The present invention relates to a method of increasing the nutrient use efficiency of a plant by applying to the plant, plant part, plant extract, plant propagation material, or a plant growing locus, a compound of formula (I). Strigolactones are plant hormones which play a pivotal role in plant growth and development, and have potential application for sustainable agriculture.

The compound of formula (I), which is known as zealactone, has recently been identified as the major strigolactone from the root exudates in corn. Zealactone is described for example in Xiaonan Xie, Takaya Kisugi, Kaori Yoneyama etal, ‘Methyl zealactonoate, a novel germination stimulant for root parasitic weeds produced by maize’, Journal of Pesticide Science 2017, 42, 58-61 , and T. V. Charnikhova, K. Gaus, A. Lumbroso et al, ‘Zealactones. Novel natural strigolactones from maize’, Phytochemistry 2017, 137, 123-131 , and its preparation is known from M. Yoshimura, M. Dieckmann, P-Y. Dakas etal. Total Synthesis and Biological Evaluation of Zealactone 1 a/b’, Helv. Chim. Acta 2020, 103, 2000017.

The skilled person will appreciate that zealactone, the compound of formula (I), exists in two naturally occurring forms known as zealactone 1a (also referred to herein as a compound of formula (la)), and zealactone 1 b (also referred to herein as a compound of formula (lb)), respectively. Both forms may be used for the improvement in nutrient use efficiency either individually, or in a mixture of the two forms at any ratio:

In addition, the following publication relates specifically to the role of zealactone in response to nitrogen use efficiency in corn: The Control of Zealactone Biosynthesis and Exudation is Involved in the Response to Nitrogen in Maize Root’, Plant and Cell Physiology, Volume 60, Issue 9, September 2019, Pages 2100-2112.

Many agricultural soils across the world are deficient in one or more of the essential nutrients needed to support the growth and development of healthy plants, and there is often a mismatch between the demand from the crops for these nutrients, and the availability of these nutrients within the soil.

Synthetic fertilizers and nutrients are often added to the soil to increase the levels of essential nutrients available to promote plant growth. Plants often use fertilizers inefficiently, and generally, more than 50% of the nitrogen applied to the soil or plant in terms of fertilizer is not absorbed, and/or used by the plant, see Tilman et al. (2002), Nature, 418, 671. Plants that are efficient in the absorption, mobilization, and utilization of nutrients from the soil significantly improve the efficiency of applying synthetic fertilizers and nutrients. Improving crop nutrient use efficiency is essential to limit the impact of fertilization and to improve sustainability.

Arbuscular mycorrhizal fungi (AMF) play a key role in plant performance and nutrition due to their capacity to improve plant mineral uptake. AMF symbiosis is particularly important for enhancing the uptake of relatively immobile and insoluble phosphate ions in soil. The basis of this symbiosis is the capacity of AMF to develop a network of external hyphae (extraradical hyphae) capable of extending the surface area (up to 40 times) and also the explorable soil volume for nutrient uptake, throughout the production of enzymes and/or excretions of organic substances. AMF can secrete phosphatases to hydrolyse phosphate from organic P compounds, and thus improving crop productivity under low input conditions (i.e. phosphorus deficiency). The extraradical hyphae are also important to increase the uptake of ammonium, nitrate, immobile micro nutrients such as Cu and Zn, and other soil-derived mineral cations (K ⁺ , Ca ²⁺ , Mg ²⁺ , and Fe ³⁺ ) (Rouphael et al. Scientia Horticulurae, 2015, 196, 91-108 & references therein).

The plant microbiome is a key determinant of plant health and productivity (Berendsen et al. The rhizosphere microbiome and plant health. Trends Plant Sci. 2012, 17: 478-486). Manipulation of the plant microbiome has the potential to reduce the incidence of plant disease, increase agricultural production, reduce chemical inputs, and reduce emissions of greenhouse gases, resulting in more sustainable agricultural practices (Turner et al. The plant microbiome. Genome Biol 14, 209 (2013).

The rhizospheric microbiome is a region of rich, largely soil-derived, microbial diversity, influenced by deposition of plant mucilage and root exudates (Kent et al. Microbial communities and their interactions in soil and rhizosphere ecosystems. Annu Rev Microbiol. 2002, 56: 211-236).

The endospheric microbiome is the internal region of the microbiome (i.e., within the plant tissues). Microbes in the rhizosphere and endosphere can establish beneficial, neutral or detrimental associations of varying intimacy with their host plants (Turner et al. The plant microbiome. Genome Biol 14, 209 (2013).

In a first aspect, there is provided a method of improving the nutrient use efficiency of plants, said method comprising applying an effective amount of a compound of formula (I), to the plant, plant part, plant extract, plant propagation material, or to the locus thereof. In another aspect, there is provided a method of improving the nutrient uptake efficiency of plants, said method comprising applying an effective amount of a compound of formula (I), to the plant, plant part, plant extract, plant propagation material, or to the locus thereof.

In another aspect, there is provided a method of improving the nutrient utilization efficiency of plants, said method comprising applying an effective amount of a compound of formula (I), to the plant, plant part, plant extract, plant propagation material, or to the locus thereof.

In another aspect, there is provided a method of improving the nitrogen use efficiency of plants, said method comprising applying an effective amount of a compound of formula (I), to the plant, plant part, plant extract, plant propagation material, or to the locus thereof.

In another aspect, there is provided a method of improving the nitrogen uptake efficiency of plants, said method comprising applying an effective amount of a compound of formula (I), to the plant, plant part, plant extract, plant propagation material, or to the locus thereof.

In another aspect, there is provided a method of improving the nitrogen utilization efficiency of plants, said method comprising applying an effective amount of a compound of formula (I), to the plant, plant part, plant extract, plant propagation material, or to the locus thereof.

In another aspect, there is provided a method of improving the phosphorus use efficiency of plants, said method comprising applying an effective amount of a compound of formula (I), to the plant, plant part, plant extract, plant propagation material, or to the locus thereof.

In another aspect, there is provided a method of improving the phosphorus uptake efficiency of plants, said method comprising applying an effective amount of a compound of formula (I), to the plant, plant part, plant extract, plant propagation material, or to the locus thereof.

In another aspect, there is provided a method of improving the phosphorus utilization efficiency of plants, said method comprising applying an effective amount of a compound of formula (I), to the plant, plant part, plant extract, plant propagation material, or to the locus thereof.

In another aspect, there is provided a method of increasing plant yield, said method comprising applying an effective amount of a compound of formula (I), to the plant, plant part, plant extract, plant propagation material, or to the locus thereof.

In another aspect, there is provided a method of increasing the levels of nutrients in the leaf tissue of a plant, in particular, a method of increasing the levels of nitrogen in the leaf tissue of a plant, said method comprising applying an effective amount of a compound of formula (I), to the plant, plant part, plant extract, plant propagation material, or to the locus thereof.

In another aspect, there is provided a method of increasing the levels of chlorophyll in the leaf of a plant, said method comprising applying an effective amount of a compound of formula (I), to the plant, plant part, plant extract, plant propagation material, or to the locus thereof. In another aspect, there is provided a method of increasing the levels of protein in a plant, said method comprising applying an effective amount of a compound of formula (I), to the plant, plant part, plant extract, plant propagation material, or to the locus thereof.

There is also provided a method of increasing the height of a plant, said method comprising applying an effective amount of a compound of formula (I), to the plant, plant part, plant extract, plant propagation material, or to the locus thereof. In particular, there is provided a method of increasing the height of a plant, said method comprising the foliar treatment of a plant with an effective amount of a compound of formula (I). There is further provided a method of increasing the height of a corn plant, said method comprising the foliar treatment of a corn plant with an effective amount of a compound of formula (I).

In another aspect, there is provided a method of regulating or improving the growth of a plant under nutrient limited conditions, said method comprising applying an effective amount of a compound of formula (I), to the plant, plant part, plant extract, plant propagation material, or to the locus thereof.

In another aspect, there is provided a method of regulating or improving the growth of a plant under nitrogen limited conditions, said method comprising applying an effective amount of a compound of formula (I), to the plant, plant part, plant extract, plant propagation material, or to the locus thereof.

In another aspect, there is provided a method of regulating or improving the growth of a plant under phosphate limited conditions, said method comprising applying an effective amount of a compound of formula (I), to the plant, plant part, plant extract, plant propagation material, or to the locus thereof.

In another aspect, there is provided a method of regulating or improving the growth of a plant under nutrient limited conditions, said method comprising applying an effective amount of a compound of formula (I), to the plant, plant part, plant extract, plant propagation material, or to the locus thereof.

In another aspect, there is provided a method of increasing the surface area of the roots of a plant, said method comprising applying an effective amount of a compound of formula (I), to the plant, plant part, plant extract, plant propagation material, or to the locus thereof.

In another aspect, there is provided a method of increasing the length of the roots of a plant, said method comprising applying an effective amount of a compound of formula (I), to the plant, plant part, plant extract, plant propagation material, or to the locus thereof.

In another aspect, there is provided a method of improving the tolerance of a plant to abiotic stress, said method comprising applying an effective amount of a compound of formula (I), to the plant, plant part, plant extract, plant propagation material, or to the locus thereof.

In another aspect, there is provided a method of promoting the germination of seeds, said method comprising applying an effective amount of a compound of formula (I), to the seeds or to the locus containing the seeds. In another aspect, there is provided a method of regulating the growth of plants at a locus, said method comprising applying an effective amount of a compound of formula (I), to the plant locus.

In another aspect, there is provided the use of a compound of formula (I) as a plant growth regulator or a seed germination promoter.

In another aspect, there is provided a plant growth regulating or seed germination promoting composition, comprising the compound of formula (I), and an agriculturally acceptable formulation adjuvant.

In another aspect, there is provided a method of increasing soil nutrient availability to a plant, said method comprising applying an effective amount of a compound of formula (I), to the plant, plant part, plant extract, plant propagation material, or to the locus thereof.

In another aspect, there is provided a method of increasing the availability of beneficial soil microorganisms to a plant, said method comprising applying an effective amount of a compound of formula (I), to the plant, plant part, plant extract, plant propagation material, or to the locus thereof.

In another aspect, there is provided a method of stimulating nitrogen fixing bacteria in the soil, said method comprising applying an effective amount of a compound of formula (I), to the plant, plant part, plant extract, plant propagation material, or to the locus thereof.

In another aspect, there is provided a method of increasing the number of beneficial bacterial or fungal families in the soil microbiome, said method comprising applying an effective amount of a compound of formula (I), to the plant, plant propagation material, or to the locus thereof. In one embodiment there is provided a method of increasing the number of beneficial bacterial fungal families in the soil microbiome, said method comprising applying an effective amount of a compound of formula (I), to the plant, plant propagation material, or to the locus thereof. In another embodiment, there is provided a method of increasing the number of beneficial fungal families in the soil microbiome, said method comprising applying an effective amount of a compound of formula (I), to the plant, plant propagation material, or to the locus thereof. Preferably, in a method of increasing the number of beneficial bacterial or fungal families in the soil microbiome, the plant to be treated is corn.

In another aspect, there is provided a method of stimulating symbiosis between a plant and arbuscular mycorrhizal fungi in the soil, said method comprising applying an effective amount of a compound of formula (I), to the plant, plant part, plant propagation material, or to the locus thereof.

In another aspect, there is provided a method of increasing the arbuscular mycorrhizal fungi spore density in the soil, said method comprising applying an effective amount of a compound of formula (I), to the plant, plant part, plant extract, plant propagation material, or to the locus thereof.

In another aspect, there is provided a method of increasing arbuscular mycorrhizal fungi root colonization of a plant, said method comprising applying an effective amount of a compound of formula (I), to the plant, plant part, plant propagation material, or to the locus thereof. In another aspect, there is provided a method of increasing the hyphal branching of arbuscular mycorrhizal fungi in the soil, said method comprising applying an effective amount of a compound of formula (I), to the plant, plant part, plant extract, plant propagation material, or to the locus thereof.

In another aspect, there is provided a method of stimulating phosphate solubilizing bacteria in the soil, said method comprising applying an effective amount of a compound of formula (I), to the plant, plant part, plant extract, plant propagation material, or to the locus thereof.

In another aspect, there is provided a method of amplifying the useful response of beneficial microorganisms, said method comprising applying an effective amount of a compound of formula (I), to the plant, plant part, plant extract, plant propagation material, or to the locus thereof.

In another aspect, there is provided a method of increasing the solubilization of phosphates available to a plant, said method comprising applying an effective amount of a compound of formula (I), to the plant, plant part, plant extract, plant propagation material, or to the locus thereof.

In another aspect, there is provided a method of increasing the levels of soluble phosphates available to a plant, said method comprising applying an effective amount of a compound of formula (I), to the plant, plant part, or to the locus thereof.

In another aspect, there is provided a method of reducing the time to flowering of a plant, said method comprising applying an effective amount of a compound of formula (I), to the plant, plant part, plant extract, or to the locus thereof.

In another aspect, there is provided a method of synchronizing male and female flowering of plant, said method comprising applying an effective amount of a compound of formula (I), to the plant, plant part, plant extract, or to the locus thereof.

In particular, there is provided a method of improving the nutrient use efficiency of plants, said method comprising applying an effective amount of a compound of formula (I), to the plant, plant part, plant propagation material, or to the locus thereof, wherein the plant is corn (maize), rice, wheat, or soybean.

Corn: Wherein the plant to which the effective amount of a compound of formula (I) is applied is corn, there is provided a method of increasing the yield of contacted corn. There is also provided a method of increasing the yield of contacted corn, wherein the average kernel mass (w/w) of the contacted corn is increased. There is also provided a method of increasing the yield of contacted corn, wherein the average ear volume (v/v) of the contacted corn is increased. There is also provided a method of increasing the yield of contacted corn, wherein the average relative hydration of silks or mass of the silk (w/w) of the contacted corn is increased.

Wherein the plant to which the effective amount of a compound of formula (I) is applied is corn, there may also be provided a reduction in the yellowing of leaves of the corn plant during corn de- tasseling. Wherein the plant to which the effective amount of a compound of formula (I) is applied is corn, there is also provided a method of shortening the anthesis silking interval (ASI).

Wheat: Wherein the plant to which the effective amount of a compound of formula (I) is applied is wheat, there is provided a method of increasing the yield of contacted wheat. There is also provided a method of increasing the yield of contacted wheat, wherein the number of ears per unit area is increased. There is also provided a method of increasing the yield of contacted wheat, wherein the number of spikelets per ear is increased. There is also provided a method of increasing the yield of contacted wheat, wherein the grain weight is increased.

Rice: Wherein the plant to which the effective amount of a compound of formula (I) is applied is rice, there is provided a method of increasing the yield of contacted rice. There is also provided a method of increasing the yield of contacted rice, wherein the rice plant height is increased. There is also provided a method of increasing the yield of contacted rice, wherein the length of the panicle is increased. There is also provided a method of increasing the yield of contacted rice, wherein the length of the seed is increased. There is also provided a method of increasing the yield of contacted rice, wherein the number of rice grains per panicle is increased. There is also provided a method of increasing the yield of contacted rice, wherein the number of panicles per plant is increased. There is also provided a method of increasing the yield of contacted rice, wherein the number of filled grains per panicle is increased. There is also provided a method of increasing the yield of contacted rice, wherein the grain weight is increased.

Soybean: Wherein the plant to which the effective amount of a compound of formula (I) is applied is soybean, there is provided a method of increasing the yield of contacted soybean. There is also provided a method of increasing the yield of contacted soybean, wherein the number of soybean plants per unit area is increased. There is also provided a method of increasing the yield of contacted soybean, wherein the number of soybean pods per plant is increased. There is also provided a method of increasing the yield of contacted soybean, wherein the number of seeds per soybean pod is increased. There is also provided a method of increasing the yield of contacted soybean, wherein the size of the soybean seed is increased.

According to a further aspect of the invention, there is provided an agrochemical composition comprising an effective amount of a compound of formula (I) according to the present invention. Such an agricultural composition may further comprise at least one additional active ingredient and/or an agrochemically-acceptable diluent or carrier.

It is also possible to use compound of formula (I) as dressing agents for the treatment of plant propagation material, e.g., seed, such as fruits, tubers or grains, or plant cuttings (e.g., rice), for the protection against fungal infections, as well as against phytopathogenic fungi occurring in the soil. The propagation material can be treated with a composition comprising a compound of formula (I) before planting: seed, e.g., can be dressed before being sown.

The compound of formula (I) can also be applied to grains (coating), either by impregnating the seeds in a liquid formulation or by coating them with a solid formulation. The composition can also be applied to the planting site when the propagation material is being planted, e.g., to the seed furrow during sowing. The invention relates also to such methods of treating plant propagation material and to the plant propagation material so treated.

Within the scope of present invention, target crops and/or plants to be protected typically comprise perennial and annual crops, such as berry plants for example blackberries, blueberries, cranberries, raspberries and strawberries; cereals for example barley, maize (corn), millet, oats, rice, rye, sorghum, triticale and wheat; fibre plants for example cotton, flax, hemp, jute and sisal; field crops for example sugar and fodder beet, coffee, hops, mustard, oilseed rape (canola), poppy, sugar cane, sunflower, tea and tobacco; fruit trees for example apple, apricot, avocado, banana, cherry, citrus, nectarine, peach, pear and plum; grasses for example Bermuda grass, bluegrass, bentgrass, centipede grass, fescue, ryegrass, St. Augustine grass and Zoysia grass; herbs such as basil, borage, chives, coriander, lavender, lovage, mint, oregano, parsley, rosemary, sage and thyme; legumes for example beans, lentils, peas and soya beans; nuts for example almond, cashew, ground nut, hazelnut, peanut, pecan, pistachio and walnut; palms for example oil palm; ornamentals for example flowers, shrubs and trees; other trees, for example cacao, coconut, olive and rubber; vegetables for example asparagus, aubergine, broccoli, cabbage, carrot, cucumber, garlic, lettuce, marrow, melon, okra, onion, pepper, potato, pumpkin, rhubarb, spinach and tomato; and vines for example grapes.

The term “nutrient use efficiency” is to be understood as a measure of how well plants use available mineral nutrients. It can be defined by the yield of crop per unit of available nutrient (applied nutrient and nutrients from the soil). It can also be defined as the product of nutrient uptake efficiency and nutrient utilization efficiency.

The term “nutrient uptake efficiency” is to be understood as the amount of nutrient taken up by the plant divided by the amount of nutrient available in the soil (i.e., soil content + fertilizer input).

The term “nutrient utilization efficiency” is to be understood as the yield of plant divided by the amount of nutrient taken up by the plant from the soil.

As used herein, the term “nitrogen” is to be understood to mean any nitrogen-containing compound that could be taken up by a plant. Examples of “nitrogen” in the context of the present invention include, but are not limited to, nitrate and ammonium.

As used herein, the term “phosphorus” is to be understood to mean any phosphorus-containing compound that could be taken up by a plant. Examples of “phosphorus” in the context of the present invention include, but are not limited to, phosphate.

The term "plants" is to be understood as also including plants that have been rendered tolerant to herbicides like bromoxynil or classes of herbicides (such as, for example, HPPD inhibitors, ALS inhibitors, for example primisulfuron, prosulfuron and trifloxysulfuron, EPSPS (5-enol-pyrovyl-shikimate- 3-phosphate-synthase) inhibitors, GS (glutamine synthetase) inhibitors or PPO (protoporphyrinogen- oxidase) inhibitors) as a result of conventional methods of breeding or genetic engineering. An example of a crop that has been rendered tolerant to imidazolinones, e.g. imazamox, by conventional methods of breeding (mutagenesis) is Clearfield® summer rape (Canola). Examples of crops that have been rendered tolerant to herbicides or classes of herbicides by genetic engineering methods include glyphosate- and glufosinate-resistant maize varieties commercially available under the trade names RoundupReady®, Herculex I® and LibertyLink®.

The term " plants" is also to be understood as including plants which have been so transformed by the use of recombinant DNA techniques that they are capable of synthesising one or more selectively acting toxins, such as are known, for example, from toxin-producing bacteria, especially those of the genus Bacillus.

The term "crops" is to be understood as including also crop plants which have been so transformed by the use of recombinant DNA techniques that they are capable of synthesising one or more selectively acting toxins, such as are known, for example, from toxin-producing bacteria, especially those of the genus Bacillus.

Toxins that can be expressed by such transgenic plants include, for example, insecticidal proteins from Bacillus cereus or Bacillus popilliae; or insecticidal proteins from Bacillus thuringiensis, such as d-endotoxins, e.g. CrylAb, CrylAc, Cry1 F, Cry1 Fa2, Cry2Ab, Cry3A, Cry3Bb1 or Cry9C, or vegetative insecticidal proteins (Vip), e.g. Vip1 , Vip2, Vip3 or Vip3A; or insecticidal proteins of bacteria colonising nematodes, for example Photorhabdus spp. or Xenorhabdus spp., such as Photorhabdus luminescens, Xenorhabdus nematophilus; toxins produced by animals, such as scorpion toxins, arachnid toxins, wasp toxins and other insect-specific neurotoxins; toxins produced by fungi, such as Streptomycetes toxins, plant lectins, such as pea lectins, barley lectins or snowdrop lectins; agglutinins; proteinase inhibitors, such as trypsin inhibitors, serine protease inhibitors, patatin, cystatin, papain inhibitors; ribosome-inactivating proteins (RIP), such as ricin, maize-RIP, abrin, luffin, saporin or bryodin; steroid metabolism enzymes, such as 3-hydroxysteroidoxidase, ecdysteroid-UDP-glycosyl-transferase, cholesterol oxidases, ecdysone inhibitors, HMG-COA-reductase, ion channel blockers, such as blockers of sodium or calcium channels, juvenile hormone esterase, diuretic hormone receptors, stilbene synthase, bibenzyl synthase, chitinases and glucanases.

In the context of the present invention there are to be understood by d-endotoxins, for example CrylAb, CrylAc, Cry1 F, Cry1 Fa2, Cry2Ab, Cry3A, Cry3Bb1 or Cry9C, or vegetative insecticidal proteins (Vip), for example Vip1 , Vip2, Vip3 or Vip3A, expressly also hybrid toxins, truncated toxins and modified toxins. Hybrid toxins are produced recombinantly by a new combination of different domains of those proteins (see, for example, WO 02/15701). Truncated toxins, for example a truncated CrylAb, are known. In the case of modified toxins, one or more amino acids of the naturally occurring toxin are replaced. In such amino acid replacements, preferably non-naturally present protease recognition sequences are inserted into the toxin, such as, for example, in the case of Cry3A055, a cathepsin-G- recognition sequence is inserted into a Cry3A toxin (see WO 03/018810).

Examples of such toxins or transgenic plants capable of synthesising such toxins are disclosed, for example, in EP-A-0 374 753, WO 93/07278, WO 95/34656, EP-A-0427 529, EP-A-451 878 and WO 03/052073. The processes for the preparation of such transgenic plants are generally known to the person skilled in the art and are described, for example, in the publications mentioned above. Cryl-type deoxyribonucleic acids and their preparation are known, for example, from WO 95/34656, EP-A-0 367 474, EP-A-0 401 979 and WO 90/13651.

The toxin contained in the transgenic plants imparts to the plants tolerance to harmful insects. Such insects can occur in any taxonomic group of insects, but are especially commonly found in the beetles (Coleoptera), two-winged insects (Diptera) and butterflies (Lepidoptera).

Transgenic plants containing one or more genes that code for an insecticidal resistance and express one or more toxins are known and some of them are commercially available. Examples of such plants are: YieldGard® (maize variety that expresses a CrylAb toxin); YieldGard Rootworm® (maize variety that expresses a Cry3Bb1 toxin); YieldGard Plus® (maize variety that expresses a CrylAb and a Cry3Bb1 toxin); Starlink® (maize variety that expresses a Cry9C toxin); Herculex I® (maize variety that expresses a Cry1 Fa2 toxin and the enzyme phosphinothricine N-acetyltransferase (PAT) to achieve tolerance to the herbicide glufosinate ammonium); NuCOTN 33B® (cotton variety that expresses a CrylAc toxin); Bollgard I® (cotton variety that expresses a Cry1 Ac toxin); Bollgard II® (cotton variety that expresses a CrylAc and a Cry2Ab toxin); VipCot® (cotton variety that expresses a Vip3A and a CrylAb toxin); NewLeaf® (potato variety that expresses a Cry3A toxin); NatureGard®, Agrisure® GT Advantage (GA21 glyphosate-tolerant trait), Agrisure® CB Advantage (Bt11 corn borer (CB) trait) and Protecta®.

Further examples of such transgenic crops are:

1. Bt11 Maize from Syngenta Seeds SAS, Chemin de I'Hobit 27, F-31 790 St. Sauveur, France, registration number C/FR/96/05/10. Genetically modified Zea mays which has been rendered resistant to attack by the European corn borer ( Ostrinia nubilalis and Sesamia nonagrioides) by transgenic expression of a truncated CrylAb toxin. Bt11 maize also transgenically expresses the enzyme PAT to achieve tolerance to the herbicide glufosinate ammonium.

2. Bt176 Maize from Syngenta Seeds SAS, Chemin de I'Hobit 27, F-31 790 St. Sauveur, France, registration number C/FR/96/05/10. Genetically modified Zea mays which has been rendered resistant to attack by the European corn borer ( Ostrinia nubilalis and Sesamia nonagrioides) by transgenic expression of a CrylAb toxin. Bt176 maize also transgenically expresses the enzyme PAT to achieve tolerance to the herbicide glufosinate ammonium.

3. MIR604 Maize from Syngenta Seeds SAS, Chemin de I'Hobit 27, F-31 790 St. Sauveur, France, registration number C/FR/96/05/10. Maize which has been rendered insect-resistant by transgenic expression of a modified Cry3A toxin. This toxin is Cry3A055 modified by insertion of a cathepsin-G- protease recognition sequence. The preparation of such transgenic maize plants is described in WO 03/018810.

4. MON 863 Maize from Monsanto Europe S.A. 270-272 Avenue de Tervuren, B-1150 Brussels, Belgium, registration number C/DE/02/9. MON 863 expresses a Cry3Bb1 toxin and has resistance to certain Coleoptera insects. 5. IPC 531 Cotton from Monsanto Europe S.A. 270-272 Avenue de Tervuren, B-1150 Brussels, Belgium, registration number C/ES/96/02.

6. 1507 Maize from Pioneer Overseas Corporation, Avenue Tedesco, 7 B-1160 Brussels, Belgium, registration number C/NL/00/10. Genetically modified maize for the expression of the protein Cry1 F for achieving resistance to certain Lepidoptera insects and of the PAT protein for achieving tolerance to the herbicide glufosinate ammonium.

7. NK603 x MON 810 Maize from Monsanto Europe S.A. 270-272 Avenue de Tervuren, B-1150 Brussels, Belgium, registration number C/GB/02/M3/03. Consists of conventionally bred hybrid maize varieties by crossing the genetically modified varieties NK603 and MON 810. NK603 ^c MON 810 Maize transgenically expresses the protein CP4 EPSPS, obtained from Agrobacterium sp. strain CP4, which imparts tolerance to the herbicide Roundup® (contains glyphosate), and also a Cry1 Ab toxin obtained from Bacillus thuringiensis subsp. kurstaki which brings about tolerance to certain Lepidoptera, include the European corn borer.

The term “locus” as used herein means fields in or on which plants are growing, or where seeds of cultivated plants are sown, or where seed will be placed into the soil. It includes soil, seeds, and seedlings, as well as established vegetation.

The term “plant part” refers to all physical parts of a plant, including seeds, seedlings, saplings, roots, tubers, stems, stalks, foliage, and fruits.

The term “plant extract” refers to a substance or an active with desirable properties that is removed from the tissue of a plant. Examples of “plant extracts” include, but are not limited to, plant microRNA (miRNA).

The term “plant propagation material” is understood to denote generative parts of the plant, such as seeds, which can be used for the multiplication of the latter, and vegetative material, such as cuttings or tubers, for example potatoes. There may be mentioned for example seeds (in the strict sense), roots, fruits, tubers, bulbs, rhizomes and parts of plants. Germinated plants and young plants which are to be transplanted after germination or after emergence from the soil, may also be mentioned. These young plants may be protected before transplantation by a total or partial treatment by immersion. Preferably “plant propagation material” is understood to denote seeds.

The term “anthesis silking interval (ASI)” is the difference in days between male flowering, when 50% of the plants have tassels releasing pollen, and female flowering, when 50% of the plants have visible style-stigmas in the ears.

The term “beneficial soil microorganisms” refers to microorganisms responsible for driving nutrient and organic matter cycling, soil fertility, soil restoration, plant health, and ecosystem primary production. Examples of “beneficial soil microorganisms” include, but are not limited to, microorganisms that create symbiotic associations with plant roots (e.g. rhizobia, mycorrhizal fungi, actinomycetes, diazotrophic bacteria), promote nutrient mineralization and availability, produce plant growth hormones, and are antagonists of plant pests, parasites or diseases (biocontrol agents). A formulation, e.g. a composition containing the compound of formula (I), and, if desired, a solid or liquid adjuvant or monomers for encapsulating the compound of formula (I), may be prepared in a known manner, typically by intimately mixing and/or grinding the compound with extenders, for example solvents, solid carriers and, optionally, surface active compounds (surfactants).

Advantageous rates of application are normally from 0.5g to 2000g of active ingredient (a.i.) per hectare (ha), preferably from 1g to 1000g a.i./ha, more preferably from 5g to 600g a.i./ha, and more preferably still from 10g to 500g a.i./ha. Particularly preferred application rates are between 0.5g to 50g a.i./ha, even more preferably, between 1g to 20g a.i./ha. When used as seed drenching agent, convenient dosages are from 5mg to 1g of active substance per kg of seeds, preferably from 10mg to 1g of active substance per kg of seeds.

When the combinations of the present invention are used for treating seed, rates of 0.001 to 50 g of a compound of formula (I) per kg of seed, preferably from 0.01 to 10g per kg of seed are generally sufficient.

The compositions of the invention may be employed in any conventional form, for example in the form of a powder for dry seed treatment (DS), an emulsion for seed treatment (ES), a flowable concentrate for seed treatment (FS), a solution for seed treatment (LS), a water dispersible powder for seed treatment (WS), a capsule suspension for seed treatment (CF), a gel for seed treatment (GF), an emulsion concentrate (EC), a suspension concentrate (SC), a suspo-emulsion (SE), a capsule suspension (CS), a water dispersible granule (WG), an emulsifiable granule (EG), an emulsion, water in oil (EO), an emulsion, oil in water (EW), a micro-emulsion (ME), an oil dispersion (OD), an oil miscible flowable (OF), an oil miscible liquid (OL), a soluble concentrate (SL), an ultra-low volume suspension (SU), an ultra-low volume liquid (UL), a technical concentrate (TK), a dispersible concentrate (DC), a wettable powder (WP) or any technically feasible formulation in combination with agriculturally acceptable adjuvants.

Such compositions may be produced in conventional manner, e.g., by mixing the active ingredients with appropriate formulation inerts (diluents, solvents, fillers and optionally other formulating ingredients such as surfactants, biocides, anti-freeze, stickers, thickeners and compounds that provide adjuvancy effects). Also conventional slow release formulations may be employed where long lasting efficacy is intended. Particularly formulations to be applied in spraying forms, such as water dispersible concentrates (e.g. EC, SC, DC, OD, SE, EW, EO and the like), wettable powders and granules, may contain surfactants such as wetting and dispersing agents and other compounds that provide adjuvancy effects, e.g. the condensation product of formaldehyde with naphthalene sulphonate, an alkylarylsulphonate, a lignin sulphonate, a fatty alkyl sulphate, and ethoxylated alkylphenol and an ethoxylated fatty alcohol.

The compound of formula (I) may be applied directly to the plant (i.e., via foliar application to the plant leaves), to the plant locus (i.e., the fields in or on which the plant is growing, or where a seed is or has been sown), to plant propagation material, or directly to the seed. In a preferred embodiment, the compound of formula (I) may be applied directly to the plant (i.e., via foliar application to the plant leaves). A seed dressing formulation is applied in a manner known per se to the seeds employing the combination of the invention and a diluent in suitable seed dressing formulation form, e.g., as an aqueous suspension or in a dry powder form having good adherence to the seeds. Such seed dressing formulations are known in the art. Seed dressing formulations may contain the single active ingredients or the combination of active ingredients in encapsulated form, e.g. as slow release capsules or microcapsules.

In general, the formulations include from 0.01 to 90% by weight of active agent, from 0 to 20% agriculturally acceptable surfactant and 10 to 99.99% solid or liquid formulation inerts and adjuvant(s), the active agent consisting of at least the compound of formula (I) together with, for example, components (B) and/or (C), and optionally other active agents, particularly microbiocides or conservatives or the like. Concentrated forms of compositions generally contain in between about 2 and 80%, preferably between about 5 and 70% by weight of active agent. Application forms of formulation may for example contain from 0.01 to 20% by weight, preferably from 0.01 to 5% by weight of active agent. Whereas commercial products will preferably be formulated as concentrates, the end user will normally employ diluted formulations.

Zealactone may be the sole active ingredient of a composition or it may be admixed with one or more additional active ingredients such as a pesticide, fungicide, synergist, herbicide, plant growth regulator, or plant extract, where appropriate. An additional active ingredient may, in some cases, result in unexpected synergistic activities. Wherein zealactone is admixed with a plant extract, said plant extract may be enriched with micro RNA.

Formulation Examples

Wettable powders a) b) c) active ingredient [compound of formula (I)] 25 % 50 % 75 % sodium lignosulfonate 5 % 5 % sodium lauryl sulfate 3 % 5 % sodium diisobutylnaphthalenesulfonate 6 % 10 % phenol polyethylene glycol ether 2 % (7-8 mol of ethylene oxide) highly dispersed silicic acid 5 % 10 % 10 % Kaolin 62 % 27 % The active ingredient is thoroughly mixed with the adjuvants and the mixture is thoroughly ground in a suitable mill, affording wettable powders that can be diluted with waterto give suspensions of the desired concentration.

Powders for dry seed treatment a) b) c) active ingredient [compound of formula (I)] 25 % 50 % 75 % light mineral oil 5 % 5 % 5 % highly dispersed silicic acid 5 % 5 %

Kaolin 65 % 40 %

Talcum 20 %

The active ingredient is thoroughly mixed with the adjuvants and the mixture is thoroughly ground in a suitable mill, affording powders that can be used directly for seed treatment.

Emulsifiable concentrate active ingredient [compound of formula (I)] 10 % octylphenol polyethylene glycol ether 3 %

(4-5 mol of ethylene oxide) calcium dodecylbenzenesulfonate 3 % castor oil polyglycol ether (35 mol of ethylene oxide) 4 %

Cyclohexanone 30 % xylene mixture 50 %

Emulsions of any required dilution, which can be used in plant protection, can be obtained from this concentrate by dilution with water.

Dusts a) b) c)

Active ingredient [compound of formula (I)] 5 % 6 % 4 % talcum 95 %

Kaolin 94 % mineral filler 96 % Ready-for-use dusts are obtained by mixing the active ingredient with the carrier and grinding the mixture in a suitable mill. Such powders can also be used for dry dressings for seed.

Extruder granules

Active ingredient [compound of formula (I)] 15 % sodium lignosulfonate 2 % carboxymethylcellulose 1 %

Kaolin 82 %

The active ingredient is mixed and ground with the adjuvants, and the mixture is moistened with water. The mixture is extruded and then dried in a stream of air.

Coated granules

Active ingredient [compound of formula (I)] 8 % polyethylene glycol (mol. wt. 200) 3 %

Kaolin 89 % The finely ground active ingredient is uniformly applied, in a mixer, to the kaolin moistened with polyethylene glycol. Non-dusty coated granules are obtained in this manner. Suspension concentrate active ingredient [compound of formula (I)] 40 % propylene glycol 10 % nonylphenol polyethylene glycol ether (15 mol of ethylene oxide) 6 %

Sodium lignosulfonate 10 % carboxymethylcellulose 1 % silicone oil (in the form of a 75 % emulsion in water) 1 %

Water 32 %

The finely ground active ingredient is intimately mixed with the adjuvants, giving a suspension concentrate from which suspensions of any desired dilution can be obtained by dilution with water. Using such dilutions, living plants as well as plant propagation material can be treated and protected against infestation by microorganisms, by spraying, pouring or immersion.

Flowable concentrate for seed treatment active ingredient [compound of formula (I)] 40 % propylene glycol 5 % copolymer butanol PO/EO 2 % tristyrenephenole with 10-20 moles EO 2 %

1 ,2-benzisothiazolin-3-one (in the form of a 20% solution in water) 0.5 % monoazo-pigment calcium salt 5 %

Silicone oil (in the form of a 75 % emulsion in water) 0.2 %

Water 45.3 %

The finely ground active ingredient is intimately mixed with the adjuvants, giving a suspension concentrate from which suspensions of any desired dilution can be obtained by dilution with water. Using such dilutions, living plants as well as plant propagation material can be treated and protected against infestation by microorganisms, by spraying, pouring or immersion.

Slow Release Capsule Suspension 28 parts of a combination of the compound of formula (I) are mixed with 2 parts of an aromatic solvent and 7 parts of toluene diisocyanate/polymethylene-polyphenylisocyanate-mixture (8:1). This mixture is emulsified in a mixture of 1 .2 parts of polyvinyl alcohol, 0.05 parts of a defoamer and 51 .6 parts of water until the desired particle size is achieved. To this emulsion a mixture of 2.8 parts 1 ,6-diaminohexane in 5.3 parts of water is added. The mixture is agitated until the polymerization reaction is completed. The obtained capsule suspension is stabilized by adding 0.25 parts of a thickener and 3 parts of a dispersing agent. The capsule suspension formulation contains 28% of the active ingredients. The medium capsule diameter is 8-15 microns. The resulting formulation is applied to seeds as an aqueous suspension in an apparatus suitable for that purpose.

The present invention will now be described with reference to the following examples, which are by way of illustration and do not limited the scope of the invention in any way.

The following examples demonstrate the ability of zealactone (the compound of formula (I)) to increase the nutrient use efficiency of a plant.

List of Abbreviations

AMF = arbuscular mycorrhizal fungus, ASV = amplicon sequence variant, °C = degrees Celsius, DMSO = dimethylsulfoxide, HBI = hyphal branching index, ITS = internal transcribed spacers, L = litre, mg = milligrams, pM = micromolar, mM = millimolar, ng = nanograms, OD = optical density, Oϋboo = optical density of a sample measured at wavelength 600nm, PCR = polymerase chain reaction, PPFD = photosynthetic photon flux density, pL = microlitre

EXAMPLES

Example 1 : Stimulatation of Arbuscular Mycorrhizal Fungi Hyphal Branching by Zealactone

The efficacy of zealactone on enhancing hyphal branching in an arbuscular mycorrhizal fungus (AMF) was tested using Gigaspora rosea grown in vitro. To induce the development of second and third order hyphal branches zealactone was applied on the agar media close to a first order branch by pipetting the zealactone compound dissolved in 100% acetone. The synthetic strigolactone GR24 was used as a positive control as its capacity to activate arbuscular mycorrhizal fungi has been previously demonstrated in several publications, for example in Akiyama, K et al. ^' Plant sesquiterpenes induce hyphal branching in arbuscular mycorrhizal fungi ^' , Nature 435, 824-827 (2005) and Borghi, L., Screpanti, C., Lumbroso, A. et al. ^' Efficiency and bioavailability of new synthetic strigolactone mimics with potential for sustainable agronomical applications ^' Plant Soil 465, 109-123 (2021).

Gigaspora rosea spores were surface sterilized by keeping them in a solution containing 0.2% NaCIO + 0.05% Triton™ X-100 (f-octylphenoxypolyethoxyethanol) for 3 minutes and rinsing them with sterile water three times. Single spores were placed in petri dishes containing a thin layer of 0.2% BioReagent Phytagel™ (an agar substitute gelling agent) + 6mM MgSC .

Plates were vertically incubated in a CO2 incubator (4% CO2, 32°C) for 5-7 days until first order branches were observed. Compounds were then dissolved in 100% acetone and filter sterilized. A small hole in the agar was made close to the first order hypha and 2 pL containing the appropriate amount of test compound pipetted in. Two different first order branches per hypha were treated. GR24 (200 ng) was used as a positive control. All procedures were performed under sterile conditions. Treated plates were incubated vertically for 72 or more hours under the same conditions (4% CO2, 32°C).

Images of the plates were taken immediately following application of zealactone (time point 0), and also at 72 hours following application of zealactone, with a light microscope. The number of new secondary and third order branches were counted after 72 hours and the hyphal branching index (HBI) was calculated according to the formula:

HBI = Log (1 + 2*number of 2 ^nd order branches + 3*number of 3 ^rd order branches)

The mean values of 10 replicates pertreatment are represented in Figure 1 as a ‘fold-change’ compared to the untreated control.

Figure 1 shows that that both the positive control (GR24) and zealactone induce AMF hyphal branching compared to the untreated control. Zealactone was the most potent treatment at inducing AMF hyphal branching and showed a dose-dependent response. At 200 ng rate, zealactone showed a 40% higher HBI compared to GR24.

Example 2: Increase in AMF Colonization and Plant Height Following Foliar Application of Zealactone

The capacity of zealactone foliar treatments to enhance AMF colonization and plant height was studied under greenhouse conditions. Corn plants (cultivar NK Falkone), grown in silty sandy soil, were treated with a foliar application of zealactone 14 days after sowing. Plants were harvested 42 days after sowing. Plant heights were recorded, and the percent colonization was measured using the methods described in McGonigle et al, New phytologist, 1990, 115, 495-501. The roots were briefly washed, boiled in 10% KOH for 45 minutes, stained with a mixture of 5% blue ink and 5% acetic acid for 20 minutes, and then stored in 10% glycerol before enumeration. Following foliar application of zealactone to corn plants, AMF colonization was increased from an average colonization rate of 27% to 36%, representing an increase of approximately 30% compared to untreated plants, as shown in Figure 2.

Figure 2 shows that a significant increase in AMF colonization was observed in zealactone treated plants compared to the untreated control.

Furthermore, foliar application of zealactone also increased plant height. Following foliar application of zealactone to corn plants, plant height was increased from an average of 53.2 cm to 54.6 cm, representing an increase of approximately 2.5% compared to untreated plants, as shown in Figure 3. Figure 3 shows that plant height is significantly increased for zealactone treated plants compared to the untreated control.

Example 3: Effect of Zealactone on Wheat Yield Components

Wheat ( Triticum aestivum) plants were grown in 1 .5 L growth columns in mixture of field soil and peat. Throughout the experiment, each plant received 215 mg nitrogen. Growth conditions were 22/12°C (day/night) air temperature, 60/70% (day/night) relative humidity, and a PPFD of 600 pmol nr ² s ^_1 at 14 hours photoperiod. Zealactone was applied twice by foliar application as an emulsified concentrate at 250 pM when plants were at growth stage BBCH39 (flag leaf stage) and at BBCH51 (beginning of heading). Kernel weights were analyzed at physiological maturity.

As demonstrated in Table 1 , the application of zealactone results in a statistically significant increase of the individual kernel weight (expressed as thousand kernel weight). The increase of the thousand kernel weight is one important component for overall yield increase.

Table 1 : The effect of foliar application of zealactone on wheat thousand kernel weight.

Values represent the mean value ± the standard deviation of 10 replicates.

Example 4: Zealactone Foliar Treatment Induces Modifications in the Below Ground Plant Microbiome

The capacity of zealactone foliar treatments to induce modifications in the below-ground microbial assembly has been studied by amplicon sequencing.

Corn plants (cultivar NK Falkone) grown on two different soils (sandy loam and silty loam) were treated with a foliar application of zealactone 14 days after sowing. Plants were harvested 35 days after sowing.

To detect microbial community modifications associated with the foliar treatment of zealactone, PCR amplification and successive high-throughput sequencing of bacterial and fungal taxonomical marker genes (the small-subunit ribosomal RNA gene (16S) and the ribosomal internal transcribed spacers (ITS) respectively) were performed on rhizosphere and root endosphere samples.

Microbiome shifts connected with the treatment have been observed in both the rhizosphere and endosphere of plants growing in each selected soil. Marker gene analysis at amplicon sequence variants (ASVs) level detected 91 bacterial taxa (Table 2) and 40 fungal taxa (Table 3) associated with the treatment. Moreover, at family level, 10 distinct bacterial families and 10 distinct fungal families have been found to be associated with the foliar treatment, with increments in relative abundance that range from + 21 % to + 2285 %. The rhizosphere was the compartment where the influence of the foliar treatment was prominent on both bacteria and fungi. Furthermore, in this compartment the bacterial families Erythrobacteraceae, Nocardiaceae and Verrucomicrobiaceae have been found consistently associated with the foliar treatment independently of the soil substrate. These three families comprise known plant growth promoting bacteria according to J. Hamedi et al., ^' F. Biotechnological application and taxonomical distribution of plant growth promoting actinobacteria ^' , J. Ind. Microbiol. Biotechnol. 2015, 42, 157-171 , T. Tang et al., ^' Erythrobacter aureus sp. nov., a plant growth-promoting bacterium isolated from sediment in the Yellow Sea, China ^' , 3 Biotech, 2019, 9, 1-9 and W. Biinger et al., ^' Novel cultivated endophytic Verrucomicrobia reveal deep-rooting traits of bacteria to associate with plants ^' , Sci. Rep. 2020, 10, 1-13.

Table 2: Bacterial Amplicon Sequencing Variants (ASVs) and Bacterial Families Associated with Zealactone Foliar Treatment in Corn

No sequences belonging to the Bacteriovoacaceae family were found in untreated control samples. * Compared to untreated control samples.

Table 3: Fungal Amplicon Sequencing Variants (ASVs) and Fungal Families Associated with Zealactone Foliar Treatment in Corn

Example 5: In Vitro Phosphate Solubilization Assay

The protocol is adapted from Nautiyal C.S. ^' An efficient microbiological growth medium for screening phosphate solubilizing microorganisms ^' , FEMS Microbiol Lett. 1999, 170(1):265-70. A 50 mL liquid Nutrient Broth (NB) media was inoculated with a loop containing the phosphate solubilizing bacteria Bacillus megaterium ( Culti-Loops ™ Bacillus megaterium ATCC™ 14581™) and incubated overnight at 30°C. After centrifugation, the cell pellet was washed twice with sterile ddhhO and its Oϋboo measured. Erlenmeyer flasks containing 20 mL of National Botanical Research Institute’s phosphate growth medium (NBRIP), which contains exclusively insoluble Ca3(PC> ₄ ) ₂ as phosphorus source, were inoculated with the B. megaterium culture to have a final OD of 0.02. Filter sterilized compounds dissolved in 100% DMSO were added to the flasks at the desired concentrations (the corresponding amount of DMSO without compound was used as control). NBRIP media cultures were then incubated at 30°C for 4 days. After 4 days, a 5 mL aliquot of each culture was collected, centrifuged and the supernatant passed through a 0.2 pm filter. The cleared supernatant was analyzed for inorganic phosphate content using the colorimetric EnzChek™ Phosphate Assay Kit (Invitrogen™) according to the manufacture instructions. The remaining supernatant fraction was used for pH measurements. The change in inorganic phosphorus content and media acidification of the compound containing cultures compared to the DMSO control was used to quantify the influence of compounds on the soil phosphorus solubilizing bacteria.

No sequences belonging to the Bacteriovoacaceae family were found in untreated control samples. * Compared to untreated control samples.

Table 3: Fungal Amplicon Sequencing Variants (ASVs) and Fungal Families Associated with Zealactone Foliar Treatment in Corn

Example 5: In Vitro Phosphate Solubilization Assay

The protocol is adapted from Nautiyal C.S. ^' An efficient microbiological growth medium for screening phosphate solubilizing microorganisms ^' , FEMS Microbiol Lett. 1999, 170(1):265-70. A 50 mL liquid Nutrient Broth (NB) media was inoculated with a loop containing the phosphate solubilizing bacteria Bacillus megaterium ( Culti-Loops ™ Bacillus megaterium ATCC™ 14581™) and incubated overnight at 30°C. After centrifugation, the cell pellet was washed twice with sterile ddhhO and its Oϋboo measured. Erlenmeyer flasks containing 20 mL of National Botanical Research Institute’s phosphate growth medium (NBRIP), which contains exclusively insoluble Ca3(PC> ₄ ) ₂ as phosphorus source, were inoculated with the B. megaterium culture to have a final OD of 0.02. Filter sterilized compounds dissolved in 100% DMSO were added to the flasks at the desired concentrations (the corresponding amount of DMSO without compound was used as control). NBRIP media cultures were then incubated at 30°C for 4 days. After 4 days, a 5 ml_ aliquot of each culture was collected, centrifuged and the supernatant passed through a 0.2 pm filter. The cleared supernatant was analyzed for inorganic phosphate content using the colorimetric EnzChek™ Phosphate Assay Kit (Invitrogen™) according to the manufacture instructions. The remaining supernatant fraction was used for pH measurements. The change in inorganic phosphorus content and media acidification of the compound containing cultures compared to the DMSO control was used to quantify the influence of compounds on the soil phosphorus solubilizing bacteria.