baja, E-31006, Pamplona, Navarra, Spain), as employee of the first applicant, on behalf and representation of the three applicants (Universidad Publica de Navarra, Consejo Superior de Investigaciones Científicas, Instituto de Ecología A.C.). Bibliographic references Arrizubieta, M., Williams, T., Caballero, P., Simón, O., 2014. Selection of a nucleopolyhedrovirus isolate from Helicoverpa armigera as the basis for a biological insecticide. Pest Management Science 70, 967-976. Barrera, G., Simón, O., Villamizar, L, Williams, T., Caballero, P., 201 1. Spodoptera frugiperda multiple nucleopolyhedrovirus as a potential biological insecticide: genetic and phenotypic comparison of field isolates from Colombia. Biological Control 58, 113-120. Bernal, A., Williams, T., Hernández-Suárez, E., Carnero, A., Caballero, P., Simón, O., 2013a. A native variant of Chrysodeixis chalates nucleopolyhedrovirus: The basis for a promising bioinsecticide for control of C. chalcites on Canary Islands' banana crops. Biological Control 67, 101-110. Bernal, A., Simón, O., Williams, T., Munoz, D., Caballero, P., 2013b. A Chrysodeixis chalcites single nucleopolyhedrovirus population from the Canary Islands is genotypically structured to maximize survival. Applied and Environmental Microbiology 79, 7709-7718. Caballero, P., Zuidema, D., Santiago-Aivarez, C, Viak, J.M., 1992. Biochemistry and biological characterization of four isolates of Spodoptera exigua nuclear polyhedrosis virus. Biocontrol Science and Technology 2, 145-157. Caballero, P., Williams, T., López-Ferber, M., 2001. Structure and classification of baculoviruses, pp. 15-46. En: Caballero, P., Williams, T., López-Ferber, M. (Eds.). Baculoviruses and their applications as bioinsecticides in the biological control of plagues. Phytoma-Espana, Valencia, Espana. Chapman, J.W., Williams, T., Escribano, A., Caballero, P., Cave, R.D., Goulson, D., 1999. Age-related cannibalism and horizontal transmission of a nuclear polyhedrosis virus in larval Spodoptera frugiperda. Ecological Entomology 24, 268-275. Chen, X., Li, M., Sun, Archives of Virology 145, 2539-2555. Chen, X., IJkel, W.F.J., Tarchini, R., Sun, Z., 2001. The sequence of the Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus genome. Journal of General Virology 82, 241-257. Cherry, A., Williams, T., 2001, Control of insect pests by baculovirus, pp. 389-450. En: Caballero, P., Williams, T., López-Ferber, . (Eds.). Baculoviruses and their applications as bioinsecticides in the biological control of plagues. Phytoma-Espana, Valencia, Espana. Clavijo, G., Williams, T., Munoz, D., Caballero, P, López-Ferber, M., 2010. Mixed genotype transmission bodies and virions contribute to the maintenance of diversity in an insect virus. Proceedings of the Royal Society B 277, 943-951. Cory, J.S., Green, B.M., Paul, R.K., Hunter-Fujita, F., 2005. Genotypic and phenotypic diversity of a baculovirus population within an individual insect host. Journal of Invertebrate Pathology 89, 101-111. Crawiey, 1993. GLIM for ecologists. Blackwell Scientific Publications, Oxford, UK. Cunningham, J.P., Zalucki, M.P., West, S.A., 1999. Learning in Heiicoverpa armigera (Lepidoptera: Noctuidae): a new look at the behavior and control of a polyphagous pest. Bulletin of Entomological Research 89, 201-207. Erlandson, M., Newhouse, S., Moore, K., Janmaat, A., Myers, J., Theilmann, D., 2007. Characterization of baculovirus isolates from Trichoplusia ni in populations from vegetable greenhouses. Biological Control 41, 256-283. Figueiredo, E., Munoz, D., Escribano, A., Mexia, A., Viak, J.M., Caballero, P., 1999. Biochemical identification and comparative insecticidal activity of nucleopolyhedrovirus isolates pathogenic for Heliothis armigera (Lep. Noctuidae) larvae . Journal of Applied Entomology 123, 165-169. Figueiredo, E., Munoz, D., Murilio, R., Mexia, A., Caballero, P., 2009. Diversity of Iberian nucleopolyhedrovirus wild-type isolates infecting Heiicoverpa armigera (Lepidoptera: Noctuidae). Biological Control 50, 43-49. Gelernter, W.D., Federici, B.A., 1986. Isolation, identification and determination of virulence of a nuclear polyhedrosis virus from the beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae). Environmental Entomoiogy 15, 240-245. Granados, R., Fu, Y., Corsaro, B., Hughes, P., 2001. Enhancement of Bacillus thuringiensis toxicity to lepidopterous species with the enhancement from Trichopiusia ni granulovirus. Biological Control 20, 153-159. Greene, G.L., Leppia, N.C., Dickerson, W.A., 1976. Velvetbeam caterpillar: a rearing procedure and artificial medium. Journal of Economic Entomoiogy 69, 487-488. Gróner, A., 1986. Specificity and safety of baculoviruses, pp, 77-202. En: Granados, R.R., Federici, B.A. (Eds,). The biology of baculoviruses: biological properties and molecular biology. CRC Press, Boca Ratón, Florida. Guo, Z,, Ge, J., Wang, D., Shao, Q., Zhang, C, 2006. Biological comparison of two genotypes of Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus. Biological Control 51, 809-820. Gupta, R.K., Raina, J.C., onobruliah, M.D., 2007. Optimization of in vivo production of nucleopolyhedrovirus in homologous host larvae of Helicoverpa armigera. Journal of Entomology 4, 279-288. Hara, K., Funakoshi, M., Kawarabata, T., 1995. In vivo and in vitro characterization of several isolates of Spodopfera exigua nuclear polyhedrosis virus. Acta Viroiogica 39, 215-222. Harrison, R.L., Bonning, B.C., 1999. The nucleopolyhedrovirus of Rachoplusia ou and Anagrapha falcifera are isolates of the same virus. Journal of General Virology 80, 2793-2798. Harrison, R.L., Popham, H.J.R., Breitenbach, J.E., Rowley, D.L., 2012. Genetic variation and virulence of Autographa californica multiple nucleopolyhedrovirus and Trichoplusia ni single nucleopolyhedrovirus isolates. Journal of Invertebrate Pathology 1 10, 33-47. Hughes, P.R., Wood, H.A., 1981. A synchronous peroral technique for the bioassay of insect viruses. Journey! of Invertebrate Pathology 37, 154-159. Jehle, J.A., Blissard, G.W., Bonning, B.C., Cory, J.S., Herniou, E.A., Rohrmann, G.F., Theilmann, D.A., Thiem, S. ., Viak, J. ., 2006. On the classification and nomenclature of baculoviruses: a proposal for revision. Archives of Virology 151:1, 257-266. Kalia, V., Chaudhari, S., Gujar, G., 2001. Optimization of production of nucleopolyhedrovirus of Helicoverpa armigera throughout larval stages. Phytoparasitica 29, 23-28. King, L.A., Possee, R.D., 1992. The baculovirus expression system. A laboratory guide. Chapman & Hall, London, UK. Lasa, R., Ruiz-Portero, C, Alcázar, M.D., Belda, J.E., Caballero, P., Williams, T., 2007. Efficacy of optical brightener formulations of Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) as a biological insecticide in greenhouses in southern Spain. Biological Control 40, 89-96. Le Ora Software, 1987. POLO-PC a user's guide to do probit or logit analysis. Berkeley, California, USA. López-Ferber, M., Simón, O., Williams, T., Caballero, P., 2003. Defective or effective? Mutualistic interactions between virus genotypes. Proceedings of the Royal Society B 270, 2249-2255. Moscardi, F., 1999. Assessment of the application of baculoviruses for control of Lepidoptera, Annual Review of Entomology 44, 257-289. Munoz, D., Castillejo, J.I., Caballero, P., 1998. Naturally occurring deletion mutants are parasitic genotypes in a wild-type nudeopolyhedrovirus population of Spodoptera exigua. Applied and Environmental Microbiology 64, 4372-4377. Munoz D., Martinez, A.M., Muriiio, R., Ruiz de Escudero, I., Vilaplana, L. 2001. Basic techniques for the characterization of baculovirus, pp. 479-518. En: Caballero, P., Williams, T., López-Ferber, M. (eds.) Los Baculovirus e sus Aplicaciones como Bioinsecticidas en el Control Biológico de Plagas. Phytoma-Espana, Valencia, Espana. Ogembo, J.G., Kunjeku, E.C., Sithanantham, S., 2005. A preliminary study on the pathogenicity of two isolates of nucleopolyhedroviruses infecting the African bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae). International Journal of Tropical Insect Science 25, 218-222. Ogembo, J.G., Chaeychomsri, S., Kamiya, K., Ishikawa, H., Katou, Y., Ikeda, M., Kobayashi, M., 2007. Cloning and comparative characterization of nucleopolyhedroviruses isolated from African Bollworm, Helicoverpa armigera, (Lepidoptera: Noctuidae) in different geographic regions. Journal of insect Biotechnology and Sericology 76, 39-49. Ogembo, J.G., Caoili, B.L., Shikata, M., Chaeychomsri, S., Kobayashi, M., ikeda, M., 2009. Comparative genomic sequence analysis of novel Helicoverpa armigera nudeopolyhedrovirus (NPV) isolated from Kenya and three others previously sequenced Helicoverpa spp. NPVs. Virus Genes 39, 261-272, Polis, G.A., 1981. The evolution and dynamics of intraspecific predation. Annual Review of Ecology, Evolution and Systematics 12, 225-251. Reed, W., Pawar, C.S., 1982, Heliothis: a global problem, pp. 9-14. En: Reed, W., Kumble, V. (Eds.). Proceedings of the International Workshop on Heliothis Management. ICRISAT, Pantanchera, India. Shieh, T.R., 1989. Industrial production of viral pesticides. Advances in Virus Research 36, 315-343. Simón, O., Wiliiams, T., López-Ferber, M., Caballero, P., 2005. Functional importance of deletion mutant genotypes in an insect nucleopolyhedrovirus population. Applied and Environmental Microbiology 71, 4254-4282. Subramanian, S., Santharam, G., Sathiah, N., Kennedy, J.S., Rabindra, R.J., 2006. Influence of incubation temperature on productivity and quality of Spocioptera Mura nucleopolyhedrovirus. Biological Conírol 37, 367-374. Systat, 1990. Systat: the system for statistics. Systat incorporation, Evaston, Illinois. Theilmann, D. A., Biissard, G. W., Bonning, B., Jehle, J. A., O'Reilly, D. R., Rohrmann, G. F., Thiem, S., Vlak, J. ., 2005. Baculoviridae, pp. 177-185. En: Fauquet, C.M., Mayo, M.A., Maniloff, J., Desselberger, U., Ball, L.A. (Eds.). Eight Report of the International Committee on Taxonomy of Viruses. Academic Press, San Diego, California. Torres-Vila, L.M., Rodríguez-Molina, M.C., Palo, E., Bielza, P., Lacasa, A., 2000. Insecticide resistance of Helicoverpa armigera Hübner in Spain: available data. Plague Plant Health Bulletin 26, 493-501. Torres-Vila, L.M., Rodríguez-Molina, M.C., Lacasa-Plasencia, A., 2003. Impact of Helicoverpa armigera larval density and crop phenology on yield and quality losses in processing tomato: developing fruit count-based damage thresholds for IPM decision-making. Crop Protection 22, 521-532. Washburn, J. O., Kirkpatrick, B. A., Haas-Stapieton, E., Volkman, L. E., 1998. Evidence that the stilbene-derived optical brightener M2R enhances Autographa californica M nucleopolyhedrovirus infection of Trichoplusia ni and Heliothis virescens by preventing sloughing of infected midgut epithelial cells . Biological Control 11, 58-69. Zhang G., 1994. Research, development and application of Heliothis viral pesticide in China. Resource and Environment in the Yangtze Valley 3, 1-6. Zhang, C.X., Ma, X.C., Guo, Z.J., 2005. Comparison of complete genome sequence between C1 and G4 isolates of the Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus. Virology 333, 190-199.

Claims (17)

1. Occlusion body, characterized by the fact that it comprises occlusion-derived virions of two different genotypes that are co-occluded in the same occlusion body, and, wherein the genotypes are selected from the group of simple nucleopolyhedroviruses of Helicoverpa armigera consisting of HearSNPV -SP1B (CNCM I-4806) and HearSNPVLB6 (CNCM I-4807). 2. Composition characterized by the fact that it comprises at least one occlusion body of claim 1. 3. Composition according to claim 2, characterized by the fact that the genotypes HearSNPV-SP1B (CNCM I-4808) and HearSNPV-LB6 (CNCM I-4807) are in the ratio HearSNPVSP1B:HearSNPV-LB6 1:1. 4. The composition according to claim 3, characterized in that the virions are present in occlusion bodies containing co-occluded virions and in which the co-occluded virions of the same nucleopolyhedrovirus belong to the same genotype or different genotypes. 5. Composition according to claim 2, characterized by the fact that it additionally contains an excipient or vehicle suitable in the agricultural sector. 6. Composition according to claim 2, characterized by the fact that the simple nucleopolyhedroviruses of Helicoverpa armigera are mixed with one or more fertilizers, fertilizers, or pesticides. 7. Process for producing occlusion bodies of claim 1, characterized by the fact that it comprises the steps of: i) feeding larvae of Helicoverpa armigera with an artificial diet comprising occlusion bodies of the H. armigera nucleopolyhedrovirus that contain virions of any one of the genotypes HearSNPV-SP1B (CNCM I-4806) and HearSNPV-LB6 (CNCM I-4807) or mixtures thereof; ii) keep the larvae at 23-30°C until death; iii) purify the occlusion bodies generated in the larvae by crushing the larval corpses in water, filtering the resulting suspension, sedimenting the occlusion bodies, washing the sediment and re-sedimenting them; iv) resuspend the final sediment in water at neutral pH; and v) optionally, store the suspension obtained in one of the following conditions: a) at room temperature, b) in refrigeration, or freezing, or c) lyophilize the suspension and store it at room temperature. 8. Process according to claim 7, characterized by the fact that the H. armigera larvae are fifth stage larvae. 9. Process according to claim 7, characterized by the fact that the occlusion bodies with which the larvae are fed are present at a concentration in the range of 2.00 x 107 to 1.00 x 109 occlusion bodies/ml . 10. Method for identifying in a sample the presence of a H. armigera simple nucleopolyhedrovirus of a genotype selected from HearSNPV-SP1B (CNCM I-4806) and HearSNPV-LB6 (CNCM I-4807), characterized by the fact that it comprises the steps of: i) amplifying by PCR the DNA extracted from said sample using a pair of primers selected from those formed by: a) SEQ ID NO: 1 (F-hr1) and SEQ ID NO:2 (R-hr1), or b) SEQ ID NO:3 (F-hr5) and SEQ ID NO:4 (R-hr5); ii) analyze the amplified fragment to determine its size or sequence; iii) digest the amplified fragment with the NdeI endonuclease; iv) analyze the fragments generated after NdeI digestion to determine the number of fragments and the size of each of them; v) conclude that one of the HearSNPV-SP1B (CNCM I-4806) or HearSNPV-LB6 (CNCM I-4807) genotypes is present if: a) the fragment is amplified by the pair of SEQ ID NO: 1 and SEQ ID NO:2 has: i) a size of 2,177 (HearSNPV-SP1B) or 2,117 (HearSNPV-LB6) nucleotides; ii) digestion of said fragment with the endonuclease Ndel generates 6 fragments of 857, 508, 381, 306, 78 and 47 nucleotides (HearSNPV-SP1B) or 5 fragments of 1,210, 475, 307, 78 and 47 nucleotides (HearSNPV-LB6 ); iii) the sequence represented by SEQ ID NO:5 (HearSNPV-SP1B) or SEQ ID NO:6 (HearSNPV-LB6); or, alternatively, b) the fragment amplified by the pair of SEQ ID NO:3 and SEQ ID NO:4 has: i) a size of 2,326 (HearSNPV-SP1B) or 2,330 (HearSNPV-LB6) nucleotides; ii) digestion of said fragment with the Ndel endonuclease generates 4 fragments of 1,120, 917, 211 and 78 nucleotides (HearSNPV-SP1B) or 3 fragments of 1,120, 998 and 212 nucleotides (HearSNPV-LB6); iii) the sequence represented by SEQ ID NO:7 (HearSNPV-SP1 B) or SEQ ID NO:8 (HearSNPV-LB6). 11. Method for controlling insect pests, characterized by the fact that it comprises applying to plants a composition according to claim 2 where the pests are of the genera Helicoverpa or Heliothis. 12. Method according to claim 11, characterized by the fact that it is for controlling pests of Helicoverpa armigera. 13. Composition, characterized by the fact that it comprises: i) HearSNPV deposited in the National Collection of Microorganism Cultures (CNCM) with deposit numbers CNCM I-4806 (HearSNPV-SP1B) and CNCM I-4807 (HearSNPV-LB6), or ii) genotypes whose genome is represented by SEQ ID NO: 13 (HearSNPV-SP1B) and SEQ ID NO: 14 (HearSNPV-LB6). 14. Method for controlling insect pests, characterized by the fact that it comprises applying to plants a composition according to claim 13, where the pests are of the genus Helicoverpa or Heliothis. 15. Method, according to claim 14, characterized by the fact that the pests of the Helicoverpa genera are larvae of Helicoverpa armigera. 16. Composition according to claim 13, which further comprises an excipient or inert vehicle suitable for the agricultural sector. 17. Composition, according to claim 13, characterized by the fact that any of the simple nucleopolyhedroviruses of Helicoverpa armigera is mixed with one or more compounds, fertilizers or pesticides.