Composition containing mixture of DNA molecule, use thereof as biological inhibitor and producing method

Abstract

To provide a composition containing a mixture of DNA molecules suitable for inhibiting targeted species, a method and use using the composition, as well as a method for producing the composition.SOLUTION: A composition for inhibiting targeted species selected from a group consisting of plants, fungi, insects, yeast, bacteria, archaea, algae, nematodes, mites, viruses, and protozoa, which have DNA sequences of different origins, wherein the mixture includes several first DNA sequences that are DNA fragments randomly generated from the chromosomal DNA sequences of the source species, and several second DNA sequences that are the second DNA sequences from host species that are different from the source species, and wherein the mixture of DNA molecules includes chimeric DNA molecules in which the DNA sequences of the source species are adjacent to non-source species' DNA sequences at at least one terminal.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to compositions comprising a mixture of DNA molecules suitable for the inhibition of target species, methods and uses of said compositions, as well as methods for producing said compositions, which may find utility, inter alia, in pest and / or disease control in the fields of human and / or veterinary medicine, agriculture or other fields in which the inhibition of one or more target species is beneficial. [Background technology]

[0002] In recent years, it has become known that fragmented DNA of a certain species is inhibitory to the species from which it originates and to phylogenetically similar species with similar genomes. The first publications to disclose this auto-inhibitory effect of DNA fragments are International Patent Application WO2014 / 020624 and its Italian priority application NA2012A000046. These documents mainly focus on the newly discovered effect of auto-inhibitory DNA fragments. According to these publications, the fragments are produced by sonication, heat treatment, or pyrolysis of the isolated total DNA from the species to be inhibited (or from a phylogenetically similar species), or by random DNA fragment synthesis starting from the total DNA of said species (or from a phylogenetically similar species). Summary of the Invention [Problem to be solved by the invention]

[0003] In order to use DNA fragments to inhibit a species more efficiently, further methods of producing the DNA fragments in an effective form are desired. It has now been surprisingly found that the DNA fragments of the species to be inhibited are effective after being incorporated into DNA not originating from the species to be inhibited. Based on this surprising discovery, the inventors of the present invention have found that the inhibitory DNA fragments of a species can also be effectively produced in a host species unrelated to the species to be inhibited. This opens up new inventive possibilities for producing the inhibitory DNA fragments. [Means for solving the problem]

[0004] The present invention according to a first aspect relates to a composition comprising a mixture of DNA molecules with DNA sequences from different sources, the mixture comprising some first DNA sequences, preferably chromosomal DNA sequences, of a source species, and some second DNA sequences, of a host species, different from the source species. In the composition, the mixture of DNA molecules optionally comprises chimeric DNA molecules in which the source species DNA sequence is flanked at at least one end by a non-source species DNA sequence selected from host species DNA sequences or DNA sequences from artificial DNA constructs. Such a composition is beneficial for the inhibition of a target species selected as source species or selected from species phylogenetically similar to the source species, in that inhibitory DNA fragments can be produced in a host species phylogenetically distant from the target species.

[0005] A further aspect of the present invention relates to the composition of the present invention for use in medicine.Given the fact that the composition of the present invention may be used to inhibit parasitic and / or pathogenic organisms, said composition has utility in human and / or veterinary medicine.

[0006] Furthermore, a further aspect of the present invention relates to the use of the composition according to the present invention as a product for inhibiting target species.This use may be in human and / or veterinary medicine, or may be non-medical use.An example of non-medical use may include, for example, agricultural use, which may be useful for inhibiting organisms that are parasitic and / or pathogenic and / or pests of plants, and / or pests of livestock animals, such as cattle or poultry, or organisms such as weeds that would otherwise interfere with agricultural production.

[0007] Alternatively, the compositions could be used as preservatives, for example in products such as foods that are subject to microbial spoilage, personal health care products, or other products that contain compounds suitable as microbial substrates.

[0008] The present invention according to a further aspect relates to a method of inhibiting a species which is a target species, comprising exposing said target species to a composition according to the invention. The method may be a non-therapeutic method or a therapeutic method.

[0009] According to a further aspect, the present invention provides a method for producing a method for treating a pulmonary circulation comprising the steps of: - providing a source of DNA from a source species, optionally including fragmented source species DNA; - providing cells of a host species which is some species different from said source species; - subjecting the host species to conditions that allow the host species to take up source species DNA in a replicable form; - optionally isolating the host species cells; - optionally isolating DNA from the source seed cells; - optionally fragmenting the DNA optionally isolated from the source seed cells; The present invention also relates to a method for producing a composition according to the present invention, comprising:

[0010] The method is advantageous in that inhibitory DNA for a target species can now be effectively produced outside the target species or a phylogenetically similar species. Autoinhibitory DNA can now be specifically produced in a species phylogenetically unrelated to the target species.

[0011] Yet a further aspect of the invention relates to a method for producing agricultural products, in which the compositions of the invention are used to inhibit organisms that are infectious and / or parasitic and / or pests of plants or livestock animals such as cattle or poultry, or that would otherwise interfere with agricultural production. Inhibition of organisms that are infectious and / or parasitic and / or pests of plants or livestock animals may improve agricultural production by such production organisms. [Brief description of the drawings]

[0012] [Figure 1]Figure 1: Results of the inhibition test for experiment 1. The effect of an Arabidopsis thaliana commercial library on homospecific seedling survival is shown. Data refer to the mean and standard deviation of 10 replicates (dishes containing 10 seedlings) per treatment. Significant differences compared to the control are marked with asterisks (*, p<0.05; **, p<0.01; ***, p<0.001). [Diagram 2] Figure 2. Inhibition study of experiment 2. The effect of Cyperus aesculentus genomic BAC library on homogenous seedling growth on two different substrate types is shown. Data refer to the mean and standard deviation of 10 replicate pots per treatment. Significant differences compared to the control are marked with asterisks (*, p<0.05; **, p<0.01; ***, p<0.001). [Diagram 3] Figure 3: Results of the inhibition study from experiment 3. The effect of a Caenorhabditis elegans genomic BAC library in growth medium on homozygous embryonic lethality (top) and developmental defects (bottom) compared to an E. coli strain (EPI300) containing heterologous DNA but no self-DNA genomic library is shown. Data refer to the mean and standard deviation of 12 replicates per treatment. [Figure 4] Figure 3: Results of the inhibition test for experiment 4. The effect of Spodoptera littoralis commercial library on conspecific larval survival is shown. Data refer to the mean and standard deviation of 10 replicates per treatment. Significant differences compared to the control are marked with asterisks (*, p<0.05; **, p<0.01; ***, p<0.001). [Diagram 5] FIG. 14. Experiment 9, total number of purslane plants in different treatment blocks at the end of the study. [Figure 6] Figure 1 shows the phylogenetic tree of angiosperms from AGP (2009) used in experiment 10 to determine phylogenetic distances. [Figure 7]Figure 1 shows the root growth of seven target plant species treated with either self-DNA or heterospecific DNA extracted from heterospecifics. Each bar represents the mean and standard deviation of four replicate bioassays. In each panel, the plant species used for DNA extraction are ranked by their phylogenetic distance from the target species, determined as described in the methods of experiment 10. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The composition of the present invention comprises a mixture of DNA molecules with DNA sequences from different sources. The mixture of DNA molecules comprises some first DNA sequences of a source species and some second DNA sequences of a host species different from the source species. As those skilled in the art will understand, the term "species" refers to an abstract concept, and as such species cannot be inhibited. Reference to species should therefore be interpreted as meaning a plurality of individuals or organisms of a species, i.e., individuals or organisms of a species, such as a population.

[0014] As the term mixture suggests, the composition comprises different DNA molecules. The DNA molecules in the composition have different sequences and preferably different sizes. The DNA sequences of the DNA molecules in the composition differ by being from different sources. In particular, a first set of DNA molecules has sequences of a source species, which is a first species, and a second set of DNA molecules has sequences of a host species, which is a second species different from the source species. Furthermore, the number of source species DNA sequences is also different. Furthermore, the number of host species DNA sequences is also different. Thus, there is variation within the population of source species DNA sequences and within the population of host species DNA sequences.

[0015] In the composition, the source species DNA sequence can be integrated into the DNA molecule of the host species' native genome, such as a chromosome from the host species cell's native genome or a native plasmid, preferably as a subsequence (fragment) of chromosomal DNA, or into an artificial DNA construct introduced into the host species cell, thus forming a chimeric DNA. Alternatively, the source species DNA sequence in the composition is destructively released from such a chimeric DNA, for example by fragmentation. The DNA molecule in the composition can thus comprise one or more of the following: the complete (intact) native chromosome of the species, the complete (intact) native extrachromosomal DNA of the species (such as a natural plasmid), the complete (intact) artificial DNA construct (such as a plasmid, cosmid, fosmid or artificial chromosome), the fragmented native chromosome of the species, the fragmented native extrachromosomal DNA of the species (such as a natural plasmid), or the fragmented artificial construct (such as a plasmid, cosmid, fosmid or artificial chromosome). In the composition of the present invention, such DNA molecules can also be present in combination. These alternative DNA molecules may vary in sequence and size, and it will be apparent to one of skill in the art how they may vary.

[0016] The DNA molecules comprising the source species DNA sequence and the DNA molecules comprising the host species DNA sequence are preferably DNA fragments. The skilled artisan will understand that the term "fragmented DNA" and related terms such as "DNA fragments" are substrands (or subsequences) of a larger DNA molecule. DNA fragments may be obtained by DNA fragmentation (degradation of a DNA molecule) or by synthesis of DNA starting from a polynucleotide template. DNA fragments are therefore DNA molecules that have a subsequence of a larger DNA molecule. In the context of the present invention, the inhibitory DNA fragments are preferably obtained by DNA fragmentation. In the context of the present invention, the term "comprising" and related terms such as "comprises" include "consists of".

[0017] The DNA fragments may be generated by any suitable means, in particular by techniques for random DNA fragment generation. WO2014 / 020624 discloses several suitable techniques for generating self-inhibiting DNA fragments. Among others, sonication, heat treatment, or pyrolysis of DNA isolated from a source species are disclosed. Alternatively, random DNA fragment synthesis starting from the DNA of a source species can also be used for random generation of DNA fragments. These and other techniques for generating DNA fragments are known to those skilled in the art and can be used based on the available common general knowledge. Further reference may be made to Mann and Krull (Mann and Krull 2004. Biosensors and Bioelectronics 20:945-955), Tong et al. (Tong et al. 2006. Nat. Protoc, 1(2):729-748), Fan et al. (Fan et al. (2008). Nucleic Acid Research, 36(19): e125) and Standard protocols DNA shearing for Bioruptor® Pico (available from the diagenode website www.diagenode.com). Alternatively, endonucleases such as DNAse I, endonuclease V or restriction enzymes may be used to fragment the source species DNA.

[0018] DNA fragmentation is also frequently used in (next-generation) DNA sequencing methods. In this context, the skilled person can also make use of the body of knowledge developed in the context of DNA fragmentation.

[0019] Furthermore, DNA synthesis methods that synthesize a portion of a genomic sequence can also be used to obtain DNA fragments. For example, PCR methods, such as random PCR, may be suitable. Also, cDNA molecules obtained by reverse transcription of mRNA may be considered as DNA fragments within the context of the present invention, since such cDNA molecules represent a partial sequence of a larger DNA molecule. The synthesis of suitable cDNA molecules is within the knowledge of a person skilled in the art.

[0020] Furthermore, processes such as (biological) digestion, decomposition, and similar processes acting on DNA-containing biological material, such as cells, can also result in fragmentation of the DNA contained in the biological material. Thus, it is not required that the DNA molecule comprising the source species DNA sequence is a DNA fragment, since DNA fragments can also be released from larger molecules by such processes.

[0021] The technique selected for generating DNA fragments is preferably a technique suitable for generating random DNA fragments. Random DNA fragments should be understood to mean DNA fragments generated randomly. It is a DNA fragment generated without a predetermined pattern.

[0022] From research on DNA fragmentation in the context of (next-generation) DNA sequencing, knowledge has emerged that certain techniques for random DNA fragmentation, such as (partial) restriction enzyme digestion and sonication, may have a certain degree of bias towards certain regions and / or sequences of the genome. As the skilled person will understand from the inhibition of self-DNA disclosed in WO2014 / 020624, this bias is not related to the inhibitory effect of randomly generated fragments of self-DNA.

[0023] In the compositions of the present invention, the DNA molecule is preferably provided as a double-stranded DNA molecule (dsDNA). This does not necessarily mean that the DNA molecule performs its biological inhibitory function in the double-stranded state.

[0024] Without wishing to be bound by this theory, it seems plausible that for double-stranded DNA molecules, the single-stranded DNA sequences resulting from the (partially) unwound dsDNA are responsible for the inhibitory effect. DNA molecules in double-stranded form may therefore exert an inhibitory effect via a (partially) DNA unwinding mechanism.

[0025] The compositions are from about 50 bases (50 bp) to about 2 Mb (2 * 10 6bp), for example, 50 to 3000 bp, 100 to 1500 bp, or 100 to 2000 bp, 100 to 2500 bp, or 0.3 to 15 kb, for example, 1 to 10 kb, for example, 5 to 10 kb, or 15 to 60 kb, for example, 15 to 40, 25 to 40 kb, 24 to 40 kb, or 40 to 340 kb, for example, 50 to 200 kb, for example, 120 to 300 kb, etc. The DNA molecules may include DNA molecules having source seed DNA sequences ranging from 200 to 300 kb, or 250 kb to 2 Mb, such as 400 kb to 1500 kb or 400 kb to 1000 kb, or 0.3 kb to 2 Mb, such as 1 kb to 2 Mb, such as 10 kb to 2 Mb, such as 100 kb to 2 Mb, such as 360 kb to 1 Mb. Depending on whether the source seed DNA sequences are free fragmented DNA sequences or whether they are incorporated into a larger DNA molecule containing non-source seed DNA sequences, their sizes may vary. Free fragmented DNA sequences will preferably have more sizes towards the lower range. Source seed DNA sequences that are incorporated into a larger DNA molecule will have a maximum size range depending on what can be stably incorporated into the respective DNA molecule. Those skilled in the art will understand and appreciate that different cloning and / or expression vectors can accommodate different sizes of DNA inserts. Table 1 provides an overview of the various cloning / expression vector systems envisaged for use in certain embodiments of the invention, with the respective insert sizes according to these embodiments. When the composition comprises DNA of an artificial DNA construct (vector, in particular a cloning vector), the artificial DNA construct is preferably selected from a plasmid, a cosmid, a fosmid, or an artificial chromosome, such as a bacterial artificial chromosome, a yeast artificial chromosome, or a fungal artificial chromosome. [Table 1]

[0026] The compositions preferably include fragments of chromosomal DNA from a source species and / or a host species. Those skilled in the art are aware of the scope of the term "chromosomal DNA" and will be able to provide the chromosomal DNA of a particular organism from which the DNA fragments may be derived.

[0027] The species from which the inhibitory DNA sequence is derived is referred to herein and in the claims as the "source species". It will be clear that the term "source species" is used as a reference term. The term is used to easily distinguish the source species from the "host species" and the "target species" which are discussed further below.

[0028] According to certain embodiments, in the composition of the present invention, the DNA molecule may be free DNA. The term "free DNA" refers to a state in which the DNA fragment is not incorporated into an envelope such as a cell or a virus particle. The free DNA fragment may be provided in any suitable form. For example, the free DNA fragment may be in solution in a suitable solvent such as water or an aqueous mixture. The solvent may be a single-phase solution or part of a multi-phase such as an emulsion or dispersion. Alternatively, the free DNA fragment may be present in the composition of the present invention in a solid. The solid containing the free DNA may be a solid in a single phase or a solid in a multi-phase such as a dispersion.

[0029] According to a particular embodiment, the mixture of DNA molecules in the composition of the present invention comprises chimeric DNA molecules in which a source species DNA sequence is flanked at at least one end by a non-source species DNA sequence. Those skilled in the art will be aware of the meaning of the term "chimeric DNA molecule" and will understand that this term refers to a DNA molecule that comprises DNA sequences from at least two different sources, such as from a first species and from at least one different source (e.g., from a second species different from the first species). The source species DNA sequence and at least one flanking non-source species DNA sequence are thus in a single DNA molecule. Alternatively, chimeric DNA molecules may be referred to as hybrid DNA molecules.

[0030] In a chimeric DNA molecule, a source species DNA sequence is flanked at at least one end by a non-source species DNA sequence. The term "at least one" in the present description is equivalent to "several" and means "one or more" whenever either of these terms are used, and vice versa. In certain preferred embodiments, these terms mean a plurality, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10, as appropriate. Those skilled in the art will recognize that due to the structure of DNA molecules, a source species DNA sequence may be flanked at no more than two ends by a non-source species DNA sequence in a chimeric DNA molecule.

[0031] As one of skill in the art will understand, flanking DNA sequences occupy positions beside each other. Thus, the term "flanked" is used in the definition of the present invention to mean, and can be interchanged with, "border" or "adjoin" or "abut." Although this is already suggested by the context in which the term "flanking" (and any of its equivalent terms) is used, for clarity, it is specified that a source species DNA sequence is linked to at least one adjacent non-source species in a single DNA molecule.

[0032] A non-source species DNA sequence is a DNA sequence that originates from a source species other than the source species. A non-source species DNA sequence may be a naturally occurring DNA sequence from a species (such as from a host species) different from the source species, such as a chromosomal DNA sequence. Alternatively, a non-source species DNA sequence may be an artificial DNA sequence, such as from an artificial DNA construct, such as a DNA sequence from an artificial plasmid, cosmid, fosmid or artificial chromosome. Within the context of the present invention, an artificial chromosome may be selected from a yeast or fungal artificial chromosome or a bacterial artificial chromosome.

[0033] Chimeric DNA molecules comprising a source species DNA sequence flanked at at least one end by a non-source species DNA sequence can be obtained by procedures known to those skilled in the art. According to certain embodiments, where the source species DNA sequence is a chromosomal DNA sequence and the non-source species DNA sequence is a sequence derived from an artificial plasmid, the DNA of the chromosomal source species (possibly together with the DNA of other parts of the total genome) can be isolated and (randomly) fragmented, for example by (partial) restriction enzyme digestion. The digested source species DNA can then be ligated to an artificial plasmid cut with a compatible restriction enzyme, thus producing a population of chimeric plasmids with different subsequences (fragments) of the integrated source species DNA. Compositions comprising such a mixed population of plasmids, as well as equivalent or derived compositions, where for example plasmids are (randomly) fragmented into DNA fragments, are embodiments of the compositions of the present invention. According to other embodiments, where the source species DNA sequence is a chromosomal DNA sequence and the non-source species DNA sequence is also a chromosomal DNA sequence, the chromosomal source species DNA (possibly together with the DNA of other parts of the total genome) may be isolated and (randomly) fragmented, for example by (partial) restriction enzyme digestion, sonication or other means of mechanical shearing, and the fragmented source species DNA may be contacted with cells of the host species that can absorb and integrate the foreign DNA fragments into their genome. There is increasing scientific knowledge about horizontal (or lateral) gene transfer between organisms of different species, and it has been shown that, in particular, microorganisms, and in particular prokaryotes, are capable of absorbing and integrating foreign DNA into their genome, including their chromosomal DNA. Furthermore, transposable elements (or transposons) can facilitate DNA transfer between cells of different species, and the source species DNA sequence may be integrated into the transposable element to facilitate integration into the host species genome. When foreign DNA is integrated into the DNA of the recipient organism during horizontal gene transfer, a chimeric DNA molecule is formed in which the foreign (source) DNA is adjacent to the DNA of the recipient (host) organism.Compositions comprising such mixed populations of host organisms that have integrated source species DNA sequences into their chromosomal DNA, and equivalent or derived compositions in which, for example, host species chromosomal DNA has been isolated (possibly together with DNA from other parts of the total genome) and (randomly) fragmented into DNA fragments, are therefore alternative embodiments of the compositions of the present invention.

[0034] It should be understood that the chimeric region of a chimeric DNA molecule (the region where the source species DNA sequence is adjacent to the non-source species DNA sequence) can be very small compared to the length of the source species DNA sequence and / or the length of the non-source species DNA sequence. Thus, when a chimeric DNA molecule is fragmented, there may be a high probability that the DNA fragments contain only source species DNA sequences or only non-source species DNA sequences. Thus, the number of chimeric DNA fragments may be very small.

[0035] The source species is preferably selected such that the fragmented DNA sequence derived therefrom is inhibitory to the target species. As will be clear to those skilled in the art after the publication of WO2014 / 020624, the DNA fragments that are inhibitory to the target species may be derived from the target species itself or from a species that is phylogenetically similar. In the context of the present invention, the source species may therefore be selected from the target species or a species that is phylogenetically similar to the target species. As will be understood by those skilled in the art, the term "inhibition" in the context of inhibition of a target species refers to preventing, slowing or even stopping the development of individuals of the target species and / or populations of the target species. The inhibitory effect of the inhibitory self-DNA may be expected to exert its effect by interfering with the physiological function of the target species at the cellular level. Self-DNA should be understood to mean the DNA of a species or a species that is phylogenetically similar.

[0036] The target species may be a species selected from plants, fungi, insects, yeasts, bacteria, archaea, algae, nematodes, acaridae, viruses and protozoa, preferably a species that may cause health and / or economic and / or environmental damage. Such target species may for example be disease-associated species, such as species that act as pathogenic species, parasitic species or disease vectors, or may be an infesting species, or may be a species associated with the deterioration of products, such as food and / or cosmetics and / or pharmaceuticals and / or other products containing organic matter. Disease-associated species may cause and / or facilitate the spread of disease to animals, in particular humans and / or livestock animals, or to plants, in particular crops. Ectoparasitic species may be any species, such as insect species, or higher animal species, or plant species, whose individuals are present in a place or site (target area) in greater numbers than desired. Ectoparasitic species at least cause harm and may (potentially) cause damage or damage. Ectoparasitic species according to certain embodiments may therefore be considered as pests. As the skilled artisan will appreciate, biological species can cause product deterioration in various ways. The mere presence of an individual of a certain species in a food or the like is often undesirable, especially if the species can produce off-flavors and / or toxins. Furthermore, the transformation of organic matter present in a product can lead to a decrease in the quality of the product, such as by the product not complying with the product specifications and / or by (partial) loss of product functionality. It will be clear to the skilled artisan that the terms "disease-associated species", "pathogenic species", "parasitic species", "species acting as disease vectors", "ectoparasitic species" and "species associated with product deterioration" are not mutually exclusive, and that there is a degree of overlap between two or more of these terms. The terms are merely used to identify domains in which inhibition of the target species may be beneficial and in which the present invention is preferably used.

[0037] If the target species is selected as a pathogenic species, the pathogenic species may be Acinetobacter baumannii, or Actinomyces israelii, or Actinomyces gerencseriae, or Propionibacterium propionicus, or Trypanosoma brucei, or HIV (human immunodeficiency virus), or Entamoeba histolytica, or Anaplasma species, or Angiostrongylus species, or Anisakis species, or Bacillus anthracis, or Arcanobacterium haemolyticum, or Junin virus, or Ascaris lumbricoides, or lumbricoides, or Aspergillus spp., or Astroviridae spp., or Babesia spp., or Bacillus cereus, or Bacteroides spp., or Balantidium coli, or Bartonella spp., or Baylisascaris spp., or BK virus, or Piedraia hortae, or Blastocystis spp., or Blastomyces dermatitidis, or Machupo virus, or Clostridium botulinum, or Sabia virus, or Brucella spp., or Yersinia Pestis, or Burkholderia cepacia, or other Burkholderia spp., or Mycobacterium ulcerans, ulcerans, or Caliciviridae, or Campylobacter species, or Candida albicans, or other Candida species, or Capillaria philippinensis, or Capillaria aerophila, or Bartonella basiliformis,bacilliformis, or Bartonella henselae, or Group A Streptococcus spp., or Staphylococcus spp., or Trypanosoma cruzi, or Haemophilus ducreyi, or Varicella zoster virus (VZV), or an alphavirus, or Chlamydia trachomatis, or Chlamydophila pneumoniae, or Vibrio cholerae, or Fonsecaea pedrosoi, or Batrachochytrium dendrabatidis, or Clonorchis sinensis, or Clostridium difficile, difficile, or Coccidioides immitis and Coccidioides posadasii, or Colorado tick fever virus (CTFV), or Rhinovirus spp., or Coronavirus, or PRNP, or Crimean-Congo hemorrhagic fever virus, or Cryptococcus neoformans, or Cryptosporidium spp., or Ancylostoma braziliense, or Cyclospora cayetanensis, or Taenia solium solium, or Cytomegalovirus spp., or Dengue viruses (DEN-1, DEN-2, DEN-3 and DEN-4), or Flavivirus spp., or Chlorophyceae, or Desmodesmus armatus, or Dientamoeba fragilis, or Corynebacterium diphtheriae, or Diphyllobothria, or Dracunculus guinea worm,medinensis, or Ebola virus (EBOV), or Echinococcus spp., or Ehrlichia spp., or Enterobius vermicularis, or Enterococcus spp., or Enterovirus spp., or Rickettsia prowazekii, or Parvovirus B19, or Human herpesvirus 6 (HHV-6), or Human herpesvirus 7 (HHV-7), or Fasciola hepatica, or Fasciola gigantica, or Fasciolopsis buski, or PRNP, or Filaria superfamily, or Clostridium perfringens, perfringens, or multiple, or Fusobacterium spp., or Clostridium perfringens, or other Clostridium spp., or Geotrichum candidum, or Giardia lamblia, or Burkholderia mallei, or Gnathostoma spinigerum, or Gnathostoma hispidum, or Neisseria gonorrhoeae, or Klebsiella granulomatis, or Streptococcus pyogenes, or Streptococcus agalactiae, or Haemophilus influenzae influenzae, or enteroviruses such as Coxsackie A virus and enterovirus 71 (EV71), or Sin Nombre virus, or Heartland virus, or Helicobacter pylori, or Escherichia coli O157:H7, O111 and O104:H4, or species from the Bunyaviridae family, or Hendra virusvirus, or Hepatitis A virus, or Hepatitis B virus, or Hepatitis C virus, or Hepatitis D virus, or Hepatitis E virus, or Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), or Histoplasma capsulatum, or Ancylostoma duodenale and Necator americanus, or Human bocavirus (HBoV), or Ehrlichia ewingii, or Anaplasma phagocytophilum, or Human metapneumovirus (hMPV), or Ehrlichia chaffeensi, chaffeensis, or one of the human papillomaviruses, or human parainfluenza virus (HPIV), or Hymenolepis nana and Hymenolepis diminuta, or Epstein-Barr virus (EBV), or Orthomyxoviridae, or Isospora belli, or Kingella kingae, or Lassa virus, or Legionella pneumophila, or Legionella varicella, or Leishmania species, or Mycobacterium leprae, or Mycobacterium lepromatosis, or Leptospira species, or Listeria monocytogenes, or Borrelia burgdorferi, or Borrelia garinii, garinii, or Borrelia afzelii, or Wuchereria bancrofti, or Brugiamalayi, or Lymphocytic choriomeningitis virus (LCMV), or Plasmodium species, or Marburg virus, or Measles virus, or Middle East respiratory syndrome coronavirus, or Burkholderia pseudomallei, or Neisseria meningitidis, or Metagonimus yokagawai, or Microsporidia phylum, or Molluscum contagiosum virus (MCV), or Monkeypox virus, or Mumps virus, or Rickettsia typhi, or Mycoplasma pneumoniae, or Mycoplasma genitalium, genitalium, or Actinomycetoma spp., or Eumycetoma spp., or Chlamydia trachomatis and Neisseria gonorrhoeae, or Nipah virus, or Norovirus, or Nocardia asteroides, or other Nocardia spp., or Onchocerca volvulus, or Opisthorchis viverrini, or Opisthorchis felineus, or Paracoccidioides brasiliensis, or Paragonimus westermani, or other Paragonimus spp., or Pasteurella spp., or Pediculus humanus capitis, or Pediculus humanus corporis, or Pthirus pubis, or Bordetella pertussis, or Yersinia pestis, or Streptococcus pneumoniae, or Pneumocystis jirovecii, or poliovirus, or Prevotella species, or Naegleria fowleri, or JC virus, or Chlamydophilapsittaci, or Coxiella burnetii, or rabies virus, or Borrelia hermsii, Borrelia recurrentis, or other Borrelia species, or respiratory syncytial virus (RSV), or Rhinosporidium seeberi, or rhinovirus, or Rickettsia species, or Rickettsia akari, or Rift Valley fever virus, or Rickettsia rickettsii, or rotavirus, or rubella virus, or Salmonella species, or SARS coronavirus, or Sarcoplasmic reticulum, or ptes scabiei, or Group A Streptococcus species, or Schistosoma species, or Shigella species, or Varicella zoster virus (VZV), or Variola major virus, or Variola minor virus, or Sporothrix schenckii, or Staphylococcus species, or Strongyloides stercoralis, or Measles virus, or Treponema pallidum, or Cestode species, or Clostridium tetani, or Trichophyton species, or Trichophyton tonsurans, or Epidermophyton floccosum, or Trichophyton rubrum, or Trichophyton mentagrophyton mentagrophytes, or Trichophyton rubrum, or Hortaea werneckii, or Malassezia spp., or Toxocara canis, or Toxocara cati, or Toxoplasma gondii, or Chlamydia trachomatis, or Trichinella spiralis, or Trichomonas vaginalis, or Trichuris trichiura, or Mycobacterium tuberculosis, or Francisella tularensis, or Salmonella enterica, or Serovar typhi, or Rickettsia, or Ureaplasma urealyticum, urealyticum, or Coccidioides immitis, or Coccidioides posadaci, or Venezuelan equine encephalitis virus, or Guanarito virus, or Vibrio vulnificus, or Vibrio parahaemolyticus, or West Nile virus, or Trichosporon beigelii, or Yersinia pseudotuberculosisThe strain may be selected from the group consisting of Yersinia pseudotuberculosis, Yersinia enterocolitica, Yellow fever virus, Zeaspora fungus, Zika virus, Mucorales, and Entomomycota.

[0038] It should be understood that the inhibition of the pathogenic target species does not have to be in or on the body of an animal (including a human). Instead, the inhibition may also be outside the context of the animal's body, for example to inhibit the target species in culture (in vitro). The selection of the pathogenic target species from local pathogenic target species and / or local target species, especially from the list directly presented above, is preferred. The skilled person will understand that the term local in the context of human and veterinary medicine means to relate to a particular surface of the body, especially the skin or mucous membrane (mucosa). Local pathogenic target species should therefore be considered to be related to the skin and / or nails, and / or mucous membranes, including the mucous membranes of the eyes, mouth, vagina, urinary tract, gastrointestinal tract, respiratory tract, including the lungs. The term local is therefore not limited to the external surfaces of the animal's body, but includes reference to internal surfaces such as the lungs and gastrointestinal tract. The local pathogenic target species is most preferably a skin pathogen and / or a nail pathogen, and / or a mucous membrane pathogen. Within the context of the present invention, the selection of pathogenic target species from archaea, bacteria, fungi (including yeasts) and protists is further preferred, in particular from the list of archaea, bacteria, fungi (including yeasts) and protists directly presented above.

[0039] When the target species is selected as the parasitic species, the parasitic species may be Acanthamoeba spp., or Balamuthia mandrillaris, or Babesia B. divergens, or Babesia bigemina, or Babesia equi, or Babesia microfti, or Babesia duncani, or Balantidium coli, or Blastocystis spp., or Cryptosporidium spp., or Cyclospora cayetanensis, or Dientamoeba, or Entamoeba histolytica, or Giardia lamblia, or Isospora warblers, or Leishmania spp., or Naegleria fowleri, or Plasmodium falciparum, or Plasmodium vivax, vivax, or Plasmodium ovale curtisi, or Plasmodium ovale Wallikeri, or Plasmodium malariae, or Plasmodium knowlesi, or Rhinosporidium seeberi, or Sarcocystis bovihominis, or Sarcocystis suihominis, or Toxoplasma gondii, or Trichomonas vaginalis, or Trypanosoma brucei, or Trypanosoma cruzi, or Cestoda, or Taenia, or Diphyllobothrium broad-segmentum latum), or Echinococcus granulosus, or Echinococcus multilocularis, or Echinococcus vogeli, or Echinococcus lycopersicon (E. vogeli), or E.oligarthrus, or Diminuta oligarthus, or Diminuta oligarthus, or Taenia saginata, or Taenia solium, or Bertiella mucronata, or Bertiella studeri, or Spirometra, or Erinaceieuropaei, or Schistosoma haematobium, or Schistosoma japonicum, or Schistosoma mekongi, or Echinostoma echinatum, or Trichobilharzia regenti, or Schistosoma family, or Ancylostoma duodenale, or Necator americanus, or Angiostrongylus spp., or costaricensis, or Anisakis, or Ascaris spp., or Ascaris lumbricoides, or Baylisascaris procyonis, or Brugia timori, or Dioctophyme renale, or Dracunculus medinensis, or Enterobius vermicularis, or Enterobius gregorii, or Gnathostoma spinigerum, or Gnathostoma hispidum, or Halicephalobus gingivalis, or Loa loa filaria, or Mansonella Streptocerca, or Onchocerca volvulus, or Strongyloides stercoralis, or Thelazia californiensis, or Thelazia callipaeda, or Roundworm of the Dog, or Roundworm of the Cat, or Trichinella spiralis.

[0040] As noted in the context of pathogenic target species, it should be understood that the inhibition of the parasitic target species does not have to be in or on the body of an animal (including human). Instead, inhibition may also be outside the context of the animal's body, for example to inhibit the target species in culture. The selection of the parasitic target species is preferred from skin parasites and / or digestive tract parasites and / or mucosal parasites, in particular from the list directly presented above. The selection of the parasitic target species is preferred from protozoa or nematodes, in particular from the list directly presented above of protozoa and nematodes.

[0041] According to certain embodiments, the target species is a fungus, or an oomycete, or a bacterium, or a virus, or a protist, or a Fusarium spp., or a Thielaviopsis spp., or a Verticillium spp., or a Magnaporthe spp., or a Magnaporthe grisea, or a Sclerotinia spp., or a Sclerotinia sclerotiorum, or a Phytophthora spp., or a Pythium spp., or a Spongospora spp., or a phytopathogenic bacillus. bacilli), or Erwinia spp., or Agrobacterium spp., or Burkholderia spp., or Proteobacteria, or Xanthomonas spp., or Pseudomonas spp., or Phytoplasma spp., or Spiroplasma spp.

[0042] If the target species is selected as an ectoparasitic species, the ectoparasitic species may be an agricultural pest arthropod (e.g., Acalymma, or Acyrthosiphon kondoi, or Acyrthosiphon gossypii, or Acyrthosiphon pisum, or African armyworm, or African honey bee, or Agromyzidae, or Agrotis ipsilon, or Agrotis munda, or Agrotis orthogonia, or Agrotis porphyricollis, or Akkaia taiwana, or Aleurocanthus citri, or Aleurocanthus spp.). woglumi, or Aleyrodes proletella, or Alphitobius diaperinus, or Alsophila aescularia, or Altica chalybea, or Anasa tristis, or Anisoplia austriaca, or Anthonomus pomorum, or Anthonomus signatus, or Aonidiella aurantii, or Aonidiella citrina, or Aonidiella orientalis, or Apamea apamiformis apamiformis, or Apamea niveivenosa, or Aphid gossypii, or Aphis gossypii, or Aphis nasturtii, or Apple fly, or Argentine ant, or Army cutworm, or Armycutworm, or Armyworm, or Arotrophora arcuatalis, or Ash whitefly, or Astegopteryx bambusae, or Astegopteryx insularis, or Astegopteryx minuta, or Asterolecanium, or Asterolecanium coffeae, or Atherigona reversura, or Athous haemorrhoidalis, or Cucumber beetle, or Aulacorthum solani, or Australian grasshopper, or potato psyllid, or Bactericera cockerelli, or fruit fly, or Bactrocera, or Bactrocera correcta, or Bagrada hilaris, or Knulliana, or armyworm, or black bean aphid, or Blepharidopterus chlorionis, or bogong moth, or boll weevil, or bollworm, or radish aphid, or Brown locust, or locust, or brown planthopper, or diamondback moth, or green caterpillar, or Callosobruchus maculatus, or carrot fly, or Cerataphis brasiliensis, or Ceratitis aliena, or Ceratitis andranotobaka, or Ceratitiscapitata, or Ceratitis flexuosa, or Ceratitis grahami, or Ceratitis ovalis, or Ceratitis penicillata, or Ceratitis rosa, or Ceratoglyphina bambusae, or Ceratopemphigus zehntneri, or Ceratovacuna lanigera, or Cereal leaf beetle, or Chaetosiphon tetrarhodum, or Chlorops pumilionis, pumilionis, or Asian longhorn beetle, or Coccus hesperidum, or Coccus viridis, or Codling moth, or Coffee flea beetle, or Colias eurytheme, or Colorado potato beetle, or Common blossom thrips, or Red flour beetle, or Cotton bush budworm, or Crambus, or Cucumber beetle, or Curculio elephas, or Curculio nucum, or Curculio occidentis, or Cutworm, or Cyclocephala borealis borealis, or Dargiada diffusa, or Dasineura brassicae, or Date stone beetle, or Delia fly, or Onion fly, or Delia antiqua, or Delia floralis, or Seed fly, or Delia platura, or Cabbage fly, or Deliaradicum, or Dermestes ater, or Dermolepida albohirtum, or Desert grasshopper, or Diabrotica, or Diabrotica balteata, or Diabrotica speciosa, or Diamondback moth, or Diaphania indica, or Diaphania nitidalis, or Diaphorina citri, or Diaprepes abbreviatus, or Diatraea saccharalis, or Differential grasshopper, grasshopper, or Diparopsis castanea, or Dociostaurus maroccanus, or Drosophila suzukii, or Dryocosmus kuriphilus, or Dysaphis crataegi, or Dysmicoccus brevipes, or Earias perhuegeli, or Epicauta vittata, or Epilachna, or Epitrix cucumeris, or Epitrix tuberis, or Erionota thrax, thrax, or Eriosoma lanigerum, or Aphididae, or Euleia heraclei, or Eumetopina flavipes, or European corn borer, or Eurydema oleracea, or Eurygaster integricepsintegriceps, or Ferrisia virgata, or Forest bug, or Frankliniella tritici, or Galleria mellonella, or Garden dart, or Sugarcane aphid, or Geoica lucifuga, or Glassy-winged sharpshooter, or Greenhouse whitefly, or Greenidea artocarpi, or Greenidea formosana, or Greenideoida ceyloniae, or Gryllotalpa orientalis, or Gryllotalpa vinae, vinae, or American gypsy moth, or Helicoverpa armigera, or Helicoverpa gelotopoeon, or Helicoverpa punctigera, or Helicoverpa zea, or Heliothis virescens, or Henosepilachna vigintioctomaculata, or Henosepilachna vigintioctopunctata, or wheat gall midge, or Heteronychus arator, or Hyalopterus pruni, or Hysteroneura setariae, or Icerya scale insect, purchasi, or Ipuka dispersum, or Jacobiasca formosana, or Kaltenbachiellaelsholtriae, or Kaltenbachiella japonica, or the small red-spotted grasshopper, or Lampides boeticus, or the leaf-mining insect, or Lema daturaphila, or Lepidiota consobrina, or the citrus persimmon scale (Lepidosaphes beckii), or the apple persimmon scale (Lepidosaphes ulmi), or Leptocybe invasa, or Leptoglossus zonatus, or Leptopterna dolabrata, or the lesser wax moth, or the genus Leucoptera, or the coffee leaf miner (Leucoptera caffeina), or the light brown apple moth, or the light brown apple moth controversy, or the false radish aphid (Lipaphis erysimi), or the black-legged leaf miner (Liriomyza huidobrensis), or the rice water weevil (Lissorhoptrus oryzophilus, or Listronotus bonariensis, or Long-tailed skipper, or Lygus, or Lygus hesperus, or Macrodactylus subspinosus, or Macrosiphoniella pseudoartemisiae, or Macrosiphoniella sanborni, or Macrosiphum euphorbiae, or Oryzophilus or Manduca sexta, or Matsumuraja capitophoroides, or Mayetiola hordei, hordei, or mealybug, or Megacopta cribraria, or Melanaphis sacchari, or Melittobia australica, or Metcalfa pruinosapruinosa, or ladybug, or Micromyzus judenkoi, or Micromyzus kalimpongensis, or Micromyzus niger, or moth, or leek moss, or American armyworm, or Mythimna unipuncta, or Myzus ascalonicus, or Myzus boehmeriae, or Myzus cerasi, or Myzus obtusirostris, or Myzus ornatus, or Myzus persicae, or Nematus, or Nematus leucotrox leucotrochus, or Nematus ribesii, or Nematus spiraeae, or Neomyzus circumflexus, or Neotoxoptera oliveri, or Nezara viridula, or Oak processionary, or Oebalus pugnax, or Olive fruit fly, or Ophiomyia simplex, or Opisina arenosella, or Opisina arenosella, or Opomyza florum, or Orseolia oryzae, or Oryzaephilus mercator, or Oscinella frit, or Ostrinia furnacalis, or Oxycarenus hyalinipennis, or Papilio demodocusdemodocus, or Paracoccus marginatus, or Paratachardina pseudolobata, or Paropsisterna selmani, or Patanga succincta, or Pemphigus betae, or Pentalonia nigronervosa, or Hemiptera, or Peridroma saucia, or Phorodon humuli, or Phthorimaea operculella, or Phylophaga, or Phyllotreta nemorum, nemorum, or the Phylloxeroidea, or Phytomyza horticola, or Pieris brassicae, or the pink bollworm, or Planococcus citri, or Platynota idaeusalis, or Plum curculio, or Prionus californicus, or the genus Pristiphora, or Pseudoregma bambucicola, or Pseudotheraptus wayi, or Psylliodes chrysosephala, chrysocephala, or Ptinus fur, or Pyralis farinalis, or Raphidopalpa foveicollis, or Fire ants, or Red grasshoppers, or Rhagoletis cerasi, or Rhagoletis indifferens, or Rhagoletis mendakismendax, or Rhodobium porosum, or Rhopalosiphoninus latysiphon, or Rhopalosiphum maidis, or Rhopalosiphum padi, or Rhopalosiphum rufiabdominale, or Rhyacionia frustrana, or Rhynchophorus ferrugineus, or Rhynchophorus palmarum, or Rhynchophorus vulneratus, or Rhizopelta, or Oreochromis orophyllus or Russian wheat aphid, or Olive scale, or Saissetia oleae, or San Jose scale insect, or Scale insect, or American grasshopper (Schistocerca americana), or Green wheat aphid (Schizaphis graminum), or Schizaphis hypersiphonata, or Schizaphis minuta, or Schizaphis rotundiventris, or Schoutedenia lutea, or Sciaridae, or Scirtothrips dorsalis, or Scutiphora pedicellata, or Bean leafminer, or White-browed cutworm, or Cotton aphid (Shivaphis celti, or Silver whitefly, or Silver leaf whitefly, or Sinomegoura citricola, or Sipha flava, or Sitobionavenae, or Sitobion lambersi, or Sitobion leelamaniae, or Sitobion miscanthi, or Sitobion pauliani, or Sitobion phyllanthi, or Sitobion wikstroemiae, or Sitona lepidus, or Sitona lineatus, or Southwestern corn borer, or Southwestern corn borer, or soybean aphid, or cutworm, or common cutworm, or melon leaf beetle, or spotted lanternfly, or squash vine borer, or stem beetle, or Stenotus binotatus, or Strauzia longipennis, or flea beetle, or sunn pest, or sweet potato bug, or Synanthedon exitiosa, or rusty white bug, or Tecia solanivora, or Tetranychus urticae, or other Tetranychus species, Tetraneura nigriabdominalis, or Tetraneura yezoensis, or Thrips, or Thrips angusticeps, or Thrips palmi, or Thrips simplex, or Thrips tabaci, or Thysanoplusia orichalcea, or Tinocalliskahawaluokalani), or Toxoptera aurantii, or Toxoptera citricida, or Toxoptera odinae, or Trichobaris trinotata, or Trioza erytreae, or Turnip moth, or Tomato tuber moth, or Tuta absoluta, or Turmeric moth The insect pest may be selected from agricultural pests such as Uroleucon minutum, or Varied carpet beetle, or Rhododendron aphid, or Vesiculaphis caricis, or Viracola isocrates, or waxworm, or Western corn rootworm, or Western flower thrips, or wheat fly, or wheat weevil, or whitefly, or winter moth, or Xylotrechus quadripes, or sunflower beetle (Zygogramma exclamationis). According to certain embodiments, the selection of target species from the order Lepidoptera is preferred, in particular from the family Tortricidae, such as for example the genus Choristoneura (in particular Choristoneura orae, Choristoneura fumiferana or Choristoneura freemani), or for example the genus Spodoptera (in particular Spodoptera frugiperda, Spodoptera litura, Spodoptera littoralis, Spodoptera cilium or Spodoptera ornithogalli), or from the family Pyralidae, for example from the genera Plodia or Ephestia, or from other species from this genus listed directly above. The agricultural pest species can also be selected from phytophagous terrestrial gastropod species.

[0043] Pest species may further be selected from disease vectors, such as disease vectors selected from arthropods. Disease vectors may be involved in the spread of animal diseases, including human diseases, or may carry plant diseases. The disease vectors carrying animal diseases may be selected from blood-feeding (haematophagous) or hemolymph-feeding arthropods, preferably hematophagous arthropods, for example selected from Culicidae (such as Aedes), or Ceratopogonidae (such as Stomach flies), or Tabanidae, or Simuliidae (such as Austrosimulium), or Tsetse fly (such as Tsetse fly), or Triatominae (such as Triatoma infestans or Rhodnius prolixus), or Siphonoptera (such as Publicidae), or Phthiraptera (such as Anoplophora), or Ixodidae, or Argilla (such as Ixodidae).

[0044] The arthropod vectors responsible for the spread of plant diseases are Acyrthosiphon pisum, or Agromyzidae, or Anastrepha grandis, or Anastrepha obliqua, or Anthomyiidae, or Aphids, or Bark beetles, or Sugar beetle, or Radish aphid, or Cacopsylla melanoneura, or Cacopsylla ulmi, or Ceratitis podocarpi, or Strawberry aphid, or Cicadulina mbila, or Common brown leafhopper ... leafhopper, or Cryptococcus fagisuga, or Curculionidae, or Diabrotica balteara, or Empoasca decedens, or Eumetopina flavipes, or Euscelis plebejus, or Frankliniella tritici, or Grassy-winged sharpshooter, or Haplaxius crudus, or Hyalestes obsoletus, or Black pine beetle, or Hylastes ater, or a leaf beetle, or a leafhopper, or a false radish aphid, or Macrosteles quadrilineatus, or a mealybug, or a melon fly, or a curculionidae, or a Pegomya hyoscyami, or a Pissodes, or Pissodes strobi or Pissodini, or a planthopper, or Pseudococcus maritimus,maritimus, or orange mealybug (Pseudococcus vibumi), or Psylla pyri, or Psylla pterygium, or Rabdophaga clavifex, or Rhynchophorus palmarum, or Scaphoideus titanus, or tea tree thrips, or silverleaf whitefly, or Tephritidae, or Thripidae, or Thripidae, or Thripidae, or Thripida palmi, or Tomicus piniperda, or Asian black aphid, or Treecutter, or Psylla pterygium, or Western flower thrips, or Xyleborus glabratus.

[0045] According to certain preferred embodiments, the pest species is in particular selected from the genus Meloidogyne (such as M. arenaria, M. incognita, M. javanica, or M. hapla), or from the genus Heterodera (such as Heterodera glycines, Heterodera avenae, and H. filipjevi), or from the genus Globodera (such as Globodera spp., ... pallida, or G. rostochiensis), or from the genus Pratylenchus (e.g. P. penetrans, P. thornei, P. neglectus, P. zeae, P. vulnus, or P. coffeee), or from the genus Radopholus (e.g. Radopholus similis).

[0046] According to certain embodiments, the ectoparasitic species may be selected from weed species. Weed species which are considered as target species within the present invention are, for example, weed species from the following families: Albustaceae, or Apiaceae, or Asteraceae, or Amaranthaceae, or Cactaceae, or Caryophyllaceae, or Chenopodiaceae, or Cendulaceae, or Commelinaceae, or Poaceae, or Portulaceae, or Euphorbiaceae, or Fabaceae (Leguminosae), or Rubiaceae, or Aconitaceae, or Azollaceae, or Salvinaceae, or Iridaceae, or Liliaceae, or Pontedriaceae, or Melastomataceae, or Myrtaceae, or Polygonaceae, or Crabaceae, or Rosaceae, or Acanthaceae, or Orobanchaceae, or Scrophulariaceae, or Convolvulaceae, or Cuscutellaceae, or Solanaceae, or Umbelliferae.

[0047] The specific weed species considered as target species are Sagittaria sagittifolia Linnaeus, or Giant hogweed (Heracleum mantegazzianum Sommier & Levier), or Ageratina adenophora (Spreng.) King & HERobins, or Ageratina riparia (Regel) King & HERobins, or Arctotheca calendula (L.) Levyns, or Carthamus oxyacanthus M. Bieberstein, or Crupina vulgaris Cass., or Inula britannica L.), or Mikania cordata (Burm.f.) BL Robins), or Mikania micrantha Kunth, or Onopordum acaulon L., or Onopordum illyricum L., or Senecio inaequidens DC., or Senecio madagascariensis Poir., or Tridax procumbens L., or Alternanthera sessilis (L.) R.Br.ex DC., or Opuntia aurantiaca Lindley), or Drymaria arenarioides Humboldt & Bonpland, or Salsola vermiculata L., or Caulerpa taxifolia (Vahl) C. Agardth, or Commelina benghalensis L.), or Avena sterilis Linnaeus, or Chrysopogon aciculatus (Retz.) Trin., or Digitaria abyssinica (A. Rich) Stapf, or Digitaria velutina (Forsk.) Beauv., or Imperata brasiliensis Trinius, or Imperata cylindrica (L.) Beauv., or Ischaemum rugosum Salisbury, or Leptochloa chinensis (L.) Nees, or Nassella trichotoma Hackel ex Arech., or Oryza longistaminata longistaminata A.Chev.& Roehr., or Oryza punctata Kotzchy ex Steud., or Oryza rufipogon Griffiths, or Paspalum scrobiculatum Linnaeus, or Pennisetum clandestinum Hochst.ex Chiov., or Pennisetum macrourum Trinius, or Pennisetum pedicellatum Trinius, or Pennisetum polystachion(Linnaeus)Schultes, or Rottboellia cochinchinensis (Lour.) WDClayton), or Saccharum spontaneum L., or Setaria pumila ssp. pallidefusca (Schumacher) BKSimon or Urochloa panicoides Beauvois, or Euphorbia terracina L., or Acacia nilotica (L.) Willd. ex Delile, or Galega officinalis L., or Mimosa diplotricha C. Wright ex Sauvalle, or Mimosa pigra L., or Prosopis alpataco RAPhilippi, or Prosopis argentina Burkart, or Prosopis articulata S. Watson, or Prosopis burkartii Munoz, or Prosopis caldenia Burkart, or Prosopis calingastana Burkart, or Prosopis campestris Griesbach, or Prosopis castellanosii Burkart, or Prosopis denudans Bentham, or Prosopis elata (Burkart) Burkart, or Prosopis farcta (Banks & Soland.) JFMacbr.), or Prosopis ferox Griesbach, or Prosopis fiebrigii Harms, or Prosopis hassleri Harms ex Hassler, or Prosopis humilis Gillies ex Hooker & Arnott, or Prosopis kuntzei Harms ex Hassler, or Prosopis pallida(Humb.& Bonpl. ex Willd.) Kunth, or Prosopis palmeri S. Watson, or Prosopis reptans Benth., or Prosopis rojasiana Burkart, or Prosopis ruizlealii Burkart, or Prosopis ruscifolia Griesbach, or Prosopis sericantha Gillies ex Hook. & Arn., or Prosopis strombulifera (Lamarck) Bentham, or Prosopis torquata (Cavan ex Lagasca y Segura) DC., or Spermacoce alata Aubl., or Hydrilla verticillata (Lf) Royle, or Lagarosiphon major (Ridley) Moss, or Ottelia alismoides(Linnaeus)Pers.), or Azolla pinnata R.Brown), or Salvinia auriculata Aublet, or Salvinia biloba Raddi, or Salvinia herzogii de la Sota, or Salvinia molesta DSMitchell, or Moraea collina Thunb., or Moraea flaccida (Sweet) Steud., or Moraea miniata Andrews, or Moraea ochroleuca (Salisb.) Drapiez, or Moraea pallida (Baker) Goldblatt, or Asphodelus fistulosus Linnaeus), or Eichhornia azurea (Swartz) Kunth), or Monochoria hastata (L.) Solms), or Monochoria vaginalis (Burm.f.) K.Presl ex Kunth), or Melastoma malabathricum L.), or Melaleuca quinquenervia (Cav.) Blake), or Emex australis Steinhall), or Emex spinosa (Linnaeus) Campdera), or Lygodium flexuosum flexuosum (L.) Sw. (1801) (Mobot) or Lygodium microphyllum (Cav.) R.Br. or Rubus fruticosus L. or Rubus moluccanus L. or Hygrophila polysperma (Roxb.) T.Anders, or Aeginetia spp. L., or Alectra spp. Thunb., or Orobanche spp. (nonnative) L., or Limnophila sessiliflora (Vahl) Blume, or Striga spp. Lour., or Ipomoea aquatica Forssk., or Cuscuta spp. L., or Lycium ferocissimum Miers, or Solanum tampicense Dunal, or Solanum torvum Sw., or Solanum viarum Dunal), or Sparganium erectum L. .

[0048] Species causing product deterioration that may be selected as target species may be selected from spoilage microorganisms such as bacteria such as gram-negative bacilli (e.g. Pseudomonas spp., Shewanella spp.), gram-positive spore bacteria (e.g. Bacillus spp., Clostridium spp.), lactic acid bacteria and other gram-positive bacteria (e.g. Brochothrix spp., Micrococcus spp.), or Enterobacteriaceae, fungi (e.g. Zygomycetes from the genus Penicillium or Aspergillus, or yeasts such as Zygosaccharomyces spp., Saccharomyces spp., Candida spp., Dekkera (Brettanomyces) spp.). Alternatively, target species causing product deterioration may be selected from acarids such as those selected from the suborder Acaridae, such as those selected from the family Sarcoglossidae, or the family Saccharomyces.

[0049] According to certain preferred embodiments of the compositions of the invention, the composition comprises cells from some species, the host species, different from the source species, in which the DNA molecule is within the host species cells.

[0050] As already mentioned above, the term "host species" is used only as a reference term. A host species in the context of the present invention is generally a species different from the source species, preferably a phylogenetically different species in whose cells the source species DNA sequence has been integrated. A phylogenetically different (distant) species according to certain embodiments is a species from a different taxonomic order, such as from a different class, phyla, kingdom, or domain. According to certain embodiments, a phylogenetically different species is a species from a different family, such as from a different order, class, phyla, kingdom, or domain. A host species may be selected from any species that is capable of incorporating and replicating the foreign DNA of the source species, in particular a fragment of the source species DNA, such as a fragment of the source species integrated in a cloning and / or expression vector. As will be clear from the present specification, it is not required that an individual of the host species incorporates and replicates the entire genome of the source species. Instead, the inhibitory effect of the self-DNA on the target species may be brought about by a plurality of host species cells and / or individuals, each different cells and / or individuals replicating a different part of the genome of the source species, such as in a DNA library. A single cell of a population of host species cells therefore contains only a portion of the entire source species DNA sequence present in the composition. The host species can be selected from, for example, a multicellular organism or a microorganism. For example, a cell of a multicellular plant can be used as the host species cell. As those skilled in the art will appreciate, the source species DNA can be introduced into the cell of a multicellular plant by various techniques, including, for example, particle bombardment of embryonic cell cultures, Agrobacterium-mediated transformation, and transformation mediated by a viral vector(s). When selected as a plant, the host species may be selected from crops, preferably food crops (e.g. selected from cereals, maize, sugar beet, rapeseed, peas, soybean, etc.), oil crops (e.g. rapeseed, Ethiopian mustard, sunflower, etc.), starch crops (e.g. (sugar) sorghum, etc.), fibre crops (e.g. flax or hemp, etc.), lignocellulosic crops (e.g. grass reed, bamboo, switchgrass, miscanthus, cardoon, etc.), short rotation forest crops (e.g. willow, poplar, or eucalyptus, etc.).

[0051] Alternatively, helminths used in helminth therapy, such as Trichuris suis, American hookworm, Trichuris trichiura, Diminuta nigricans, Ascaris lumbricoides, Strongyloides stercoralis, Pinworm or Diminuta nigricans, can be used as host species. The helminth species used as host species is preferably a human mutualistic species (especially Trichuris suis, American hookworm, Trichuris nigricans or Diminuta nigricans). Horizontal gene transfer events have been observed for nematode species (see, among others, PLoS Negl Trop Dis. 2007 October;1(1):e35;doi:10.1371 / journal.pntd.0000035), and horizontal gene transfer can be induced so that therapeutic helminths integrate source species DNA into their genome.

[0052] According to another alternative embodiment, the host species is selected from microorganisms. As will be understood by those skilled in the art, microorganisms are microscopic organisms that may be in unicellular form or may exist in colonies of cells. Within the context of the present invention, the microorganism is preferably selected from prokaryotes (such as archaea or bacteria), or eukaryotes (such as fungi, including yeast), or microphytes (microalgae). The host species selected from microorganisms may be microorganisms that can be grown in liquid culture (such as, for example, Escherichia coli, in particular the strain DH5a or a derived strain, or the strain DH10B or a derived strain), or yeast species (such as, for example, Saccharomyces cerevisiae, for example the strain AB1380 or a derived strain), or Aspergillus species (such as, for example, Aspergillus niger, Aspergillus oryzae, or Aspergillus nidulans), or Bacillus species, or Pseudomonas species, or Lactobacillus species (such as, for example, Lactobacillus species, Leuconostoc species, Pediococcus species, Lactococcus species, Enterococcus species, or Streptococcus species), or Cyanobacteria (such as, for example, Arthrospira species, in particular Arthrospira plantensis, The host species may be selected from the following: Arthrospira maxima, Arthrospira plantensis, Arthrospira maxima ...According to certain preferred embodiments, the host species is selected from biocontrol agents, such as entomopathogenic biocontrol agents (e.g., microbial control agents, in particular bacterial biocontrol agents such as Bacillus thuringiensis or Bacillus subtillus), or fungal biocontrol agents (e.g., Beauveria bassiana, Isaria fumosorosea, Lecaninicilium spp., or Metarhizium spp.). The biocontrol agent may alternatively be selected from, for example, Trichoderma spp. or Ampelomyces quisqualis. The host species may further be selected from microbial plant growth promoters, such as Bacillus spp., Trichoderma spp., or Mycorrhyza spp. According to certain highly preferred embodiments, when the host species is a microbial insect pathogenic biocontrol agent, the target species is an arthropod, preferably an arthropod selected from agricultural pest arthropods or arthropod vectors involved in the spread of plant diseases. Specific examples of organisms from these groups are given above.

[0053] As explained, in the embodiment where the source species DNA sequence is in the host species cell, the source species DNA sequence is either integrated into a replicable artificial DNA construct, such as an artificial plasmid or artificial chromosome, introduced into the host species cell, or integrated into the native genome of the host species cell. This integration of the source species DNA sequence into an artificial DNA construct introduced into the host cell or into the native genome of the host cell allows the source species DNA sequence to be replicated together with the replication of the host species DNA by the DNA replication machinery of the host species cell. The surprising discovery by the inventors that the inhibitory DNA fragments of the species remain inhibitory when presented in association with the (production) host species DNA opens new possibilities for producing and presenting self-inhibitory DNA fragments.

[0054] The incorporation of DNA sequences into DNA constructs and the introduction of DNA constructs into host cells have become standard techniques in various fields of biochemistry. Thus, those skilled in the art can easily apply such procedures within the present invention. Furthermore, it has become known that various organisms can take up DNA molecules from their environment and integrate these DNA molecules into their natural genome. In particular, microorganisms, especially prokaryotes, are naturally capable of such horizontal gene transfer. Moreover, certain known genetic engineering techniques, such as biolistics, rely on the integration of DNA fragments into the natural genome of cells, such as plant cells.

[0055] Additionally, transposable elements (i.e., transposons) can facilitate DNA transfer between cells of different species.

[0056] It is preferred that the coverage of the genome of the source species in the mixture of DNA molecules is between 20-100%, such as 20-90%, 30-100%, 30-90%, 40-100%, 40-90%, 50-100%, 50-90%, etc., and preferably 60-100%, such as 60-90%, 70-100%, 70-90%, 80-100%, 80-90%, etc. Based on the experimental observations by the present inventors, it can be derived that within these ranges there is good inhibitory activity of the source species DNA sequences in the composition of the present invention. Those skilled in the art will know and understand that the coverage of the genome of the source species in the mixture of DNA molecules correlates with the percentage of source species DNA sequences, particularly chromosomal DNA sequences, present in the total (population) of DNA molecules present in the composition. Given the fact that experimental results show that parts of the entire genome are inhibitory, it can also be expected that (fragmented) cDNA sequences corresponding to parts of the entire DNA sequence of the genome will have inhibitory activity against the species from which they originate and against phylogenetically similar species. However, based on the available knowledge in the context of chromosomal structure, it can be expected that there will be a higher species specificity for non-coding region sequences, so if the source species genome contains a significant amount of non-coding region sequences, it is preferred that the source species DNA sequence contains such non-coding region sequences. In view of this, it is preferred that the source species DNA sequence comprises randomly fragmented chromosomal DNA.

[0057] By replicating the source species DNA sequence of the (chimeric) DNA molecule in the host species cell, the source species DNA sequence becomes associated with a significant amount of host species DNA sequences and possibly with a significant amount of DNA sequences derived from the artificial DNA construct. The inventors have surprisingly found that the source species DNA sequence maintains its autoinhibitory activity when it is associated with a relatively large amount of these non-source species DNA sequences. Hence, exclusion of the host species DNA sequences and / or DNA sequences derived from the artificial construct from the composition is not necessary. According to certain preferred embodiments, the composition of the invention therefore comprises non-source species DNA such that the non-source species DNA exceeds the source species DNA, preferably at a ratio of non-source species DNA:source species DNA between 10:1 and 1000:1, such as between 20:1 and 500:1, between 50:1 and 500:1, or between 100:1 and 500:1. The non-source species DNA comprises, and preferably consists essentially of, host species DNA sequences and optionally DNA sequences derived from an artificial DNA construct (e.g., a cloning and / or expression vector, e.g., selected from a plasmid, a BAC, a YAC, or an artificial fungal chromosome).

[0058] The source species is preferably selected so that the inhibitory DNA sequence derived therefrom is inhibitory to the target species as discussed above.As will be clear to those skilled in the art after the publication of WO2014 / 020624, the inhibitory DNA sequence may be derived from the target species itself or a phylogenetically similar species.In the context of the present invention, the source species may therefore be selected from the target species or a phylogenetically similar species to the target species.

[0059] In the context of the present invention, phylogenetically similar species are species with similar genomes. Those skilled in the art will understand that species that are closely related phylogenetically have more similar genomes than species that are distantly related phylogenetically. Phylogenetic similarity therefore means having a close phylogenetic relationship. Phylogenetic similarity can therefore be determined based on known phylogenetic relationships. Thus, according to certain preferred embodiments, phylogenetically similar species are species within the same taxonomic order. Within a certain order, phylogenetically similar species are preferably from the same monophyletic group (clade), such as the same family, subfamily, tribe, subtribe, genus, etc. Most preferably, phylogenetically similar species are from the same taxonomic family, such as the same subfamily, tribe, subtribe, genus.

[0060] Furthermore, techniques for determining genomic similarity (or relatedness) are readily available. Genomic similarity can be determined, for example, by determining the renaturation / reassociation kinetics of single-stranded DNA (ssDNA) fragments of genomes from both species. Alternatively, or in addition, the denaturation (melting) of double-stranded DNA (dsDNA) fragments renatured from a mixture of genomic ssDNA fragments from both species may be investigated. The latter technique allows the determination of the melting temperature T m , i.e., the temperature at which half of the DNA strands are in the ssDNA state, and the associated T 50This allows the definition of H. Approaches involving the evaluation of renaturation / denaturation kinetics and melting profiles were introduced in the early 70s to determine the relatedness of bacteria (see de Ley et al. Eur J Biochem. January 1970; 12(1): 133-42), but these approaches involving melting temperature profile analysis have also been used to determine the relatedness of eukaryotic species (see, for example, Sibley and Ahlquist, J Mol Evol (1984) 20: 2-15). As is further known since the publication of WO2014 / 020624, the phylogenetic similarity of species can be determined based on whether inhibitory DNA fragments from one species are also inhibitory for another species. According to certain other embodiments, phylogenetically similar species are therefore species for which DNA obtained by random fragmentation of the extracted total DNA or by random fragment synthesis starting from the total DNA is inhibitory for the target species. Based on this functional definition, it will be clear to the skilled artisan that the phylogeny can be determined by tests similar to those presented in WO2014 / 020624 and in the experiments attached thereto. The DNA obtained from the source species by random fragmentation of the total extracted DNA or by random fragment synthesis starting from the total DNA will be inhibitory to the target species, so that the source species will be phylogenetically similar to the target species within the same taxonomic order as well.

[0061] The composition of the present invention is intended to inhibit the target species. The target species may be the same as the source species or may be a species phylogenetically similar to the source species. To enhance the inhibition of the target species, the composition may be combined with the use of some other (natural) biocides, such as (natural) pesticides, such as fungicides, insecticides, nematicides, acaricides, artropodicides, bactericides or algicides. The additional (natural) biocide may be a biological control agent, such as a microbial control agent, such as a bacterial or fungal biological control agent, in particular an insect pathogenic biological control agent. According to certain embodiments, the additional (natural) biocide is present in the composition. When the additional (natural) biocide is a microbial control agent, such as an insect pathogenic biological control agent, it is preferred that the living cells of the microbial control agent function as host cells that contain and replicate the source species DNA. For example, the source species may be selected from the target species or species phylogenetically similar to the target species, where the target species is selected from the order Lepidoptera, in particular from the family Tortricidae, such as the genus Chorystoneura (particularly Chorystoneura olae, Chorystoneura fumiferana, or Chorystoneura flehemani), or from the family Noctuidae, such as the genus Spodoptera (particularly Nectaria, Spodoptera litura, Nectaria cinerea, Nectaria litura, Nectaria litura, Nectaria litura), or from the family Pyralidae, such as the genus Plodia or Ephestia, or from the orders Diptera, Coleoptera, or Hymenoptera, most preferably from the genus Chorystoneura. The composition may then further comprise a biocontrol agent for the target species, such as a bacterial biocontrol agent (e.g., Bacillus thuringiensis or Bacillus subtilis), or a fungal biocontrol agent, such as an entomopathogenic fungus (e.g., Bacillus subtilis, Isaria fumosorosea, Lecaninicilium spp., or Metarhizium spp.). The biocontrol agent may be a viable biomass of a bacterial or fungal biocontrol agent, preferably a Bacillus thuringiensis biomass. The viable biomass, such as a Bacillus thuringiensis cell, may then also have the source species DNA incorporated in a replicable form, and thus function as a host species cell.

[0062] The composition can be administered to the target species by any suitable means, such as surface contact, cytotropic administration, systemic administration (e.g., by injection, ingestion or inhalation, or adsorption). The composition can be formulated in a form for dry or liquid processing selected from the group consisting of dispersions (e.g., in the form of an aerosol), suspensions, wettable powders or soluble powders, emulsions in water or other solvents, dispersible granules, suspensions of microcapsules, emulsifiable concentrates, fluid pastes, macroemulsions, oil dispersions, baits. Solvent systems including water or deep eutectic solvent (DES) systems, such as natural deep eutectic solvent (NADES) systems, can be used.

[0063] It is within the knowledge of one of ordinary skill in the art to determine the concentration range at which the DNA of the composition of the present invention is inhibitory to the target species. One of ordinary skill in the art will understand that the required concentration may depend on factors such as the ability of the DNA in the composition to inhibit the target species, the desired level of inhibition, or whether additional biocides are applied, and / or the route of application to the target species. For many applications, suitable concentrations may range from 1 to 1500 ppm, e.g., 2 to 1300 ppm, 2 to 1000 ppm, 5 to 1000 ppm, 10 to 1000 ppm, 50 to 1000 ppm, 100 to 1000 ppm, 200 to 1000 ppm, 500 to 1000 ppm, etc. For other applications, higher concentrations may be desired.

[0064] As is evident from WO2014 / 020624, the inhibitory DNA fragments may be used in medicine. Among others, WO2014 / 020624 shows the inhibition of Aspergillus niger, which may cause infections such as otomycosis. Similarly, the compositions of the invention comprising DNA fragments or precursors thereof may also be used in medicine for inhibiting Aspergillus niger, or other parasitic and / or pathogenic organisms such as nematodes, parasites, etc. In medical applications of the compositions, the source species DNA is an inhibitor or precursor thereof, since if the source species DNA is in a non-fragmented form, it may be converted to a fragmented form in situ, such as in the digestive tract. A further aspect of the invention is therefore the composition of the invention for use in medicine, or in other words for use as a medicament. The pharmaceutical use in the context of the present invention relates to human and / or veterinary medicine, preferably human medicine. Reference to an animal in the context of the present invention includes reference to a human, unless a different meaning follows from the context in which it is used. The composition may be used, for example, in medicine as an anti-pathogenic composition to inhibit the spread and / or growth of, or even kill, pathogenic organisms. Pathogenic target organisms that may be inhibited using the composition of the invention are, for example, local pathogens, enteric pathogens, pulmonary pathogens or systemic pathogens. Pathogens may be selected, for example, from microbial pathogens (e.g., selected from bacteria, fungi or viruses, etc.), parasites (e.g., selected from protozoa, etc.), or helminths (e.g., nematodes, cestodes, trematodes or roundworms). The medical use of the composition of the invention is particularly directed to the pathogenic and / or parasitic target species set forth above in the discussion of the composition of the invention, particularly local pathogens and / or parasites associated with the skin, nails or mucous membranes. For use as a medicine, the composition is in a pharmaceutically acceptable form.

[0065] A further aspect of the invention relates to the use of the composition of the invention as a product for inhibiting a target species. The target species may correspond to the source species or be a species phylogenetically similar to the source species. In a product, the composition of the invention may be used as an active ingredient for inhibiting a target species. The intended inhibition of the target species may be in the product, for example to improve the biological safety of the product, or alternatively the intended inhibition of the target species may be the target surface or the target area to which the product is applied. The product may comprise further components, including further components that are inhibitory against parasitic, pathogenic and ectoparasitic species, including one or more selected from biocides, herbicides, fungicides, insecticides, acaricides, nematicides, arthropodicides, algicides, bactericides. The product may be a medical product or a non-medical product. According to certain embodiments, the product is a non-medical product, such as an agricultural product, a personal health care product, or a hygiene product such as a soap or detergent.

[0066] In certain preferred embodiments of the use of the composition, the source species DNA sequence may be in the chimeric DNA molecule in the host species cell. For some applications, it is sufficient to indicate the chimeric DNA molecule in the host species cell. The reason is that the chimeric DNA molecule in the host species cell may be released from the host species cell by the conditions to which the host species cell is exposed. For example, if the product is for inhibiting a target species for which the host species cell is a suitable food source, when the chimeric DNA molecules containing the source species DNA sequence are eaten by the target species, they are released from the host species cell in the intestine of the target species. In this process, the source species DNA sequence may also be released from the chimeric DNA molecule. Also, in the natural degradation process of the host species cell, the chimeric DNA molecule contained in the host species cell may be released from the host species cell, and the source species DNA sequence may be released from the chimeric DNA molecule by fragmentation. In an alternative use of the present invention, the host species cell is destroyed by a cell destruction method, such as a cell destruction method that fragments DNA, and the material from the destroyed host species cell is used for the inhibition of the target species. For this purpose, the DNA may be destroyed and optionally isolated from the host species cell by known methods. Alternatively, the disrupted cell population may be used for inhibition of the target species without further isolation of DNA.

[0067] A further aspect of the present invention relates to a method for producing a composition according to the present invention, comprising the steps of: providing some host species; - providing a source of DNA from a source species, optionally including fragmented source species DNA; - subjecting the host species cells, together with a source of DNA from a source species, to conditions that allow the host species cells to take up the source species DNA in a replicable form; - growing host species cells containing the source species DNA in a replicable form; - optionally isolating host species cells containing the source species DNA in replicable form; - optionally isolating DNA from the host species cells, in particular DNA containing source species DNA; - optionally fragmenting the DNA of the host species cells, in particular the DNA containing the source species DNA; Includes.

[0068] In the first step of the method, a number of host species are provided. Based on what has been discussed in connection with the compositions of the invention, and using the present invention and available common general knowledge, a person skilled in the art will know and understand how to provide a suitable host species cell.

[0069] In the second step, a source of DNA from the source species (optionally including fragmented DNA) is provided. The source of DNA from the source species may be any suitable source. For example, the cells of the source species may be used as such. Alternatively, DNA released from the source species cells may be used, for example DNA released from cells disrupted by a cell disruption method, such as a cell disruption method that fragments the DNA. The released DNA may or may not be isolated. The DNA may or may not be fragmented. The fragmented DNA from the source species may be presented as such or may be incorporated into an artificial DNA construct, such as an artificial plasmid, cosmid, artificial chromosome (such as a YAC or BAC or fungal artificial chromosome), or a chimeric DNA molecule constructed in association with a transposable element.

[0070] In a third step, the host species cells containing the source of DNA from the source species are subjected to conditions that allow the host species cells to take up the source species DNA in a replicable form. Taking up the source species DNA in a replicable form causes the host species cells to replicate the source species DNA with their DNA replication machinery. Thus, the conditions must be such that the source species DNA, when provided, is incorporated into the host species cells so that it is functionally available to the DNA replication machinery of the host species cells to be replicated. The skilled artisan will be able to select appropriate conditions to allow the host species cells to take up the source species DNA in a replicable form.

[0071] For example, if the source species DNA is provided incorporated into artificial DNA constructs such as plasmids, cosmids, artificial chromosomes, or constructed chimeric DNA molecules associated with transposable elements, one of skill in the art would know how to condition the host species cells to efficiently take up these artificial DNA constructs so that they can be efficiently replicated.

[0072] If the source species DNA is provided as source species cells, the skilled person will know that the required conditions must allow the source species cells to release the source species DNA and for the source species DNA to be fragmented. It has been found that in the process of litter decomposition, the conditions are such that DNA from plant material in the litter can be transferred to the soil microbiome. Based on these findings, the skilled person will expect that DNA from non-plant sources contained in the litter or similar decomposing biological material can also be incorporated into the soil microbiome. Suitable conditions can therefore be those of litter decomposition, in particular where the host species is a soil microorganism selected from Alphaproteobacteria species, Betaproteobacteria species, Deltaproteobacteria species, Actinobacteria species, Thermoleophilum species, Rubrobacter species, Chlorachidobacterium species, Acidobacterium species, or Solibacter species.

[0073] If the source species DNA is provided as DNA released from source species cells, one of skill in the art will understand that the released source DNA may be taken up by suitable cells of the host species through natural processes, or by forced methods such as particle bombardment (or gene shotgun).

[0074] Fourth, step 4 is optional and includes isolating the host species cells. From the entire specification, including the examples, the skilled artisan will understand that isolation of the host species cells is not required for all applications. If isolation of the host species cells is required or desired, the skilled artisan will be knowledgeable about suitable techniques for isolating the host species cells.

[0075] The fifth step is also optional and involves isolating DNA from the host species cells. From the entire specification, including the examples, the skilled artisan will understand that isolation of DNA from the host species cells is not required for all applications. If isolation of DNA from the host species cells is required or desired, the skilled artisan will be knowledgeable about suitable techniques for isolating DNA from the host species cells.

[0076] The sixth step is a further optional step, which includes fragmenting the DNA of the host species cells. From the entire specification, including the examples, the skilled person will understand that fragmenting the DNA from the host species cells is not required for all applications. If fragmentation of the host species DNA is required or desired, the skilled person will be knowledgeable about the appropriate method for fragmenting the DNA from the host species cells. When the DNA from the host species cells is fragmented, it is preferred that the chimeric DNA molecule is fragmented. DNA fragmentation by random fragmentation method is preferably used.

[0077] The invention will now be further illustrated by the following non-limiting experiments. EXAMPLES

[0078] (Experiment 1) Arabidopsis Library A commercial microbial genomic library of Arabidopsis was purchased from TAIR (https / / www.arabidopsis.org / ) and engineered in both yeast and E. coli.

[0079] The YAC library CD4-21P was a pooled yeast artificial chromosome (YAC) genomic library in the pYAC4 vector; DNA isolated from Columbia plant nuclei; the original library consisted of 1,152 clones; average insert size = 420 kb; 21% of the clones contained chloroplast DNA; the library consisted of 81 pools; cloning enzymes: EcoRI and EcoRI.

[0080] BAC library CD4-21P binary cosmid genomic library in POCA18-hyg vector; generated from a Wassilewskija (Ws) genomic DNA fragment generated by partial digestion with TaqI; corresponds to 10 genome equivalents; the vector carries the hygromycin resistance gene under the control of the CaMV35S promoter; cloning enzymes: ClaI and ClaI.

[0081] Each library was used to obtain a microbial solution prepared as follows: YAC library CD4-21P: Each of the 80 stocks of the library was inoculated into a tube containing YPD medium (1% yeast extract, 2% bactopeptone, 1% glucose) and incubated at 28°C for 48 hours. After incubation, the tubes were pooled and the cell suspension was centrifuged (4000 rpm, 5 min) and resuspended in 10 ml NaCl 0.9% (pooled yeast suspension). The pooled YAC culture was then performed in a bench stirred reactor (New Brunswick) containing 1 L of YPD medium at 28°C, 200 rpm, and 1 vvm aeration speed. The reactor was inoculated with an appropriate aliquot of the pooled yeast suspension to obtain an initial OD590=0.1. After 48 hours, the yeast biomass was harvested, washed, and resuspended in distilled water to obtain a final concentration of 50 mg cell dry weight per ml.

[0082] For yeast cells used as a control, the same procedure was followed, but a preculture of the Saccharomyces cerevisiae strain CEN.PK113-7D in YPD medium was used as inoculum for the reactor. Again, the produced biomass was harvested, washed and resuspended in distilled water to the same cell density reported above.

[0083] BAC library CD4-21P: Each of the 80 stocks of the library was diluted to 50 mg / ml -1Tubes containing Tryptone Soya Broth (TSB) with hygromicin were inoculated and incubated at 37°C for 48 hours. After incubation, the tubes were pooled and the cell suspension was centrifuged (10,000 rpm, 10 min) and resuspended in 10 ml NaCl 0.9% (pooled bacterial suspension). Cultivation of the bacterial library was then carried out in a bench stirred reactor (New Brunswick) containing 1 L of TSB medium with hygromycin at 37°C, 200 rpm, and 1 vvm aeration rate. The reactor was inoculated with an appropriate aliquot of the pooled bacterial suspension to obtain an initial OD590=0.01. After 48 hours, the bacterial biomass was harvested, washed, and resuspended in distilled water to obtain a cell density of 50 mg cell dry weight per ml.

[0084] For the bacterial cells used as a control, the same procedure was followed, but a preculture of E. coli ATCC 10536 in TSB was used as the inoculum for the reactor. Again, the produced biomass was harvested, washed and resuspended in distilled water to the same cell density reported above.

[0085] Inhibition test The microbial solutions of the two libraries were sonicated and the resulting cell homogenates were incubated for 15 days at room temperature under aerobic conditions, with distilled water being added to replenish water evaporation. Different aliquots of the homogenates were diluted 1:1, 1:10, and 1:100 in distilled water and used to moisten filter paper in Petri dishes with three replicates containing 10 plants per concentration level. Arabidopsis seedlings were prepared by germinating seeds in Petri dishes containing standard nutrient solution on filter paper. Healthy seedlings of similar size and at least one week old were selected for use in the inhibition tests. The experiments were performed by exposing seedlings to the various solutions in Petri dishes (10 seeds per dish, 10 dishes per treatment). Controls were assessed by seedlings that were maintained only in distilled water for the duration of the experiment. Treatments consisted of exposure to either the libraries or the corresponding microbial solutions without the genomic library. Seedling survival was assessed after 10 days by counting the number of live plants. The experiments were carried out in a growth chamber maintained at optimal levels of temperature and air humidity.

[0086] result The results of the inhibition studies demonstrated the inhibitory effect of mixtures of heterologous and self-DNA on homologous seedling viability by testing Arabidopsis genomic microbial libraries (BAC and YAC). Indeed, seedling viability was significantly lower under exposure to YAC and BAC libraries of genomic self-DNA compared to both the distilled water-treated control and the corresponding microbial solutions containing heterologous DNA but not the self-DNA genomic library (Figure 1). This pattern was consistent across three levels of treatment dilution. Moreover, treatments containing heterologous DNA but not self-DNA did not generally affect seedling viability, which was not different from that of the control, except for the Saccharomyces cerevisiae material, which showed a nonspecific inhibitory effect at the highest concentration level.

[0087] (Experiment 2) Nutsher Library A genomic BAC library of C. monadelpha was constructed by Bio S&T (Montreal, Canada) in E. coli EPI300 using the vector pSmart-HindIII (chloramphenicol resistance). Libraries were pooled in 1× 96-well plates with each well containing 123 primary clones for a total of 11,808 clones (>2× genome coverage). The average insert size (110 Kb) was determined using test random clones digested with NotI. The library was stored at −80°C.

[0088] The library was used to obtain a microbial solution prepared as follows: For amplification, each well was inoculated from a frozen stock in a tube containing Tryptone Soya Broth (TSB) with chloramphenicol at 12.5 mg / L and incubated at 37°C for 48 hours. After incubation, 96 tubes were pooled and the cell suspension was centrifuged (10,000 rpm, 10 min) and resuspended in 10 ml NaCl 0.9% (pooled bacterial suspension). Cultivation of the bacterial library was then performed in a bench stirred reactor (New Brunswick) containing 1 L of TSB medium with chloramphenicol at 37°C, 200 rpm, and 1 vvm aeration speed. The reactor was inoculated with an appropriate aliquot of the pooled bacterial suspension to obtain an initial OD590=0.01. After 48 hours, the bacterial biomass was harvested, washed, and resuspended in distilled water to obtain a cell density of 50 mg cell dry weight per ml.

[0089] For the bacterial cells used as a control, the same procedure was followed, but a preculture of E. coli EPI300 in TSB was used as the inoculum for the reactor. Again, the produced biomass was harvested, washed and resuspended in distilled water to the same cell density reported above.

[0090] Inhibition test The microbial solution was sonicated and the resulting cell homogenate was incubated for 15 days at room temperature under aerobic conditions, with the addition of distilled water to replenish water evaporation. Different aliquots of the homogenate were then diluted 1:10 and 1:100 v / v in each of two different substrates for plant growth (quartz sand and standard potting soil). Seedlings of Cyperus moniliforme were prepared by germinating seeds in Petri dishes containing standard nutrient solution on filter paper. Healthy seedlings of similar size and at least 10 days old were selected for use in the inhibition test. The experiment was performed by exposing seedlings to different substrate mixtures in small plastic pots of 15 mL capacity (one seedling per pot, 20 pots per treatment). Treatments included factorial combinations of substrate type (two levels, either sand or potting soil), E. coli material type (two levels, either with or without the BAC library), and solution concentration (two levels, either 1:10 or 1:100). Pots containing unmixed substrate were considered as controls (10 pots per substrate type).Cyperus gracilis grown under controlled conditions (day / night T 22 / 16°C, photoperiod 16:8 h, ambient air humidity, daily watering to field capacity of pots) were assessed 10 days after potting seedlings by destructive sampling of plant material followed by oven drying (60°C x 48 h) and weighing of total biomass.

[0091] result The results demonstrated the inhibitory effect of the BAC genomic library of C. mongolica on the homogenous seedling growth. Indeed, seedling biomass was significantly lower under exposure to the BAC library of genomic self-DNA compared to both the control treated with distilled water and the corresponding microbial material containing heterologous DNA but without the self-DNA genomic library (Figure 2). The inhibitory effect was dose-dependent and consistent across the two levels of treatment concentration. Treatments containing heterologous DNA but not self-DNA did not affect seedling growth, which was not different from that of the control.

[0092] (Experiment 3) Caenorhabditis elegans Library A Caenorhabditis elegans fosmid library was purchased (commercially available) from Source BioScience (http: / / www.sourcebioscience com / ). The library contained 41 × 384 plates with a total of 15,744 clones. A large insert (average size 43.3 kb) library was constructed in E. coli EPI300-T1R using the fosmid CopyControl vector pCC1FOS (chloramphenicol resistant).

[0093] Two E. coli strains were used: OP50 and EPI300-T1R. E. coli strains were grown in LB growth medium. When required, chloramphenicol at a final concentration of 12.5 μg / ml and arabinose at a final concentration of 0.01% weight / volume were added to the growth medium. The Caenorhabditis elegans strain was wild type Bristol (N2). The Caenorhabditis elegans strains were maintained at 20 °C on NGM agar plates (without arabinose) plated with E. coli OP50 as a food source. E. coli EPI300-T1R and EPI300-T1R transformed with a pCC1FOS-based library were used as a food source on NGM plates (with arabinose) as specified.

[0094] For fosmid amplification, each microtiter plate containing 384 library clones was inoculated from a frozen stock on an LB agar plate (150 mm Petri dish) containing chloramphenicol. The resulting 41 plates were incubated overnight at 37°C. After incubation, bacteria were harvested in 12 ml of LB broth containing chloramphenicol and spread onto each plate using a spatula. The resulting bacterial mixture was transferred to a 15 ml culture tube (41 tube). 100 μl was inoculated into 4 ml LB broth containing chloramphenicol and arabinose and incubated overnight at 37°C (41 tube). The next day, 100 μl of the bacterial culture was transferred to 2 ml LB broth containing chloramphenicol and arabinose and incubated overnight at 37°C (41 tube).

[0095] Inhibition test The resulting 41 bacterial cultures were pooled and 20 μl of these pooled bacteria were used on each NGM plate (35 mm) containing arabinose. Untransformed E. coli EPI300-T1R was used as a negative control. In this case, bacteria were grown using the same procedure followed for fosmid amplification, but chloramphenicol was omitted. For generation I screening, a single L4 stage Caenorhabditis elegans worm was transferred to each NGM plate containing arabinose, inoculated with bacteria, and maintained at 20°C. The worms were transferred to fresh identical plates every 12 hours to allow for full egg laying (3 days).

[0096] The following parameters were monitored: embryonic lethality (ratio of nonviable eggs to laid eggs) and aberrant phenotypes (ratio of aberrant phenotypes to hatched eggs). The same parameters were monitored for generation II screening. For generation II screening, a single non-defective L4 stage Caenorhabditis elegans worm from generation I was transferred to each NGM plate containing arabinose, inoculated with bacteria, and treated as described above for generation I screening.

[0097] Inhibition by parts of the Caenorhabditis elegans genome was also tested. For this, worms were fed with clone A01 (WRM0610aA01) of the Caenorhabditis elegans library (chromosome I; clone start: 13249863 to clone end: 13287543 [size 37680]), clone B01 (WRM0610cA02) of the Caenorhabditis elegans library (chromosome X; clone start: 12671842 to clone end: 12705840 [size 33998]), and a combination of clone AO1 and clone BO1. Embryonic lethality (ratio of nonviable eggs to laid eggs) and abnormal phenotypes (ratio of abnormal phenotypes to hatched eggs) were tested in the first generation progeny.

[0098] result In this experiment, 11 worms fed EPI300-T1R containing the entire Caenorhabditis elegans fosmid library and 12 worms fed non-transformed EPI300-T1R were analyzed in a generation I screen (2574 and 3055 eggs, respectively). Worms fed EPI300-T1R containing the entire Caenorhabditis elegans fosmid library showed an increase in embryonic lethality that was significantly different from worms fed EPI300-T1R (3.85% and 0.82%, respectively, p<0.0001). The ratio of males to hatched eggs did not differ between treatments due to the absence of males in both progenies. A significantly higher frequency (6.32%) of developmental abnormalities was observed in the progeny of worms fed EPI300-T1R containing the entire Caenorhabditis elegans fosmid library compared to worms fed EPI300-T1R (p<0.0001, Figure 3).

[0099] Notably, the negative effect of the entire Caenorhabditis elegans fosmid library was exacerbated in generation II compared to generation I (Figure 3).

[0100] The results of inhibition by portions of the genome are shown in Table 2 below. [Table 2]

[0101] The results show that a small portion of the genome can produce a significant effect on both embryonic lethality and the appearance of abnormal phenotypes. Combining clones produces an additive effect (higher than additive). By combining with further clones, it can be expected that the inhibitory effect may be cumulative.

[0102] Example 4 Egyptian Armyworm Library A genomic BAC library of Spodoptera (cotton leafworm) was constructed using vector Copy control pCC1BACH (BamH I Hind III, EcoR I Cloning Ready Vector-8128bp) in E. coli DH10B after extraction and digestion (restriction enzyme Hind III) of high molecular weight genomic DNA from laboratory-grown Spodoptera larvae into the Hind III site, then frozen. Clones were grown and selected on plates containing Luria Bertani (LB) + chloramphenicol solid substrate. Clones were screened, picked, induced for expression to high copy number, and then transferred to wells of a 96 deep-well plate. Each well contained 290 primary clones (total of 27,840 clones) (>10× genome coverage). The average insert size (132Kb) was determined using test random clones digested with NotI. An empty vector control was also prepared and grown in the same conditions of the library vector. The library and empty vector were stored at -80°C.

[0103] The library was used to produce plasmid DNA as follows: a single sterile toothpick was dipped into each single-well culture, wetted, then transferred to a 2 mL microcentrifuge tube containing 1 ml of LB with chloramphenicol (12.5 μg / ml) and used for inoculation. Once inoculated, the microcentrifuge tube cultures were incubated overnight at 37°C with orbital agitation. All 96 starter cultures (96 ml) were pooled together to obtain a starter inoculum representative of the entire library. Ten liters of LB with chloramphenicol were prepared, divided into glass Erlenmeyer flasks, and sterilized. Each substrate recipient was inoculated with the starter inoculum and incubated overnight at 37°C with orbital agitation.

[0104] To extract plasmid DNA from both the libraries and the empty vector control, the QIAGEN Plasmid Giga Kit was used according to the manufacturer's instructions. The resulting DNA concentrations were 688 ng / μl for the library, approximately 800 μl volume (total yield DNA=550 μg), and 268 ng / μl for the empty vector.

[0105] Inhibition test Third instar Spodoptera larvae were maintained under controlled conditions (T22–24°C, 16:8 h photoperiod) in 15 cm Petri dishes and fed as described previously (Bergomaz and Boppre, 1986). Treatments included solutions of DNA fragments obtained from salmon (Salmo salar) and Spodoptera by extraction and sonication as described in Mazzoleni et al. (2015b), as well as a mixture of autologous and heterologous DNA obtained by extraction from the BAC library described above.

[0106] Each of the three treatment solutions was diluted to a DNA concentration of 200ng / μl and 4mL of each solution was included in the daily diet of the larvae. Twenty replicates (Petri dishes, each containing 30 larvae) per treatment were maintained in controlled conditions for 20 days following the start of the growth experiment. Control replicates (N=10) were also maintained under the same conditions and fed a standard untreated diet. Larval survival for all treatments was assessed at the end of the 20-day growth period.

[0107] result The results of the study in experiment 4 showed that the survival of Spodoptera larvae was significantly affected by exposure to self-DNA by ingestion, both in the presence and absence of heterologous DNA (i.e., DNA of the microbial strain used as the BAC library vector) in the feeding pellets. Indeed, in these two cases, 5% and 0% survival rates were observed after 20 days (Figure 4). Conversely, in the case of feeding pellets containing only heterologous DNA (i.e., salmon DNA), no significant reduction in survival was observed compared to untreated controls, indicating that heterologous DNA alone does not induce an inhibitory effect on the larvae (Figure 4).

[0108] Example 5 In a confirmatory experiment, to further confirm the findings from the above experiments that fragmented self-DNA is inhibitory to individuals of a species when presented in association with foreign DNA (e.g., DNA from the host organism used for DNA amplification), we tested the inhibition of Drosophila melanogaster by self-DNA obtained by amplification in yeast, a suitable food source for Drosophila melanogaster.

[0109] Self-DNA Amplification Drosophila melanogaster cultures were obtained using standard rearing methods. Yeast cells containing fragments of Drosophila melanogaster DNA were engineered at Bio S&T (Montreal, Canada). For this total DNA isolated from an individual Drosophila melanogaster, the culture was partially digested using Sau3 AI. The digested DNA fragments were cloned into the pGADT7 vector using the BamHI cloning site. E. coli DH10B host cells were transformed with the pGADT7 vector containing the cloned Drosophila melanogaster fragments, and the resulting library was grown overnight and amplified on agar medium. Approximately 120,000 original clones were plated in large Petri dishes (total of 10 dishes) in semi-solid medium. The colonies were then washed and collected in 10 tubes. A maxiprep was performed to isolate the library DNA.

[0110] The isolated DNA from the E. coli library was then used to transform Saccharomyces cerevisiae Y187 host cells. The library was amplified on agar medium by overnight culture and plated on semi-solid medium, approximately 120,000 original clones per large Petri dish (total of 10 dishes). Colonies were then washed and collected in 10 tubes. Insert size determination was performed on 10 randomly selected clones by gel chromatography, showing an average size of 4k with a range of 1-6kb.

[0111] Inhibition test The yeast library was used as a component of the standard feeding diet (Sang (1978)) for Drosophila from hatching to the pupal stage. Treatments consisted of the following: self-DNA treatment: fed with the yeast library; control 1: fed with the same yeast strain without the vector as a control; control 2: fed with the same yeast strain containing the empty vector. The results are shown in Table 3 below. [Table 3]

[0112] Results showed significant inhibition for larvae fed yeast libraries containing Drosophila DNA. Significant prolongation of development time (twice as long as controls) and mortality (40% at 3rd instar and 83% at pupae) were observed during larval development. No differences were observed between control 1 using standard diet and control 2 using yeast containing an empty library vector, thus demonstrating that inhibition (as determined by life stage duration and mortality levels) was due to Drosophila DNA insertion into the yeast library.

[0113] Example 6 Inhibition of Aleppo pine (Pinus halepensis) and cottonwood (Populus nigra) Based on the effective inhibition of target species by (parts of) self-DNA fragments integrated into artificial DNA constructs that replicate in the host organism, it can be expected that self-DNA integrated into the native genome of the host species will be similarly effective in inhibiting the target species. Horizontal gene transfer is known to occur widely in nature, including in the soil microbiome.

[0114] To confirm the inhibition of the target species by self-DNA integrated into the native genome of the host species in this experimental soil microbiome, two different litter materials (Aleppo pine and poplar leaves) were inoculated with soil microbes from an oak forest soil where the two species are absent. Decomposing litter was used for inhibition tests as in Mazzoleni et al. 2015. Decomposing litter material showing homotypic inhibition was used for metagenomic analysis of microbiome DNA to confirm the integration of plant DNA into the soil microbiome DNA.

[0115] In detail, fresh leaves of Aleppo pine and poplar were collected from the field and used in the decomposition experiments in the following way: nets were placed under randomly selected plants (n>20) and freshly fallen leaves were collected in the natural stands, dried at room temperature in a ventilated chamber until constant weight was reached, and then stored at room temperature. The decomposition experiments were carried out in a growth chamber under optimal water availability and temperature conditions. Litter was watered with distilled water to water-holding capacity every 7 days, and the temperature was 18±2°C at night and 24±2°C during the day. Dry leaf litter (100 g of each species in three replicates) was placed in plastic trays (size 30×50×50 cm). Microbial inocula collected from two species-free oak forests were prepared and sectioned according to Bonanomi et al. (2011). Litter was collected 120 days after inoculation and dried in paper bags (at 40°C until constant weight was reached).

[0116] To confirm the species-specific inhibitory effect of decomposed litter materials, a bioassay test was performed as follows: 8 cm wide sterile filter paper strips were placed in square Petri dishes (size 12 × 12 × 1.5 cm). Pre-germinated seeds of each species (5 per dish) were placed on top of the paper strips previously amended with different decomposed litter materials. A root observation plate was placed at 45° and covered with an opaque sheet. Root length was evaluated after 28 days.

[0117] To separate the microbial population from the decomposed material, 1 g of litter sample was placed in a 5 ml tube. 3 ml of phosphate-buffered saline (PBS) was added to each sample. The samples were vortexed for 60 seconds to homogenize, then centrifuged at 10,000 rpm for 1 minute to separate the microbial cells from the litter residue. After centrifugation, the supernatants of the separated microbial cells were transferred to a new 2 ml tube. After the pooling step, DNA was extracted from the microbial cells by the CTAB method. At the end of the extraction step, the samples were treated with polyvinylpyrrolidone (PVP) to remove phenol to avoid possible inhibition to PCR. The extracted DNA was further purified with ethanol, then dried in a microbiological laminar airflow chamber, and eluted with nuclease-free PCR-grade water.

[0118] The metagenomics analysis of the decomposing litter was performed by standard shotgun sequencing methods. The paired-end reads from the metagenomics were then analyzed according to the bioinformatics procedures described below to find recombinant fragments belonging to both microbial species and DNA from plant species of the decomposing litter.

[0119] To find recombinant DNA fragments that could belong to both the abovementioned plants and the corresponding microbiome species, the following procedure was followed: Paired-end reads from the metagenomics experiments were merged using the BBmerge function of BBmap (Bushnell (2016)) with the kmer option applied to reduce false positives (extend2=20, iteration=5). Unmerged paired-ends were fused by inserting 0N between the forward and reverse reads using the fuse.sh function of BBmap (Bushnell 2016). The merged and fused reads were then aligned against the host reference genome or related species if the genome was not available using BLASTn (Camacho et al. 2009). The following reference genomes were used: loblolly pine (Pinus taeda) (1760464 scaffolds, ~22 GB) and cottonwood (Populus trichocarpa) (1694 scaffolds, ~500 MB). All reads that had at least one hit with the host genome were considered as matches. To confirm matches with reference species, reads matching the host genome were independently aligned against the NCBI nonredundant database using BLASTn (Camacho et al. 2009).

[0120] result Bioassay experiments confirmed species-specific inhibitory effects of decomposing litter for Aleppo pine and Poplar: root growth of germinating seeds of Aleppo pine was 50% less in its own litter compared to that in Poplar litter; root growth of Poplar was 70% less in its own decomposing litter compared to Aleppo pine litter.

[0121] Analysis of metagenomic data from decomposing litter clearly showed the presence of several recombinant DNA fragments between plant species and microorganisms. Starting with 44,431,532 integrated reads, 108,288 reads matched the loblolly pine genome scaffold. The 108,288 reads were then compared against the NCBI Nucleotide Database (NT), and 100% of the reads found at least one match with 41,533 unique IDs in the NT database. We selected a random subset of 10,000 IDs from the unique IDs to obtain detailed taxon annotation. From the total number of matching sequences (108,288), annotation was confirmed for a subset of 29,149 sequences, and 4,595 sequences were confirmed to match loblolly pine sequences in NT. Of these 4,595 sequences, 1,359 sequences had lower than 80% coverage. This means that the sequence can be considered as a recombination sequence between pine and microbial species. To confirm the specificity of the pine genome incorporation by microorganisms in pine litter, we also checked the match with cottonwood, and only 139 sequences were found. Similar results are found in the case of poplar litter. The annotation results are summarized in Tables 4 and 5.

[0122] The presented analysis by a shotgun approach clearly confirms the enrichment in the metagenomics data of fragments with partial similarity to the reference genomes of decomposing plant litter. In other words, the results show the specific inclusion of DNA fragments originating from the litter matrix, thereby giving rise to a microbiome characterized by plants exhibiting an inhibitory effect.

[0123] [Table 4] [Table 5]

[0124] To confirm these remarkable findings, the experiment was repeated and the analysis expanded. Leaf collection, litter production, and material handling were as described above. Litter material was incubated and decomposed under optimal conditions for 120 days, and samples were collected after 30 days and at the end of the experiment. Fresh litter and decomposed material from 30 and 120 days were used for DNA extraction using standard commercial kits as described by Mazzoleni et al. (2015) and the extracted DNA was analyzed. We compared the reads obtained from the shotgun sequencing approach on litter (0, 30, 120 days decomposition) from host plant species (Pinus and Populus) against the indicated reference genomes.

[0125] Bioinformatic analysis was performed according to the following procedure: Merge paired-end reads from metagenomics experiments using the BBmerge function in BBmap (Bushnell (2016)) applying the kmer option to reduce false positives (extend2=20, iteration=5).

[0126] The integrated reads were then aligned against the host genome using BLASTn (Camacho et al. (2009)). All reads with at least one hit are listed as a whole set of reads with matches, and are grouped according to their coverage. Potential recombinant fragments are those with matches between 20-80 or 90% coverage. Filtering at 90% identity is also considered. Further details on the bioinformatics procedures can be found in Bushnell (2016) and Camacho et al. (2009).

[0127] The results are shown in Table 6. The results show that the frequency of overall plant DNA fragments found in the samples decreases with time, given that the amount of free plant DNA in the litter decreases as decomposition proceeds. On the other hand, the number of partial fragments matching plant genomes (meaning that they are fragments made up of parts of plant DNA and parts of some other DNA) increases as decomposition proceeds (Table 7), peaking after 120 days. It is important to note that litter material decomposed for 120 days shows the greatest inhibitory effect on the germination of seeds of the same species (i.e., Pinus and Populus).

[0128] Data were analyzed for the number of sequence matches in each sample (Table 6) and the relative coverage of each read from a portion of the genome (Table 7). Partial coverage reads are assumed to reflect chimeras in the read sampling.

[0129] [Table 6] Table 6 shows that examination of the match frequencies from the blast search analysis against the Pinus and Populus genomes, respectively, yields higher numbers for day 0 litter than day 120 litter, meaning that the similarity against the host plant decreases due to sequence collapse.

[0130] [Table 7] Table 7 shows that there is a relative decrease in high coverage reads (decrease in 99–100% coverage values) and an increase in partial coverage sequences (20–90% coverage) as the litter matures relative to the host plant.

[0131] Similarity to the host genome was also examined at 90% identity to discard unlikely similarities to the host genome. Reduced coverage indicates the presence of chimeras in the reads sequenced from the litter.

[0132] It is very interesting to note that, on the one hand, the number of matches with plant genomes shows a very high decrease in the fast decomposing Populus litter, whereas the slower decomposition of the Pinus litter corresponds to a very slow disappearance of Pinus sequences. On the other hand, both species materials show a continuous increase in the number of mixed fragments in which species can still be recognized, reflecting natural incorporation into microorganisms.

[0133] These results support the formation of natural libraries similar to that described further in this application for monocultures of microalgae (see below), where the phenomenon of natural library formation occurs in natural soil microbiomes where the DNA of plant litter in the environment is enriched, thus resulting in a species-specific inhibitory effect on the plant or plants that produce the litter.

[0134] (Experiment 7) Arthrospira platensis natural library To further confirm the inhibition of the target species by self-DNA integrated into the natural genome of the host species, Arthrospira platensis, a species belonging to the cyanobacteria, was also incubated with DNA extracted from Arabidopsis thaliana to induce natural uptake of DNA, and the transformed Arthrospira platensis cells were used to inhibit Arabidopsis thaliana.

[0135] Before incubation, pure cultures of cyanobacteria were grown in Zarrouk's growth medium under controlled conditions (T22–24°C, photoperiod 12:12 h, irradiance 20 μmol photons m -2 s -1 ) and kept without gassing for at least 4 hours prior to incubation.

[0136] Arabidopsis DNA for incubation was prepared by standard methods for whole genome extraction from fresh leaves and randomly fragmented by sonication bursts (details are given in the protocol reported by Mazzoleni et al. (2015a)). Samples were sonicated for 30 min with 45 sec / 15 sec on / off cycles at 20% amplitude in a VC 505 SONICS Ultrasonic processor with the microtip immersed in a 1.5 ml microtube in a water bath with cold water.

[0137] Incubation of Arthrospira platensis with Arabidopsis DNA To reduce intracellular and extracellular DNAse activity in cyanobacteria and promote the retention and integration of foreign DNA, Arthrospira platensis was pre-incubated with Mg ++ The cells were cultured in a deficient growth medium (Cao et al., 1999). During incubation, concentrated Arthrospira platensis cells (up to 5 × 10 cells obtained by gentle centrifugation) were cultured in a deficient growth medium (Cao et al., 1999). 7 Three hundred microliters of the lysed lysed Arabidopsis cells were added to a 15 mL centrifuge tube containing 300 mg of glass beads (diameter 500 μL), 100 μL of Arabidopsis DNA prefragmented by bursts of sonication, and 100 μL of Mg ++ 100 μL of starved growth medium was added to the incubation tube, which was then vortexed for 15 seconds at top speed.

[0138] Zarrouk's Mg ++ The sample volume was increased to 2.5 ml in the same tube using starved medium and incubated for 24 h in an oscillator under LD light cycle.

[0139] The volume was then gradually increased to 10 L with complete Zarrouk's medium in the photobioreactor to obtain a cell density of 1 g cell dry weight per L. After 90 days, the biomass was centrifuged and harvested to obtain a pellet of material with a cell density of 100 g cell dry weight per L.

[0140] Inhibition test The cyanobacterial pellet was sonicated and the resulting cell homogenate was incubated at room temperature for 15 days with the addition of distilled water to replenish water evaporation. Different aliquots of the homogenate were then diluted 1:1, 1:10, and 1:100 in distilled water and used to moisten filter paper in Petri dishes with three replicates containing 10 plants per concentration level. Arabidopsis seedlings were prepared and the experiment was performed as in experiment 1.

[0141] result We observed a color change in Arthrospira platensis cultures after incubation with Arabidopsis DNA, which is an indirect indication of the integration of Arabidopsis DNA into Arthrospira platensis cells. Indeed, this observed color change indicates a change in gene expression that is probably not a result of alterations in the Arthrospira platensis genome due to the integration of Arabidopsis DNA.

[0142] The results of the inhibition study demonstrated the inhibitory effect of a mixture of heterologous and self DNA on homospecific seedling viability by testing a cyanobacterial natural library of the Arabidopsis genome. At high concentrations, seedling viability was significantly lower under exposure to the natural library of Arthrospira platensis imbibed with Arabidopsis DNA compared to the control with a microalgae solution lacking Arabidopsis DNA (i.e., containing heterologous DNA but not the plant DNA) (Table 8).

[0143] Although it is most likely that only a portion of the Arabidopsis DNA is integrated into the Arthrospira platensis genome (and therefore replicated by the Arthrospira platensis cells), there is considerable inhibition of Arabidopsis seedlings. Given the results of the tests on inhibition by a portion of the Caenorhabditis elegans genome (see experiment 3), it can be expected that the inhibitory effect may be increased by combining the inhibitory transformed Arthrospira platensis culture with further cultures obtained by a similar transformation procedure. [Table 8]

[0144] Example 8 To further confirm the inhibition of self-DNA integrated into the native genome of the host species, a confirmatory experiment was performed in which two different species of cyanobacteria (photosynthetic microalgae) were grown in bioreactors, sheared Arabidopsis DNA was added to the growth medium, and inhibition of disrupted biomass from the cyanobacteria was tested.

[0145] The cyanobacteria selected were Arthrospira platensis (as in Example 7) and Synechocystis sp. PCC 6803. Prior to incubation, all cultures are maintained at the culture conditions shown in Table 1 without gassing for at least 4 h prior to incubation. [Table 9]

[0146] DNA isolated from harvested cells was randomly fragmented by sonication bursts. Samples were sonicated for 30 minutes with 45sec / 15sec on / off cycles at 20% amplitude in a VC 505 SONICS Ultrasonic processor with the microtip immersed in a 1.5ml microtube in cold water in a water bath.

[0147] Incubation of Arthrospira platensis with Arabidopsis DNA To reduce intracellular and extracellular DNAse activity in cyanobacteria and promote the retention and integration of foreign DNA, Arthrospira platensis was pre-incubated with Mg ++ The cells were cultured in a deficient growth medium (Cao et al., 1999). During incubation, concentrated Arthrospira platensis cells (up to 5 × 10 cells obtained by gentle centrifugation) were cultured in a deficient growth medium (Cao et al., 1999). 7Three hundred microliters of the cells were added to a 15 ml centrifuge tube containing 300 mg of glass beads (500 μm diameter) as described by Dunahay et al. (1997). Then, 100 ul of Arabidopsis DNA, prefragmented by a burst of sonication (approximately 10 ug), and Mg ++ 100 ul of starved growth medium was added to the incubation tube and the tube was agitated using a vortex mixer at top speed for 15 seconds.

[0148] Zarrouk's mg in the same tube ++ The sample volume is increased to 2.5 ml with starvation medium and incubated for 24 h on an oscillator under LD light cycles, then the volume is gradually increased with complete Zarrouk's medium.

[0149] Incubation of Synechocystis sp. PCC6803 with Arabidopsis DNA As described by Zang et al. (2007), Ca ++ To reduce dependent DNAse activity, cells were cultured in BG11 medium modified with EDTA (2 mM) for 2 days prior to incubation.

[0150] During the incubation, concentrate Synechocystis sp. PCC6803 cells (up to 5 × 10 cells obtained by gentle centrifugation and washed with BG11 EDTA-free medium) 7 400 μl of the culture medium (cells) is added to a 15 ml centrifuge tube containing 100 μl of Arabidopsis DNA prefragmented by sonication bursts (approximately 10 μg). The tube is then incubated for 5 hours on an oscillator under an LD light cycle, then transferred to a flask and diluted to 2.5 ml with BG11 medium, then the volume is gradually increased.

[0151] experiment Microalgae exposed to Arabidopsis DNA according to the protocol above were used as inoculum in two 5 liter bioreactors and grown under standard growth conditions until maximum cell density was reached. The algal biomass of the two species was then extracted from the medium to form a pellet, carefully washed, and then dried.

[0152] The algal pellets were inoculated with 1 g of forest soil and kept at room temperature with weekly moistening to allow natural decomposition for up to 60 days. Decaying material was sampled every 15 days and added to filter paper in Petri dishes at optimal laboratory conditions to assess germination and root growth of Arabidopsis plants on the plates.

[0153] result Undegraded pellets had no significant inhibitory effect on Arabidopsis seed germination and root elongation, but significant inhibition of germination was observed in treatments with degraded pellets from both species after 30 and 60 days of decomposition. After 90 days of decomposition, the inhibitory effect of degraded pellets showed a slight decrease. This indicates that the decomposition of cyanobacterial pellets has an obvious inhibitory effect on plant germination. This is because the plant DNA contained in the algae genome is released by the decomposition process and eventually further amplified by the decomposition soil microorganisms. [Table 10]

[0154] Bioinformatic analysis We sequenced and analyzed the genomes of the two cyanobacteria used as natural libraries in the seed germination experiments described above. We used the BBmerge function in BBmap (Bushnell (2016)) to merge paired-end reads from the metagenomics experiments, applying the kmer option to reduce false positives (extend2=20, iteration=5).

[0155] The integrated reads are then aligned against the host genome using BLASTn (Camacho et al. (2009)). All reads with at least one hit are listed, grouped according to their coverage, with the list creator listing them as all reads with matches. Potential recombinant fragments are those with matches between 20-80 or 90% coverage. Filtering at 90% identity is also considered. Further details on the bioinformatics procedures can be found in Bushnell (2016) and Camacho et al. (2009).

[0156] [Table 11] Table 11 shows the number of sequences found in DNA from the cyanobacterial samples and indicates that the matched Arabidopsis genomes differed significantly not only between samples but also between the different matching classes defined according to the percent coverage by the plant and cyanobacterial genomes.

[0157] Classes 1 and 2 showed extremely low numbers, likely related to the uncertainties inherent in the sequencing and bioinformatics methods. Class 3 showed the highest inter-sample differences, likely related to differences in genome size, chromosome copy number, and data availability of blast in the reference genome database between the two cyanobacteria. Class 4 data shows the occurrence of chimeric sequences in both sample types that may partially match both plant and cyanobacterial genomes. Class 5 showed null values ​​indicating that both sample types did not contain free Arabidopsis DNA from the chimeric sequences.

[0158] Notably, compared to Arthrospira platensis samples, Synechocystis sp. PCC 6803 samples that showed a greater inhibitory effect on Arabidopsis seedlings also showed a greater number of DNA sequences matching the Arabidopsis genome, either when considering the number of sequences of all classes pooled together or when considering the number of sequences for each meaningful class separately. This confirms that the inhibitory effect is associated with self-DNA fragments and that these inhibitory effects of self-DNA fragments are maintained when associated with host species DNA. Based on this experiment, we further confirm that host species cells can effectively replicate the inhibitory self-DNA of a phylogenetically distant target species in its native genome.

[0159] (Experiment 9) Further validation experiments were performed by testing inhibition of the weed purslane (Portulaca oleracea) by fragmented self-DNA that had been degraded and absorbed by the soil microbiome in test plots of spinach grown on a farm where purslane ectoinfestation is a recurring problem.

[0160] The Portulaca oleracea BAC library was originally generated by Bio S&T (Montreal, Canada) with methodology similar to that used for the Croton sieboldii library discussed in Experiment 2. An overnight culture of the library (Terrific Broth (TB) as culture medium, 250 rpm agitation, T=37° C.) was used as a starter culture for a 60 L bioreactor fermentation with a working volume of 40 liters (1% of the working volume of the bioreactor). Nutrient broth (NB) was used as the substrate for large-scale liquid fermentation. In addition, 0.1% L-arabinose was added to the total volume of the culture medium to induce the pSmart-Hindlll plasmid. Liquid fermentation was carried out for 2 days under controlled conditions of temperature, agitation, aeration, and pH to obtain a high cell density fermentation broth and stimulate the production of plasmid DNA. The concentration reached was OD 600 As estimated by 8 cells / ml.

[0161] Inoculation and maturation of compost The resulting fermentation broth was subjected to boiling for about 15 minutes and then added to peat in three different concentrations, as previously described: 1.30 mL / kg(1) 2.3mL / kg (dilution 1:10) 3.0.3mL / kg (dilution 1:100)

[0162] The inoculated soil was allowed to mature in air for about 20 days, and the resulting product was then used in the field experiment.

[0163] Field experiment Field trials were carried out on farms in the province of Salerno (Italy) where purslane infestation is a recurrent problem.

[0164] Two days before sowing of spinach (mid-July), the products were distributed according to the following scheme: Outdoor experiment (block area approx. 2 x 2.4 m) [Table 12]

[0165] To experimentally evaluate the effectiveness of the products, a battle against purslane weeds was carried out in a tunnel next to the products prepared for the experimental test, using conventional farming methods and herbicides approved by current laws that bring about further regulations.

[0166] In particular, a common combating practice adopted by farmers provides for the use of metam sodium, an agricultural general-purpose pesticide mainly used as a pre-planting broad-spectrum soil fumigant to suppress weeds, weed seeds, fungi, nematodes, and soil insects. Treatment with metam sodium is expensive and harmful to the soil, resulting in a loss of rhizosphere biodiversity.

[0167] The first survey was conducted at the time of spinach emergence (approximately 10 days after sowing) and other evaluations were made at weekly intervals thereafter until the end of the cultivated spinach life cycle. To ascertain the inhibitory effect of the products on purslane germination rate, purslane plants were uprooted, counted and the roots checked for the presence of necrosis during the field evaluation.

[0168] result As shown in Figure 5, there was a significant decline in the number of purslane plants in block treatment 1, determined at the end of the study. Additionally, as expected, the number of sprouted spinach seeds was significantly higher in blocks with fewer purslane plants.

[0169] This experiment further confirms that the inhibitory effects are associated with self-DNA fragments, and that these inhibitory effects of self-DNA fragments are maintained when they are amplified in the host species and associated with the host species DNA. In light of the experimental protocol used, and taking into account the experimental results presented above, it is reasonable to expect that the amplification of purslane DNA fragments by the peat microbiome must have been due to an inhibitory effect.

[0170] (Experiment 10) Inhibition of DNA fragments from phylogenetically related species The inhibition of DNA fragments from phylogenetically similar species was tested using fragmented DNA (not associated with the host species DNA sequence) according to WO2014 / 020624. In light of the results from other experiments, if an inhibitory effect is observed for DNA fragments according to WO2014 / 020624, it can be expected that the effect will also be observed for DNA when fragmented in the composition according to the invention.

[0171] Extraction and quantification of DNA from plant material DNA was extracted from plant leaf material using the DNeasy Plant Maxi Kit as described by the manufacturer (QIAGEN, Valencia, CA) and the processing solution was prepared using the protocol by Fulton et al. (1995) (Appendix E). 100 grams of fresh weight starting material was harvested and frozen at -20°C. Samples were then immersed and stirred in 200 ml fresh microprep buffer and then filtered through cheesecloth. DNA was resuspended in pure sterile water. DNA extracted from leaves was fragmented by sonication to a target size range of 200-500 bp. This was done with a Bandelin Sonopulse (Bandelin, Berlin, DE) for 12 minutes at 0.9 sec pulses and 90% power. Verification of sonicated band sizes was done by visual inspection on 3% MetaPhor™ agarose gels (Lonza scientific, NJ) with Sybr® Safe (Invitrogen).

[0172] Bioassays with purified DNA in target plants The autotoxic and phytotoxic effects of purified DNA extracted from plant leaves were evaluated in eight target species (Table 12). The experimental design was: 1. Control with sterile distilled water; 2. Addition of total extracted and randomly fragmented self-DNA; 3. Addition of total extracted and randomly fragmented DNA from a single heterologous species (Table 1); DNA was added at 200 μg ml -1 The plants were applied at 4 ml / well. In vitro bioassays were performed using surface-sterilized seeds (n=20 in each plate) of each target species (four replicates) placed on sterile filter paper in 9 cm Petri dishes soaked with 4 ml of test solution. Seedling root length was measured after variable days since the start of the experiment depending on the target species (Table 12). Petri dishes were arranged in a growth chamber in a completely randomized design (temperature 22±2°C, watered every 2 days with distilled water). [Table 13-1] [Table 13-2]

[0173] For each bioassay, the phylogenetic distance distinguishing the target species from the plant species used as the source for DNA extraction was assessed in the following way: -Zero distance and homogeneous -Species that belong to the same family with distance=1 -Species belonging to different families within the same order with distance=2 -Species from different orders with distance = 2 + N, where N is equal to the number of tree nodes distinguishing plant orders in a reference phylogenetic tree (APG 2009, Fig. 6) reporting the phylogeny of the taxonomic orders of angiosperms (i.e. flowering plants). All plant species, families and orders used in the bioassays, as well as the phylogenetic distances obtained for all pairs of target and DNA source plants, are reported in Table 12.

[0174] Bioassay results (i.e., root growth of the target species) were expressed as a percentage of the distilled water-treated control and plotted as a function of the phylogenetic distance distinguishing the DNA source from the target species.

[0175] result Bioassay results show a clear inhibitory effect of the target species' own DNA on root growth (Figure 7). A similarly significant inhibitory effect was observed when DNA from species phylogenetically similar to the target species (within the same taxonomic order) was tested, but it was lower than the effect of DNA from the target species itself. The effect of foreign DNA from species distant from the target species was not significant. (References) JPEG2025060642000015.jpg226155 JPEG2025060642000016.jpg146155

Claims

1. 1. A composition comprising a mixture of DNA molecules having DNA sequences from different sources, said mixture comprising some first DNA sequences, preferably derived from chromosomal DNA sequences, of a source species, and some second DNA sequences, preferably derived from chromosomal DNA sequences, of a host species, which is a species different from said source species, optionally wherein said mixture of DNA molecules comprises chimeric DNA molecules in which a source species DNA sequence is flanked at at least one end by a non-source species DNA sequence, said non-source species DNA sequence being selected from host species DNA sequences, preferably chromosomal DNA sequences derived from a naturally occurring genome of said host species, or DNA sequences derived from an artificial DNA construct selected from a plasmid, cosmid or artificial chromosome, etc.

2. 2. The composition according to claim 1, comprising a population of cells of a host species, preferably from a host species selected from a microbial species, and wherein the DNA molecule is present in the host species cells, preferably live host species cells.

3. 3. The composition of any one of claims 1 to 2, wherein the DNA molecule comprises a non-fragmented chimeric DNA molecule and the source species DNA sequence comprises a partial DNA sequence, preferably a randomly generated partial DNA sequence of chromosomal DNA, or a cDNA sequence reverse transcribed from the transcriptome of the host species integrated into the chimeric DNA molecule.

4. The composition of claim 1 , wherein the DNA molecule is a fragmented DNA molecule.

5. 5. The composition of any one of claims 1 to 4, wherein the source species DNA sequences comprise chromosomal DNA sequences and the coverage of the source species genome in the mixture of DNA molecules is between 20-100%, such as 20-90%, 30-100%, 30-90%, 40-100%, 40-90%, 50-100%, 50-90%, preferably 60-100%, such as 60-90%, 70-100%, 70-90%, 80-100%, 80-90%.

6. 6. The composition of any one of claims 1 to 5, wherein the non-source species DNA, preferably the host species DNA, exceeds the source species DNA, preferably such that the ratio of non-source species DNA:source species DNA is between 10:1 and 1000:1, such as between 20:1 and 500:1, 50:1 and 500:1, 100:1 and 500:

1.

7. 7. The composition according to any one of claims 1 to 6, wherein the host species is selected from microbial species such as bacterial species, or species from the Ascomycota, or species from the Archaea, or multicellular organisms such as microphytes, multicellular plants, or helminth species, soil microorganisms, GRAS status microorganisms, microbial control agents.

8. the bacterial species is selected from E. coli, Bacillus species, Pseudomonas species, Lactobacillus species (such as Lactobacillus species, Leuconostoc species, Pediococcus species, Lactococcus species, or Streptococcus species), Cyanobacterial species (such as Arthrospira species, in particular Arthrospira plantenis or Arthrospira maxima); Microbial species from the Ascomycetes can be yeast species, such as, for example, Saccharomyces cerevisiae, or Aspergillus species, such as, for example, Aspergillus niger, Aspergillus oryzae, or Aspergillus nidulans, or lactic acid bacteria, such as, for example, Lactobacillus species, Leuconostoc species, Pediococcus species, Lactococcus species, Enterococcus species, or Streptococcus species, or cyanobacteria, such as, for example, Arthrospira species, in particular Arthrospira plantenis, or Arthrospira maxima, or lactic acid bacteria, such as, for example, Lactobacillus species, Leuconostoc species, Pediococcus species, Lactococcus species, Enterococcus species, or Streptococcus species, or cyanobacteria, such as, for example, Arthrospira species, in particular, Arthrospira plantenis, or Arthrospira maxima, or lactic acid bacteria, such as, for example, Lactobacillus species, Leuconostoc species, Pediococcus species, Lactococcus species, Enterococcus species, or Streptococcus species ... maxima) the multicellular plant is selected from crops, preferably food crops (e.g. selected from cereals, maize, sugar beet, rapeseed, pea, soybean, etc.), oil crops (e.g. rapeseed, Ethiopian mustard, sunflower, etc.), starch crops (e.g. (sugar) sorghum, etc.), fibre crops (e.g. flax or hemp, etc.), lignocellulosic crops (e.g. grass reed, bamboo, switchgrass, miscanthus, cardoon, etc.), short rotation forest crops (e.g. willow, poplar, or eucalyptus, etc.); the helminth species is selected from helminths used in helminthic therapy (e.g. Trichuris suis, Necator americanus, Trichuris trichiura, Hymenolepis diminuta, Ascaris lumbricoides, Strongyloides stercoralis, Enterobius vermicularis, or Hymenolepis nana); the soil microorganism is selected from Alphaproteobacteria spp., Betaproteobacteria spp., Deltaproteobacteria spp., Actinobacteria spp., Thermoleophilia spp., Rubrobacter spp., Chloracidobacteria spp., Acidobacteria spp., or Solibacteres spp.; The composition according to claim 7, wherein the microbial control agent is selected from bacterial biocontrol agents, in particular Bacillus thuringiensis or Bacillus subtilis, or fungal biocontrol agents, such as insect pathogenic fungi (such as Beauveria bassiana, Isaria fumosorosea, Lecaninicilium spp. or Metarhizium spp.), or for example Trichoderma spp. or Ampelomyces quisqualis.

9. 9. The composition according to any one of claims 1 to 8, wherein the source species is selected from a target species or a species phylogenetically similar to the target species, the target species being a disease-associated species, such as a pathogenic species, a parasitic species or a species acting as a disease vector, or an ectoparasitic species or a species associated with the deterioration of a product, such as food and / or cosmetics and / or pharmaceuticals and / or other products containing organic matter.

10. 10. The composition according to claim 9, wherein the target species is selected from pathogenic species, preferably local pathogenic target species, most preferably pathogenic target species selected from archaea, bacteria, fungi (including yeasts) and protozoa, parasitic species (especially species parasitic on humans and / or livestock and / or pet animals, preferably parasitic target species selected from skin parasites and / or digestive tract parasites and / or mucosal parasites), plant pathogenic species (e.g. plant pathogens selected from fungi, oomycetes, bacteria, viruses or protozoa), agricultural pest arthropods, disease vectors of animal diseases, preferably disease vectors selected from arthropods, disease vectors of plant diseases, preferably disease vectors selected from arthropods, plant parasitic nematode species, weed species or species causing product deterioration.

11. 11. The composition of any one of claims 1 to 10, wherein the host species comprises a food source for the target species, e.g. the host species is selected from a bacterial species and the target species is selected from a bacterial-feeding nematode species, or the host species is a multicellular plant and the target species is a phytophagous species, such as a phytophagous arthropod species or a terrestrial gastropod species.

12. the size of said source seed DNA sequence is from about 50 bp to about 2 Mb, such as 50-3000 bp, 100-1500 bp, or 100-2000 bp, 100-2500 bp, etc., alternatively from 0.3 to 15 kb, such as 1-10 kb, such as 5-10 kb, alternatively from 15 to 60 kb, such as 15-40, 25-40 kb, etc., alternatively from 40 to 340 kb, such as 50-200 kb; 12. The composition of any one of claims 1 to 11, wherein the length is from 120 to 300kb, such as from 200 to 300kb, or from 250kb to 2Mb, such as from 400kb to 1500kb or such as from 400kb to 1000kb, or from 0.3kb to 2Mb, such as from 1kb to 2Mb, such as from 10kb to 2Mb, such as from 100kb to 2Mb, such as from 360kb to 1Mb.

13. 13. The composition of claim 1, wherein when DNA of an artificial DNA construct is present, the composition comprises DNA of an artificial DNA construct selected from a plasmid, a cosmid, a fosmid, or an artificial chromosome, such as a bacterial artificial chromosome, a yeast artificial chromosome, or a fungal artificial chromosome.

14. 14. The composition of any one of claims 1 to 13, wherein the source species and the host species are phylogenetically distant, preferably by being selected from different taxonomic orders, such as being selected from different classes, different phyla, different kingdoms, or different domains.

15. The source species is selected from the target species or species phylogenetically similar to the target species, the target species being in particular selected from the family Tortricidae, such as for example the genus Choristoneura (in particular Choristoneura orae, Choristoneura fumiferana or Choristoneura freemani) or from the genus Spodoptera (in particular Spodoptera frugiperda, Spodoptera litura, Spodoptera littoralis, Spodoptera the target species is selected from the order Lepidoptera, for example from the family Noctuidae, such as from the genus Plodia or Ephestia, or from the order Diptera, Coleoptera, or Hymenoptera, most preferably from the genus Chorystoneula, and the composition ... and the composition is selected from the order Lepidoptera, for example from the order Lepidoptera, Coleoptera, or Hymenoptera, most preferably from the genus Noctuidae, and the composition is selected from the order Lepidoptera, for example from the order Lepidoptera, Coleoptera, or 15. The composition of any one of claims 1 to 14, further comprising a biocontrol agent such as Bacillus thuringiensis, Bacillus subtilis ...

16. 16. The composition of any one of claims 1 to 15 in a pharma- ceutically acceptable form.

17. 16. The composition of any one of claims 1 to 15, which is an agricultural composition.

18. 17. The composition according to any one of claims 1 to 16, wherein the source species is selected as a target species or is a species phylogenetically similar to the target species, preferably the species are phylogenetically similar if they are from the same taxonomic order such as the same family or genus, for use as a medicament, in particular for the treatment of infections by the target species selected from parasites and / or pathogens, preferably infections by pathogens and / or parasites of the skin, nails or mucous membranes, in particular the mucous membranes of the eyes, mouth, gastrointestinal tract, vagina, urinary tract, respiratory tract including the lungs.

19. 17. Use, such as non-medical or medical, of a composition described in any one of claims 1 to 16 as a product to inhibit a species that is a target species, the source species being selected from the target species or a species that is phylogenetically similar to the target species, preferably when the species are from the same taxonomic order such as the same family or genus, the species being phylogenetically similar.

20. 20. The use according to claim 19, wherein the composition comprises a host species cell, the DNA molecule is in a host species cell, and in said use the host species cell is disrupted and material from the disrupted host species cell is used to inhibit the target species, optionally after isolation of DNA from the disrupted cell.

21. 20. A method for inhibiting a species which is a target species, the source species being selected from a target species or selected from species which are phylogenetically similar to the target species, preferably where the species are from the same taxonomic order such as the same family or genus, the species being phylogenetically similar, comprising exposing the target species to a composition according to any one of claims 1 to 17.

22. 22. The method of claim 21, wherein the method is a non-therapeutic method or a therapeutic method.

23. - providing a source of DNA from a source species, optionally including fragmented source species DNA; - providing cells of a host species which is some species different from said source species; - subjecting said host species cells, together with a source of DNA from said source species, to conditions allowing said host species cells to take up said source species DNA in a replicable form; - optionally isolating the host species cells; - optionally isolating DNA from said host species cells; - optionally fragmenting the DNA content of said host species cells; 18. A method for producing the composition of claims 1 to 17, comprising:

24. 1. A method for producing agricultural products from a number of production species, other than human species, susceptible to infection by a pathogenic or pest species and / or susceptible to being infested or ecto-infested by a pathogenic or pest species, comprising: - providing several individuals of said several production species in an area that is a target area; - providing a composition according to any one of claims 1 to 15 or 17 to an individual of said production species or to said target area; - providing suitable nutritional and environmental conditions to said individuals of said several production species to produce agricultural products; The method comprises:

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