FIELD OF THE INVENTION

The invention relates to a method for disinfestation of soil, especially infested with fusarium oxysporum f.sp. cubense (Foe). BACKGROUND OF THE INVENTION

Disinfestation of soil in asparagus cultivation has been described for example in Acta Horticulturae, 776 (2008), 135, where the authors studied the combating of Fusarium oxysporum f.sp. asparagi and Fusarium redolens f.sp. asparagi. Although fusarium is not fully destroyed, the asparagus plants planted out in the decontaminated plots seemed to do better. The authors also reported a better quality and a higher yield of asparagus over several years after this biological soil decontamination.

SUMMARY OF THE INVENTION

Global consumption banana culture is a monoculture of the variety Cavendish. The Cavendish variety was "discovered" by accident after the most recent outbreak of the Panama Disease (the fungus Foe) in the 1950's, wiping out almost completely all Gros Michel banana plantations in Mid and Central America and making the more tasty variety Gros Michel almost extinct. The Cavendish- variety was resistant towards the Fusarium-fungus Tropical Race 1 (Foe TRl ) and was cloned rapidly in order to save the global banana culture.

Since the 1990s a new strain, the so-called Tropical Race 4 (TR4), of Fusariu oxysporum f.sp. cubense (Foe), the causal agent of Panama disease, is disseminating widely in East and Southeast Asia where upwards of 100,000 hectares have been lost. TR4 has recently been identified in the Middle East, the Indian subcontinent and Africa triggering alarms in Latin American and the Caribbean where seven of the top ten exporting countries are based. Cavendish clones are extremely susceptible to TR4, which also is pathogenic on a wide variety of other bananas that are destined for local markets. Foe produces resting spores, which survive for decades in the soil, thereby disabling further banana production unless resistant banana varieties are cropped. At the moment no resistant varieties matching the productivity and market acceptance of Cavendish and other susceptible local cultivars are known. Other Foe pathogenic strains, endemic globally, affect important market cultivars and thereby complicate the challenge of monitoring the spread of TR4, since no diagnostic tools for these strains are currently available. However, these strains offer opportunities to build readiness for TR4 through more effective exclusion, containment and management of other Foe genotypes where already present.

Although host resistance is the preferred approach to these threats, banana breeding has hitherto not delivered widely accepted new cultivars. Therefore, on the short and medium term, disease management practices and more effective measures to slow, prevent and contai n incursions into new areas are the focus of innovation.

Fusarium wilt may in many soils for different types of produce successfully be combatted by known methods like steaming, chemical fumigation, biofumigation, and solarization

Although Fusarium wilt may be defeated for many different types of produce, combatting Fusarium oxysporum f.sp. cubense in bananas appears very hard or is, especially for TR4, not possible using prior art solutions. Presently used methods, essentially, do not seem to be able to solve the problem. Known fungicides seem largely ineffective. Chemical sterilization of the soil with methyl bromide may significantly reduce incidence of the disease but as found to be effective for only three years after which the pathogen had recolonized the fumigated areas. Injecting the host plants with carbendazim and potassium phosphonate appears to provide some control but results have been inconclusive. Heat treatment of soil has also been tried in the Philippines but the pathogen is likely to reinvade the treated area. Moreover, at. their website http://www.promusa.org/, ProMusa (a recognized network of scientists and other stakeholders working on banana) pays a lot of attention to the problems with Tropical Race 4 (TR4) of the the fungus Fusarium oxysporum f. sp. cubense (Foe). ProMusa acknowledges the problem with respect to controlling TR4, and describe that like all other so l-dwelling Foe strains, TR4 cannot be controlled using fungicides and cannot be eradicated from soil using fumigants (see Tropical race 4 (20 June 2018) In Mmapedia, the banana knowledge compefufium Retrieved 5 July 201 8, from http : //www. promu sa. org/Tropi cal+race+4+~+TR4) . Furthermore, also the Government of Quensland informs their banana growers about the fact that at present there is no known cultural, biological or chemical control or cure for Panama disease tropical race 4. If Panama disease tropical race 4 is confirmed, infected banana plant remnants along with any other plant material is injected with a combination of chemicals to die the plants. Dead plants and plant material is sprinkled with urea to speed up decomposition. The entire area is securely covered in thick high grade plastic. Any future approved use of these sites is currently under consideration by Biosecurity Queensland. It will, however, not involve the production of bananas (see Department of Agriculture and Fishesries, Biosecurity Queensland, Panama Disease Tropical Race 4; Frequently Asked Questions (Version 2, August 2016) In Panama disease tropical race 4 Growers Kit. Retrieved 5 July 2018 from https://pL3blications.qld .gov. au d.ataset panama-disease-iropical-race~4-grovver- kit/resource/94304dc3-f5bb-4494-adf6-f3543 cae2 b05).

Hence, it is an aspect of the invention to provide an alternative method for disinfestation of a soil, such as in embodiments infested with Fusarium oxysporum f.sp. cnbense, that preferably further at least partly obviate(s) one or more of the above- described drawbacks. In a further aspect, the invention provides a method for planting a banana plant in a plot after disinfestation of the soil of the plot, that preferably further at least partly obviate(s) one or more of the above-described drawbacks.

The present invention may have as object to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.

In a first aspect, the invention provides method for disinfestation of a soil, wherein the method comprises an introduction stage and a treatment stage. The introduction stage comprises introducing a product comprising organic material into (a determined volume of) the soil, especially wherein the product comprises a protein. The treatment stage comprises applying a barrier (layer) to the soil, especially covering the soil comprising the product with a barrier (layer), during a treatment time, especially wherein an anaerobic condition is provided and maintained (in the soil). The (infested) soil may be infested with any kind of harmful (pathogenic) organisms, such as nematodes, fungi and bacteria, protozoa. In embodiments, the soil (to be disinfested) is infested with Fusarium oxysporum f.sp. cnbense. Further, especially the soil is configured as soil for banana plants.

The treatment time may comprise at least 1 week, such as at least 2 weeks, especially at least 3 weeks, such as at least 4 weeks. The treatment time preferably is equal to or less than 3 month, such as equal to or less than 12 week, especially equal to or less than 10 weeks. In embodiments, the treatment time is selected from the range of 2-10 weeks, such as 4-10 weeks.

The method may especially reduce the number of harmful organisms, such as fungi and inactivates or kills spores of e.g. the Fusarium ox sponim f.sp. cubense present in the soil. Although known prior art solutions do not seem to be capable of inactivating or killing the spores op Fusarium oxysponim f.sp. cubense present in the soil, it was surprisingly found that using the method of the invention these spores may successfully be inactivated and/or killed The method may allow to plant new banana plants that may stay free from the Panama Disease.

Herein, the method is especially exemplified in relation to bananas, banana fields, and the Panama Disease (see further below). The invention however, (also) provides the method for other types of diseases and harmful organism, and contaminated soil. The invention also provides the use of the product described herein to disinfest infested soil as described herein (i.e. infested with Foe as well as with other harmful organism).

Especially, in the treatment stage, a barrier is applied to the soil, and especially the barrier is maintained. The barrier may comprise a barrier (layer) at the soil, especially between the soil and air (above the soil). Hence, the barrier may relate to a barrier layer, especially covering the soil. Alternatively or additionally, the barrier may also be comprised by part of the soil, especially a top layer of the soil. For instance, the barrier may comprise water. Water may form a layer on top of the soil. At least a part of the water may also penetrate in the soil. Below, the barrier may especially be explained by a barrier, such as a plastic layer, especially covering the soil. For such embodiments, especially the term "barrier layer" may be used. For other embodiment, the term "barrier layer" may be replaced by the term "barrier". Especially, for embodiments related to inundation (see further below) the term barrier may be used (instead of barrier layer). Yet, in embodiments related to inundation, also the term barrier layer may be used herein. Likewise, the phrase "a barrier layer applied between the soil and the air" in some and the like used in some embodiments, may in other embodiments be replaced by the phrase "a barrier applied to the soil". The phrase "a barrier applied to the soil" may comprise different types of barriers and/or different ways to arrange the barrier to the soil. The term may relate to applying the barrier layer on the soil, such as covering the soil with the barrier layer. Additionally or alternatively, the barrier layer is applied into (especially a top layer) of the soil ( see below).

The method is especially a biological method, wherein the product, especially a carbon source, comprises an organic material, especially a non-living organic material.

The term "non-living organic material" is used herein to denote an organic material that is not in the form of unprocessed plants, plant residues, animals or animal residues, where "unprocessed" means not processed or only subjected to processing that consists of cutting, such as mowing or chopping. Organic materials like grass, straw, leaves, etc. are not covered by the term "non-living organic material". On the other hand, the term does cover organic material obtained by processing plants, animals or parts thereof, where processing entails more than mere cutting; for example, proteins, lipids and carbohydrates that have been isolated are covered by the term. In one of the embodiments, organic materials such as isolated gluten are also covered by this term. In another embodiment, the term also covers organic materials chosen from a group comprising humus, compost, extracted soya bean meal, bone meal, gelatin and optionally granulated manure. The (non-living) organic material especially comprises the protein. The preferred non-living organic material is non-living biomass. The term "lipid" may relate to a fat as well as to an oil. The product, especially the protein, may comprise a plant (based) protein, such as a grain protein.

The product especially comprises processed (plant based) material. The term

"processed material" will be understood by the skilled person and relates to material (from products as well as side streams/by-products) obtained from any kind of processes applied to animal or vegetable base material after harvesting it, especially wherein the structure of the material is changed such as at least partly disrupted as a result of the applied process conditions (such as heat, pressure, shear, friction, pH, etc.). The term especially relates to primary processed and/or further processed (plant based) material. In primary processing harvested agricultural raw material may be processed to provide standardized ingredients, like oil, sugar, protein isolates, etc., for the food, feed or other industries. Further processes especially provide final food, feed or non-food products. Examples of further processes comprise the production process of a food product, a portioning process for meat products, fermentation processes, et cetera.

The treatment stage may comprise applying a barrier (layer) to the soil, especially at least partly between the soil and the air (during the treatment time). The invention therefore also relates to a method for disinfestation in which a product containing organic material is introduced into the soil, wherein the product is selected from a group comprising proteins, carbohydrates and lipids, especially wherein the product comprises a protein, and is not in the form of unprocessed plants, plant residues, animals or animal residues, and wherein the method also comprises the application of a barrier (layer) (at least partly) between the soil and the air. Especially, the carbohydrates comprise rapidly degradable carbohydrates, such as sugars, starches, pectins).

Especially, the method for disinfestation relates to disinfestation of soil infested with Fus rium oxysporum f.sp. cubense comprising a race selected from the group consisting of the races TR1, TR3 and TR4, especially comprising the race TR4.

In an advantageous embodiment according to the present invention, the method of disinfestation involves the introduction of a product into the soil, wherein the product preferably comprises one or more materials chosen from a group consisting of proteins, carbohydrates and lipids but does not contain any unprocessed fresh plants or unprocessed fresh plant residues, and where the product preferably comprises one or more of the various types of powders, granulates, liquids and agricultural by-products described below, followed by the application of a barrier to the soil.

The term "anaerobic condition" especially relates to a condition wherein an amount of oxygen in a gaseous material comprised by the soil (especially in the pores and/or between soil particles) is lower than an amount of oxygen in air. Especially, said amount of oxygen in the gaseous material comprised by the soil is less than 5 vol.%, especially less than 2 vol.%, such as less or equal to 1 vol.%, especially at most 0.5 vol.%.

The term "unprocessed fresh plants or unprocessed fresh plant residues" denotes fresh plants or plant residues that have not been subjected to any treatment other than optional cutting, such as mowed grass. The further processing of plants or plant residues can lead for example to products such as extracted rapeseed meal, extracted soya bean meal, gluten, steamed potato peelings and protamylasse.

The term "agricultural by-product" is used here to denote materials formed in farming and containing proteins and/or carbohydrates and/or lipids, an example being slaughter waste. Other examples of agricultural by-products are extracted soya bean meal, steamed potato peelings and bone meal. A further example of agricultural byproduct is brewer's spent grain. Brewer's spent grain may comprise about 20 wt% protein. Especially barley may be used to brew. Yet also other grains may be used to brew. The product, especially the protein, may especially comprise grain protein. The product preferably comprises a material chosen from a group comprising proteins, carbohydrates and lipids and is preferably non-living biomass. Especially, the product (at least) comprises a protein.

The preferred products contain at least 5 wt%, such as especially at least 10 wt% of protein on a dry-matter basis. In embodiments, the product comprises at least 15 wt% protein (on a dry-matter basis). Especially, the product comprises an amount of protein selected from the range of 10-40 wt%, especially 15-40 wt%, such as 20-35 wt%. In an embodiment, the product comprises more than 40 wt% protein, such as at least 50 wt% protein, such as at least 70 wt% protein. The product may substantially completely consist of proteins.

Herein, weight percentages especially relate to weight percentage on a dry- matter basis, unless otherwise indicated in the description or unless it will be clear to the skilled person that another basis is meant.

In a further embodiment, the method of disinfestation comprises the introduction of a particulate organic product into the soil, followed by the application of a barrier layer between the soil and the air.

In an embodiment, the introduction stage comprises introducing a product comprising organic material into the soil; wherein the product comprises a protein. In further embodiments, the treatment stage comprises covering the soil comprising the product with a barrier (layer) during a treatment time, wherein an anaerobic condition is maintained in the soil, wherein the treatment time is selected from the range of at least 1 week, especially from the range of 4 weeks- 10 weeks.

The term "particulate organic material" is used herein to denote a particulate product, essentially consisting of particles defined below (such as powders and/or granulates), wherein the particles contain one or more organic components preferably chosen from a group comprising proteins, carbohydrates and lipids. The product may further comprise a fiber. A fiber may especially comprise a slowly degradable carbohydrate, e.g. cellulose, hemicellulose, and lignin. Hence, a fiber may comprise a carbohydrate.

It has also been found that advantageous results are obtained in particular from a method of disinfestation that comprises the introduction of a particulate protein- containing product into the soil and the application of a barrier layer between the soil and the air. In a further embodiment, the product (to be introduced into the soil) is therefore in the particulate form. The particles of the product introduced into the ground preferably have a d:,.2 value of about 0.5 μιιτι- 10 mm, especially about 1 μιη-5 mm, and more especially about 0.1-5 mm. The d;,,2 value of a particle is defined as the volume/surface area mean surface or Sauter mean diameter.

Herein, the term "product" may especially relate to "the product to be introduced into the soil". If the product is introduced in the soil, characteristics of the product may change; a granular product may, e.g., fall apart or dissolve and potentially forming a liquid, et cetera.

Powder is a particulate product whose particles can range from very small (for example of the order of magnitude of about 0.5-100 μιιτι) to quite large (for example of the order of magnitude of about 0.1-1 mm). In the case of a granulate, the particle size can vary for example from about 1 to 10 mm. According to one of the advantageous embodiments, the product is a granulate. According to another advantageous embodiment, the product introduced into the soil is a powder. The product may (also) comprise a granulate and also a powder. The product may especially comprise a low moisture content. In embodiments, the moisture content is equal to or less than 15 wt%, especially equal to or less than 12 wt%, such as equal to or less than 10 wt%. Especially, the dry matter weight of the product may comprise about 85-95 %, especially 90-95%, of a weight of the product including the (weight of the) moisture.

In a further embodiment, the product comprises a liquid. The term "liquid" is used here to denote a pourable substance comprising one or more components. The person skilled in the art will realize that a liquid can be for example an emulsion, dispersion, solution, aqueous slurry, suspension and the like. An example of the liquids that can be used in the method is milk, such as cow's, calf s, goat's milk, etc. In an embodiment, the liquid or slurry preferably comprises at least 10 wt% of proteins, calculated on a dry-matter basis. In a further embodiment, the liquid or slurry comprises at least 10 wt% of protein and/or at most 90 wt% of carbohydrates and/or at most 90 wt% of lipids, on a dry-matter basis, totaling 100 wt%. In one of the embodiments, the liquid or slurry comprises at least 1 wt% of carbohydrates and/or lipids on a dry-matter basis, totaling 100 wt%. If the liquid or slurry comprises a protein in combination with a carbohydrate and/or a lipid, then the total amount of the carbohydrate and the lipids in wt% is preferably less than about ten times the amount of protein in wt%. In a further embodiment, the product (to be introduced into the soil) comprises a slurry, such as for example an aqueous slurry of one or more of the following substances: wheat gluten, thick potato sap, protamylasse, wheat yeast concentrate and liquid by-products of bio-ethanol production.

The product - notably the product comprising non-living organic material, the product comprising no unprocessed fresh plants or unprocessed fresh plant parts, the particulate organic product, or the particulate protein-containing product - preferably comprises one or more organic components (materials) chosen from a group consisting of a protein, a carbohydrate and a lipid (where the particulate protein-containing product comprises a protein by definition), for example a combination (mixture) of (different) proteins or a combination (mixture) of (different) proteins and binders and/or fibers, or combinations of one or more proteins, carbohydrates and inorganic substances.

According to an embodiment, the product comprises at least 10 wt% of organic components, for example 60-100 wt% of organic components, such as protein and/or carbohydrate and/or lipid, and preferably in any case at least 10 wt% of protein and/or at most 90 wt% of carbohydrate and/or at most 90 wt% of lipid, all on a dry-matter basis, totaling 100 wt%. In embodiments, the liquid comprises at least 1 wt% of carbohydrate and/or lipid on a dry-matter basis, totaling 100 wt%. If the product comprises a protein in combination with a carbohydrate and/or a lipid, then the total amount of carbohydrate in wt% is preferably less than about ten times the amount of protein in wt%. Especially, if the product comprises a rapidly degradable carbohydrate, an amount of rapidly degradable carbohydrate is selected to be equal to or less than an amount of protein. If the product comprises a protein in combination with a carbohydrate, wherein the carbohydrate is a rapidly degradable carbohydrate, than the amount of (rapidly degradable) carbohydrate is especially less than the amount of protein in wt%, especially less than half the amount of protein in wt%.

In further embodiments, the product comprises an amount of lipid selected from the range of 2-15 wt%, such as in the range of 2-8 wt%, especially 4-5 wt%, or in the range of 7-15 wt%, such as 8-12 wt%, especially 9-1 1 wt% (all on a dry-matter basis).

Alternatively or additionally, the product may comprise a carbohydrate, such as a sugar or a starch. Especially, an amount of carbohydrate may be selected from the range of 1-15 wt%, especially 3-15 wt%, such as 3-12 wt%, especially 5-12 wt%, even more especially 6-9 wt% (on a dry-mater basis). In further embodiments, the product comprises an amount of starch selected from the range of 1-5 wt%, especially 2-3 wt% (on a dry-matter basis). In yet further embodiments, the product comprises an amount of sugar selected from the range of 2-10 wt%, especially 3-8 wt%, even more especially 4- 6 wt% (on a dry-matter basis).

Hence, in embodiments the product comprises an amount of protein selected from the range of 15-40 wt%, especially from the range of 20-35 wt%, an amount of lipid selected from the range of 2-15 wt%, especially from the range of 4-11 wt%, an amount of sugar selected from the range of 2-10 wt%, especially from the range of 4- 6 wt%, and an amount of starch selected from the range of 1-5 wt%, especially from the range of 2-3 wt%.

In specific further embodiments, the amount of protein is selected from the range of 25-40 wt%, especially 30-35 wt% and the amount of lipid is selected from the range of 3-7 wt%, especially 4-5 wt%, and especially the amounts of sugar and starch are selected from the ranges described above.

In another specific embodiment, the amount of protein is selected from the range of 15-30 wt%, especially 20-26 wt% and the amount of lips is selected from the range of 5-15 wt%, especially 9-11 wt%, and especially the amounts of sugar and starch are selected from the ranges described above.

In a further embodiment, the product (used) only contains a small amount of carbohydrates, especially rapidly degradable carbohydrates, such as carbohydrates (sugars) comprising monosaccharides and/or polysaccharides with od→4 linkages. The product preferably comprises at most 30 wt% and more preferably at most 20 wt%, such as at most 10 wt% of rapidly degradable carbohydrates (sugars), calculated on a dry- matter basis. Higher amounts can quickly acidify the soil, inhibiting the process of disinfestation.

Herein, the terms "a protein" and "proteins" especially relate to more than one

(different) protein. Likewise the terms "a carbohydrate" and "carbohydrates", "a sugar" and "sugars", "a starch" and "starches" especially relate to more than one (different) carbohydrate, sugar, and starch, respectively, and the terms "a lipid" and "lipids" especially relate to more than one (different) lipid.

Suitable proteins are exemplified by potato protein, Protamylasse, bone meal

(protein) and gluten. Gluten forms a specific group of proteins, occurring in some seeds and grains. According to an embodiment, the product comprises the particulate or liquid protein-containing product, gluten, and - according to a further embodiment - the product preferably comprises a particulate or liquid protein-containing product selected from wheat gluten, maize gluten and a combination thereof.

The product may especially comprise other proteins, such as for example potato protein, soya bean protein, bone meal protein, an oilseed protein, or a combination thereof. Especially the product comprises a combination of gluten(s), such as for example wheat gluten, maize gluten or a combination thereof, and other proteins, such as for example potato protein, soya bean protein, bone meal protein or a combination thereof. In embodiments, the protein comprises grain protein, such as cereal grain protein. The protein may comprise a feed protein, especially a dietary protein contained in feed (products), or a protein feed, for livestock. Oilseed proteins may be the byproduct from an oil production process of the respective oilseed. The oilseed protein may comprise at least part of ground oil (press) cake, e.g. in the form of granulate or meal (powder). The oilseed may, e.g., comprise a soybeen, rapeseed (canola), sunflower, and flaxseed. Hence, the oilseed protein may comprise a rapeseed (or rape) protein. The product, especially the protein, may comprise rapeseed protein.

In a further embodiment, the product comprises a particulate or liquid product in the form of one or more proteins, with preferably at least about 10 wt% of protein (on a dry-matter basis), for example comprising 10-30 wt% of protein or especially 15- 40 wt% of protein.

According to one of the advantageous embodiments of the present invention, the product is therefore a protein-containing, preferably particulate or liquid, product. The term "protein-containing particulate or liquid product" denotes here a product that both comprises protein and is particulate, such as for example a powder or a granulate, or it is a liquid, such as for example an aqueous slurry.

The protein that is preferred in one of the embodiments comprises one or more proteins selected from a group consisting of potato protein, wheat protein, a maize (corn) protein, an oilseed protein, and a microbial protein. In further embodiments, the protein is selected from the group consisting of a potato protein, a wheat protein, a maize protein, an oilseed protein, and a microbial protein.

A suitable form of protein-containing non-living organic material is wheat gluten. Yet another suitable form of protein-containing non-living organic material is a product that contains microbial protein. The term "microbial protein" denotes a protein that is obtained from fermentation processes. An example of products containing microbial proteins is wheat yeast concentrate, which is obtained from the fermentation of wheat. Hence, the microbial protein may e.g. (also) comprise a grain protein, such as a wheat protein and vice versa. Another example of a product comprising microbial proteins is brewer's spent grain, which is obtained from the brewing process of beer and may e.g. contain fermented grains such as barley and/or wheat. The microbial protein may directly or indirectly be derived from and/or comprise one or more of the (other) proteins described herein.

Microbial protein can also be formed in the fermentation of for example maize, etc. One can therefore use for example wheat yeast concentrate and/or maize yeast concentrate as the protein-containing non-living organic material.

The protein especially comprises one or more proteins selected from the group consisting of a potato protein, gluten, a wheat protein, a maize protein, an oilseed protein, and a microbial protein. In a further embodiment, the product comprises one or more of extracted soya bean meal, oil (press) cake, steamed potato peelings, thick potato sap, wheat yeast concentrate, bone meal, brewer's spent grain, and a cereal grain.

The product may further comprise a fiber. In embodiments, an amount of fiber in the product is at least 5 wt%, such as equal to or more than 10 wt%. Especially, the amount of fiber is equal to or less than 60 wt%, such as equal to or less than 40 wt%, especially equal to or less than 20 wt%. The term "a fiber" may also relate to more than one (different) fiber. A fiber may especially comprise slowly degradable carbohydrates.

Especially, the product (further) comprises a low mineral content and a low moisture content. The product may e.g. comprise less than 7.5 wt% ash, especially equal to or less than 5 wt% ash. In embodiments, the product comprises at least 2 wt% ash, such as at least 4 wt% ash (on a dry-matter basis).

The product is introduced into the soil and especially into a top layer of the soil, that is to say, especially down to a depth of about 30-60 cm, especially 50 cm. The product can be introduced by ploughing it into the soil, but it can also be injected into the soil. The product may especially be introduced into a (top) layer of a field, a parcel or a plot to be disinfested. The top layer especially comprises a (ground) surface (of the layer) in contact with the open surrounding (environment), especially air.

Hence, in an embodiment, the method for disinfestation, especially the introduction stage comprises: introducing the product into a layer of a field comprising the soil, wherein the layer comprises a ground surface (i.e. a surface of the layer), and wherein the layer comprises a height (thickness) selected from the range of 30-60 cm. Especially, the height (thickness) of the layer is defined perpendicular to the ground surface (the surface of the layer). In further embodiments, the height of said layer is selected to be equal to or less than 1 m. Especially, the height of said layer is selected to be at least 20 cm.

The term "introduction into the soil" and similar expressions used here denote notably the introduction of the product into the soil by man, possibly with the aid of machines.

In an embodiment of the method, at least 0.1 gram of protein per liter of soil is introduced into the soil. In a further embodiment, at least 0.25 gram per liter of soil of protein is introduced into the soil. Especially, 0.5-1.5 gram, such as 0.5-1 gram per liter of soil of protein is introduced into the soil. In further embodiments, an amount of protein introduced in to the soil is selected to be less than 0.5 gram per liter soil.

Hence, in an embodiment the product comprises protein and an amount of protein introduced (into the soil) is at least 0. 1 gram per liter of soil, especially at least 0.25 gram per liter of soil such as at least 0.3 gram per liter of soil. Especially, the amount of protein introduced (in the soil) is selected from the range of 0. 1-2.5 gram per liter of soil, especially from the range of 0.25-2.5 gram per liter of soil, even more especially from the range of 0.5-2.5 gram per liter of soil. In further embodiments, the amount of protein introduced in the soil is less than 0.5 gram per liter of soil.

In a further embodiment of the present invention, 1-50 g of protein is introduced per liter of soil. In a further embodiment, 1-100 g of carbohydrate is introduced per liter of soil. In yet a further embodiment, 1-100 g of lipid is introduced into the soil. If proteins are used in combination with rapidly degradable carbohydrates and/or lipids, then the total amount of carbohydrates and lipids in wt% is preferably less than the amount of proteins in wt%.

In order to provide or create an anaerobic condition in the soil that is advantageous for disinfestation of the soil, a barrier is applied to the soil after the introduction of the product described above. The term "application of a barrier layer between the soil and the air" covers various options and denotes in particular the placement of a barrier (layer) on the ground, i.e. generally in contact with the soil, but it also includes cases where the top layer of the soil is worked, for example by compaction. It further covers the provision of a water-based barrier to the soil, such as providing an amount of water to inundate the soil. The barrier layer essentially has a very low oxygen permeability (transmission) (see below). The barrier layer is especially substantially not permeable for oxygen. Especially, the barrier layer is configured to provide a substantially oxygen-free environment in the soil (see also below).

In embodiments, the barrier layer is made of a plastic. The term "plastic" also covers film materials here. When disinfestation is effected by the introduction of plants or plant residues into the soil, an expensive plastic film with a very low oxygen permeability (transmission) may be needed to create the required anaerobic conditions and to retain these conditions for the time needed for effective decontamination to take place. However, the method of the present invention can be effected in a shorter period, partly because the amount of oxygen in the soil may decrease very quickly (the required oxygen- free state may be reached within 2 days), and partly because the products introduced into the soil are in a very readily accessible form, so they can be quickly digested. Since the anaerobic conditions may be maintained for a shorter time (for example for 2-8 weeks) a cheaper, less airtight alternative to the expensive air-tight film can be used. The plastics and films that can be used to apply a barrier layer between the soil and the air are exemplified by low- density polyethylene (LX)PE), high-density polyethylene (HDPE), nylon, multi-ply barrier film (such as Hytibarrier film), biodegradable film and plastics, as well as spray- on plastics or other spray-on film- forming products. The term "oxygen-free state", especially relates to anaerobic conditions.

Under certain conditions, it may be possible to create a barrier layer between the soil and the air by rolling the top layer of the soil or driving over it in order to compress or compact it and therefore seal it. In an embodiment, the barrier layer may therefore be applied between the soil and the air by compacting the top layer of the soil. Another possible alternative for the application of a barrier layer between the soil and the air is sealing the soil with water. According to one of the advantageous embodiments of the present invention, the barrier layer is applied by inundation. In embodiments, the barrier may comprise a water-based barrier. The water-based barrier may comprise a layer of water. The water-based barrier may consist of a layer of water. The water-based barrier may be arranged in (a top layer of) the soil, especially wherein the part of the soil is saturated by the water-based barrier. The water-based barrier may further (partly) be arranged at the soil (on the ground (surface)). Especially (a part of) the water-based barrier arranged at the soil may comprise a thickness (perpendicular to the ground surface). Said thickness may be a few millimeter. Said thickens may also be a few centimeter. Said thickness may also be substantially 0 mm, especially wherein the water- based barrier is substantially completely arranged in the soil, especially wherein a (top) layer of the soil may be saturated with the water-based barrier, especially with water.

In embodiments, the water-based barrier is substantially completely arranged in the soil. In further embodiments, at least a part of the water-based barrier is arranged at the soil. The water-based barrier may be applied by irrigation. The water-based barrier may be applied by spraying. The water-based barrier may be provided by rain fall. In further embodiments, the water-based barrier comprises rain-water and/or surface water.

In further embodiments, the water-based barrier comprises a water-based polymer coating. A water-based polymer coating may e.g. be sprayed on the ground (when providing the barrier to the soil). Such polymer coating comprises a polymer. Especially the coating comprises a dissolved polymer (especially dissolved in water). Especially the polymer (coating) comprises a bio-degradable polymer (coating), especially a bio based polymer (coating). The polymer (coating) may comprise a polysaccharide polymer (coating), especially comprising one or more compounds selected from the group of a starch, a modified starch, a glycogen a cellulose and a pectin. In embodiments, the polymer (coating) comprises a carboxy methyl cellulose (coating). In a further embodiment, the polymer (coating) comprises a poly lactic acid (coating). The polymer coating may (also) comprise a protein coating, such as a gluten (protein) coating. In embodiments, the polymer (coating) comprises a low molecular weight polyethylene oxide (coating), especially having a molecular weight equal to or lower than 1000. In further embodiments, the polymer (coating) comprises polyvinyl alcohol (PVOH). The term " ^• polymer" such as used in the phrase "water-based polymer coating" may relate to more than one (different) polymer, and/or, e.g., a (co)polymer comprising more than one different polymer. The coating may further comprise other non-polymer components.

The water-based polymer coating may especially comprise a fluid, especially the water-based coating may be flow-able (when provided at the soil). The water-based coating especially comprises a high-viscous liquid, especially having a viscosity larger than the viscosity of water, such as 10 times the viscosity of water, especially at least 100 times the viscosity of water. Hence, a viscosity of the water-based coating may be at least 100 inPa-s, especially at least 1 Pa-s, even more especially at least 10 Pa-s. In embodiments, the viscosity of the water-based coating is selected in the range of 1-1000 Pa-s, especially in the range of 1-500 Pa-s, especially 10-250 Pa-s. The water-based coating especially (also) comprises water, such as at least 10 vol.% water (related to a total volume of the water-based coating), especially at least 20 vol.% water. In further embodiments, the water based coating comprises more than 75 vol.% water. Yet, in other embodiments, the water-based coating comprises equal to or less than 50 vol.% water, especially equal to or less than 40 vol.% water. The coating may be provided and/or maintained when at least a part of the water evaporates from the water-based coating and/or when at least a part of the water penetrates into the soil, and especially the coating maintains at the soil. The coating (essentially arranged on the soil) may further be maintained by providing water to the coating. In embodiments, the coating is maintained by providing additional water to the coating, especially when an amount of water in the coating is less than 20 vol.%, such as less than 10 vol.%. The term "a barrier layer" may also comprise more than one (different) barrier layer. Hence, the coating is especially a (essentially) continuous layer on the soil, thereby providing a barrier.

In further embodiments, the method further comprises a compacting stage configured between the introduction stage and the treatment stage, wherein the compacting stage comprises compacting the soil. Hence, in embodiments the soil is compacted before it is covered with a barrier, especially with a plastic barrier layer or a water-based barrier.

The barrier layer is preferably applied 0-72 hours, and preferably 0-48 hours after the introduction of the product into the soil. It can be removed, if required, preferably about 1-12 weeks, such as 2-12 weeks, like 4-12 weeks, especially 4-10 weeks and more especially 6-8 weeks after it is applied. Especially, the barrier (layer) is maintained during the treatment time. The phrase "water-based barrier is applied" includes embodiments wherein the barrier (layer) is applied, and thereafter the barrier layer stays for the indicated time. The phrase "water-based barrier is applied" may also relate to embodiments wherein the barrier (layer) is applied, and occasionally, regularly, or continuously, the barrier (layer) is maintained by adding barrier (layer) material, such as by irrigation or by flowing additional liquid water-based barrier material over the soil (or barrier (layer) on the soil).

The method according to the invention is preferably employed without placing any plants in the soil between the introduction of the product into the soil and the application of the barrier layer. The method according to the invention is preferably employed without the presence of any plants to be grown and/or cultivated during the introduction of the product into the soil.

One of the advantages of the method according to the present invention is that the product that is introduced into the soil can have a known and, furthermore, a constant composition. In addition, the product is preferably particulate or liquid, which ensures its easy and uniform distribution in the soil.

Hence, in embodiments the product comprises a protein-containing material. More preferably, the product comprises at least 10 wt% of protein, calculated on a dry- matter basis, especially at least 15 wt% of protein, such as at least 20 wt% of it. In some specific embodiments, the product contains at least 50 wt% of protein on a dry-matter basis, and especially at least 70 wt% of protein, in the form of for example wheat protein and/or maize protein, especially wheat gluten or maize gluten.

In another embodiment, preferably at least 0.1 gram of protein, such as at least 0.25 gram of protein, especially at least 0.3 gram of protein, such as at least 0.4 gram of protein, even more especially at least 0.5 gram of protein is introduced per liter of soil, and more preferably at least 1 gram of protein per liter of soil, such as at least 1.5 grams, at least 4 grams and especially at least 10 grams of protein per liter of soil. In particular, the amount of protein introduced into the soil per liter of soil is 0.1-50 g, such as 0.15-10 g, such as 0.25-10 g, especially 0.25-5 g, especially 0.5-5 g and more especially 0.5-3 g per liter of spoil. In further embodiments, the amount of protein introduced may be 1-50 g, such as 5-40 g, especially 10-40 g of protein per liter of soil. The disinfestation may (already) be satisfactory at high atmospheric temperatures, especially above 25 °C, when (only) low amounts of protein is used, such as 0.1 g, 0.25, or 0.3 g, or even 0.4 protein per liter of soil. Hence, in embodiments, the amount of protein introduced in the soil per liter of soil is equal to or lower than 50 g, such as equal to or less than 40 g, especially equal to or less than 10 g, even more especially equal to or less than 5 g. In specific embodiments, the amount of protein introduced in the soil is equal to or less than 1 g., such as equal to or less than 0.5 g., especially equal to or less than 0.4 gram per liter soil. Further, the addition of water to the soil before covering the soil may positively affect the method and (also) enabling a satisfactory effect at said low amount amounts of protein introduced per liter of soil. Yet, it has also been surprisingly found that by applying a water-based barrier, low protein amounts provided to or obtained in the soil may provide good results. It has also been found that providing water such as by a water-based barrier, and allowing it to penetrate into the soil may reduce the treatment time. Additionally or alternatively, humidifying the soil before providing the barrier, and/or during the treatment stage may speed up the treatment and/or may allow a reduction in the amount of protein used. Without being bound to theory, it is hypothesized that humidifying the soil may stimulate a growth of the anaerobic microorganisms in the treatment stage. The anaerobic microorganisms may decompose the product and form compounds toxic to the Foe. Moreover, these toxic compounds may also be more easily transported in a humidified soil. Hence, in further embodiments, the method further comprises humidifying the soil before covering the soil comprising the product with the barrier layer. Humidifying may, e.g., comprise providing an aqueous liquid to the soil, such as by spraying or by irrigation. In further embodiments, humidifying comprises the addition of an aqueous liquid (such as water) to the product before covering the soil with the barrier layer. Especially, humidifying comprises providing an aqueous liquid (in)to the soil, especially wherein 10-250 ml water, especially 10-100 ml water, is provided per liter of soil. Yet, in further embodiments, (much) larger amounts of an aqueous liquid may be provided, for instance to saturate and/or inundate the soil, and especially to provide a water-based barrier to the soil (see above). Hence, the soil may be humidified after providing the product to the soil and before applying the barrier to the soil. The term "humidifying" especially relates to providing water (to the soil), such as to irrigate, to spray, and/or to water (the soil).

The barrier layer used is preferably essentially impervious to oxygen. A plastic film is used in particular as the barrier layer. In another embodiment, the barrier layer does not comprise any openings for the plants, so the soil can be virtually fully covered by it. The barrier layer is arranged in particular to create substantially anaerobic conditions in the soil under it. The amount of the product and the nature of the barrier layer are preferably so chosen that - in the course of preferably at least a number of days, such as at least 2 days or at least 5 days - the oxygen content of the air in the soil under the barrier layer is of the order of magnitude of at most 2 vol.% and especially at most 1 vol %, such as at most 0.5 vol.%. For example, it can drop below about 2 vol.% about 2 days after introduction and coverage, and it can remain the same preferably for at least 2 consecutive days and preferably for at least 5 consecutive days even more especially for at least 4 weeks such as at least 8 weeks. In one of the embodiments, the barrier layer used preferably has an oxygen transmission rate (OTR) of at most 2000 ml of oxygen per square meter per hour, i.e. an OTR value of at most 2000 ml/m ² /h. In a specific embodiment, the OTR value is at most 1500 ml/m ² /h. With polyethylene (PE), it is possible to achieve an OTR value of 1400 ml/m ² /h, which can create good anaerobic conditions. In a further embodiment, the barrier layer used comprises a virtually impermeable film (VIF foil). VIF foils are known in the art and especially comprise a polyamide layer, especially a nylon layer. Alternatively, totally impermeable films (TIF) may be applied. TIF foils are also known in the art and especially comprise an ethyl vinyl alcohol (EVOH) resin layer. Hence, in embodiments, the barrier layer comprises an impermeable film selected from a group consisting of a VIF foil and a TIF foil.

Hence, the invention provides in an embodiment, a method for disinfestation of a soil infested with Fiisarhim oxysporum f.sp. cubense, wherein the method comprises an introduction stage and a treatment stage, wherein (i) the introduction stage comprises introducing a product comprising organic material into the soil; wherein the product comprises a protein and wherein an amount of protein introduced (into the soil) is at least 0.1 gram per liter soil, such as at least 0.25 gram per liter soil, especially at least 0.3 gram per liter soil, even more especially at least 0.4 gram per liter soil; and (ii) the treatment stage comprises: applying (and maintaining) a (water-based) barrier to the soil comprising the product during a treatment time, wherein an anaerobic condition is maintained in the soil, wherein the treatment time is selected from the range of at least 1 week, especially selected from the range of at least 2 weeks.

The method for disinfestation may advantageously be applied to disinfest the soil of a (banana) field before new banana plants are planted in the field. Hence, in a further aspect, the invention provides a method for planting a field with a banana plant, wherein the soil of the field is disinfested according to the method for disinfestation described herein and wherein the banana plant is planted after the disinfestation of the soil. When the soil is disinfested, planting may be performed rather quickly. In embodiments, the banana plant is planted directly after the disinfestation of the soil. In further embodiments, the banana plant is planted at least one week, such as at least 4 weeks after disinfestation of the soil. In yet other embodiments, the banana plant is planted first, and then the method of the invention is applied.

The product described herein may be advantageously used to disinfest a soil infested with Fusarium oxysporum f.sp. cubense, especially a soil comprises by a top layer of a banana field. These soils and these filed may especially be infested with Fusarium oxysporum f.sp. cubense.

Hence, in an aspect, the invention provides a use of a product, especially the product described herein, to disinfest a (infested) soil, especially infested with Fusarium oxyspornm f.sp. ciibense, wherein the product comprises an amount of protein selected from the range of equal to or more than 10 wt%.

Therefore, the invention provides a method for disinfesting soil and also the use of a product for disinfesting soil. The herein described embodiments for the method also apply for the use. Not all embodiments of the method are repeated below, but a number are further elucidated below.

Especially, the product is introduced into the soil, especially in an introduction stage, to obtain (or provide) an amount of protein introduced into the soil of at least 0.1 gram per liter soil, such as at least 0.25 gram per liter soil, especially at least 0.3 gram per liter soil, even more especially at least 0.4 gram per liter soil, and wherein, especially in a treatment stage, a (water-based) barrier is applied (and especially (also) maintained) to the soil comprising the product during a treatment time. Especially, the treatment time is selected to be equal to or more than 2 weeks. The treatment time is especially selected to be equal to or more than 1 day, such as at least 1 week, like at least 2 weeks, such as 4-12 weeks.

In an embodiment, after introduction into the soil of the product, the soil is compacted, and then the water-based barrier is applied to the compacted soil comprising the product during the treatment time. In an embodiment (of the use of the product) the water-based barrier comprises a layer of water. In a further embodiment, the water-based barrier comprises a water-based (bio-degradable, especially bio-based) polymer coating. In an embodiment the amount of protein introduced in the soil is less than 0.5 gram per liter of soil.

Especially, the invention also provides the use of a product, especially the product described herein, to disinfest a soil infested with Fusarium oxysporum f.sp. ciibense, especially Fusarium oxysporum f.sp. ciibense comprising a race selected from the group consisting of the races TR1, TR3 and TR4, especially comprising the race TR4.

In an embodiment, the product is used to disinfest a soil infested with Fusarium oxysporum f.sp. ciibense, especially wherein the product comprises an amount of protein selected from the range of equal to or more than 10 wt%. The protein especially comprises one or more proteins selected from the group consisting of a potato protein, gluten, a wheat protein, a maize protein, an oilseed protein, and a microbial protein. In an embodiment, the product is introduced into the soil to obtain an amount of protein introduced into the soil of at least 0.25 gram per liter of soil. Hence, an amount of protein introduced (into the soil) is at least 0.25 gram per liter soil. Especially a barrier is applied to the soil comprising the product during a treatment time, especially wherein an anaerobic condition is maintained in the soil (during the treatment time). The treatment time is especially selected to be equal to or more than 2 weeks. In a further embodiment, the soil comprising the product is humidified before applying the barrier to the soil comprising the product.

In an embodiment, the product comprises an amount of protein selected form the range of at least 5 wt%, such as at least 10 wt%, such as 10-40 wt%. In further embodiments, the product comprises (also) an amount of fat selected from the range such of 2-15 wt% fat, and/or an amount of sugar selected from the range of 2-10 wt%, and an amount of starch selected from the range of 1-5 wt%.

In a further embodiment, the product is used to disinfest the soil, wherein the soil is configured as a soil for a banana plant. Especially, the product is used to disinfest a soil infested with Fiisarhim oxysporiim f.sp. cubense preceding planting a banana plant in the soil.

The term "substantially" herein, such as in "substantially consists", will be understood by the person skilled in the art. The term "substantially" may also include embodiments with "entirely", "completely", "all", etc. Hence, in embodiments the adjective substantially may also be removed. Where applicable, the term "substantially" may also relate to 90% or higher, such as 95% or higher, especially 99% or higher, even more especially 99.5% or higher, including 100%. The term "comprise" includes also embodiments wherein the term "comprises" means "consists of. The term "and/or" especially relates to one or more of the items mentioned before and after "and/or". For instance, a phrase "item 1 and/or item 2" and similar phrases may relate to one or more of item 1 and item 2. The term "comprising" may in an embodiment refer to "consisting of but may in another embodiment also refer to "containing at least the defined species and optionally one or more other species".

Furthermore, the terms "first", "second", "third" and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein. The devices herein are amongst others described during operation. As will be clear to the person skilled in the art, the invention is not limited to methods of operation or devices in operation.

It should be noted that the above-mentioned embodiments illustrate rather than limit the invention, and that those skilled in the art will be able to design many alternative embodiments without departing from the scope of the appended claims. In the claims, any reference signs placed between parentheses shall not be constnied as limiting the claim. Use of the verb "to comprise" and its conjugations does not exclude the presence of elements or steps other than those stated in a claim. The article "a" or "an" preceding an element does not exclude the presence of a plurality of such elements. The invention may be implemented by means of hardware comprising several distinct elements, and by means of a suitably programmed computer. In the device claim enumerating several means, several of these means may be embodied by one and the same item of hardware. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to an advantage.

The invention further applies to a device comprising one or more of the characterizing features described in the description and/or shown in the attached drawings. The invention further pertains to a method or process comprising one or more of the characterizing features described in the description.

The various aspects discussed in this patent can be combined in order to provide additional advantages. Furthermore, some of the features can form the basis for one or more divisional applications. EXPERIMENTAL

A simple test using Anaerobic Soil Resetting (ASR) of soil infested with Foe TR4 was set-up in the Lelystad-lab of PPO. This test yielded very promising results with killing rates of 99,9+%, which was unique in the world as there is no effective method available against Foe TR4. Both parties decided that the initial tests had to be followed- up soon by field trials in the Philippines in order to assess the value of the results on a small scale in practice. This field trial again gave good results in killing of Foe TR4 in soil.

The potential of Anaerobic Soil Disinfestation (ASD) using products with two different compositions (Product A and Product B) has been investigated in naturally TR4 infested fields in Mindanao, Philippines, at three experimental sites with two replicated plots each in commercial Cavendish banana plantations of Tadeco, NEH and Unifrutti. The treatments were subdivided into two sub-trials in naturally infested soils and naturally infested soils added with nylon mesh bags containing Foc-TR4 chlamydospores inside a bucket that was incorporated in the field. In addition, a separate trial was conducted following the same set-up where buckets with nylon mesh bags containing Foc-TR4 chlamydospores were maintained under laboratory conditions.

The size of each experimental plot was 10 x 10 m plot (gross test plot) including a 5 x 5 m plot (net test plot), with different dosages of the products in the gross or net plot. The products were incorporated in the soil up to 50 cm below the surface, either mechanically or manually, and were covered with plastic for eight weeks. The plastic was then removed and the plots subsequently planted to susceptible Cavendish bananas at one and four weeks post treatment.

We quantified the presence of TR4 prior and after the treatment to determine efficacy of ASD. A significant reduction of Foc-TR4 inoculum levels was recorded in the net plots (100-fold reduction) and gross plots (20-fold reduction) relative to the controls. The application/incorporation of the products significantly affects the efficacy of ASD. None of the planted Cavendish bananas were infected with Fusarium wilt at the NEH and Unifrutti plots while 25% disease incidences were recorded at the Tadeco plots. Disease incidence will be monitored for two cropping cycles. A fine-tuning experiment is necessary to further confirm the applicability of ASD as a sustainable and effective management strategy for Fusarium wilt.

Introduction

In these trials, anaerobic soil disinfestation (ASD) comprises an organic product amendment as carbon source in a humid soil, followed by mixing through the soil, and subsequently compressing the soil and covering with airtight plastic for several weeks. The combination of an anaerobic condition of the soil, volatile and/or toxic product accumulation from anaerobic decomposition of the organic product and most likely antagonism and/or control by facultative and strict anaerobic microorganisms may result in the reduction of soil-borne pathogens.

The efficacy of ASD against Fusarium oxysporum f.sp. cubense Tropical Race 4 (Foc-TR4) causing Fusarium wilt or Panama disease in banana was determined. ASD was first tested in a laboratory experiment under controlled conditions at 24 °C temperature at Applied Plant Research, Wageningen University and Research (WUR), Lelystad, The Netherlands. After four weeks, ASD reduced the Foc-TR4 inoculum (amount of viable spores) to 0.01%. Based on these results it was decided to test the efficacy of ASD under field conditions in a pilot study in Mindanao, The Philippines, at three sites with different agro-ecological conditions, two replicates each, which are naturally infested with Foc-TR4 as well as in second laboratory trial using the infested field soils.

Materials and methods

Carbon products used

Composition Product A:

- 30-35 wt% protein, and further:

100% plant origin (sources: wheat and corn);

low mineral content (4-5 wt % ash);

low moisture (90-95% dry weight);

10 wt% fiber;

- minor ingredients are fats/oils (4-5 wt%), sugars (4-6 wt%) starches (2-3 wt%).

Composition Product B:

20-26 wt% protein, and further:

100%) plant origin (source: rape seed);

low mineral content (4-5 wt% ash);

- low moisture (90-95% dry weight);

>10 wt% fiber;

minor ingredients are fats/oils (9-11 wt%), sugars (4-6 wt%) starches (2- 3 wt%).

Location

The field pilot was performed in Mindanao, southern Philippines, at sites of commercial Cavendish banana producers viz. Tadeco (Farm 24- GPS reading North 07° 29. 765' East 125° 37.484'), Unifrutti Philippines (MADC farm - GPS reading North 07° 57'44" East 125° 5'28") and NEH (DFADI farm - GPS reading North 06° 17' 29" East 125° 15' 30" and SAVI farm - GPS reading North 06° 2' 49" East 125° 5' 2"). At each farm we designed two experimental plots (six plots in total).

Field characteristics and Foc-TR4 infestation

The soil at Tadeco trial site was a clay loam (0-50 cm soil layer: pH = 6.7 and organic matter = approximately 1.4%). At NEH DFADI, a heavy clay soil at 0-50 cm soil layer, pH= 7.3 and organic matter = 1.9% while NEH SAVI has a sandy loam soil, pH= 7.5 and organic matter of 0.6%. At Unifrutti trial site, a field of clay soil (0-50 cm soil layer: pH = 6.6 and organic matter = 3.5%) were recorded. Organic matter may play an essential role in stimulating biological activity, important for decomposition of the carbon products. During the eight weeks treatment, atmospheric temperature (presented here as average temperature) were recorded in all sites: Tadeco = 27.7 °C, NEH DFADI

= 26.9 °C, NEH SAVI = 27.5 °C and Unifrutti = 27.1 °C. Rainfall were documented for the entire eight-weeks period as follows (1) Tadeco: high = 25.4 mm and low = 0.25 mm; (2) NEH DFADI: high = 50.3 mm and low = 0.8 mm, NEH SAVI only one time rainfall at 1.96 mm; (3) Unifaitti: high = 115.6 mm and low = 3.1 mm.

All the three trial sites were previously identified that had high natural infestation of Foc-TR4. Prior to the treatment, soil (0-50 cm and 51-100 cm layer) were collected to check for the presence of Foc-TR4 inoculum by direct DNA extraction and

TaqMan probe-based Real-Time qPCR. All sites were tested positive for Foc-TR4 both at 0-50 cm and 51-100 cm except for NEH DFADI, only detected at 0-50 cm soil layer.

Experimental design

Each ASD treatment was subdivided into two sub-trials namely: (1) naturally infested soils and (2) naturally infested soils + nylon mesh bags containing Foc-TR4 chlamydospores inside a bucket that was incorporated in the soil. In addition, a separate trial with the same set-up as #2 was conducted under laboratory conditions.

Naturally infested soil (1)

The size of the experimental plots was 10 x 10 m plot (gross test plot) including a 5 x 5 m plot in the center (net test plot). A combination of Product A and Product B were incorporated in the soil. Soil was dug out until 50 cm depth in the gross test plot prior to the experiment.

1. Products A and B were incorporated in the soil in a layer of 50 cm (five subsequent applications at 10 cm each) at the different dosage given in Table 1 either mechanically (Unifrutti farm) or manually (NEH and Tadeco farms), the net test plot was treated first and then the entire plot, the remaining 2.5 m margins, was treated.

Table 1. Product dosages applied to both net and gross plot for anaerobic soil disinfestation field trial. 2. Airtight plastic film (thickness: Plastic A (comprising a VIF foil) = 40 microns, Plastics B (polyethylene-based plastic):Tadeco = 152.4 microns, NEH = 76.2 to 82.2 microns, and Unifrutti = 76.2 microns) layers were used to cover the net test plot and subsequently the entire gross test plot was covered with one layer of Plastic A and another two layers of Plastic B provided by the respective companies for eight weeks.

3. At week eight, plastic was removed ensuring that the surrounding soil (outside the test plot, untreated) would not mix with the soil of the treated plot. Soil samples were collected from the 50 cm treated soil and in the untreated soil (51-100 cm) for both the net test plot and the remaining margins of the gross test plot. The treated test plots were considered a quarantine area to prevent the introduction of Foc-TR4 infected soil.

4. Cavendish cv. Williams bananas were randomly planted at the treated plots. We planted both in the net and gross plot at one and four weeks after treatment (WAP). Disease severity is recorded at weekly intervals.

Naturally infested soils + nylon mesh bags containing Foc-TR4 chlamydospores inside a bucket and in a laboratory set-up (2)

This experiment was conducted as a control set-up either in the field (f) or kept inside a building, a laboratory setting, which is similar to the initial trial (b). The same dosage of Products A and B as in the net plot (Table 1) were added to each bucket. Basically, the experiment comprised the following:

Treatment l - infested soil, treated Product A;

Treatment 2 - infested soil, treated Product B;

Treatment 3 - infested soil, treated Product A + Product B;

Treatment 3 _f - infested soil, treated Product A + Product B;

Treatment \ - infested soil, untreated (positive control);

Treatment 5 _b - non-infested soil, untreated soil (negative control).

1. There were three buckets for each treatment and nylon mesh bags containing Foc-TR4 in soil was incorporated in the bucket with naturally infested soil. The mesh with inoculum was placed in the bottom of the soil layer.

2. For Treatment 3f, the containers were not sealed off and were placed in the soil in the field at -50 cm for both net and gross test plots. Other treatments were sealed with a lid and set aside in a building for an eight weeks' period. Total number of buckets: 3 buckets x 5 treatments = 15 buckets/ plot x 6 plots = 90 buckets.

3. Buckets were removed from the field plots (5 buckets for the net test plots and four buckets for the gross test plots) and the meshes were collected for Foc-TR4 quantity at Wageningen University and Research. Total number of buckets: 9 buckets x 6 plots = 54 buckets

Assessment of Foc-TR4 inoculum levels

Naturally infested soils were collected prior and post treatment (0-50 cm and 51-100 cm soil layer) to check for the presence of Foc-TR4. DNA extractions were conducted for each of the collected soils using the MoBio kit (MoBio laboratories, USA) that is compatible with the Kingfisher DNA extraction robot (KingFisher™ Flex Purification System, Thermo Scientific ^* , USA). Extracted DNA was analyzed using the developed TaqMan probe-based PCR. Spiked soils represent a chlamydospores (isolate Phil 2.6c, Foc-TR4 from Philippines) infested Dutch sandy soil prepared at Wageningen UR that was used as positive control. The non-infested soil is a Dutch sandy soil (autoclaved twice) that was used as negative control.

To enumerate the viable chlamydospores inside the nylon mesh bags, spiral plating was conducted employing dW Scientific spiral plater (Smets Technology Alliance Group, The Netherlands). For each sample, approximately 10 grams of chlamydospore-infested soil were diluted in 95 ml 0.1% sodium pyrophosphate (Sigma, USA) and mixed for 10 minutes. Soil suspensions were subsequently diluted in ¼ Ringers solution (Honeywell Fluka, Germany), and spread on Komada agar plates and incubated at 25 °C. After three days, the number of Foc-TR4 colonies were counted. The initial chlamydospores density was estimated at 8 x 10 ⁶ chlamydospores/gram soil.

Data analysis

The means of Foc-TR4 population densities were subjected to Analysis of Variance (ANOVA) on log-transformed data (log CFU + 1) followed by REML variance analysis (Genstat 64-bit version 8.1, VSN International Ltd.). Contrast analyses were performed between buckets and individual treatments and the experimental location. Results

Effect of ASD on inoculum densities of Foc-TR4 chlamydospores in nylon mesh bags

As determined by spiral plating, the inoculum densities (log ¹⁰ number of chlamydospores/gram) of Foc-TR4 chlamydospores in nylon mesh bags inside the bucket buried at 50 cm for eight weeks ASD treatment are shown in Table 2.

Treatment Log ¹⁰ number of chlamidospores/gram

Product A 5.15 ± 0.25 ^b

Product B ^h 4.95 ± 0.25 ^a ' Product A+B ^b 4.55 ± 0.25 ^a - ^b

Product A+B ⁸ 4.95 ± 0.25 ^{a b}

Product A+B ¹¹ 4.2 ± 0.25 ^a

Positive control 6.2 ± 0.25°

Table 2 Effect of different treatments [Treatment in the building (superscript b) (Product A alone, Product B alone and Product A+B) Treatment in the field (Product A+B in gross plot (superscript g) and Product A+B in net plot (superscript n))] applied on naturally infested soil on the survival of Fusarium oxysporum f.sp. ciibeme Tropical Race 4 chlamydospores (CFU g ^"1 soil) after eight weeks' treatment. Values are presented as means ± SEM of log transformed numbers. Error margins indicate standard error of the mean. Values with different letter (a, b, c) are significantly different at confidence level of 95% (Genstat 64-bit v8.1). There were no sufficient values to conduct statistical analysis from EH (zero bucket scores i.e. buckets without Foc-TR4 growth on Komada plates), we omitted these data and continue with Tadeco and Unifrutti (Table 3). For the ASD treatment in the building, the inoculum levels significantly decreased for Product A alone, Product B alone and combination of both compared with the control (P < 0.05). Although, it appeared that the combination of both products did not significantly contribute to the reduction of inoculum levels. For the field experiment, both net and gross plot had a significant reduction of Foc-TR4 inoculum level compared with the control (P < 0.05). The net plot demonstrated a 100-fold reduction of inoculum level while the gross plot had a 20-fold reduction relative to the control. There was no significant interaction between location and treatment (P < 0.05). This might suggest that location (excluding NEH trial site) i.e. soil type and agro-ecological conditions did not significantly affect the ASD experiment. Nevertheless, the treatment itself i.e. type of product and the manner of applying the product significantly affect the ASD experiment.

A qualitative data analysis showing the number of buckets from the field with zero scores of Foc-TR4 chlamydospores is presented in Table 3. In here, NEH had a significant number of buckets with zero scores for all treatments relative to Tadeco and Unifrutti. To validate that Foc-TR4 chlamydospores were present in the buckets with zero scores, DNA extraction and TaqMan probe-based Real-Time qPCR were performed in all samples. An extraction and qPCR control (PLRV plasmid DNA) were employed to further check if there is inhibition in the sample. We confirmed that Foc-TR4 chlamydospores were present in the buckets but were killed due to ASD treatment (data not shown).

Table 3. Binary table showing the number of buckets with zero scores of Foc-TR4 chlamydospores relative to the total number of buckets used; : treatment in the building; ⁸ : treatment in the gross plot; ⁿ : treatment in the net plot

Effect of ASD on naturally infested soil and incidence of Fusarium wilt

The Foc-TR4 naturally infested soil samples were collected at different depths from each trial plot before and after ASD treatment, at 0-50 cm and 51-100 cm in the gross and net plots. These samples were analyzed by soil dilution plating to investigate the diversity of Fusarium spp. in the soil and to have an impression if ASD can alter the microbial diversity in the soil. These Fusarium and non-Fusarium species isolated from the ASD-treated soil might take part in the suppression of Foc-TR4 in the soil similar to those reducing F. oxysporum f.sp. lycopersici. The isolated Fusarium spp. can also be an indication that antagonists and biological microorganisms may not be killed by ASD.

The soil samples were also examined by DNA extraction and TaqMan probe- based Real-Time qPCR. It was verified that Foc-TR4 DNA was present both in the treated net and gross plot for Tadeco and NEH, but absent for Unifrutti. The untreated soil (51-100 cm soil depth) was also analyzed and results proved that Foc-TR4 DNA was present in the net plot of the Unifrutti farm, but absent in the gross plot. The NEH and Tadeco farms tested positive for Foc-TR4 DNA in the gross plot, see table 4.

Net plot Gross plot

Pilot location 0-50cm 51-100 cm 0-50 cm 51-100 cm NEH yes no yes yes

Tadeco yes no yes yes

Unifrutti no yes no no

Table 4. Foc-TR4 diagnostics of the naturally infested soils treated with the products for eight weeks utilizing a TaqMan probe-based Real-Time qPCR assay. PLRV plasmid DNA were employed as extraction and qPCR reaction control. Spiked soils represent a chlamydospores infested Dutch sandy soil was used as positive control. The non-infested Dutch sandy soil (autoclaved twice) was used as negative control.

After eight weeks of ASD treatment, Cavendish cv. Williams plants were planted to both net and gross plots at one and four weeks after treatment (WAT). Different timing of planting was performed as soil biology needs time to recover and related biological suppresiveness of Foc-TR4 might occur over time (several months). The interval between the end of the soil treatment and planting of the susceptible crop should be sufficient to reestablish the soil microbiota that initiate disease suppression like in the case of Verticillium wilt caused by Veticilliiim dahliae. Table 5 shows the Fusarium wilt disease incidence on the planted banana three months after planting. None of the plants were infected both for NEH and Unifrutti plot. For Tadeco, plants at gross plot showed Fusarium wilt symptoms i.e. yellowing of leaves and splitting of the pseudostem planted at one and four WAT. Of all the test plots, we used a different plastic at Tadeco (Plastic B only) and not the Plastic A (with a better oxygen barrier) as the provided plastic was lost in the warehouse. This might be one reason why an early appearance of Foc-TR4 symptoms occurred.

Table 5. Fusarium wilt disease incidence on planted Cavendish banana after eight weeks ASD treatment; ^* WAT: week after treatment : treatment in the gross plot; ⁿ : treatment in the net plot Summary, future perspectives and fine tuning

The ASD field pilot utilizing products A and B was successfully conducted in naturally TR4 infested commercial field in the Philippines. Three experimental sites (two plots each) were situated in commercial Cavendish banana plantations of producers Tadeco, NEH and Unifrutti. There was a significant reduction of Foc-TR4 inoculum levels both in the net (100-fold reduction) and gross (20-fold reduction) plots compared with the control. The manner of applying the products significantly seems to affect the result of the experiment. As of three months after planting, none of the planted Cavendish bananas were infected with Fusarium wilt for NEH and Unifrutti plots while 25% disease incidence were recorded at the Tadeco plot. The plants will be monitored for up to two years after finishing the ASD treatment.

A fine-tuning experiment, comprising a larger area, may be further carried out to confirm the applicability of ASD as a sustainable and effective management strategy for Fusarium wilt. In that experiment we may optimize the product concentrations and professionalize and mechanize the incorporation in the soil as this seems to affect the efficacy of the treatment. Furthermore, we may experiment with one type of plastic, or test a sealing product that can be sprayed onto the soil.