ES2976115B2 - METHOD TO PROTECT AGAINST STRESS AND INCREASE PLANT GROWTH - Google Patents

Description

[0001] DESCRIPTION

[0003] METHOD TO PROTECT AGAINST STRESS AND INCREASE GROWTH

[0004] OF THE PLANTS

[0006] FIELD OF THE INVENTION

[0008] The present invention belongs to the field of the agronomic sector, in particular to the field of methods for promoting the growth and development of plants with emphasis on root development, both under optimal growth conditions and under different types of stress.

[0010] BACKGROUND OF THE INVENTION

[0012] In the agricultural sector, there is enormous interest in increasing plant growth while minimizing the use of chemical fertilizers and promoting greater sustainability. This interest is particularly important when it comes to improving tolerance or alleviating stress experienced by plants when they are not growing under optimal conditions. Stress prevents crops from reaching their full genetic potential and causes significant yield losses worldwide. It is well known that good root development is crucial for the efficient absorption of water and nutrients by the plant, especially in the early stages of development, particularly under stressful conditions such as drought, salinity, and extreme temperatures. Improving plant root development can therefore be a key factor in enhancing plant tolerance to stresses caused by unfavorable nutritional and environmental conditions (Koevoets, I. T. et al., 2016, Front. Plant Sci. 7, 1335. doi: 10.3389/fpls.2016.01335). For example, the angle of development of maize roots modifies their nitrogen uptake capacity (Dathe, A et al., 2016, Ann Bot. 2016 118(3), 401-14- doi: 10.1093/aob/mcw112). On the other hand, phosphorus uptake is related to a higher density of lateral roots in the maize taproot (Jia, X. et al., 2018, J Exp Bot. 69(20). 4961-4970. Doi: 10.1093/jxb/ery252).

[0014] Plants in nature establish symbiotic associations with microorganisms called mutualists, which confer benefits to their growth, survival, and reproduction. These microorganisms can be isolated and sometimes used to improve crop yields. For example, the colonization of rapeseed roots by Serendipita vermifera, Alternaria alternata, or Leptosphaeria biglobosa significantly increases root and stem biomass (Dolatabadi, H. K. and Goltapeh, E. M., 2013, J. Hortic. Res. 21, 115–124. doi: 10.2478/johr-2013-0030; Zhang, Q. et al., 2014, Biol. Control 72, 98–108. doi: 10.1016/J.BIOCONTROL.2014.02.018). This improved growth may be due to an improvement in the plant's efficiency in capturing or assimilating nutrients. For example, Aspergillus niger and Trichoderma harzianum in wheat roots produce ammonium and transfer it to the plant, increasing its growth (Ripa et al., 2019, Biomed Res Int 2019:6105865–6105812. doi.org/10.1155/2019/6105865). Metarhizium brunneum increases root development and shoot biomass, as well as phosphorus content, in potato plants (Krell Vet al., 2018, Fungal Ecol 34:43–49. doi.org/10.1016/j.funeco.2018.04.002). Another advantage conferred by beneficial microorganisms can be the increased tolerance of plants to stresses such as salinity or toxicity of the environment, or extreme nutrient scarcity. For example, *T. virensa* increases the number of lateral roots in *Arabidopsis*, making the plant more tolerant to salinity (Contreras-Cornejo, H. A. et al., 2009, *Plant Physiol.* 149, 1579–1592. doi: 10.1104/PP.108.130369). As these examples illustrate, different microorganisms can have distinct mechanisms of action that serve to improve specific aspects of plant growth. It is therefore necessary to find microorganisms that can improve plant growth under different conditions, especially if these conditions are not optimal for plant growth, in order to alleviate the stress produced under those conditions and restore productivity. Among the modes of action of the microorganism to improve the stress tolerance of plants can be included the increase in the development of the root system.

[0016] Mohamed Tarroumet et al. (2021), Plants, 10, 784, https://doi.org/10.3390/plants10040784, presents a set of six fungal strains from different species isolated from the rhizosphere of a halophytic grass, Aeluropus littoralis, and analyzes their growth-promoting activities on tobacco plants. The six strains improved the growth of cultivated tobacco plants when added to the hydroponic growing medium. Furthermore, when cell-free culture filtrates (CFF) were added to 0.5 NS (nutrient solution) in a closed hydroponic system, the greatest effects on tobacco seedlings (shoot and root dry weight, number of leaves, and root length) were observed when adding CFF from strains A5.1 and A8, which presumably belong to the species Byssochlamys spectabilis and Penicillium melinii, based on the similarity of a single genetic marker (ITS), which is not entirely conclusive in taxonomic identification, with the sequences of these species obtained from the NCBI database of Genebank. It was found that the CFF from all strains, when added to 0.5 NS, can replace some of the chemical inputs, with biomass production equal to or significantly better than that produced in full NS. The authors attribute the ability to promote plant growth to the production of indoleacetic acid-related substances (auxins), a growth hormone, by the fungi. Furthermore, the authors show that the treated tobacco plants also exhibit induced expression of the Tryp1 and YUCCA6-like genes, involved in auxin biosynthesis. Based on these results, this study proposes the use of the A8 strain of P. meliniio and its filtrates to replace some of the chemical fertilization necessary for optimal plant growth, but not as a stress reliever. The only root parameters measured were related to growth (weight or length); other developmental parameters related to lateral root formation were not analyzed. This effect was also not measured under stress conditions. In this regard, the publication shows that high temperature (40ºC) or high salinity conditions (greater than or equal to 100 mM of NaCl in the culture medium) inhibit the growth of strain A8 of the species P. melinii.

[0018] WO2014046553A1 (BIOCONSORTIA INC) discloses a method for selecting microorganisms capable of imparting beneficial properties to plants. Example 4 describes the use of this method to obtain microorganisms that improve the growth of ryegrass plants cultivated under favorable growing conditions. Among the microorganisms tested is P. melinii (Table 2), although this microorganism did not produce the best results and was therefore not selected for the next stage. Furthermore, the conditions used for selecting the microorganisms did not involve stressing the plants, as they were cultivated under favorable growing conditions.

[0020] BRIEF DESCRIPTION OF THE FIGURES.

[0022] Figure 1. Increase in root growth in plants treated with CECT 21242 compared to untreated plants (control), under nutritional stress due to inorganic phosphorus (Pi) deficiency.

[0024] Figure 2. A. Phylogenetic tree using the maximum likelihood method of the concatenated regions RPB2, CAM, and BenA of 94 strains of the genus Penicillium and other closely related genera to determine the taxonomic position of strain CECT 21242, following the methodology described in Houbraken et al., 2020 (Studies in Mycology 95: 5–169). Hamigera avellanea was selected as an outgroup. B. Detail of the cluster where strain CECT 21242 groups with the sequences of other strains of the species Penicillium melinii. Figure 3. Relative increase in prebranching sites (PBS) in roots of untreated plants (White) and plants treated with CECT 21242 grown with different concentrations of inorganic phosphorus in the medium (Pi) (A, B). Increased auxin signaling in the root of DR5::LUC plants inoculated with different fungi (C).

[0026] Figure 4. Treatment with CECT 21242 increases root growth in plants subjected to the combination of light stress to the root and stress from excess (625μM) or deficiency (20μM) of inorganic phosphorus in the culture medium.

[0028] Figure 5. The extract of CECT 21242 (mycelium) or the filtrate of the CECT 21242 culture (filtrate) increases the length (Figure 5A) and number of lateral roots (Figure 5B) of Arabidopsis thaliana plants grown in phosphate deficiency and under light stress from root illumination.

[0030] Figure 6. Treatment with CECT 21242 increases root development in plants grown at 32°C (high temperature stress) and under conditions of phosphate (Pi) deficiency in the culture medium (20 μM).

[0032] Figure 7. Increase in root growth (Figure 7A and C) and aerial biomass (Figure B and D) in Arabidopsis thaliana plants treated with CECT 21242 and grown at 22°C or 32°C.

[0034] Figure 8. Treatment with CECT 21242 increases the thickness and length of lateral roots (8A), the area occupied by the root system (8B) and the biomass of the aerial part (8C) of barley plants.

[0036] Figure 9. Treatment with CECT 21242 increases the development of the aerial part in barley (A and B) and maize (C and D) plants grown in pots in a greenhouse for three weeks.

[0038] Figure 10. Growth curves of maize plants grown in agricultural soil treated with CECT 21242 or with water (control) and kept in a greenhouse for 6 weeks.

[0039] DESCRIPTION OF THE INVENTION

[0040] The present invention has identified, through screening of 10 strains of different fungal species, the CECT 21242 strain of the species Penicillium melinii, which surprisingly increases the development of lateral roots in plants and serves as a stress reliever. The increased root development occurs under stress conditions caused by phosphorus deficiency or excess, light stress on the roots, or heat stress, or a combination of these stresses. Furthermore, the application of extracts or filtrates of the fungus also increased the length and number of lateral roots in plants under stress conditions. Therefore, the data indicate that plants treated with Penicillium melinii, its extracts, or filtrates acquire tolerance to different types of stress. Furthermore, plants treated with the CECT 21242 strain of Penicillium melinii show increased root development and shoot growth under both stress and optimal conditions in in vitro culture or when growing the plants in agricultural soil.

[0042] The species identification was carried out following the description published by Houbraken et al. 2020 Studies in Mycology 95: 5–169, using partial sequences of the genes encoding the proteins beta-tubulin (BenA Partial beta-tubulin), calmodulin (CaM), and RNA polymerase II (RPB2). The genetic marker similarity (ITS) method was also used with nucleotide sequences obtained from the NCBI database of Genebank, obtaining the greatest similarity (100% nucleotide identity) with the sequence with accession number NR_077155.1, “Penicillium melinii FRR 2041 ITS region; from TYPE material” (as of 22/03/2024). The sequences used for the identification of the strain of the present invention are those of the three markers used for phylogeny (SEQ. ID. No. 1 to 3) and that of the ITS (SEQ. ID. No. 4).

[0044] Nothing in the prior art suggested that a treatment based on this microorganism could play a preventive or stress-relieving role in plants. Surprisingly, the increase in lateral roots in the plant is not caused by an increase in auxin activity, contrary to what was described by Mohamed Tarroumet et al. (2021), who indicated an increase in the biosynthesis of this hormone, both by the fungus and by the treated plant.

[0046] Furthermore, there was evidence that would have discouraged the use of this treatment, either because of the absence of particularly notable effects of Penicillium melinii on the growth of ryegrass plants (WO2014046553A1), as well as because of the negative role that different types of stress play on the growth of this microorganism (Mohamed Tarroumet et al.(2021).

[0048] A first aspect of the present invention is a method of protecting or relieving plants from various types of stress and/or increasing the number of lateral roots in plants, characterized by comprising a step of contacting said plants with a composition comprising a microorganism of the species Penicillium melinii, and/or strains of said species, and/or extracts of said species or strain, and/or filtrates of said species or strain.

[0050] Hereafter this is the method of the invention, and the term “microorganism of the invention” refers to the microorganism of the species Penicillium meliniiy/or the CECT 21242 strain of said species.

[0052] Increased lateral root formation in the plant means a greater length of the root system, comprising the main root and lateral roots, and/or a greater number of areas capable of forming a lateral root (pre-branching sites, PBS) along the main root.

[0054] Stress is understood as the set of processes that a plant undergoes when growing in unsuitable environmental conditions, resulting in reduced growth compared to what it would have achieved under optimal conditions. An example of stress is an extreme lack of nutrients, such as phosphorus. Optimal conditions are those that allow for adequate plant growth, as any expert in the field can attest.

[0056] A second aspect of the present invention consists of the use of a microorganism of the species Penicillium melinii, and/or strains of said species, and/or extracts of said species or strain, and/or filtrates of said species or strain, for promoting plant growth, both under optimal growth conditions and under various types of stress. The microorganism itself may be used, or extracts of said microorganism, and/or filtrates thereof.

[0058] Plant growth promotion is understood as an increase in the growth rate and absolute growth of the aerial part of the plant and/or the root system (biomass or length).

[0059] A third aspect of the present invention is constituted by the CECT 21242 strain of the species Penicillium melinii, deposited in the Spanish Collection of Type Cultures, CECT (Paterna, Valencia, Spain), following the rules of the Treaty of Budapest, with the deposit number CECT 21242).

[0062]

[0065] A fourth aspect of the present invention is a substrate for growing plants comprising a microorganism of the species Penicillium melinii, and/or strains of said species, in particular strain CECT 21242, and/or extracts of said species or strain, and/or filtrates of said species or strain.

[0067] A fifth aspect of the present invention consists of the use of the substrate comprising a microorganism of the species Penicillium melinii, and/or strains of said species, in particular strain CECT 21242, and/or extracts of said species or strain, and/or filtrates of said species or strain, to obtain stress-tolerant plants and/or increase the growth of plants under stress.

[0069] In this document, "plant growing medium" means any solid or liquid material, natural or synthetic, on which a plant can grow. Examples of growing media include, but are not limited to, soil, earth, sand, humus, or peat, or mixtures thereof. Examples of synthetic growing media include, but are not limited to, Murashige and Skoog (MS) nutrient media, Hoagland media, or any other medium comprising plant nutrients, in liquid form for use in hydroponic cultivation or solidified with gelling agents such as agar, for use in in vitro culture. Other examples include materials for use in hydroponic cultivation, such as germination paper, rockwool, or coconut fiber.

[0071] A sixth aspect of the present invention is a plant propagation material comprising a microorganism of the species Penicillium melinii, and/or strains of said species, in particular strain CECT 21242, and/or extracts of said strain, and/or filtrates of said strain.

[0072] In this document, “propagation material” means any type of cellular material from which a plant can germinate or develop. Examples of propagation material include, but are not limited to: seeds, seedlings, young plants, cuttings, bulbs and tubers, cell suspensions, callus culture, tissue culture, protocorms, explants, or germplasm.

[0074] Propagation material may have various origins. For example, it may be freshly collected or derived from a stock, such as a seed sample or a frozen cell stock. Preferably, it may be selected from the group comprising seeds, seedlings, young plants, cuttings, bulbs, and tubers.

[0076] The present invention can be applied to any type of plant, preferably gymnosperms, angiosperms, monocots, and dicots. Some preferred, though not limiting, examples of plants are Arabidopsis, and in particular Arabidopsis thaliana, barley, and maize.

[0077] Plant growth promotion can occur under various types of stress conditions as well as under optimal growth conditions, and in various artificial or natural substrates, including, but not limited to, hydroponic and in vitro cultivation. In the present invention, the plant can be a natural, transgenic, or genetically edited plant.

[0079] Preferred aspects of the present invention consist of a substrate for plant cultivation, and a plant propagation material, comprising the CECT 21242 strain of the microorganism Penicillium melinii and/or extracts of said microorganism and/or filtrates of said microorganism.

[0081] The present invention shows how the CECT 21242 strain of the fungus Penicillium melinii increases root length and lateral root development in treated plants. This increased root development occurs under stress conditions, whether due to phosphorus deficiency or excess, light stress, heat stress, or a combination of these stresses, as well as under optimal growing conditions. Data indicate that plants treated with the CECT 21242 strain of Penicillium melinii acquire stress tolerance. Furthermore, plants treated with the CECT 21242 strain of Penicillium melinii exhibit increased growth rate and absolute shoot growth (biomass or length) under all these conditions in in vitro culture, on germination paper, or when grown in agricultural soil.

[0082] Although the examples of the present invention have been made with the CECT 21242 strain of Penicillium melinii, the present system is universal and is extensible to any other type of strain of Penicillium melinii.

[0084] Stress conditions can be of any type. For example, but not limited to, stress conditions can include deficiency or excess of phosphorus or other nutrients, water stress, toxicity stress, light stress, heat stress, biotic stress, or a combination of several of these stresses. Among the conditions where the treatment has shown particular efficacy are: phosphorus deficiency or excess, root light stress, high temperatures, or a mixture of one or more of these stress conditions.

[0086] Phosphorus deficiency refers to phosphate concentrations below 25 µM in in vitro culture, which result in significantly less plant growth than under optimal conditions. Optimal conditions are those that allow for adequate plant growth, as any expert in the field can attest. Specifically, for A. thaliana cultured in vitro, the optimal phosphate level is 300 µM.

[0088] Excess phosphorus refers to phosphate concentrations higher than the optimum, which produce less plant growth than under optimum conditions.

[0090] Root light stress refers to roots that are exposed to light radiation conditions that, due to the underground nature of the root, are not perceived as optimal by it, and produce less plant growth.

[0092] Water stress is understood as the lack or excess of water during the vegetative cycle of the plant, which produce different types of osmotic, physiological or metabolic disruption, producing reduced plant growth compared to optimal conditions and which can even lead to cells, tissues or the plant suffering serious damage or death.

[0094] Toxicity stress refers to the presence of substances that are harmful to the plant, producing different types of osmotic, physiological, or metabolic disruption, resulting in reduced plant growth compared to optimal conditions and potentially causing serious damage to cells, tissues, or the plant itself, or even death. Toxic substances can be inorganic or organic elements or molecules and may be present in the environment where the plant grows or produced by other organisms (such as other plants, pests, or pathogens) in contact with or near the plant.

[0096] Heat stress refers to a temperature below or above the plant's optimum growth temperature, resulting in inhibited development or reduced growth in the aerial parts and/or root system. Optimum conditions are those that allow for adequate plant growth, as any expert in the field can attest. Specifically, for A. thaliana cultivated in vitro, the optimum temperature is 22°C.

[0098] Biotic stress is understood as the consequence for the plant of suffering damage caused by other organisms, such as phytophagous pests and pathogens (such as fungi, bacteria and other microorganisms, viruses or nematodes) or the interference or allelopathy produced by other plants, which hinders the optimal growth and development of the plant and its ability to take advantage of water and nutrients, resulting in reduced plant growth compared to optimal conditions and producing in many cases different types of physiological or metabolic disruption that can even lead to the death of cells and tissues of the plant, or the entire plant.

[0100] In the method of the invention, a plant is brought into contact with a composition comprising the microorganism Penicillium melinii. The composition can be applied to the whole plant or to any of its parts, such as leaves, shoots, flowers, fruits, ears of corn, seeds, bulbs, tubers, roots, and seedlings. The composition can be applied to the plant at any stage, for example, to the seed before sowing, during sowing, after sowing, and before or after emergence, during the vegetative period, such as during seedbed cultivation, or at the time of transplanting seedlings, or at the time of taking or rooting cuttings, or during the growth stage in a plantation, or even during the reproductive period before flowering, during flowering, or during the fruit ripening process.

[0102] Therefore, an embodiment is the method of the invention, wherein said composition is applied to the seeds of said plant.

[0104] Another embodiment is the method of the invention, wherein said composition is applied to the aerial parts of said plant.

[0105] Another embodiment is the method of the invention, wherein said composition is applied to the roots of said plant or to other underground parts of said plant.

[0107] The method of the invention includes spraying or misting whole plants or any part thereof with a suitable dilution of the composition according to the invention, or immersing whole plants or any part thereof in such dilution. The method of the invention also includes dry dusting whole plants or any part thereof with a composition according to the invention. The method of the invention includes pelleting, or coating seeds with a thin film of a composition according to the invention. The composition according to the invention may also be mixed with the irrigation liquid. The method of the invention also includes treatment by means of agar fragments with mycelium that are placed in contact with a part of the plant, whether root, stem, or leaves, or even on the surface of the soil near the roots of the crop.

[0109] Another embodiment is the method of the invention, wherein said composition comprises fertigation salts, fertilizers, insecticides, nematicides, fungicides, bactericides and herbicides, minerals, organic materials, organic compounds, inorganic compounds, metabolites, fermentation or reaction by-products, liquid diluents, alcohols, ketones, vegetable oils or esters, aliphatic hydrocarbons, esters and other mineral solvents, water, anionic surfactants, non-ionic surfactants, cationic surfactants, amphoteric surfactants, water-soluble polymers, polysaccharides, preservatives, coloring agents, thickening agents, and/or stabilizing agents.

[0111] Another embodiment is the method of the invention, wherein said composition is a liquid, a solid, a paste, or a gel.

[0113] Another embodiment is the method of the invention, wherein said composition is a powder, tablet, compressed, granulated or emulsifiable concentrate.

[0115] Another embodiment is the method of the invention, wherein said composition is applied by spraying, spritzing, immersion, irrigation or dusting.

[0117] The present invention also relates to plants that, according to the method of the invention, have been brought into contact with a composition comprising the microorganism Penicillium melinii strain of the invention, and/or extracts of said microorganism and/or filtrates of said microorganism, and to products produced from the harvested parts of said plants.

[0119] The microorganism of the invention can be cultivated on a wide variety of natural or synthetic substrates. For example, it can be grown in different types of solid or liquid culture media, such as Potato Dextrose Agar (PDA) or Broth (PDB), and can be propagated by techniques known to experts. It can also grow on various natural sources, such as leaves of various plants, pollen grains, oatmeal, potato, carrot, and cellulose. It can also grow on artificial sources such as paper or cardboard and polymers.

[0121] The culture medium can be left at rest or be constantly agitated during cultivation, for example at approximately 1 rps. In addition, the culture temperature can be set in the range between 15 and 35 °C.

[0123] Another embodiment is the method of the invention, wherein said microorganism is in the form of spores, hyphae, mycelium or sclerotia.

[0125] Another embodiment is the method of the invention, where said composition is applied to the substrate for the cultivation of said plant.

[0127] The substrate is preferably treated so that the microorganism of the invention is cultivated in it before the substrate is used for plant cultivation. Examples of substrate treatment include perfusing a liquid into the substrate (by irrigation, injection, or dripping), spraying, dusting, or direct mixing with the substrate. The method of the invention also comprises treating a hydroponic medium for hydroponic cultivation. The method of the invention comprises treating the substrate or hydroponic medium with a composition comprising the appropriate concentration of mycelium and/or spores and/or any other part of the microorganism of the invention, the culture medium, or the filtrate according to the invention in liquid form and/or in solid form as granules or powder.

[0129] The substrate for plant cultivation may comprise the spore, the mycelium or any other part of the microorganism of the invention or the culture medium or filtrate thereof or any of the possible combinations of some of these components. The substrate may be liquid or solid.

[0130] Examples of substrates for plant cultivation are solidified natural or synthetic media for growing plants, especially in vitro. Other examples include soil, sand, humus, peat, or mixtures thereof.

[0132] Another embodiment is the method of the invention, where said microorganism is applied in the form of an extract.

[0133] The extract can be obtained from the microorganism alone or together with the culture medium. There are several ways to obtain extracts known to experts in the field, such as, among others: grinding the microorganism with or without culture medium, lyophilizing the microorganism with or without culture medium, subjecting the microorganism with or without culture medium to low or high temperatures, or mixing the microorganism with or without culture medium with different solvents of aqueous or organic nature and separating the different fractions produced by different methods, such as by particle or molecular size (filtration, centrifugation), or by chemical or biochemical affinity.

[0135] Another embodiment is the method of the invention, where filtrates of the microorganism are applied. In this specification, "filtrate" means a liquid culture medium obtained from the cultivation of the microorganism of the invention. It is possible to obtain a liquid culture medium that is free or essentially free of the solid material of the microorganism of the invention. This culture medium can be prepared by first cultivating the microorganism of the invention in a solid (subsequently mixed in a solvent) or liquid culture medium and then separating the culture medium from the microorganism of the invention. The separation can be carried out by various methods known to a person skilled in the art, for example, by centrifugation or filtration. It is possible, for example, to heat the medium containing the microorganism of the invention twice to approximately 80°C for 30 minutes and then remove the solid fungal material by centrifugation.

[0137] Preferably, the filtrate is obtained by filtering the culture medium through a filter with a pore size of no more than 2 μm, preferably through a filter with a pore size of no more than 0.2 μm. The filtration step allows the extraction of essentially all the hyphae of the microorganism of the invention; more preferably, the filtration would also remove the spores, and even more preferably, it should remove all types of solid fungal material.

[0138] The following are a series of examples that serve to illustrate the nature of the present invention. These examples are included for illustrative purposes only and should not be construed as limitations on the invention claimed herein.

[0139] PREFERRED MODES OF REALIZATION.

[0141] Example 1. Screening of fungi that increase root development in plants.

[0143] During the years 2008 to 2011, Arabidopsis thaliana (A. thaliana) plants were collected growing in natural conditions in different areas of central Spain (García et al.

[0144] 2013. Fungal Diversity 60, 71-89). These plants lacked disease symptoms such as chlorosis, leaf spots, and other types of pathogen-induced lesions. Fragments of the collected plants were disinfected by immersing them in 20% commercial bleach (1% active chlorine) and gently agitating for 5 minutes. The fragments were then rinsed twice in sterile water and placed in a humid chamber at room temperature (20-24°C). The fragments were inspected periodically, and emerging mycelium was isolated on Petri dishes containing potato dextrose agar (PDA) with 200 mg/L chloramphenicol. From the isolates obtained, a screening was performed to identify fungi that were beneficial in increasing root length in plants under phosphorus stress.

[0146] Surface-sterilized seeds of A. thaliana accession Col-0 were sown in modified Murashige and Skoog (MS) medium 1/2 (without sucrose or vitamins and with an optimum phosphate concentration of 300 μM or a deficiency concentration of 5 μM), stratified at 4 °C for 2 days, and then maintained in a controlled growth chamber at 23 °C, under a long cycle (16 hours of light per day) within the D-Root system, which prevents light from reaching the plant's root system. After 7 days, the plants were transplanted to another plate with the same modified MS medium 1/2 where each of the tested fungi had been cultured for 4 days under the same conditions as the plants. In the control treatment, the plants were transplanted to another plate where no fungi were growing.

[0148] Plants grown in the presence of the CECT 21242 strain showed a significant increase (P<0.05) in root growth in plants grown in MS medium with 5 μM, both in the length of the main and lateral roots and in total length compared to untreated control plants (Table 1, Figure 1), showing that plants treated with CECT 21242 become tolerant to stress caused by inorganic phosphorus deficiency. Other fungi produced significant reductions in the length of the main root or root system or did not produce significant changes.

[0149] Table 1.

[0150] Length (mean ± standard error) of the main root and root system of plants treated with different fungi as described in Example 1. Data in bold indicate significant differences between treated and control plants (P<0.05, Student's t).

[0152]

[0155] (1) The length of the root system is calculated as the sum of the length of the main root and all lateral roots. Plants were grown with an optimum phosphate concentration of 300μM (2) or a deficiency concentration of 5μM (3).

[0157] Example 2. Taxonomic identification of strain CECT 21242.

[0159] The morphology of strain CECT 21242 indicated that the strain belongs to the genus Penicillium. To determine the species, the methodology of Houbrake et al., 2020 (Studies in Mycology 95: 5–169) was followed, obtaining partial sequences of genes encoding the proteins beta-tubulin (BenA Partial beta-tubulin), calmodulin (CaM), and RNA polymerase II (RPB2). These sequences were concatenated, aligned, and phylogenetically compared with those of 94 other reference strains from different species of the genus Penicillium and other closely related genera obtained from the NCBI database. Alignment was performed using the MAFFT program, and phylogenetic comparison was performed using the maximum similarity method with IQtree (Figure 2A). Strain CECT21242 clusters significantly with all strains of the species P. melinii, indicating that strain CECT21242 belongs to this species (Figure 2B). The authors of the methodology described in Houbraken et al. (2020) belong to the Westerdijk Fungal Biodiversity Institute (Utrecht, Netherlands) and are leading experts in fungal taxonomy. For the identification of strain CECT21242, the genetic marker similarity (ITS) method was also used with nucleotide sequences obtained from the NCBI database of GeneBank. The greatest similarity (100% nucleotide identity) was obtained with the sequence with accession number NR_077155.1, “Penicillium melinii FRR 2041 ITS region; from TYPE material” (as of 22/03/2024). The sequences used were those of the three markers used for phylogeny (SEQ. ID. No. 1 to 3) and that of the ITS (SEQ. ID. No. 4).

[0161] Example 3. The P. melinii strain CECT 21242 increases the capacity to form lateral roots, measured as the number of pre-branching sites (PBS) along the main root in A. thaliana plants transformed with the synthetic promoter DR5 fused to luciferase (DR5:LUC), a marker of lateral root formation and auxin signaling. This increase in lateral roots is observed under both optimal growth conditions (roots in darkness and 300 μM inorganic phosphate) and stress conditions of high (625 μM) or low (5 μM) inorganic phosphorus availability (Figure 3A and B). Unlike other fungi that increase auxin signaling levels along the root (Figure 3C), CECT 21242 only increases the number of PBS, thus promoting root development.

[0163] Surface-sterilized A. thaliana accession Col-0 DR5:LUC seeds were sown in modified Murashige and Skoog (MS) medium 1/2 (without sucrose or vitamins and with a phosphorus concentration of 625 μM, 300 μM, or 5 μM), stratified at 4 °C for 2 days, and then maintained in a controlled growth chamber at 23 °C, under a long cycle (16 hours of light per day) within the D-Root system, which prevents light from reaching the plant's root system. After 7 days, the plants were transplanted to another plate with the same modified MS medium 1/2 where the P. melinii strain CECT 21242 had been cultured for 4 days under the same conditions as the plants. Four days after transplanting, the plants were sprayed with the luciferase (luciferin) substrate, and the areas along the root that emitted luminescence were counted (counting from the point of contact with CECT 21242). These luminescent spots correspond to the PBS (Figure 3A). A high-sensitivity CCD camera and Indigo software were used to capture the luminescence images. Plants treated with P. melinii CECT 21242 showed a significant increase in PBS compared to untreated control plants. Specifically, approximately 1.5 times more PBS (Student's t-test, P<0.001) in plants treated with P. melinii CECT 21242 (Figure 3B). This increase occurs under both optimal inorganic phosphorus (300 μM) and conditions of excess (625 μM) or phosphorus deficiency (5 μM), implying that these plants have a greater capacity to develop lateral roots under both conditions. Furthermore, the increase in PBS in plants treated with P. melinii CECT 21242 shows that these plants become tolerant to stress caused by excess or low availability of inorganic phosphorus, precisely because of this greater capacity to produce more PBS.

[0165] Example 4.

[0167] The CECT 21242 strain of P. melinii increases the growth of the main root and secondary roots of A. thaliana plants grown in vitro under conditions of light stress in the root and deficiency or excess of inorganic phosphorus in the culture medium (Figure 4).

[0169] Surface-sterilized seeds of A. thaliana accession Col-0 were sown in modified Murashige and Skoog (MS) medium 1/2 (without sucrose or vitamins and with phosphorus concentrations of 625 μM and 20 μM), stratified at 4 °C for 2 days, and then maintained in a controlled growth chamber at 23 °C under a long cycle (16 hours of light per day) with light supplied to the plant's root system. After 7 days, the plants were transplanted to another plate with the same modified MS medium 1/2 where the P. melinii strain CECT 21242 had been cultured for 4 days under the same conditions as the plants. The light supplied to the root causes stress because this organ is adapted to underground growth in darkness, and this manifests as reduced plant growth.

[0171] Plants grown under this stress are more sensitive to excess (625 μM) or deficiency (21 μM) of inorganic phosphorus (Pi) content in the medium due to the addition of stressors. Under these conditions, plants grown in the presence of P. melinii CECT 21242 showed greater root growth in both main root length and total length compared to untreated plants, demonstrating that plants treated with P. melinii CECT 21242 become tolerant to the combined stress due to the light input to the root and the low availability of inorganic phosphorus.

[0173] Example 5.

[0175] The extract of the CECT 21242 strain of P. melinii (mycelium) or the filtrate of the CECT 21242 strain culture of P. melinii (filtrate) increases the length (Figure 5A) and number (Figure 5B) of lateral roots of A. thaliana plants grown in phosphate deficiency and with root illumination.

[0176] Extracts of CECT 21242 were obtained after cultivation for two weeks in potato dextrose broth (PDB). Fungal tissue (mycelium and spores) was separated from the culture medium by filtration. The mycelium was then frozen in liquid nitrogen and ground into a fine powder, which was suspended in sterile distilled water. The fungal tissue-free filtrate was lyophilized and suspended in sterile distilled water. This mixture was lyophilized, suspended in sterile water, and filtered through a 0.22 µm filter. For the culture medium extract control (PDB control), the same procedure was performed using PDB medium in which the fungus had not been incubated. Each extract was applied to a plate with Murashige and Skoog (MS) 1/2 phosphate-deficient medium (without sucrose or vitamins and with a low phosphorus concentration of 20 μM). Seeds of A. thaliana accession Col-0, surface-sterilized and previously stratified at 4 °C for 2 days, were sown on the culture plates where each extract had been previously applied (mycelium, filtrate, PDB control, and sterile distilled water for the control) and then kept in a long-cycle controlled growth chamber (16 hours of light per day) at 23 °C for 14 days, after which the roots were measured.

[0178] Example 6.

[0180] The CECT 21242 strain of P. melinii increases the growth of the main root and secondary roots of A. thaliana plants grown in vitro under high temperature conditions and lack of inorganic phosphorus (20μM) in the culture medium (Figure 6).

[0182] Surface-sterilized seeds of A. thaliana accession Col-0 were sown in Murashige and Skoog (MS) 1/2 medium with phosphate deficiency (without sucrose or vitamins and with a low phosphorus concentration of 20 μM), stratified at 4 °C for 2 days, and then maintained in a long-cycle controlled growth chamber (16 hours of light per day) at 23 °C. After 7 days, the plants were transplanted to another plate with the same modified MS 1/2 medium where the CECT 21242 strain of P. melinii had been cultured for 4 days under the same conditions as the plants. From that point on, the plants were maintained at 32 °C (aerial part), but the roots were maintained at a temperature gradient between 24 and 32 °C simulating soil conditions, using the TGRooZ system (González-García et al.).

[0183] 2023. Plant Comm. 4, 100514, https://doi.org/10.1016/j.xplc.2022.100514). Plants grown at 32°C are subjected to thermal stress, which manifests as reduced root system growth and lower aboveground biomass.

[0184] Plants grown in the presence of P. melinii CECT 21242 showed greater root growth than untreated plants, showing that the treated plants become tolerant to the combination of both stresses (inorganic phosphorus deficiency and high temperature).

[0186] Example 7.

[0188] The CECT 21242 strain of P. melinii increases the growth of the root system and the biomass of the aerial part of A. thaliana plants grown in vitro under optimal temperature (22ºC) or high temperature (32ºC) conditions (Figure 7).

[0190] Surface-sterilized A. thaliana accession Col-0 seeds were sown in modified Murashige and Skoog (MS) 1/2 medium (without sucrose or vitamins and with a phosphorus concentration of 625 μM), stratified at 4 °C for 2 days, and then maintained in a long-cycle controlled growth chamber (16 hours of light per day) at 23 °C. After 7 days, the plants were transplanted to another plate with the same modified MS 1/2 medium where the P. melinii strain CECT 21242 had been cultured for 4 days under the same conditions as the plants. From that point on, the plants were maintained at 22 °C or 32 °C. In this latter case, the aerial parts were at 32 °C, but the roots were maintained at a temperature gradient between 24 and 32 °C that simulates soil conditions, using the TGRooZ system (González-García et al. 2023. Plant Comm. 4, 100514, https://doi.org/10.1016/j.xplc.2022.100514). Plants grown at 32 °C are subjected to thermal stress, which manifests as reduced root system growth and lower shoot biomass.

[0192] Under both conditions, plants grown in the presence of CECT 21242 showed greater root growth (Figure 7A, C) and a significant increase in shoot biomass compared to untreated plants (75% increase for 22°C and 45% for 32°C Student's t P<0.05) (Figure 7B, D), showing that CECT 21242 promotes the growth of plants grown under optimal conditions or under thermal stress.

[0194] Example 8

[0196] The CECT 21242 strain of P. melinii increases root thickness, lateral root growth and total surface area occupied by the root system, and promotes the growth of barley plants grown using germination paper as support (Figure 8).

[0197] Barley seeds were germinated on moistened filter paper for 5 days and the resulting seedlings were immersed in a 10<5>spores/ml suspension (or water for the control) for 30 seconds. The treated seedlings were placed on germination paper within the adapted D-Root system and watered with a nutrient solution composed of 1/8 MS for 7 days.

[0199] Plants treated with P. melinii CECT 21242 showed greater root development, with thicker roots (Figure 8A), longer lateral roots (Figure 8A), and a greater total surface area occupied by the root system (32% increase; Figure 8B). This greater root development resulted in a significant increase in plant growth, measured as the weight of the plant shoot (11% increase, Student's t-test P<0.05; Figure 8C).

[0201] Example 9.

[0203] The CECT 21242 strain of P. melinii increases the growth of barley plants (Figure 9A and B) and maize grown in agricultural soil in a greenhouse (Figure 9C and D).

[0204] Barley and maize seeds were germinated on moistened filter paper for 5 days and the resulting seedlings were transplanted into 15 cm diameter pots containing 1 kg of agricultural soil. After 2 days, the soil was inoculated with 2 mL of a solution containing 10<5>spores/ml (or water for the control) and the pots were kept in the greenhouse for 6 weeks.

[0206] Plants treated with P. melinii CECT 21242 showed greater height than untreated plants in measurements taken at 3 weeks (increases of 15 and 16% for barley and maize respectively, and statistically significant, P<0.05, for maize). Treated barley plants also showed a greater leaf area than untreated plants, measured at 5 weeks (19%).

[0208] Example 10.

[0210] The CECT 21242 strain of P. melinii increases the growth rate of barley and maize plants grown in agricultural soil in a greenhouse (Figure 10).

[0212] Barley and maize seeds were germinated on moistened filter paper for 5 days, and the resulting seedlings were transplanted into 15-cm-diameter pots containing 1 kg of agricultural soil. After 2 days, the soil was inoculated with 2 mL of a solution containing 10⁵ spores/ml (or water for the control), and the pots were kept in the greenhouse for 6 weeks. Plants treated with P. melinii CECT 21242 showed a higher rate of height growth than the control plants, as measured by the slope of the growth curve (Figure 10).

Claims (20)

1. CLAIMS 1. A method for protecting plants from different types of abiotic stress and/or increasing the number of lateral roots in plants, characterized by comprising a step of contacting said plants with a composition comprising a microorganism that is the Penicillium melini strain deposited with deposit number CECT 21242, and/or lyophilized mycelium and spores of said strain, and/or filtrates of said strain. 2. Method according to claim 1, wherein the plants are Angiosperms. 3. The method of claim 2 wherein the plants are Arabidopsis thaliana, maize or barley. 4. The method according to any of claims 1 to 3 wherein the plants are grown in vitro, on germination paper or in agricultural soil. 5. The method according to any of claims 1 to 4, wherein the microorganism is in the form of spores, hyphae, mycelium or sclerotia. 6. The method according to any of claims 1 to 5 wherein the composition is applied to the seeds of the plant. 7. The method according to any of claims 1 to 5 wherein the composition is applied to the aerial parts of the plant. 8. The method according to any of claims 1 to 5 wherein the composition is applied to the roots of the plant or to other underground parts of the plant. 9. The method according to any of claims 1 to 5 wherein the composition is applied to the substrate for growing the plant. 10. The method according to any of claims 1 to 9 wherein the composition comprises minerals, organic materials, organic compounds, inorganic compounds, metabolites, fermentation or reaction by-products, liquid diluents, alcohols, ketones, vegetable oils or esters, aliphatic hydrocarbons, esters and other mineral solvents, water, anionic surfactants, non-ionic surfactants, cationic surfactants, amphoteric surfactants, water-soluble polymers, polysaccharides, preservatives, coloring agents, thickening agents, and/or stabilizing agents. 11. The method according to any of claims 1 to 10 wherein the composition is a liquid, a solid, a paste or a gel. 12. The method according to any of claims 1 to 11 wherein the composition is a powder, tablet, compressed, granulated or emulsifiable concentrate. 13. The method according to any of claims 1 to 12 wherein the composition is applied by spraying, spritzing, immersion, irrigation or dusting. 14. Use of a microorganism that is the Penicillium melini strain deposited with deposit number CECT 21242 and/or lyophilized mycelium and spores of said strain, and/or filtrates of said strain, for the promotion of plant growth, both under optimal growth conditions and under different types of abiotic stress. 15. The use of claim 14 wherein the stress conditions are one or more conditions selected from the group: absence of phosphorus, low availability or excess of phosphorus, light stress, and thermal stress. 16. Strain of the microorganism Penicillium melinii, deposited with the deposit number CECT 21242. 17. Substrate for plant cultivation comprising a microorganism that is the Penicillium melini strain deposited with deposit number CECT 21242, and/or lyophilized mycelium and spores of said strain, and/or filtrates of said strain. 18. Use of the substrate of claim 17 to obtain plants tolerant to abiotic stress and/or to increase the growth of plants subjected to abiotic stress. 19. Plant propagation material comprising a microorganism that is the Penicillium melini strain deposited with deposit number CECT 21242 and/or lyophilized mycelium and spores of said strain, and/or filtrates of said strain. 20. Propagation material according to claim 19, selected from the group comprising seeds, seedlings, young plants, cuttings, bulbs and tubers.