FIELD OF THE INVENTION

The present invention relates to a method for improving water availability in soil using a lipase. BACKGROUND OF THE INVENTION

Soil water repellency (SWR) is a condition where soil does not spontaneously wet when a drop of water is applied to the soil surface, i.e. the soil is too hydrophobic (Muller and Deuer (2011 ) Agric., Ecosystems and Environ. 144: 208-221). Hydrophobic soils occur in many countries on various lands, such as agricultural, pasture, coastal dune sands, forest, shrub lands, parks, turfgrass soils, no-till agriculture, and soils irrigated with treated wastewater. A substantial inter- est in soil water repellency soils has grown in recent times (WO 2013/181240; Dekker et al. (2005) Aust. J. of Soil Res. 43: 403-441 ).

Soil water repellency can cause undesirable consequences such as environmental deterioration and considerable losses in crop production. Soil water repellency becomes especially problem- atic on water relationships and can cause associated environmental issues, such as, but not limited to, reduction in soil water intake, uneven wetting patterns, reduced irrigation efficiency and effective precipitation, increased preferential flow that can have adverse effects on aquifer contamination, greater runoff and erosion, limited seed and vegetative establishment, and re- duced plant growth and quality (Doerr et al. (2000). Earth-Sci. Reviews 51 : 33-65; Muller and Deuer (201 1 )).

In agricultural settings, soil water repellency across large areas of crop-producing fields leads to reduction or complete loss of already planted crops as well as reduction in soil quality and wa- tering problems for the next set of seeds. On sandy turfgrass soils and grasslands, on the other hand, soil water repellency is a reoccurring problem called“localized dry spot” (LDS). In LDS, soil water repellency appears as irregular dry areas from a few centimeters to several meters diameter with the repellency usually extending from the surface of the soils into 5-10 cm depth. A second soil water repellency situation on turfgrass sites that can appear on all soil types oc- curs within the dry area of basidiomycete induced“fairy-ring” (Barton and Colmer (2011 ) Agric. Water Management 99:1-7).

The primary cause of soil water repellency is the formation of a coating of hydrophobic, organic material on soil particles. This hydrophobic organic material can include surface waxes, fatty ac- ids, and other organics such as lignin, a phenolic polymer. These materials originate from plant leaves and other decomposing organic matter, plant root exudates, fungal hyphae/exudates, and volatized organic materials condensing on soil particles following forest or grassland fires (Atanassova and Doerr (2010) Europ. J. of Soil Sci. 61 : 298-313). Sandy soils are especially susceptible to soil water repellency due to a lower particle surface area. Once sands become coated by organics and upon drying to a critical moisture level, they exhibit a hydrophobic na- ture and, thereafter, resist rewetting. The conventional remediation practice involves the use of wetting agents (surfactants). It is known in the art that single surfactants can improve soil water infiltration (SWI) and reduce soil water repellency (see, e.g., PCT/EP2018/052676). When used on specific soils, the effect of single surfactants on improving SWI and reducing soil water repellency can be very limited (Da- vies et al. (2012) Crop Updates 2012, p.358-p.362). Further, this approach is indirect, costly, and only produces short-term alleviation of the problem, requiring repeated applications of the surfactants to maintain effectiveness (Muller and Deuer (201 1 ); Moore et al. (2010) J. Hydrol. Hydrochem. 58(3): 142-148).

Biological methods involving microorganisms capable of degrading wax in soil have been de- scribed (Roper (2006) Biologia 61/Suppl. 19: S358-S362), but to see a significant effect, a great amount of microorganisms was necessary which is no longer economic. In another approach, enzymes such as laccase, pectinase, chitinase, and protease (WO 2013/181240 A2; Zeng (2014) HortScience 49(5): 662-666) have been applied to turfgrass and showed a reduction in soil water repellency, while other enzymes such as esterases and lipases did not show this ef- fect.

Hence, there is still a need for an efficient method for managing the water availability in soils, in particular in non-wetting soils.

SUMMARY OF THE INVENTION

The present inventors have surprisingly found that the application of a lipase to soil leads to a reduced soil water repellency and an increased water holding capacity of non-wetting soils. Ad- ditionally, the application of said lipase also improves the water availability in wetting soils.

Accordingly, the present invention relates to the use of an isolated polypeptide having lipase ac- tivity which is selected from the group consisting of:

(i) a polypeptide comprising the amino acid sequence according to SEQ ID NO: 1 or an enzy- matically active fragment thereof;

(ii) a polypeptide encoded by the nucleic acid sequence according to SEQ ID No. 2 or an enzy- matically active fragment thereof;

(iii) a polypeptide comprising an amino acid sequence with at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 1 or an enzymatically active fragment thereof; and

(iv) a polypeptide encoded by a nucleic acid sequence which hybridizes to the complement of the nucleic acid sequence according to SEQ ID NO: 2 under stringent conditions;

or a variant thereof having lipase activity for increasing water availability in soil.

In one embodiment the soil water repellency of non-wetting soil is reduced and/or the water holding capacity of non-wetting soils is increased. In another embodiment the water holding capacity of wetting soil is increased and/or the evapo- ration from wetting soil is decreased.

In one embodiment the variant comprises at least one amino acid insertion, amino acid deletion or amino acid substitution with respect to the sequence according to SEQ ID NO:1.

The at least one amino acid insertion, amino acid deletion or amino acid substitution may be at an amino acid residue position selected from the group of positions 23, 33, 82, 83, 84, 85, 160, 199, 254, 255, 256, 258, 263, 264, 265, 268, 308 and 31 1.

The at least one amino acid substitution may be selected from the group consisting of: Y23A, K33N, S82T, S83D, S83H, S83I, S83N, S83R, S83T, S83Y, S84S, S84N, I85A, I85C, I85F, I85H, I85L, I85M, I85P, I85S, I85T, I85V, I85Y, K160N, P199I, P199V, I254A, I254C, I254E, I254F, I254G, I254L, I254M, I254N, I254R, I254S, I2454V, I254W, I254Y, I255A, I255L, A256D, L258A, L258D; L258E, L258G, L258H, L258N, L258Q, L258R, L258S, L258T, L258V, D263G, D263K, D263P, D263R, D263S; T264A, T264D, T264G, T264I, T264L, T264N, T264S, D265A, D265G, D265K, D265L, D265N, D265S, D265T, T268A, T268G, T268K, T268L, T268N, T268S, D308A, and Y31 1 E and/or wherein the at least one amino acid insertion is selected from the group consisting of: 84Ύ, 84’L and 84’S. Preferably, the at least one amino acid substitution is D265G.

In one embodiment the variant comprises a combination of amino acid substitutions with re- spect to the sequence according to SEQ ID NO:1.

The combination of amino acid substitutions may be selected from the group consisting of: a) I85L, D265S, T268G;

b) S83H, I85L, T268G; and

c) I255A, D265S.

In one embodiment the polypeptide having lipase activity is used in combination with at least one soil additive, preferably selected from the group consisting of surfactants, fertilizers, nitrifi- cation inhibitors and/or pesticides, preferably fungicides.

In one embodiment the polypeptide having lipase activity is applied in a concentration of be- tween 0.01 kg to 600 kg of polypeptide per hectare, preferably of between 0.16 kg to 100 kg per hectare, more preferably of between 0.6 kg to 20 kg per hectare, most preferably of between 1 kg to 5 kg per hectare.

The soil may be selected from agricultural land, pasture, coastal dune sands, forest, shrub lands, parks, turfgrass soils, no-till agriculture, and soils irrigated with treated wastewater.

The present invention also relates to a method for increasing the water availability in soil corn- prising treating an area of groundcover with an isolated polypeptide having lipase activity which is selected from the group consisting of: (i) a polypeptide comprising the amino acid sequence according to SEQ ID NO: 1 or an enzy- matically active fragment thereof;

(ii) a polypeptide encoded by the nucleic acid sequence according to SEQ ID No. 2 or an enzy- matically active fragment thereof;

(iii) a polypeptide comprising an amino acid sequence with at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 1 or an enzymatically active fragment thereof; and

(iv) a polypeptide encoded by a nucleic acid sequence which hybridizes to the complement of the nucleic acid sequence according to any of SEQ ID NO: 2 under stringent conditions;

or a variant thereof.

In one embodiment the isolated polypeptide having lipase activity is as defined above.

In one embodiment the soil water repellency of non-wetting soil is reduced and/or the water holding capacity of non-wetting soil is increased.

In another embodiment the water holding capacity of wetting soil is increased and/or the water infiltration time of wetting soil is decreased.

The isolated polypeptide having lipase activity may be used in combination with at least one soil additive, preferably selected from the group of surfactants, fertilizers, nitrification inhibitors and/or pesticides, preferably fungicides.

The polypeptide having lipase activity may be applied in a concentration of between 0.01 kg to 600 kg of polypeptide per hectare, preferably of between 0.16 kg to 100 kg per hectare, more preferably of between 0.6 kg to 20 kg per hectare, most preferably of between 1 kg to 5 kg per hectare.

BRIEF DESCRIPTION OF THE DRAWINGS

The disclosure can be better understood with reference to the following drawings,

which are described in the description and examples below.

Figure 1 and Figure 2 show the results for assessing water droplet penetration time (WDPT) for lipases LIP120 and LIP173.

Figure 1 shows the water droplet penetration time (=WDPT) on soil sample No. 23 from a patting green treated with 1 water (control) or 2 the enzyme lipase LIP120 batch a; 5 sample replications, +SD (standard deviation).

Figure 2 shows the water droplet penetration time (=WDPT) on soil sample No. 31 from a pat- ting green treated with 1 water (control), or the enzymes lipase: 2 LIP120 batch a, 3 LIP120 batch b, 4 LIP173; 5 sample replications, +SD (standard deviation). DETAILED DESCRIPTION OF THE INVENTION

Although the present invention will be described with respect to particular embodiments, this de- scription is not to be construed in a limiting sense.

Before describing in detail exemplary embodiments of the present invention, definitions im- portant for understanding the present invention are given. Unless stated otherwise or apparent from the nature of the definition, the definitions apply to all methods and uses described herein.

As used in this specification and in the appended claims, the singular forms of "a" and "an" also include the respective plurals unless the context clearly dictates otherwise. In the context of the present invention, the terms "about" and "approximately" denote an interval of accuracy that a person skilled in the art will understand to still ensure the technical effect of the feature in ques- tion. The term typically indicates a deviation from the indicated numerical value of ±20 %, pref- erably ±15 %, more preferably ±10 %, and even more preferably ±5 %.

It is to be understood that the term "comprising" is not limiting. For the purposes of the present invention the term "consisting of is considered to be a preferred embodiment of the term "corn- prising". If hereinafter a group is defined to comprise at least a certain number of embodiments, this is meant to also encompass a group which preferably consists of these embodiments only.

Furthermore, the terms "first", "second", "third" or "(a)", "(b)", "(c)", "(d)" etc. and the like in the description and in the claims, are used for distinguishing between similar elements and not nec- essarily 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 illus- trated herein. In case the terms "first", "second", "third" or "(a)", "(b)", "(c)", "(d)", "i", "ii" etc. re- late to steps of a method or use or assay there is no time or time interval coherence between the steps, i.e. the steps may be carried out simultaneously or there may be time intervals of sec- onds, minutes, hours, days, weeks, months or even years between such steps, unless other- wise indicated in the application as set forth herein above or below.

It is to be understood that this invention is not limited to the particular methodology, protocols, reagents etc. described herein as these may vary. It is also to be understood that the terminol- ogy used herein is for the purpose of describing particular embodiments only, and is not in- tended to limit the scope of the present invention that will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

As discussed above, the present invention is based on the finding that the application of a li- pase to soil increases the water availability in soil. The term "water availability" refers to the amount of water which is present in a given volume of soil. This amount of water can be taken up by plants and used for growing. Hence, by improving the water availability in soil, the soil is more suitable for growing plants.

By the use and method of the present invention the water availability in soil is increased by at least 3% or 5%, preferably by at least 8% or 10%, more preferably by at least 12% and most preferably by at least 15%. This means that the amount of water which can be taken up by plants treated in accordance with the present invention is increased by at least 3% or 5%, pref- erably by at least 8% or 10%, more preferably by at least 12% and most preferably by at least 15% compared to a soil which has not been treated in accordance with the present invention.

In one embodiment, the application of the lipase reduces soil water repellency and/or increases the water holding capacity of non-wetting soil.

The term "soil water repellency" (SWR) is a condition where soil does not spontaneously wet when a drop of water is applied to the surface. It reduces the affinity of soils to water such that they resist wetting for variable periods. It may be present in a large area and therefore affect agriculture. Such large areas of water-repellent soil predominantly occur in Australia as well as in North and South America. Alternatively, soil water repellency may present as "localized dry spots" which are irregular dry areas from a few centimeters to several meters diameter and mainly occur on sandy turfgrass soils and grasslands. Also alternatively, soil water repellency may present as "fairy rings" which are caused by fungi, in particular by basidiomycetes and which have a round shape. The fairy rings can range from a few centimeters to 20 meters in diameter, particularly from 0.5 m to 5m in diameter.

The organic matter contributing to hydrophobic organic coatings in soils with soil water repel- lency arises from sources such as, but not limited to: a) plant vegetation and root exudates with certain plant species especially prone to causing SWR, such as pine, gum, and oak trees and grasses; b) decomposition products from soil microbial activity, soil microorganism biomass such as fungal hyphae, and root exudates; c) applied organic amendments; d) ashed or volatized organic materials condensing on soil particles following forest or grassland fires; and e) organic matter in treated waste water used for irrigation. Regardless of soil type or organic matter source, soil drying increases repellency with air-drying greatly enhancing SWR severity.

The typical types of organic compounds suggested to be involved in soil water repellency in- clude: a) high molecular weight, polar fatty acids and their esters (alkanes that are derived from plant and cuticular waxes); b) other alkanes (paraffin-like compounds), microbial derived waxes, alkanols, phytanols, phytanes; c) amphiphilic (partially hydrophobic) lipids, stigmasterols and plant derived sterols that have polar (hydrophilic) and non-polar (hydrophobic) groups; d) other polar molecules such as sugars, gylocsides, aromatic acids, and low molecular weight organic acids; e) humic and fulvic acids from soil microbial activity or possibly added as amendments; and f) hydrophobins, cysteine rich proteins expressed only by filamentous fungi. The soil water repellency can be measured and classified by a variety of methods. The water droplet penetration time (WDPT) test is based on the time taken for a drop of water to infiltrate into an air- or oven-dried soil sample (Dekker et al. (1998) Soil Sci. 163: 780-796). A description of this method is provided in the examples section herein. The molarity of ethanol droplet (MED) or ethanol test uses a series of aqueous ethanol solutions prepared in concentrations ranging between 0% and 36%. Drops of the various solutions are placed on the surface of soil samples, and the degree of soil water repellency is then defined as the ethanol percentage or molarity of the least concentrated ethanol solution that is absorbed by the soil in a mean time of <10 s (DeBano et al. (2000) J. Hydrol. 231 :4-32). In the intrinsic sorptivity method the sorptivity of wa- ter which is influenced by repellency is compared to the sorptivity of ethanol which is not influ enced by repellency (Tillman et al. (1999) Austr. J. Soil Res. 27: 637-644). Further, the contact angle between water and soil may be measured e.g. by the capillary rise method (Woche et al. (2005) Eur. J. Soil Sci. 56: 239-251 ).

By the use and method of the present invention the soil water repellency of non-wetting soils is reduced by at least 10% or 20%, preferably by at least 30% or 40%, more preferably by at least 50% or 60% and most preferably by at least 70% or 75%. If the soil water repellency is meas- ured using the WDPT test, the time taken for a drop to infiltrate the soil is reduced by at least 10% or 20%, preferably by at least 30% or 40%, more preferably by at least 50% or 60% and most preferably by at least 70% or 75%.

The term "water holding capacity" refers to the amount of water that a given amount of soil can hold. It has an impact on the water supply of any crops planted on the soil, but also on the re- tention of nutrients and pesticides by the soil. The water holding capacity can be determined by applying a defined amount of water onto a defined amount of soil and after a certain period of time such as two hours weighing the soil. The difference between the weight of the soil after ap- plying the water and before applying the water is the amount of water which is held by the soil. Alternatively, the water holding capacity can be determined by placing a water saturated soil sample on a porous ceramic plate which is then placed in closed chambers and a known amount of pressure is applied to the chamber which forces water out of the soil sample and into the porous plate and out of the chamber. The water holding capacity can also be determined by measuring the amount of water leached through the soil after a defined amount of soil has been watered with an amount of water which exceeds the water holding capacity. A lower amount of water which has leached from the soil indicates an increased water holding capacity.

By the use and method of the present invention the water holding capacity of non-wetting soil is increased by at least 3% or 5%, preferably by at least 8% or 10%, more preferably by at least 12% and most preferably by at least 15%. This means that the amount of water which is present in the soil after a certain period of time after application is increased by at least 3% or 5%, pref- erably by at least 8% or 10%, more preferably by at least 12% and most preferably by at least 15% compared to a soil which has not been treated in accordance with the present invention.

Preferably, by the use and method of the present invention the soil water repellency of non-wet- ting soil is reduced and the water holding capacity of non-wetting soil is increased. The soil wa- ter repellency of non-wetting soil is reduced by at least 10% and the water holding capacity of non-wetting soil is increased by at least 3%, preferably the soil water repellency of non-wetting soil is reduced by at least 30% and the water holding capacity of non-wetting soil is increased by at least 8%, more preferably the soil water repellency of non-wetting soil is reduced by at least 50% and the water holding capacity of non-wetting soil is increased by at least 12% and most preferably the soil water repellency of non-wetting soil is reduced by at least 70% and the water holding capacity of non-wetting soil is increased by at least 15%.

The term "evaporation" refers to the process of losing water over a surface, particularly a soil surface. The evaporation can be determined by adding water to a soil sample to exceed the wa- ter holding capacity and leaving the soil sample for a period of several days, for example eight days, without watering. After this period the soil sample is weighed and the difference in weight at the timepoint after adding the water and the weight after the period of leaving the soil is deter- mined. This difference is defined as evaporation.

By the use and method of the present invention the water holding capacity of wetting soil is in- creased by at least 5% or 8%, preferably by at least 10% or 12%, more preferably by at least 15% or 18% and most preferably by at least 20%. This means that the amount of water which percolates through a water-saturated soil is decreased by at least 5% or 8%, preferably by at least 10% or 12%, more preferably by at least 15% or 18% and most preferably by at least 20% compared to a soil which has not been treated in accordance with the present invention.

By the use and method of the present invention the evaporation from wetting soil is reduced by at least 1 %, preferably by at least 2%, more preferably by at least 3% and most preferably by at least 4%. This means that after adding water to a pot containing wetting soil and leaving the pot without watering for a period of five to ten days, preferably of eight days, the loss of weight is reduced by at least 1 %, preferably by at least 2%, more preferably by at least 3% and most preferably by at least 4% compared to a soil which has not been treated in accordance with the present invention.

The term "soil" refers to material forming the surface of the earth and including a mixture of or- ganic material and minerals. Soil includes materials such as mud, sand, silt, and clay. It may it- self form the surface of the earth in some areas, and in other areas it may underlie other types of groundcover, such as grass and other plants and vegetation, gravel, pebbles, and the like. Preferably, the soil is sandy soil characterized by a low particle-surface area.

The soil may be agricultural land, pasture, coastal dune sands, forest, shrub lands, parks, turfgrass soils, potting mixes and soils irrigated with treated wastewater.

In one embodiment, the soil is agricultural land. This means that the soil is used for planting crops. Preferably, the agricultural land has not been treated by tillage (so-called no-till agricul- ture). This means that the soil has not been disturbed by tillage. The agricultural land may corn- prise huge areas of non-wetting soil of several square kilometers as contrasted to localized dry spots which have a size of at most several meters. In Western Australia the total area of agricul- tural land having water repellent soil is about 5 million hectares.

In one embodiment the soil is not a turfgrass soil.

The term "turfgrass" refers to any vegetative ground covering such as, but not limited to, various species of grasses used for lawns, fields, golf course grounds, and the like.

In another embodiment the soil is turfgrass soil, for example turfgrass soil from a golf course. The turfgrass soil may have one or more localized dry spots (LDS). LDS are irregular dry areas from a few centimeters to several meters diameter with the repellency usually extending from the surface into a depth of 5 to 10 cm. Additionally or alternatively, the turfgrass soil may have one or more "fairy rings" which are caused by fungi, in particular by basidiomycetes and which have a round shape. The fairy rings can range from a few centimeters to 20 meters in diameter, particularly from 0.5 m to 5 m in diameter. In one embodiment the turfgrass soil has at least one localized dry spot and at least one fairy ring.

In one embodiment, the soil is non-wetting soil. The term "non-wetting soil" refers to a soil which does not spontaneously wet when a drop of water is applied to its surface. The non-wetting soil can be characterized by the water droplet penetration time test as explained above. The non- wetting soil has a water droplet penetration time of at least 5 seconds, preferably of at least 20 or at least 100 seconds, more preferably of at least 300 or 500 seconds, even more preferably of at least 700 or 1000 seconds and most preferably at least 1 ,200 seconds. Alternatively or ad- ditionally, the non-wetting soil can also be defined by the MED test discussed above. The non- wetting soil requires a molarity of ethanol of at least 0.2, preferably of at least 0.5, more prefera- bly of at least 1.0 and most preferably of at least 2.0 to absorb the solution into the soil. Alterna- tively or additionally, the non-wetting soil can also be defined by the contact angle between wa- ter and soil as determined for example by the capillary rise method. The non-wetting soil is de- fined by a contact angle of greater than 90°.

In one embodiment, the soil is wetting soil. The term "wetting soil" refers to a soil which sponta- neously wets when a drop of water is applied to its surface. The wetting soil can be character- ized by the water droplet penetration time test as explained above. The wetting soil has a water droplet penetration time of less than 5 seconds. Alternatively or additionally, the wetting soil can also be defined by the contact angle between water and soil as determined for example by the capillary rise method. The wetting soil is defined by a contact angle of less than 90°. Alterna- tively or additionally, the wetting soil can also be defined by the MED test discussed above. The non-wetting soil requires a molarity of ethanol of 0 to absorb the solution into the soil.

The term "isolated polypeptide" refers to a polypeptide that has been separated from its biologi cal source such as a bacterium or fungus producing the polypeptide, for example by centrifuga- tion or filtration. After separating the polypeptide from the biological source it may be purified to remove other components from the biological source or medium components. Hence, the term "isolated polypeptide" includes both purified polypeptides and polypeptides which are present in a cell culture supernatant or the like.

The isolated polypeptide used according to the present invention has lipase activity. Lipases (E.C. 3.1.1.3) are hydrolytic enzymes that are known to cleave ester bonds in lipids. Lipases in- clude phospholipases, triacylglycerol lipases, and galactolipases. The enzyme used in the method of the present invention is preferably a triacylglycerol lipase which cleaves the ester bond between glycerol and fatty acids, resulting in the release of fatty acids from the glycerol.

The lipase used in the context of the present invention is selected from the group consisting of:

(i) a lipase comprising the amino acid sequence according to SEQ ID NO: 1 or an enzymatically active fragment thereof;

(ii) a lipase encoded by the nucleic acid sequence according to SEQ ID No. 2 or an enzymati- cally active fragment thereof;

(iii) a lipase comprising an amino acid sequence with at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 1 or an enzymatically active fragment thereof; and

(iv) a lipase encoded by a nucleic acid sequence which hybridizes to the complement of the nu- cleic acid sequence according to any of SEQ ID NO: 2 under stringent conditions.

An "enzymatically active fragment" of a lipase is understood to refer to a smaller part of the li- pase which consists of a contiguous amino acid sequence found in the amino acid sequence of the lipase and which has lipase activity. The skilled person knows that for a fragment to be en- zymatically active the fragment has to comprise at least the amino acids present in the catalytic centre of the lipase. These amino acids are either known for a given lipase or can easily be identified by the skilled person, for example by homology screening or mutagenesis.

A "fragment" of a nucleic acid sequence encoding an enzymatically active fragment of a lipase is understood to refer to a smaller part of the nucleic acid sequence which consists of a contigu- ous nucleic acid sequence found in the nucleic acid sequence of the lipase and which encodes a protein having lipase activity. The skilled person knows that for a fragment of an enzyme to be enzymatically active the fragment has to comprise at least the amino acids present in the cata- lytic centre of the lipase. These amino acids are either known for a given lipase or can easily be identified by the skilled person, for example by homology screening or mutagenesis.

Within the present invention a lipase having an amino acid sequence with at least 80 % se- quence identity, preferably with at least 81 , 82, 83, 84, 85 or 86% sequence identity, more pref- erably with at least 87, 88, 89 or 90% sequence identity even more preferably with at least 91 , 92, 93, 94 or 95% sequence identity and most preferably with at least 96, 97, 98, 99 or 100% sequence identity to the complete sequence according to SEQ ID No. 1 or an enzymatically ac- tive fragment of any of these sequences may also be used. The lipases having the aforemen- tioned sequence identity to the sequence according to SEQ ID No. 1 preferably comprise the amino acids present in the catalytic centre of the lipase. “Sequence Identity,”“% sequence identity.”“% identity,” or“sequence alignment” means a com- parison of a first amino acid sequence to a second amino acid sequence, or a comparison of a first nucleic acid sequence to a second nucleic acid sequence and is calculated as a percentage based on the comparison. The result of this calculation can be described as“percent identical” or “percent ID.”

Generally, a sequence alignment can be used to calculate the sequence identity by one of two different approaches. In the first approach, both, mismatches at a single position and gaps at a single position are counted as non-identical positions in final sequence identity calculation. In the second approach, mismatches at a single position are counted as non-identical positions in final sequence identity calculation; however, gaps at a single position are not counted (ignored) as non-identical positions in final sequence identity calculation. In other words, in the second ap- proach gaps are ignored in final sequence identity calculation. The differences between these two approaches, counting gaps as non-identical positions vs ignoring gaps, at a single position can lead to variability in sequence identity value between two sequences.

In an embodiment of this disclosure, sequence identity is determined by a program, which pro- duces an alignment, and calculates identity counting both mismatches at a single position and gaps at a single position as non-identical positions in final sequence identity calculation. For ex- ample program Needle (EMBOS), which has implemented the algorithm of Needleman and Wun- sch (Needleman and Wunsch (1970) J. Mol. Biol. 48: 443-453), and which calculates sequence identity by first producing an alignment between a first sequence and a second sequence over the full length of these sequences, then counting the number of identical positions over the length of the alignment, then dividing the number of identical residues by the length of an alignment, and then multiplying this number by 100 to generate the % sequence identity [% sequence identity = (# of Identical residues / length of alignment) x 100)].

In another embodiment of this disclosure, sequence identity can be calculated from a pairwise alignment showing only a local region of the first sequence or the second sequence (“Local Iden- tity”). For example, program Blast (NCBI) produces such alignments; % sequence identity = (# of identical residues / length of alignment) x 100)].

In a preferred embodiment, a sequence alignment is calculated with mismatches at a single po- sition being counted as non-identical positions in final sequence identity calculation; however, gaps at a single position are not counted (i.e. they are ignored) as non-identical positions in final sequence identity calculation. The sequence alignment is generated by using the algorithm of Needleman and Wunsch (J. Mol. Biol. (1979) 48, p. 443-453). Preferably, the program“NEEDLE” (The European Molecular Biology Open Software Suite (EMBOSS)) is used for the purposes of the current invention, with using the programs default parameter (gap open=10.0, gap extend=0.5 and matrix=EBLOSUM62). Then, a sequence identity can be calculated from the alignment show- ing both sequences over the full length, so showing the first sequence and the second sequence in their full length (“Global sequence identity”). For example,; % sequence identity = (# of identical residues / length of alignment) x 100)]. The nucleic acid sequence hybridizing under stringent conditions with a complementary se- quence of a nucleic acid sequence according to SEQ ID No. 2 or an enzymatically active frag- ment thereof encodes a protein having lipase activity.

The term "hybridizing under stringent conditions" denotes in the context of the present invention that the hybridization is implemented in vitro under conditions which are stringent enough to en- sure a specific hybridization. Stringent in vitro hybridization conditions are known to those skilled in the art and may be taken from the literature (e.g. Sambrook and Russell (2001 ) Molec ular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, NY). The term "specific hybridization" refers to the circumstance that a mole- cule, under stringent conditions, preferably binds to a certain nucleic acid sequence, i.e. the tar- get sequence, if the same is part of a complex mixture of, e.g. DNA or RNA molecules, but does not, or at least very rarely, bind to other sequences.

Stringent conditions depend on the circumstances. Longer sequences hybridize specifically at higher temperatures. In general, stringent conditions are chosen such that the hybridization temperature is about 5°C below the melting point (T _m ) of the specific sequence at a defined ionic strength and at a defined pH value. T _m is the temperature (at a defined pH value, a defined ionic strength and a defined nucleic acid concentration), at which 50% of the molecules comple- mentary to the target sequence hybridize to the target sequence in the state of equilibrium. Typ- ically, stringent conditions are conditions, where the salt concentration has a sodium ion con- centration (or concentration of a different salt) of at least about 0.01 to 1.0 M at a pH value be- tween 7.0 and 8.3, and the temperature is at least 30°C for small molecules (i.e. 10 to 50 nucle- otides, for example). In addition, stringent conditions may include the addition of substances, such as, e. g., formamide, which destabilise the hybrids. At hybridization under stringent condi- tions, as used herein, normally nucleotide sequences which are at least 60% homologous to each other hybridize to each other. Preferably, said stringent conditions are chosen such that sequences which are about 65%, preferably at least about 70%, and especially preferably at least about 75% or higher homologous to each other, normally remain hybridized to each other. A preferred but non-limiting example of stringent hybridization conditions is hybridizations in 6 x sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washing steps in 0.2 x SSC, 0.1 % SDS at 50 to 65°C. The temperature depends on the type of the nucleic acid and is between 42°C and 58°C in an aqueous buffer having a concentration of 0.1 to 5 x SSC (pH value 7.2).

If an organic solvent, e.g. 50% formamide, is present in the above-mentioned buffer, the tem- perature is about 42°C under standard conditions. Preferably, the hybridisation conditions for DNA:DNA hybrids are, for example, 0.1 x SSC and 20°C to 45°C, preferably 30°C to 45°C. Pref- erably, the hybridisation conditions for DNA:RNA hybrids are, for example, 0.1 x SSC and 30°C to 55°C, preferably between 45°C and 55°C. The above-mentioned hybridization temperatures are determined, for example, for a nucleic acid which is 100 base pairs long and has a G/C con- tent of 50% in the absence of formamide. Those skilled in the art know how to determine the re- quired hybridization conditions using text books such as those mentioned above or the following textbooks: Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), Hames and Higgins (publ.) 1985, Nucleic Acids Hybridization: A Practical Approach, IRL Press at Oxford

University Press, Oxford; Brown (publ.) 1991 , Essential Molecular Biology: A Practical Ap- proach, IRL Press at Oxford University Press, Oxford.

Typical hybridization and washing buffers for example have the following composition:

Pre-hybridization solution. 0.5 % SDS

5x SSC

50 mM sodium phosphate, pH 6.8

0.1 % sodium pyrophosphate

5x Denhardt's solution

100 pg/mL salmon sperm DNA

Hybridization solution: pre-hybridization solution

1x10 ⁶ cpm/mL probe (5 - 10 min 95 °C)

20x SSC: 3 M NaCI

0.3 M sodium citrate

ad pH 7 with HCI

50x Denhardt's reagent: 5 g Ficoll

5 g polyvinylpyrrolidone

5 g bovine serum albumin

ad 500 mL aqua destillata

A typical procedure for hybridization is as follows:

Optional: wash blot 30 min in 1x SSC/ 0.1 % SDS at 65 °C

Pre-hybridization: at least 2 h at 50 - 55 °C

Hybridization: over night at 55 - 60 °C

Washing: 5 min 2x SSC/ 0.1 % SDS hybridization temp

30 min 2x SSC/ 0.1 % SDS hybridization temp

30 min 1x SSC/ 0.1 % SDS hybridization temp.

45 min 0.2x SSC/ 0.1 % SDS 65 °C

5 min O.lx SSC room temperature

Those skilled in the art know that the given solutions and the presented protocol may be modi- fied or have to be modified, depending on the application. The proteins encoded by the nucleic acid sequence hybridizing under stringent conditions to the complement of the sequence according to SEQ ID No. 2 preferably comprise the amino acids present in the catalytic site of the lipase.

The proteins having the abovementioned sequence identity to the sequence according to SEQ ID NO: 1 and the proteins encoded by the nucleic acid sequence hybridizing under stringent conditions to the complement of the sequence according to SEQ ID NO: 2 are called variants of this protein.

A "variant polypeptide" refers to a polypeptide which differs from its parent polypeptide in its amino acid sequence.

Other variants of the lipase according to SEQ ID No. 1 are those that comprise at least one amino acid residue insertion, deletion or substitution compared to the sequence according to SEQ ID No.1 and which have lipase activity.

An "amino acid substitution” is described by providing the original amino acid followed by the number of the position within the amino acid sequence. For example, a substitution of amino acid residue 24 means that the amino acid of the parent at position 24 can be substituted with any of the 19 other amino acid residues. In addition, a substitution can be described by provid- ing the original amino acid followed by the number of the position within the amino acid se- quence and followed by the specific substituted amino acid. For example, the substitution of iso- leucin at position 254 with serine is designated as“Ne254Ser” or“I254S”. Combinations of sub- stitutions are described by inserting commas between the amino acid residues, for example: I85L, D265S, T268G; represent a combination of three different amino acid residues substitu- tions when compared to a parent polypeptide. Variants having substitutions in the context of amino acid changes, may also be applied to nucleic acid modifications, e.g. by substitutions.

The amino acid substitution may be a conservative amino acid substitution. A“conservative amino acid substitution” or related amino acid” means replacement of one amino acid residue in an amino acid sequence with a different amino acid residue having a similar property at the same position compared to the parent amino acid sequence. Some examples of a conservative amino acid substitution include but are not limited to replacing a positively charged amino acid residue with a different positively charged amino acid residue; replacing a polar amino acid resi- due with a different polar amino acid residue; replacing a non-polar amino acid residue with a different non-polar amino acid residue, replacing a basic amino acid residue with a different basic amino acid residue, or replacing an aromatic amino acid residue with a different aromatic amino acid residue. A list of related amino acids is given in the Table below (see for example Creighton (1984) Proteins. W.H. Freeman and Company (Eds)).

Examples of conserved amino acid substitutions:

An "amino acid insertion” is described by providing the number of the position within the amino acid sequence behind which the amino acid is inserted followed by an apostrophe and the spe- cific inserted amino acid. For example, the insertion of serine behind position 84 is designated as“84'S”. Variants having insertions in the context of amino acid changes may also be applied to nucleic acid modifications, e.g. by insertions.

The amino acid residue insertion, deletion or substitution may be at an amino acid residue posi- tion selected from the group of positions 23, 33, 82, 83, 84, 85, 160, 199, 254, 255, 256, 258, 263, 264, 265, 268, 308 and 311 of SEQ ID No. 1.

Preferably, the at least one amino acid substitution is selected from the group consisting of Y23A, K33N, S82T, S83D, S83H, S83I, S83N, S83R, S83T, S83Y, S84S, S84N, I85A, I85C, I85F, I85H, I85L, I85M, I85P, I85S, I85T, I85V, I85Y, K160N, P199I, P199V, I254A, I254C, I254E, I254F, I254G, I254L, I254M, I254N, I254R, I254S, I2454V, I254W, I254Y, I255A, I255L, A256D, L258A, L258D; L258E, L258G, L258H, L258N, L258Q, L258R, L258S, L258T, L258V, D263G, D263K, D263P, D263R, D263S; T264A, T264D, T264G, T264I, T264L, T264N, T264S, D265A, D265G, D265K, D265L, D265N, D265S, D265T, T268A, T268G, T268K, T268L,

T268N, T268S, D308A, and Y31 1 E. More preferably, the at least one amino acid substitution is D265G. The numbering refers to the numbering of amino acids in the sequence according to SEQ ID No. 1.

Preferably, the at least one amino acid insertion is selected from the group consisting of 84Ύ, 84’L and 84’S. The numbering refers to the numbering of amino acids in the sequence accord- ing to SEQ ID No. 1.

The variant may also comprise a combination of two, three, four, five, six or more amino acid substitutions as described above, preferably of two or three amino acid substitutions as de- scribed above.

Preferably, the variant comprises one of the following combinations of amino acid substitutions: a) I85L, D265S, T268G; b) S83H, I85L, T268G; and

c) I255A, D265S.

The numbering refers to the numbering of amino acids in the sequence according to SEQ ID No. 1 .

The above positions and amino acid substitutions are provided with respect to the amino acid sequence according to SEQ ID No. 1 . The skilled person can easily identify corresponding posi- tions in polypeptides having an amino acid sequence with at least 80% sequence identity to the sequence according to SEQ ID No. 1 and in polypeptides encoded by a nucleic acid sequence which hybridizes to the complement of the nucleic acid sequence according to SEQ ID No. 2 under stringent conditions.

The term "lipase activity" means that a protein has the ability to cleave a suitable lipid substrate. Preferably, the variants of the lipase have essentially the same lipase activity as the wild-type lipase characterized by the specific sequence according to SEQ ID No. 1 , i.e. the activity of the variants is at least 50% or 60%, preferably at least 70% or 80%, more preferably 85% or 90% and most preferably at least 95% or 98% of the activity of the wild-type lipase characterized by the specific sequence according to SEQ ID No. 1 .

The activity of the variants can be compared with the activity of the corresponding wild-type li- pase by incubating the variant and the wild-type lipase with a suitable substrate under suitable conditions and detecting the amount of the cleavage products of the variant and the wild-type lipase. Suitable substrates for the lipase according to SEQ ID No. 1 include triacylglycerol, monoglactosyl diglyceride, digalactosyl diglyceride, N-acylphosphatidyl ethanolamine, phospha- tidyl choline, monoacyl glycerol, diacyl glycerol, free fatty acid, monogalactosyl monoglyceride and digalctosyl monoglyceride.

The lipase or an enzymatically active variant thereof as described herein is preferably recombi- nantly produced using a bacteria, fungi, or yeast expression system.“Expression system” also means a host microorganism, expression hosts, host cell, production organism, or production strain and each of these terms can be used interchangeably for this disclosure. Examples of expression systems include but are not limited to: Aspergillus niger, Aspergillus oryzae, Han- senula polymorpha, Thermomyces lanuginosus, Fusarium oxysporum, Fusarium heterosporum, Escherichia coH, Bacillus, preferably Bacillus subti/is, or Bacillus Hcheniformis, Pseudomonas, preferably Pseudomonas fluorescens, Pichia pastoris (also known as Komagataella phaffii), My- celiopthora thermophile ( C1 ^' ), Schizosaccharomyces pombe, Trichoderma, preferably Tricho- derma reesei and Saccharomyces, preferably Saccharomyces cerevisiae. In an embodiment the lipase or an enzymatically active variant thereof as described herein is produced using one of the expression systems listed above. Suitable vectors for expressing the lipase and cultivation con- ditions for producing the lipase are known to the skilled person.

The lipase or an enzymatically active variant thereof as described herein may be isolated from the host microorganism by well-known methods including centrifugation and filtration which re- move most of the host cell components. If a higher degree of purity is desired, the lipase may be subjected to further purification steps such as anion or cation exchange chromatography, hy- drophobic interaction chromatography, mixed mode chromatography or hydroxyapatite chroma- tography. The isolated lipase may be used directly or it may be subjected to a drying step such as a spray-drying step. If the isolated lipase is dried, e.g. spray-dried or lyophilized, it has to be dissolved in a suitable solvent, before it is applied to the soil.

The lipase or an enzymatically active variant thereof as described above may be used in combi- nation with another enzyme which may be useful in reducing soil water repellency and/or en- hancing water holding capacity. This other enzyme may be selected from the group consisting of amylases, cellulases, chitinases, esterases, beta-glucosidases, laccases, pectinases, prote- ases and xylanases. Preferably, the lipase or a variant thereof is used in combination with a chi- tinase, a laccase, a pectinase and/or a protease. The chitinase may be from Streptomyces griseus. The laccase may be from Pycnoporus sp. SYBC-L3. The pectinase may be from Asper gillus niger The protease may be from Aspergillus oryzae.

The lipase may be used in a composition with at least one auxiliary. The at least one auxiliary may be a liquid carrier which may be selected from the group of water and organic solvents.

The at least one auxiliary may be a solid carrier selected from the group of phytogels, hydro- gels, mineral earths, meal, cellulose powder, fumed silica or precipitated silica, polysaccharides, or compost. In a preferred embodiment the lipase is dissolved in water.

The lipase may be used in combination with at least one soil additive. The soil additive may be selected from the group consisting of surfactants, fertilizers, nitrification inhibitors, urease inhibi- tors and/or pesticides.

"Surfactant" (synonymously used herein with“surface active agent” and "wetting agent") means an organic chemical that, when added to a liquid, changes the properties of that liquid at an in- terface. According to its ionic charge, a surfactant is called non-ionic, anionic, cationic, or am- photeric. Other examples of surfactants include block polymers and polyelectrolytes.

Non-limiting examples of surfactants are disclosed McCutcheon's 2016 Detergents and Emulsi fiers, and McCutcheon's 2016 Functional Materials, both North American and International Edi- tion, MC Publishing Co, 2016 edition. Further useful examples are disclosed in earlier editions of the same publications which are known to those skilled in the art.

Non-ionic surfactant means a surfactant that contains neither positively nor negatively charged (i.e. ionic) functional groups. In contrast to anionic and cationic surfactants, non-ionic surfac- tants do not ionize in solution. Mixtures of two or more different non-ionic surfactants may also be used.

Amphoteric surfactants are those, depending on pH, which can be either cationic, zwitterionic or anionic. Mixtures of two or more different amphoteric surfactants may also be used. Anionic surfactant means a surfactant with a negatively charged ionic group. Anionic surfactants include, but are not limited to, surface-active compounds that contain a hydrophobic group and at least one water-solubilizing anionic group, usually selected from sulfates, sulfonate, and car- boxylates to form a water-soluble compound. Mixtures of two or more different anionic surfac- tants may also be used. Mixtures of non-ionic and/or amphoteric and/or anionic surfactants may also be used.

Cationic surfactant means a surfactant with a positively charged ionic group. Typically, these cationic moieties are nitrogen containing groups such as quaternary ammonium or protonated amino groups. The cationic protonated amines can be primary, secondary, or tertiary amines.

Suitable block polymers are block polymers of the A-B or A-B-A type comprising blocks of poly- ethylene oxide and polypropylene oxide or of the A-B-C type comprising alkanol, polyethylene oxide and polypropylene oxide. Preferably, the surfactant is an ethylene oxide-propylene oxide block copolymer (EO/PO) or a blend of alkyl polyglycoside and ethylene oxide-propylene oxide block copolymer (APG-EO/PO). Such surfactants are marketed as ACA 1853 (EO/PO) and ACA 1848 (APG-EO/PO), respectively.

Suitable polyelectrolytes are polyacids or polybases. Examples of polyacids are alkali salts of polyacrylic acid or polyacid comb polymers. Examples of polybases are polyvinylamines or poly- ethyleneamines.

Several surfactants are commercially available for reducing soil water repellency. These in- clude, but are not limited to, Kick ^® (available from Compo), Clearing (available from Collier Turf Care), Primer ^® 604 (available from Plant Products) and Aqueduct ^® , Revolution ^® , ACA 1853 and ACA 1848 (all available from Aquatrols).

In accordance with the invention solvents or liquid carriers, solid carriers, surfactants, adjuvants, dispersants, emulsifiers, wetters, adjuvants, solubilizers, penetration enhancers, protective col- loids, adhesion agents, thickeners, humectants, compatibilizers, bactericides, anti-freezing agents, anti-foaming agents, colorants, tackifiers, binders, preservatives, antioxidants, and odorants may be used in addition to the lipase.

Suitable solvents and liquid carriers are water and organic solvents, such as mineral oil frac- tions of medium to high boiling point, e.g. kerosene, diesel oil; oils of vegetable or animal origin; aliphatic, cyclic and aromatic hydrocarbons, e. g. toluene, paraffin, tetrahydronaphthalene, alkyl- ated naphthalenes; alcohols, e.g. ethanol, propanol, butanol, cyclohexanol; glycols; DMSO; ke- tones, e.g. cyclohexanone; esters, e.g. lactates, carbonates, fatty acid esters, gammabutyrolac- tone; fatty acids; phosphonates; amines; amides, e.g. N-methylpyrrolidone, fatty acid dimethyla- mides; and mixtures thereof. Preferably, the solvent is water.

Suitable solid carriers or fillers are mineral earths, e.g. silicates, silica gels, talc, kaolins, lime- stone, lime, chalk, clays, dolomite, diatomaceous earth, bentonite, calcium sulfate, magnesium sulfate, magnesium oxide; polysaccharides, e.g. cellulose, starch; fertilizers, e.g. ammonium sulfate, ammonium phosphate, ammonium nitrate, urea; products of vegetable origin, e.g. ce- real meal, tree bark meal, wood meal, nutshell meal, and mixtures thereof.

Suitable thickeners are polysaccharides (e.g. starch, xanthan gum, carboxymethylcellulose), anorganic clays (organically modified or unmodified), polycarboxylates, superabsorbent poly- mers and silicates.

Suitable bactericides are bronopol and isothiazolinone derivatives such as alkylisothiazolinones and benzisothiazolinones.

Suitable anti-freezing agents are ethylene glycol, propylene glycol, urea and glycerin.

Suitable anti-foaming agents are silicones, long chain alcohols, and salts of fatty acids.

Suitable colorants (e.g. in red, blue, or green) are pigments of low water solubility and water- soluble dyes. Examples are

- inorganic colorants, such as iron oxide, titan oxide, iron hexacyanoferrate,

- metal-complex dyes such as chromium-complex dyes, for example Orasol Yellow 141 ,

- organic colorants such as alizarin-, azo- and phthalocyanine colorants.

Preferred colorants are metal-complex dyes, more preferably chromium-complex dyes

Suitable tackifiers or binders are polyvinylpyrrolidones, polyvinylacetates, polyvinyl alcohols, polyacrylates, biological or synthetic waxes, and cellulose ethers.

Suitable preservatives include e.g. sodium benzoate, benzoic acid, sorbic acid, and derivatives thereof.

Suitable antioxidants include sulfites, ascorbic acid, tocopherol, tocopherol acetate, tocotrienol, melatonin, carotene, beta-carotene, ubiquinol, and derivatives thereof. Tocophercol acetate is preferred as antioxidant.

In addition, compounds which stabilize the lipase enzyme may be used, such as buffers, chela- tors, anti-oxidants, non-ionic surfactants, sugars, proteins (e.g. BSA) and heavy metal and phe- nol scavengers.

In accordance with the invention fertilizers and/or nitrification inhibitors and/or urease inhibitors may be used in addition to the lipase or a variant thereof as defined herein.

As used herein, the term“fertilizer” includes any chemical compound that improves the levels of available plant nutrients and/or the chemical and physical properties of soil, thereby directly or indirectly promoting plant growth, yield, and quality. Fertilizers are typically applied either through the soil (for uptake by plant roots) or by foliar feeding (for uptake through leaves). The term "fertilizer" can be subdivided into two major categories: a) organic fertilizers (composed of decayed plant/animal matter) and b) inorganic fertilizers (composed of chemicals and minerals). Inorganic fertilizers are usually manufactured through chemical processes (such as the Haber- Bosch process), also using naturally occurring deposits, while chemically altering them (e.g. concentrated triple superphosphate). Naturally occurring water soluble inorganic fertilizers in- clude Chilean sodium nitrate.

The fertilizer is preferably a urea-containing fertilizer, and/or P-containing fertilizer, and/or a K fertilizer (potassium-containing fertilizer), and/or a N fertilizer (nitrogen-containing fertilizer), and/or a NK fertilizer (nitrogen-potassium fertilizer), and/or a NPK (nitrogen-phosphorous-potas- sium fertilizer), and/or a single or dual element fertilizer containing S, Ca, Mg, Fe, Mn, Cu, Zn, Mo, B, Ni, Cl, or a combination thereof.

As used herein, a“urea-containing fertilizer” is defined as a fertilizer comprising at least one component selected from the group consisting of urea, urea ammonium nitrate (UAN), and sus- pensions of isobutylidene diurea (IBDU), crotonylidene diurea (CDU) and urea formaldehyde (UF), urea-acetaldehyde, and ureaglyoxal condensates.

In a preferred embodiment of the invention, the urea-containing fertilizer is urea or urea ammo- nium nitrate (UAN).

In customary commercial fertilizer quality, the urea has a purity of at least 90%, and may for ex- ample be in crystalline, granulated, compacted, prilled or ground form.

The urea-containing fertilizer may be used together with a urease inhibitor. Urease is an en- zyme which hydrolyzes urea to ammonia and carbon dioxide. In agriculture, high urease activity during treatment with urea-containing fertilizers causes significant environmental and economic problems due to the release of ammonia which may be toxic to the plants and which deprives the plants of urea. Accordingly, it is desirable to inhibit the action of urease. Inhibitors of urease activity may comprise (i) substrate structural analogs of urea as e.g. hydroxyurea or hydroxamic acid or (ii) inhibitors which affect the mechanism of the urease reaction. The later may be di- vided in the four groups of (i) phosphorodiamidates or phosphorotriamidiates as e.g. N-(n-bu- tyl)thiophosphoric triamide, (ii) thiols as e.g. cysteamine, (iii) hydroxamic acids and its deriva- tives as e.g. acetohydroxamic acid, and (iv) ligands and chelators of the nickel ion in the active center of ureases as e.g. fluoride ions. Urease inhibitors are also discussed in Upadhyay (2012) Ind. J. Biotechnol. 1 1 : 381-388 and in "Improving Efficiency of Urea Fertlizers by Inhibition of Soil Urease Activity" by Kiss and Simihaian (2002), Springer Netherlands, ISBN 978-1-4020- 0493-3.

As used herein, the“P-containing fertilizer” is any fertilizer providing any form of the chemical element phosphorus (P) or containing any chemical compounds incorporating the chemical ele- ment phosphorus (P), including but not limited to phosphate-containing fertilizers or fertilizers containing P. Preferably, the P-containing fertilizer is selected from the group consisting of a NPK fertilizer, a NP fertilizer, a PK fertilizer, or a P fertilizer. Most preferably, the P-containing fertilizer is a NPK fertilizer. Of course, also combinations of these fertilizers may be used as ad- ditional P-containing fertilizer.

P fertilizers, K fertilizers, and N fertilizers are straight fertilizers, i.e. fertilizers that contain only one of the nutritive elements P, K, and N. It is to be understood, however, that these fertilizers may additionally comprise at least one additional nutritive element selected from S, Ca, Mg, Fe, Mn, Cu, Zn, Mo, B, Ni, and Cl.

NPK fertilizers, NP fertilizers, and PK fertilizers are multinutrient fertilizers, i.e. fertilizers that comprise combinations of the nutritive elements P, K, and N as indicated by the terms“NPK”, “NP”, and“PK”. It is to be understood, however, that these fertilizers may additionally comprise at least one additional nutritive element selected from S, Ca, Mg, Fe, Mn, Cu, Zn, Mo, B, Ni and Cl.

The NPK fertilizers, NP fertilizers, and PK fertilizers may be provided as complex fertilizers or bulk-blend or blended fertilizers. The term complex fertilizer refers to a compound fertilizer formed by mixing ingredients that react chemically. In bulk-blend or blended fertilizers, two or more granular fertilizers of similar size are mixed to form a compound fertilizer.

Dual element fertilizers are preferably dual element fertilizers with Ca, Mg, Fe, Mn, Zn or Ni which may be applied as soluble salts of chloride, sulfate, nitrate or in chelated form (e.g.

MnEDTA, Fe EDTA, FeEDDHA).

Single or dual element fertilizers of Mo are available as salts of molybdate, B as boric acid or borates.

Ammonium (NH ₄ ⁺ ) compounds present in nitrogen-containing fertilizers are converted by soil microorganisms to nitrates (NO ₃ ) in a relatively short time in a process known as nitrification. The nitrification process typically leads to nitrogen leakage and environmental pollution. As a result of the various losses, approximately 50% of the applied nitrogen fertilizers are lost during the year following fertilizer addition (see Nelson and Huber; Nitrification inhibitors for corn pro- duction (2001), National Corn Handbook, Iowa State University).

As countermeasure, nitrification inhibitors, mostly together with fertilizers, can be used. Suitable nitrification inhibitors include linoleic acid, alpha-linolenic acid, methyl p-coumarate, methyl feru- late, methyl 3-(4-hydroxyphenyl) propionate (MHPP), Karanjin, brachialacton, p-benzoquinone sorgoleone, 2-chloro-6-(trichloromethyl)-pyridine (nitrapyrin or N-serve), dicyandiamide (DCD, DIDIN), 3,4-dimethyl pyrazole phosphate (DMPP, ENTEC), 4-amino-1 ,2,4-triazole hydrochloride (ATC), 1-amido-2 -thiourea (ASU), 2-amino-4-chloro-6-methylpyrimidine (AM), 2-mercapto-ben- zothiazole (MBT), 5-ethoxy-3-trichloromethyl-1 ,2,4-thiodiazole (terrazole, etridiazole), 2-sulfanil- amidothiazole (ST), ammoniumthiosulfate (ATU), 3-methylpyrazol (3-MP), 3,5-dimethylpyrazole (DMP), 1 ,2,4-triazol thiourea (TU), N-(1 H-pyrazolyl-methyl)acetamides such as N-((3(5)-methyl- 1 H-pyrazole-1-yl)methyl)acetamide, and N-(1 H-pyrazolyl-methyl)formamides such as N-((3(5)- methyl-1 H-pyrazole-1 -yl)methyl formamide, N-(4-chloro-3(5)-methyl-pyrazole-1 -ylmethyl)-forma- mide, N-(3(5),4-dimethyl-pyrazole-1 -ylmethyl)-formamide, neem, products based on ingredients of neem, cyan amide, melamine, zeolite powder, catechol, benzoquinone, sodium terta board and zinc sulfate.

Pesticides are substances which aim to control pests and include herbicides, insecticides, ne- maticides, mollluscicides, piscicides, avicdes, rodenticides, bactericides, insect repellents, ani- mal repellents, antimicrobials and fungicides.

If the water-repellent soil is a fairy ring caused by basidiomycetes, preferably a fungicide is used in combination with the lipase. More preferably, the fungizide is a fungizide which can be ap- plied through the soil.

Hence, the present invention also relates to the use of (a) an isolated polypeptide having lipase activity as disclosed herein; and (b) at least one fungicide for the treatment of at least one fairy ring.

The present invention also relates to a method for treating at least one fairy ring comprising ap- plying (a) an isolated polypeptide having lipase activity as disclosed herein; (b) at least one fun- gicide to an area of groundcover.

Suitable fungicides include strobilurins, carboximides, oxidizing agents, polyoxins and sterol de- methylation inhibitors. Specific examples include, but are not limited to, Azoxystrobin, Triflox- istrobin, Picoxystrobin, Pyraclostrobin, Sedaxane, Penthiopyrad, Penflufe, Fluopyram, Fluxapy- roxad, Boscalid, Oxathiapiprolin, Metalaxyl, Metalaxyl-M, Ethaboxam, DMM, Cyproconazole, Difenoconazole, Prothioconazole, Flutriafol, Thiabendazole, Ipconazole, Tebuconazole, Triad- imenol, Prochloraz, Fluquinconazole, TTZ, Fludioxinil, Carboxin, Silthiofarm, Ziram, Thiram, Carbendazim, TPM, Valifenalate, Oxathiapiprolin analague of BCS, Pydiflumetofen (Adepydin), Fluindapyr, Mandestrobin, Inpyrfuxam, Imazalil and Picarbutrazox. Preferably, a fungizide se- lected from Azoxystrobin, Trifloxistrobin, Picoxystrobin, Pyraclostrobin, Sedaxane, Penthiopy- rad, Penflufe, Fluxapyroxad, Boscalid, Oxathiapiprolin, Metalaxyl-M, Ethaboxam, DMM, Difeno- conazole, Prothioconazole, Flutriafol, Ipconazole, TTZ, Fludioxinil, Carboxin, Silthiofarm and Valifenalate is used in combination with the lipase.

Examples of suitable fungicides which are marketed for the treatment of fairy rings and which may be used in combination with the lipase include azoxystrobin (marketed under the name Heritage ^® ), flutolanil (marketed under the name Prostar ^® ), hydrogen dioxide (marketed under the name Zerotol ^® ), polyoxin D (marketed under the name Endorse ^® ), triadimefon (marketed un- der the name Bayleton ^® FLO), pyraclostrobin (marketed under the name Insignia ^® ), a mixture of pyraclostrobin and triticonazole (marketed under the name Pillar ^® G Intrinsic), a mixture of azoxystrobin and propiconazole (marketed under the name Headway ^® ) and a mixture of triad- imefon and trifloxystrobin (marketed under the name Tartan ^® Stressgard ^® ). Insecticides which may be used in combination with the lipase include, but are not limited to, Fipronil, BAS 450, IL-39, Clothianidin, Thiamethoxam, Acetamiprid, Dinotefuran, Imidaclophd, Thiaclophd, Sulfoxaflor, Methiocarb, Tefluthrin, Bifenthrin, Cypermethrin, Alphacypermethrin, Spinosad, Cyazypyr, Rynaxapyr, Thiodicarb, Triflumezopyrim (Mesoionic), Acephate, Chlorpyri- phos, Flupyradifurone, Tetraniliprole, IL-101 , S-1587, Oxazosulfyl, Cyclaniliprole and Fluxamet- amide. Preferably, an insecticide selected from the group consisting of Fipronil, BAS 450, IL-39, Clothianidin, Thiamethoxam, Imidacloprid, Thiacloprid, Sulfoxaflor, Tefluthrin, Spinosad, Cyazypyr, Rynaxapyr, Triflumezopyrim (Mesoionic), Acephate and Chlorpyriphos is used in combination with the lipase.

Nematicides which may be used in combination with the lipase include, but are not limited to, Thiodicarb, Abamectin, Tioxazafen, Bacillus firmus and Pasteuria nishizawae.

The polypeptide having lipase activity may be applied to the groundcover in a concentration of between 0.01 kg to 600 kg of polypeptide per hectare, preferably of between 0.16 kg to 100 kg per hectare, more preferably of between 0.6 kg to 20 kg per hectare, most preferably of be- tween 1 kg to 5 kg per hectare.

In one embodiment the lipase is dissolved in water in a concentration of 0.01 % to 30% w/v, preferably of 0.02% to 20% or 10% w/v, more preferably of 0.03% to 0.8 or 0.6% w/v, even more preferably of 0.04% to 0.4% or 0.3% w/v and most preferably of 0.05% to 0.1 %. One par- ticularly preferred concentration of the lipase is 0.06% w/v.

Typically, for large volume applications the lipase is dissolved in water in a concentration of 0.01 % to 0.1 % w/v, preferably of 0.02% to 0.08% w/v, more preferably of 0.03% to 0.07% and most preferably of 0.06% (w/v). Typically, for small volume applications the lipase is dissolved in water in a concentration of 1 % to 50% w/v, preferably of 5% to 30% w/v, more preferably of 8% to 20% and most preferably of 10% (w/v). In large volume applications the lipase is dissolved in a great volume of water for application to a great area of soil. In small volume applications the lipase is dissolved in a small volume of water for application to a small area.

Alternatively, to a lipase application dissolved or suspended in water, also an application to the ground, soil or substrate as solid may be possible with intensive watering in the range of 0.5 to 15 L Water per square meter (e.g. 5000 L/ha to 150,000 L/ha) shortly after application.

In the use and method of the present invention the lipase is applied to an area of groundcover.

The term“groundcover” as used herein includes, but is not limited to, soil, natural soil, potting soil, sand, silt, clay, turfgrasses and other plants and forms of vegetation used to cover and pro- tect the soil, as well as composites of organic materials that form within or as part of such groundcovers, such as thatch and mat layers, and also includes potting mixes. Preferably, groundcover is soil, more preferably, groundcover is water-repellent or non-wetting soil. In another preferred embodiment, groundcover is potting mix. Potting mix, which is also re- ferred to potting soil, is typically a soilless blend of ingredients that is used to grow plants, pref- erably, the potting mix comprises a combination of peat moss, vermiculite, coir fiber, perlite, pine bark, sand, compost, and further ingredients. The potting mix may also comprise native soil.

The term "applying" includes any activity by which the lipase come into contact with the area of groundcover. The lipase can be dissolved in water and applied as a solution. Alternatively, a granulate can be prepared from the lipase and the granulate is then applied to the groundcover in solid form which is dissolved when water is applied to the soil.

In one embodiment, the lipase is applied in irrigation water. In one embodiment the lipase is ap- plied extensively and non-directional to turfgrass soil. In one embodiment the lipase is applied extensively and non-directional to turfgrass soil in irrigation water. In one embodiment the lipase is applied directional to turfgrass soil using a watering can.

In one embodiment the lipase is applied to turfgrass soil as part of a topdressing which corn- prises particles to which the lipase is coupled. Topdressing refers to a material applied to the top of a ground covering, usually in order to obtain a desirable effect on the groundcover, and includes sand or other particulate material.

In another embodiment the application of the lipase serves to prepare a non-wetting soil for the seeding of crops. In one embodiment the lipase is applied selectively to those locations of the non-wetting soil where the seeds will be placed, e.g. the seeding row, so that they can prepare the soil for seed uptake. In another embodiment, the lipase is applied non-selectively to the en- tire application site (so-called blanket application).

The following examples are provided for illustrative purposes. It is thus understood that the ex- amples are not to be construed as limiting. The skilled person will clearly be able to envisage further modifications of the principles laid out herein.

EXAMPLES

Example 1

Soil samples from golf course putting greens

Soil sampling and preparation

Putting greens established with grass seed mixture Penn A-4 showing both symptoms of localized dry spot or not were sampled from a golf club near Mannheim, Germany. Soil cores were taken with an auger 8.5 cm in diameter and 10 cm in depth. Transported in firmly packed plastic bags to keep the cores intact, they were segmented with a knife into 0 - 2 cm (soil sample No. 31 ) and 2 - 4 cm (soil sample No. 23) sections. The soil sections were then air dried at room temperature and thereafter passed through a 4 mm sieve and then through a 2 mm sieve and stored at room temperature up to testing. All soil samples 0 - 2 cm and 2 - 4 cm had contact angles > 90° measured according to Bachmann et al. [Bachmann, J., Ellies, A., Hartge, K.H. (2000)“Develop- ment and application of a new sessile drop contact angle method to assess soil water repellency.” J. of Hydrology 231 , 66-75]. Contact angles > 90° indicates hydrophobicity, below 90° indicates wettability of a soil sample. The pH (0.01 M CaCh) of the samples were in the range between 6.4 to 6.8.

Testing for changes in soil water repellency due to lipase treatment of soil

One gram of dry soil was placed in 2 ml. Eppendorf cups prepared with a 2 mm hole at the bottom, which was small enough to keep the dry sandy turf soil and on the other hand large enough to drain the soil. On top of the 1 g of dry soil 0.5 ml. water as a control or test solution with enzyme was placed at room temperature. The lipase enzymes used were: LIP120, batch a, at a concen- tration of 151 mg/10 mL water, LIP120, batch b, at 205 mg/10 ml. water, and LIP173 at 157 mg/10 ml. water. Excess of the solution drained after a while. The cups were then placed in a growth room at 20 °C for 24 h. For rinsing the soil prior to drying and testing later, a 5-step procedure was employed. With the help of a vacuum pump first the incubation solution was sucked away, and then 1 mL of deionized water was given to the soil, sucking way again. This procedure was repeated four times.

Thereafter, soil drying took place at 30° C for two days within the cups. Then the soil was emptied to weighing boats at room temperature for soil water repellency analysis. The soil water repellency was measured using the Water Droplet Penetration Time (WDPT) test [Doerr, S.H. (1998) On standardizing the "water drop penetration time and the "molarity of an ethanol droplet" technique to classify soil hydrophobicity: A case study using medium textured soils.” Earth Surf. Landforms 23, 663-668]. 15 pL droplets were gently uploaded with a pipette onto the soil surface and the time taken for its complete penetration. Per sample the WDPT test was repeated at least twice and the seconds averaged per sample. Each treatment had 5 sample replications. Results are presented as arithmetic mean values with error bars indicating the standard deviation in Figure 1 and Figure 2.

Example 2

Non-naturally occurring variant lipase enzymes were created using rational design single site mutagenesis and multisite mutagenesis. The variant lipase enzymes include single point amino acid modifications, insertions, or deletions compared to a parent enzyme (LIP062, which is the amino acid sequence of SEQ ID 1 , and is encoded by nucleic acid sequence of SEQ ID NO:2) at 18 different amino acid residue positions: 23, 33, 82, 83, 84, 85, 160, 199, 254, 255, 256, 258, 263, 264, 265, 268, 308, 31 1 , or any combination thereof, wherein the variant lipase enzymes has lipase activity.

Variant lipase enzymes were also created with various combinations of the single point modifica- tions of the parent enzyme (LIP062), wherein the variant lipase enzymes have lipase activity. For example, the single point modifications and various combinations of single point modifications are listed in Table: 1.

Table: 1

Lipase Amino Acid Residue Position Numbers

Table: 1

Lipase Amino Acid Residue Position Numbers

Table: 1

Lipase Amino Acid Residue Position Numbers

LIP062_1873 L

LIP062_1872 A

LIP062_1871 N

LIP062_1870 K

UP062J869 S

UP062J868 G

UP062J867 L

UP062J866 N

UP062J865 K

UP062J864 N

LIP062_1863 D

LIP062_1862

UP062J861 A

LIP062_1860 A E

LIP062_1859 A

LIP062_1858 E

LIP062_1857 S

UP062J856 L

LIP062_1855 Y

LIP062_1854 E

LIP062_1853 Q

LIP062_1852 T

UP062J851 H

LIP062_1850 D

LIP062_1849 V

LIP062_1848 R

LIP062_1847 N

UP062J846 G

LIP062_1845 A

LIP062_1844 S

LIP062_1843 . M

LIP062_1842 . G

UP062J841 . R

LIP062_1840 F

LIP062_1839 E

LIP062_1838 . W

LIP062_1837 . L

UP062J836 . Y

LIP062_1835 S

LIP062_1834 . c

LIP062_1833 . A

LIP062_1832 . V

UP062J831 . N

LIP062_1830 . M -

LIP062_1829 S

LIP062_1828 . C - - -

LIP062_1827 N . N -

UP062J826

LIP062_1825 N - - V - - - A A G

LIP062_1824 T - - V - - - A G Table: 1

Lipase Amino Acid Residue Position Numbers

LIP062_1704 H - - - A A

UP062J703 H - - T A A

LIP062_1701 T - - V G T

LIP062_1700 S T

LIP062_1696 A T

LIP062_1695 N V A T

UP062J694 G

LIP062_1692 . A T

LIP062_1691 N - - V . S S

LIP062_1686 H - - V . A S

LIP062_1685 N - - V . N A

UP062J684 . N T

LIP062_1683 . D A

LIP062_1681 T . N T

LIP062_1680 N - - A . T

LIP062_1678 N . A

UP062J677 G T Table: 1

Lipase Amino Acid Residue Position Numbers

_

LIP062_0391

Example 3

The variant lipase enzymes were obtained by constructing expression plasmids containing the encoding polynucleotide sequences, transforming plasmids into Pichia pastor/s ( Komagataella phaffit) and growing the resulting expression strains in the following way. Fresh Pichia pastoris cells of the expression strains were obtained by spreading the glycerol stocks of sequence-con- firmed strains onto Yeast extract Peptone Dextrose (YPD) agar plates containing Zeocin. After 2 days, starter seed cultures of the production strains were inoculated into 100 ml. of Buffered Glycerol complex Medium (BMGY) using cells from these plates, and grown for 20-24 hours at 30°C and 225-250 rpm. Seed cultures were scaled up by transferring suitable amounts into 2-4 L of BMMY medium in a baffled Fermentor. Fermentations were carried out at 30°C and under 1 100 rpm of agitation, supplied via flat-blade impellers, for 48-72 hours. After the initial batch-phase of fermentation, sterile-filtered Methanol was added as feed whenever the dissolved oxygen level in the culture dipped below 30%. Alternatively, feed was added every 3 hours at 0.5% v/v of the starting batch culture. The final fermentation broth was centrifuged at 7000xg for 30 mins at 4°C to obtain the cell-free supernatant.

After filtering through cheese-cloth, the cell-free supernatants were ultrafiltered using a lab-scale tangential flow filtration (TFF) system with a molecular weight cut-off of 5 kD (Spectrum Labs). Samples were first concentrated 10-20X and then buffer-exchanged 5X into 50 mM HEPES pH 7.5. The resultant retentate was centrifuged at 27000xg for 1 hour, and then sterile filtered through 0.2 pm filters to remove any production organisms or particulate matter. Total protein content of the final samples was determined using the Braford assay. Lipases were lyophilized to form pow- der.

Example4

The activity of the variant lipase enzymes was determined using natural substrates in solution. Natural lipid substrates were prepared at 5 mM final concentration in 0.25 % sodium deoxycholate by sonication. Substrate (15 pL) was mixed with 30 uL fluorescein (0.25 pg/mL in 10 mM CaCI2) and 10 pL recovered lipase (~1 -2 pg/mL) pre-diluted in 5 mM Hepes pH 7.5. Products of lipid hydrolysis were monitored by the drop in fluorescence due to pH change (485 nm/525 nm for excitation/emission), recorded kinetically every 30 seconds for 10 min at 26°C . Activity on a log scale was proportional with the fluorescence change per min. The results are shown below in Table 2, expressed as percentage of parent (LIP062) fluorescence change at same protein con- centration.