Method for protecting against stress and increasing growth in plants

Abstract

The invention relates to a method for protecting different types of plants against abiotic stress, increasing stress tolerance in plants, promoting growth and / or increasing nutritional efficiency, based on the use of a microorganism of the species Penicillium melinii and strains thereof. Also claimed is the use of a microorganism of the species Penicillium melinii and / or strains of same to protect plants from and / or increase the tolerance thereof to different types of abiotic stress, such as an absence of phosphorus, low availability or excess phosphorus, light stress, thermal stress, water stress, toxicity stress, salt stress and nutritional deficiency.

Description

[0001] METHOD TO PROTECT AGAINST STRESS AND INCREASE PLANT GROWTH

[0002] FIELD OF INVENTION

[0003] The present invention belongs to the field of the agronomic sector, in particular to the field of methods for promoting plant growth and development with emphasis on root development, both under optimal growth conditions and under conditions of different types of stress.

[0004] BACKGROUND OF THE INVENTION

[0005] In the agricultural sector, there is enormous interest in increasing plant growth while minimizing the use of chemical fertilizers and achieving greater sustainability. This interest is particularly important when it comes to improving plant tolerance or alleviating the stress produced when plants grow under suboptimal conditions. Stress prevents crop plants from reaching their full genetic potential and causes significant yield losses worldwide. It is well known that good root development is crucial for efficient water and nutrient uptake by plants, especially in the early stages of development, and especially under stressful conditions such as drought, salinity, extreme temperatures, or low nutrient availability.Improving plant root development may therefore be a key factor in improving plant tolerance to stresses caused by unfavorable nutritional and environmental conditions (Koevoets, IT et al., 2016, Front. Plant Sci. 7, 1335. doi: 10.3389 / fpls.2016.01335). For example, the angle at which maize roots develop 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 main root of corn (Jia, X. et al., 2018, J Exp Bot. 69(20). 4961 -4970. Doi: 10.1093 / jxb / ery252).

[0006] Plants in nature establish symbiotic associations with microorganisms called mutualists that confer benefits on their growth, survival and multiplication. These microorganisms can be isolated and sometimes used to improve crop yield. For example, the colonization of rapeseed roots by Serendipita vermifera, Alternaria alternata or Leptosphaeria biglobosa, significantly increases root and shoot biomass (Dolatabadi, HK 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 growth enhancement may be due to an improvement in the plant's efficiency at 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.Metarhizium brunneum increases root development and shoot biomass, as well as phosphate content, in potato plants (Kreil V et al., 2018, Fungal Ecol 34:43-49. doi.org / 10.1016 / j.funeco.2018.04.002). Another advantage conferred by beneficial microorganisms may be increased plant tolerance to stresses such as salinity or toxicity of the environment or extreme nutrient shortage. For example, T. virens increases the number of lateral roots in Arabidopsis thaliana, making the plant more tolerant to salinity (Contreras-Cornejo, H. A et al., 2009, Plant Physiol. 149, 1579-1592. doi: 10.1 104 / PP.108.130369). As these examples illustrate, different microorganisms may have distinct mechanisms of action that serve to improve different aspects of plant growth in particular ways.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, it is necessary to alleviate the stress produced under these conditions and restore plant productivity. Microorganisms' modes of action to improve plant stress tolerance may include increasing root system development.

[0007] Mohamed Tarroum et al. (2021 ), Plants, 10, 784, https: / / doi.org / 10.3390 / plants10040784, presents a set of six fungal strains of different species isolated from the rhizosphere of a halophytic grass, Aeluropus littoralis, and analyzes their promoting activities on the growth of tobacco plants. All six strains improved the growth of cultivated tobacco plants when added to the hydroponic culture medium. Furthermore, when cell-free culture filtrates (CFFs) were added to 0.5 NS (nutrient solution) in a closed hydroponic system, the greatest effects on tobacco seedlings (shoot and root dry weight, leaf number, and root length) were observed when CFFs of A5 strains were added.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 completely decisive for taxonomic identification, with the sequences of these species obtained from the NCBI Genebank database. It was found that CFFs from all strains, when added to 0.5 NS, can replace part of the chemical inputs, with biomass production equal to or significantly better than that produced by complete NS. The authors attribute the ability to promote plant growth to the production of substances related to indoleacetic acid (auxins), a growth hormone, by the fungi. Furthermore, the authors show that the expression of the Trypl and YUCCA6-like genes, involved in the biosynthesis of the phytohormone auxin, is also induced in treated tobacco plants.Based on these results, this work proposes the use of P. melinii strain A8 or its filtrates to replace part of the chemical fertilization required for optimal plant growth, but not as a stress reliever. The only root parameters measured were growth-related (weight or length), but 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 (40. e C) or high salinity conditions (greater than or equal to 100 mM NaCl in the culture medium), inhibit the growth of strain A8 of the species P. melinii.

[0008] Borrego López R. et al., Botrysclero 2020 (https: / / elaborate.uca.es / wp-content / uploads / 2022 / 06 / BotrySclero2022.pdf) shows a strain of P. melinii with antifungal effect against Botrítys cinerea in the co-culture of both fungi in vitro, although it does not show any data on experimentation in plants or in the field.

[0009] 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 grown under favorable conditions for plant growth. Among the microorganisms tested, P. melinii is cited (Table 2), although this microorganism is not one of those that presents the best results, so it is not selected for the next stage. Furthermore, the conditions used for the selection of the microorganisms did not include the application of stress to the plants, since they were grown under favorable growth conditions.

[0010] BRIEF DESCRIPTION OF THE FIGURES.

[0011] Figure 1. Increased root growth in plants treated with CECT 21242 compared to untreated plants (control), under nutritional stress due to phosphate (Pi) deficiency. (T-test compared to control, *: p < 0.05). Figure 2. A. Phylogenetic tree using the maximum likelihood method of the concatenated RPB2, CAM and BenA regions of 94 strains of the genus Penicillium and other 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.

[0012] Figure 3. Relative increase in pre-branching sites (PBS) in roots of untreated plants (blank or control) and plants treated with CECT 21242 grown with different phosphate (Pi) concentrations in the medium (A, B). Increased auxin signaling in the roots of DR5::LUC plants inoculated with different fungi (C). (T-test compared to control, *: p < 0.05).

[0013] Figure 4. Increased root growth (Figure 4A and C) and shoot biomass (Figure 4B and D) in A. thaliana plants treated with CECT 21242 and grown at 22 °C. e C or 32 e C. (T-test compared to the control, *: p < 0.05).

[0014] Figure 5. Treatment with CECT 21242 increases root development in plants grown at 32°C. e C (high temperature stress) and under phosphate deficiency conditions (20pM P¡) in the culture medium.

[0015] Figure 6. Treatment with CECT 21242 increases root growth in plants subjected to the combination of light stress to the root and stress due to excess (625pM) or deficiency (20pM) of phosphate (Pi) in the culture medium.

[0016] Figure 7. Plants treated with CECT 21242 under salinity conditions exhibited lower levels of free amino acids. (T-test compared to the mock control, *: p < 0.05).

[0017] Figure 8. The expression of genes in the abiotic stress category decreases in plants treated with CECT 21242. (T-test with respect to the control ****: p < 0.0001).

[0018] Figure 9. Treatment with CECT 21242 increases lateral root thickness and length (9A), root area (9B), and shoot biomass (9C) of barley plants grown on germination paper under general nutrient deficiency conditions. Figure 10. Treatment with CECT 21242 increases shoot development in barley (10A and 10B) and maize (10C and 10D) plants grown in agricultural soil in pots in a greenhouse for three weeks. (T-test vs. control, *: p < 0.05).

[0019] Figure 1 1. Growth curves of corn plants grown in agricultural soil treated with CECT 21242 or with water (control) and maintained in a greenhouse for 6 weeks.

[0020] Figure 12. Treatment with CECT 21242 increases root development (12A) and total length of the root system (12B) of tomato plants growing in peat with optimal fertilization conditions (T-test compared to control, *: p < 0.05).

[0021] Figure 13. Treatment with CECT 21242 increases root weight of tomato plants var. Moneymaker under phosphate deficiency (P¡) conditions (T-test vs. control, *: p < 0.05).

[0022] Figure 14. Treatment with CECT 21242 increases plant fresh (14A) and dry (14B) weight, number of marketable first and second category tomato fruits (14C), marketable fruit weight (14D), and total marketable yield (tonnes / hectare) obtained from the total crop (14E) in field trials under optimal fertilization (Optimum P¡) and phosphate deficiency stress (P¡ Deficiency) conditions measured on untreated (control) and treated (CECT 21242) plants. Different letters indicate significant differences in an ANOVA (p<0.15, Post-hoc Tukey).

[0023] Figure 15. Increase in foliar phosphorus content in tomato plants treated with CECT 21242 compared to untreated plants (control) in field trials under optimal phosphate (P¡) fertilization conditions.

[0024] Figure 16. Treatment with CECT 21242 increases the partial factor of phosphorus productivity under phosphate-deficient conditions (P¡).

[0025] Figure 17. Relative fresh weight increase compared to the control (Mock) of the root (A) and shoot (B) of tomato plants grown under phosphate (Pi) deficiency stress 15 days post-treatment with different P. melinii strains. (T-test compared to the mock, ns: p > 0.05, *: p < 0.05; p < 0.01; ***: p < 0.001). Figure 18. Relative increase compared to the control (mock) of pre-branching sites (PBS) in roots of untreated A. thaliana plants (control or mock) and plants treated with different P. melinii strains. (T-test compared to the mock control, *: p < 0.05; **: p < 0.01).

[0026] Figure 19. Freeze-dried mycelium and spore extract of CECT 21242 (mycelium) or culture filtrate of CECT 21242 (filtrate) increase the length of the main root (19A) and number of lateral roots (19B) of A. thaliana plants grown under phosphate deficiency (P¡) and with light stress due to root illumination. (T-test with respect to the control, ns: p > 0.05, *: p < 0.05; **: p < 0.01; ***: p < 0.001, p < 0.0001).

[0027] Figure 20. Treatment with different types of CECT 21242 extracts increases plant growth in fresh weight (20A, 20B and 20D) and root length (20B and 20C) in plants under nutritional stress due to phosphate deficiency (P¡). Different letters indicate significant differences in an ANOVA (p<0.05, Post-hoc Tukey).

[0028] Figure 21. Treatment with aqueous extract of CECT 21242 increases the number of barley stems under optimal growth conditions.

[0029] DESCRIPTION OF THE INVENTION

[0030] The present invention provides a method for increasing plant growth by enhancing root development, both under stressful and optimal growing conditions. The method allows plants to be protected from various types of abiotic stress and / or to increase plant growth. The method also allows plants to increase their tolerance to various stressful conditions. The method also increases the plant's nutritional efficiency. Increased plant growth is determined by one or more of the following parameters: number of pre-branching sites (PBS) in the root, area occupied by the root, root weight, shoot length, shoot weight, number of stems, leaf area, number of fruits, and / or fruit weight, and / or the total yield of all the plants comprising a crop area.Protection or tolerance to stress in plants is determined by one or more of the following parameters: increased growth or development of the plant or one of its parts under stress conditions, decreased expression of plant genes related to abiotic stress, and / or decreased content of free amino acids in the plant. Greater nutritional efficiency of the plant is determined by increased content of these nutrients in the plant tissues or by increased partial productivity factor of the nutrient applied to the set of plants that make up a crop area. The method consists of contacting the 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.

[0031] The present invention has identified, by screening 10 strains of different fungal species, the Penicillum melinii strain CECT 21242, which, surprisingly, increases the plant's capacity for lateral root development and acts as a plant stress reliever. This increase in root development occurs under stress conditions caused by a lack or excess of phosphorus, by light stress on the root, or by heat stress, by salt stress, or by a combination of these stresses. The ability of CECT 21242 to alleviate abiotic stress in plants is also evidenced by the expression of genes related to abiotic stress in A. thaliana plants inoculated with CECT21242, where the expression of these genes is reduced compared to control plants, whether under specific stress conditions (e.g., phosphorus deficiency) or non-specific stress conditions (without applying a specific stress to the plant).Furthermore, the application of extracts or filtrates of the fungus also increased the length and number of lateral roots of the plant under stress conditions.

[0032] The same effect obtained with Penicillum melinii strain CECT 21242 has been achieved with other Penicillum melinii strains, including:

[0033] Strain CBS 139142, isolated from South Africa, Stellenbosch in 2014 from soil;

[0034] CBS 218.30 strain isolated from the USA in 1930 from a forest soil; and

[0035] Strain CBS 285.65 isolated from England, Lancashire (Grange-over-Sands) in 1965 from Querqus sp. litter in an acid pH (4.5) field.

[0036] The data of the present invention indicate that plants treated with Penicillium melinii and / or its strains CECT 21242, CBS 139142, CBS 218.30, CBS 285.65, and / or their extracts or filtrates, acquire tolerance to different types of stress. Furthermore, plants treated in this manner increase the development and / or growth of the root and / or aerial parts (leaves, stems and / or fruits) both under stress conditions and under optimal conditions in in vitro culture or by cultivating the plants in peat or agricultural soil. Furthermore, plants treated in this manner increase the phosphorus content in their tissues. Plants treated with CECT 21242 decrease the expression of genes normally induced under abiotic stress conditions, which demonstrates the general state of lower abiotic stress in these plants. Furthermore, plants treated with CECT 21242 show a lower content of free amino acids.A lower free amino acid content is correlated with decreased protein degradation due to abiotic stress, as described in López-Hidalgo et al. 2021. Plant Cell Environ. 44: 1977–1986. https: / / doi.org / 10.1 11 1 / pce.14007.

[0037] The taxonomic classification of strains CBS 139142, CBS 218.30, CBS 285.65 within the species Penicillum melinii has been confirmed by the Westerdijk Fungal Biodiversity Institute (Utrecht, The Netherlands “https: / / wi.knaw.nl / About”), an institution recognized worldwide as an authority in the study and taxonomic identification of fungal strains, and in whose collection of fungal cultures (CBS, “https: / / wi.knaw.nl / Collection”), they are deposited.

[0038] The identification of the species corresponding to the isolate of strain CECT 21242 has been carried out following the description published by Houbraken et al. 2020 Studies in Mycology 95: 5-169, using partial sequences of the genes that code for the proteins beta-tubulin (BenA Partial beta-tubulin), calmodulin (CaM) and RNA polymerase II (RPB2). The genetic marker similarity (ITS) method has also been used with the nucleotide sequences obtained from the NCBI Genebank database, obtaining the highest similarity (100% nucleotide identity) with the sequence with accession number NR 077155.1, “Penicillium melinii FRR 2041 ITS region; from TYPE material” (as of 03 / 22 / 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).

[0039] 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 Mohamed Tarroum et al. (2021) described, which indicated an increase in the biosynthesis of this hormone, both by the fungus and the treated plant.

[0040] Furthermore, there was evidence that would have discouraged the use of this treatment, either due to the absence of particularly notable effects of Penicillum melinii on the growth of ryegrass plants (WQ2014046553A1 ), as well as the negative role played by different types of stress on the growth of said microorganism (Mohamed Tarroum et al. (2021 ).

[0041] A first aspect of the present invention is a method for protecting or relieving plants from different types of abiotic stress and / or increasing the tolerance of plants to different types of abiotic stress, 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 strains, and / or filtrates of said species or strains.

[0042] A second aspect of the invention is the use of a microorganism of the species Penicillium melinii, and / or strains of said species, and / or extracts of said species or strains, and / or filtrates of said species or strains, to protect plants from different types of abiotic stress and / or obtain plants tolerant to stress, and / or increase the tolerance of plants subjected to different types of abiotic stress.

[0043] Stress is understood as the set of processes that a plant undergoes when growing in unsuitable environmental conditions, manifesting in a reduction in growth compared to what it would have achieved under optimal conditions. Furthermore, when a plant is subjected to stress, it triggers a series of processes that involve the expression of certain genes. These processes can be demonstrated through the analysis of genes and physiological markers whose relationship with stress has been described in the scientific community. An example of stress is an extreme lack of nutrients, such as phosphorus. Optimal conditions are those that allow for proper plant growth, as any expert in the field can appreciate.

[0044] Abiotic stress refers to any type of stress caused by the environmental conditions in which a plant grows. More specifically, abiotic stress refers to any type of stress other than biotic stress. Biotic stress refers to the consequences for the plant of suffering damage or disease directly caused by other living organisms, such as phytophagous pests and pathogens. Some of the conditions that cause abiotic stress include a deficiency or excess of phosphorus or other nutrients, water stress, toxicity stress, salinity stress, light stress, heat stress, or a combination of several of these stresses. Among the conditions where the treatment has proven particularly effective are: phosphorus deficiency or excess, root light stress, high temperatures, salinity, or a combination of one or more of these stress conditions.In a preferred embodiment of the present invention, the stress conditions are one or more conditions selected from the group: absence of phosphorus, low availability or excess of phosphorus, light stress, heat stress, water stress, toxicity stress, salinity stress and nutritional deficiency.

[0045] In a preferred embodiment of the present invention, the protection of plants from different types of abiotic stress and / or the tolerance of plants to different types of abiotic stress is determined by one or more of the following parameters: increase in the growth or development of the plant or of some of its parts under stress conditions, decrease in the expression of plant genes related to abiotic stress and / or decrease in the content of free amino acids in the plant.

[0046] An "extract" refers to any preparation of the microorganism, which may be present alone or together with the culture medium. It may be a naturally occurring form, such as spores or mycelium, or it may also be the result of a procedure applied to a natural medium containing the microorganism.There are various ways of obtaining extracts known to those skilled in the art, such as, among others and without being limited to: grinding the microorganism with or without culture medium, freeze-drying the microorganism with or without culture medium, subjecting the microorganism with or without culture medium to low or high temperatures or high pressures or vacuum, or mixing the microorganism with or without the culture medium with different solvents of an aqueous or organic nature and separating the different fractions produced by different methods, such as by the size of the particles or molecules (filtration, centrifugation), or by chemical or biochemical affinity.

[0047] In a preferred embodiment of the use of the present invention, the microorganism is the Penicillium melinii strain, deposited under deposit number CECT 21242.

[0048] A third aspect of the present invention is constituted by plant propagation material comprising a microorganism of the species Penicillium melinii, and / or strains of said species, and / or extracts of said species or strains, and / or filtrates of said species or strains.

[0049] As used herein, "propagation material" refers to 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.

[0050] The propagation material can be of various origins. For example, it can be freshly collected or derived from a stock, such as a seed sample or a frozen cell stock. Preferably, the propagation material of the present invention can be selected from the group comprising seeds, seedlings, young plants, cuttings, bulbs, and tubers.

[0051] A fourth and fifth aspect of the invention are constituted respectively by a substrate for the cultivation of plants comprising a microorganism of the species Penicillium melinii, and / or strains of said species, and / or extracts of said species or strains, and / or filtrates of said species or strains, and by the use of said substrate to obtain plants tolerant to stress and / or increase the tolerance of plants subjected to stress.

[0052] In a preferred embodiment of the present invention, the propagation material and the substrate of the above aspects comprise the microorganism is the CECT 21242 strain of the Penicillium melinii species, and / or extracts of said strain, and / or filtrates of said strain.

[0053] A sixth aspect of the invention is constituted by the strain CECT 21242 of the species Penicillium melinii, deposited in the Spanish Collection of Type Cultures, CECT (Paterna, Valencia, Spain), following the rules of the Budapest Treaty, with the deposit number CECT 21242).

[0054] A seventh aspect of the present invention consists of a method for promoting plant growth and / or increasing the nutritional efficiency of plants under both optimal growth conditions and stress conditions, characterized in that it comprises a step of contacting said plants with a composition comprising the CECT 21242 strain of the Penicillium melinii species, and / or extracts of said strain, and / or filtrates of said strain. An eighth aspect consists of the use of the CECT 21242 strain of the Penicillium melinii species, for promoting plant growth and / or increasing their nutritional efficiency, both under optimal growth conditions and under stress conditions. The use can be of the microorganism itself, or of extracts of said microorganism, and / or filtrates thereof.

[0055] Plant growth promotion is understood to mean an increase in the growth rate and / or absolute growth of the aerial part of the plant and / or the root system (biomass, area, number or length) and / or an increase in fruit yield (number, or weight or marketable yield of fruit).

[0056] Preferably, plant growth may be determined by one or more of the following parameters: number of branching initiation sites (or its term in English “pre-branching sites” or PBS) in the root, area occupied by the root, root weight, length of the aerial part, weight of the aerial part, leaf area, number of stems, number of fruits, weight of the fruits, and / or total yield of all the plants that make up a crop extension.

[0057] Increased lateral root formation of the plant refers to a longer root system length, comprising the main root and lateral roots, and / or a larger area occupied by the root system and / or a greater number of lateral root-forming zones (LRPs) along the main root.

[0058] Nutritional efficiency is understood as the capacity of a plant to absorb, assimilate, and / or utilize the nutrients available in the medium in which it is growing. Nutritional efficiency can be measured in different ways. For example, but not limited to, one way to measure nutritional efficiency is by determining the content of a given nutrient in plant tissues, and another way to measure nutritional efficiency is by calculating the partial productivity factor (PFP) of a given nutrient. PFP can be determined based on the recommendations described in technical specifications such as CEN / TS 17700-1-2022 and CEN / TS 17700-2-2022 for the determination of nutritional efficiency for biostimulants.More specifically, the PFP can be calculated using the formula: PFP=Y / F, where Y is the yield of the set of plants present in a crop area and F is the amount of nutrient available to the crop through fertilization and / or other environmental sources, such as the initial content in the soil. In a preferred embodiment of the present invention, the nutritional efficiency of the plants is determined by the nutrient content in the plant tissues or by the increase in the partial factor of the productivity of a nutrient.

[0059] The ninth and tenth aspects of the present invention relate respectively to a substrate for growing plants comprising the CECT 21242 strain of the Penicillium melinii species, and / or extracts of said strain, and / or filtrates of said strain, as well as to the use of said substrate to increase plant growth, increase the nutritional efficiency of plants, obtain stress-tolerant plants and / or increase the tolerance of plants subjected to stress.

[0060] As used herein, "plant growing substrate" refers to any solid or liquid material, natural or synthetic, on which a plant can grow. Examples of substrates include, but are not limited to, soil, earth, sand, vermiculite, perlite, humus, peat, or mixtures thereof. Examples of synthetic substrates include, but are not limited to, Murashige and Skoog (MS) nutrient media, Hoagland, minimal medium, or any other medium comprising plant nutrients, either in liquid form for use in hydroponic culture or solidified with gelling agents, such as agar, for use in in vitro culture. Other examples include materials for use in hydroponic culture, such as germination paper, rock wool, or coconut fiber.

[0061] The present invention can be applied to any type of plant, preferably gymnosperms, angiosperms, monocots, and dicots. In a preferred embodiment of the invention, the plants are angiosperms. Some preferred, but non-limiting, examples of plants are tomato (e.g., Solanum lycopersicum), Arabidopsis (e.g., Arabidopsis thaliana), barley (e.g., Hordeum vulgare), corn (e.g., Zea mays), and wheat (specifically, species of the genus Triticum or Triticum spp.).

[0062] Some especially preferred varieties are Solanum lycopersicum variety (var.) Moneymaker, Solanum lycopersicum var. Optima, Solanum lycopersicum var. N-283, Solanum lycopersicum var. Malpika F1, Solanum lycopersicum var. Globe Trotter, Hordeum vulgare var. Rubiana and Zea mays var. LG3490.

[0063] Plant growth promotion and / or increased nutritional efficiency can occur under various 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, germination paper, peat, or agricultural soil.

[0064] In the present invention, the plant may be a natural or transgenic or genetically edited plant.

[0065] Preferred aspects of the present invention consist of a substrate for growing plants, 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.

[0066] The present invention shows how the Penicillium melinii strain CECT 21242 increases root length and the capacity for lateral root development in plants treated with it. This increase in root development occurs both under stress conditions, whether due to phosphorus deficiency or excess, light stress, heat stress, salt stress, or a combination of these stresses, as well as under optimal growth conditions. The data indicate that plants treated with the Penicillium melinii strain CECT 21242 acquire stress tolerance. Furthermore, plants treated with the Penicillium melinii strain CECT 21242 increase their growth rate and absolute growth of the aerial part (biomass or length or area, or number of stems or number or weight of fruits) under all these conditions in in vitro culture, on germination paper, or by cultivating the plants in peat or agricultural soil.Furthermore, plants treated with Penicillium melinii strain CECT 21242 increase their nutritional efficiency under all these conditions.

[0067] Although most of the examples of the present invention correspond to the Penicillium melinii strain CECT 21242, experiments carried out with other strains such as CBS 139142, CBS 218.30, CBS 285.65, indicate that the present system is universal and can be extended to any other type of Penicillium melinii strain.

[0068] Stress conditions can be any type of abiotic stress. Abiotic stress refers to any type of stress produced by the environmental conditions in which the plant grows. More specifically, abiotic stress refers to any type of stress other than biotic stress. Biotic stress refers to the consequences for the plant of suffering damage or disease directly caused by other living organisms, such as phytophagous pests and pathogens. Abiotic stress can produce different types of osmotic, physiological, or metabolic disruption, resulting in reduced plant growth compared to optimal conditions. Furthermore, when a plant is subjected to abiotic stress, it triggers a series of processes that involve the expression of certain genes. These processes can be demonstrated through the analysis of genes and physiological markers whose relationship with abiotic stress has been described in the scientific community.Therefore, greater tolerance to stress can be evidenced by improved growth or development of the plant or some of its parts under these stress conditions, or by lower expression of stress-related genes, or by a lower presence of stress-related physiological markers. One type of stress-related marker is the amount of free amino acids, produced by protein degradation due to stress, as described in López-Hidalgo et al. 2021. Plant Cell Environ. 44: 1977–1986. https: / / doi.Org / 10.11 11 / pee.14007.

[0069] Conditions that cause abiotic stress include, but are not limited to, phosphorus or other nutrient deficiency or excess, water stress, toxicity stress, salinity stress, light stress, heat stress, or a combination of these stresses. Conditions where the treatment has proven particularly effective include phosphorus deficiency or excess, general nutrient deficiency, root light stress, high temperatures, salinity, or a combination of one or more of these stress conditions.

[0070] Phosphorus deficiency refers to phosphorus concentrations below 25 pM in in vitro culture, which result in significantly lower plant growth than under optimal conditions. Optimal conditions are those that allow for adequate plant growth, as can be appreciated by any expert in the field. Specifically, for A. thaliana grown in vitro, optimal phosphorus conditions are 300 pM.

[0071] Excess phosphorus refers to phosphorus concentrations greater than optimum, which produce less plant growth than under optimal conditions.

[0072] Phosphorus is generally supplied to the culture medium or to the plant in the form of phosphate, preferably inorganic phosphate, more preferably inorganic potassium phosphate (KH2PO4) or P¡, or organic phosphate. Therefore, a deficiency or excess of phosphorus can also be expressed as a deficiency or excess of phosphate, inorganic phosphate, inorganic potassium phosphate (KH2PO4) or P¡, or organic phosphate.

[0073] General nutrient deficiency refers to nutrient concentrations in the growing medium that are lower than those that would produce optimal plant growth. For example, the amounts included in a minimal growth medium, such as that described by Mahdi et al. (2022) https: / / academic.oup.eom / ismei / article / 16 / 3 / 876 / 7474249.

[0074] Root light stress refers to roots that are exposed to light radiation conditions that, due to the subterranean nature of the root, are not perceived as optimal by the root, resulting in reduced plant growth.

[0075] Water stress refers to the lack or excess of water during the plant's vegetative cycle, which produces different types of osmotic, physiological or metabolic disruption, resulting in reduced plant growth compared to optimal conditions and which can even lead to cells, tissues or the plant itself suffering serious damage or death.

[0076] Toxicity stress refers to the presence of substances that are harmful to the plant, producing various types of osmotic, physiological, or metabolic disruption. This leads to reduced plant growth compared to optimal conditions and can even lead to serious damage or death to cells, tissues, or the plant itself. Toxic substances can be inorganic or organic elements or molecules present in the medium where the plant grows or produced by other organisms (such as other plants, pests, or pathogens) in contact with or near the plant. A particular case of this stress is salinity, caused by high amounts of salts that cause death by toxicity or osmotic stress. A particular case of salt stress is that caused by concentrations of 100 pM or higher of NaCl.

[0077] Thermal stress refers to a temperature that is lower or higher than the optimum for plant growth, inhibiting its development or slowing down the growth of the aerial parts and / or the root system. Optimal conditions are those that allow for adequate plant growth, as can be appreciated by any expert in the field. Specifically, for A. thaliana grown in vitro, the optimal temperature conditions are 22°C. e C.

[0078] In the method of the invention, a plant is contacted with a composition comprising the microorganism Penicillium melinii. The composition can be applied to the entire plant or to any of its parts, such as leaves, shoots, flowers, fruits, ears, seeds, bulbs, tubers, roots, and seedlings. The composition can be applied to the plant at any stage, and for example, it can be applied 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 cuttings or rooting of cuttings, or at the time of growth in a plantation, or even in the reproductive period before flowering or during flowering or during the fruit ripening process.

[0079] In a preferred embodiment of the method of the invention, the composition is applied to the seeds of said plant.

[0080] In another preferred embodiment of the method of the invention, the composition is applied to the aerial parts of said plant.

[0081] In another preferred embodiment of the method of the invention, the composition is applied to the roots of said plant or to other underground parts of said plant.

[0082] In another preferred embodiment of the method of the invention, the composition is applied to the substrate where said plant grows or is going to grow.

[0083] The method of the invention includes treatment by spraying or pulverization onto whole plants or any parts thereof with a suitable dilution of the composition according to the invention, or by immersing whole plants or any parts thereof in said dilution. The method of the invention also includes treatment by dry dusting whole plants or any parts 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 can also be mixed with the irrigation liquid. The method of the invention also includes treatment by means of agar fragments with mycelium that are brought into contact with a part of the plant, be it root, stem or leaves, or even on the surface of the soil near the roots of the crop.

[0084] In another embodiment of the method of the invention, the composition comprises fertigation salts, fertilizers, insecticides, nematocides, 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.

[0085] In another embodiment of the method of the invention, the composition is a liquid, a solid, a paste or a gel.

[0086] In another embodiment of the method of the invention, the composition is a powder, tablet, tablet, granule or emulsifiable concentrate.

[0087] In another embodiment of the method of the invention, the composition is applied by injection, spray, spraying, immersion, irrigation or dusting.

[0088] The present invention also relates to plants which, according to the method of the invention, have been brought into contact with a composition comprising the microorganism Penicillium melinii and strains 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.

[0089] Hereinafter, the term “microorganism of the invention” refers to the microorganism of the species Penicillium melinii and / or strains thereof, preferably the Penicillium melinii strain deposited under deposit number CECT 21242.

[0090] The microorganism of the invention can be cultivated on a wide variety of natural or synthetic substrates. For example, it can be cultivated in different types of solid or liquid culture media, such as Potato / Dextrose / Agar (PDA) or Broth (PDB) medium, and can be propagated using techniques known to those skilled in the art. It can also grow on various natural sources, such as plant leaves, pollen grains, oat flour, potato, carrot, and cellulose. It can also grow on waste from industrial processes, such as brewer's waste, beet waste, or algae. It can also grow on artificial sources such as paper or cardboard and polymers.

[0091] The culture medium may be at rest or constantly agitated during the culture, for example at approximately 1 rpm. Furthermore, the culture temperature may be in the range of 15 to 35 °C. In a preferred embodiment of the method of the invention, the microorganism is in the form of spores, strands, mycelium, or sclerotia.

[0092] Another embodiment is the method of the invention, wherein said composition is applied to the substrate for the cultivation of said plant.

[0093] The substrate is preferably treated so that the microorganism of the invention is cultivated therein before the substrate is used for plant cultivation. Examples of treatment of the substrate include infusion of a liquid into the substrate (by irrigation, injection or dripping), spraying, dusting or directly mixing with the substrate. The method of the invention also comprises the treatment of a hydroponic medium, for hydroponic cultivation. The method of the invention comprises the treatment of 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 such as granules or powder.

[0094] The substrate for growing plants may comprise the spore, mycelium, or any other part of the microorganism of the invention, or the culture medium or filtrate thereof, or any possible combination of these components. The substrate may be liquid or solid.

[0095] Examples of substrates for plant cultivation include solidified natural or synthetic media and those for plant cultivation, especially in vitro. Other examples include soil, sand, humus, peat, or mixtures of these.

[0096] Another embodiment is the method of the invention, where said microorganism is applied in the form of an extract.

[0097] The extract can be obtained from the microorganism alone or together with the culture medium. There are various ways of obtaining extracts known to those skilled in the art, such as, among others and not limited to: grinding the microorganism with or without culture medium, freeze-drying the microorganism with or without culture medium, subjecting the microorganism with or without culture medium to low or high temperatures, or high pressures or vacuum, or mixing the microorganism with or without the culture medium with different solvents of an 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. Another embodiment is the method of the invention, where filtrates of the microorganism are applied.As used herein, the term "filtrate" refers to a liquid culture medium obtained from the cultivation, growth, or fermentation of the microorganism of the invention. It is possible to obtain a liquid culture medium, free or essentially free of solid material from the microorganism of the invention. This culture medium can be prepared by first culturing the microorganism of the invention in a solid (later mixed with 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 those skilled in the art, for example, by centrifugation or filtration. It is possible, for example, to heat the medium with the microorganism of the invention twice to about 80°C for 30 minutes and then remove the solid fungal material by centrifugation.

[0098] 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 for the extraction of essentially all traces of the microorganism of the invention; more preferably, the filtration would also remove spores, and even more preferably, this should remove all solid fungal material.

[0099] The following are a few examples intended 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.

[0100] PREFERRED EXECUTION MODES.

[0101] Example 1 .

[0102] Screening for fungi that increase plant root development.

[0103] During the years 2008 to 2011, Arabidopsis thaliana (A. thaliana) plants growing under natural conditions were collected from different areas of central Spain (García et al. 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 shaking for 5 minutes. The fragments were then rinsed twice in sterile water and placed in a humid chamber at room temperature (20-24 eC). The fragments were periodically inspected, isolating the emerging mycelium on PET plates with potato dextrose agar (PDA) medium containing 200 mg / L of chloramphenicol. Among the isolates obtained, a screening was performed to find fungi that were beneficial by increasing the length of the root system in plants subjected to phosphorus deficiency stress.

[0104] Surface-sterilized seeds of A. thaliana accession Col-0 were sown on Murashige and Skoog (MS) medium. 1 Modified Z> (without sucrose or vitamins and with an optimal phosphate (Pi) concentration of 300 pM or deficiency of 5 pM), were stratified to 4 e C for 2 days and then kept in a controlled growth chamber at 23 e C, in a long cycle (16 hours of daily light) within the D-Root system that prevents light from reaching the plant's root system. After 7 days, the plants were transplanted to another plate with the same MS medium.1 Z> modified, where each of the tested fungi had been grown 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.

[0105] Plants grown in the presence of strain CECT 21242 showed a significant increase (P<0.05) in root growth in plants grown on MS medium with 5pM phosphate (Pi), both in primary and lateral root length and in total length compared to untreated control plants (Table 1 , Figure 1 ), showing that plants treated with CECT 21242 become tolerant to phosphate stress. Other fungi produced significant reductions in the length of the primary root or root system or did not produce significant changes. Table 1 .

[0106] 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).

[0107] _ Length (cm) _

[0108] Main root Root system (T)

[0109] (1 ) Root system length is calculated as the sum of the length of the main root and all lateral roots. Plants were grown at an optimal phosphate (Pi) concentration of 300 pM (2) or a deficiency of 5 pM (3).

[0110] Example 2.

[0111] Taxonomic identification of strain CECT 21242.

[0112] The morphology of strain CECT 21242 indicated that the strain belongs to the genus Penicillium. To determine the species, the methodology of Houbraken et al., 2020 (Studies in Mycology 95: 5-169) was followed, obtaining the 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 of different species of the genus Penicillium and other closely related genera obtained from the NCBI database. The alignment was performed using the MAFFT program, and the phylogenetic comparison was performed using the maximum similarity method (Maximum Likelihood) using IQtree (Figure 2A). Strain CECT21242 significantly clusters with all P. melinii strains used in this study, 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, The Netherlands) and are leading experts in fungal taxonomy. For the identification of strain CECT21242, the genetic marker similarity (ITS) method was also used with the nucleotide sequences obtained from the NCBI Genebank database, obtaining the highest 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 were those of the three markers used for phylogeny (SEQ. ID. No. 1 to 3) and that of the ITS (SEQ. ID No. 4).

[0113] Example 3.

[0114] P. melinii strain CECT 21242 increases the ability to form lateral roots, measured as the number of pre-branching sites (PBS) along the primary root, in A. thaliana plants transformed with the synthetic DR5 promoter fused to luciferase (DR5::LUC), a marker of lateral root formation and / or auxin signaling. This increase in lateral roots was obtained both under optimal growth conditions (roots in darkness and 300 pM phosphate (Pi)) and under stress conditions of high (625 pM Pi) or low phosphate availability (5 pM Pi) (Figure 3A and B).Contrary to what is observed with other fungi that increase the levels of auxin signaling in a nonspecific manner along the root as observed in Figure 3C, CECT 21242 only specifically increases the number of PBS, favoring root development (Figure 3A and B).

[0115] Surface-sterilized seeds of A. thaliana accession Col-0 DR5::LUC were sown on Murashige and Skoog (MS) medium. 1 Modified Z> (without sucrose or vitamins and with a phosphate concentration of 625 pM, 300 pM or 5 pM Pi), were stratified to 4 e C for 2 days and then kept in a controlled growth chamber at 23 e C, in a long cycle (16 hours of daily light) within the D-Root system that prevents light from reaching the plant's root system. After 7 days, the plants were transplanted to another plate with the same MS medium. 1 / 2 modified in which the P. melinii strain CECT 21242 had been grown for 4 days under the same conditions as the plants. Four days after transplantation, the plants were sprayed with the luciferase substrate (luciferin) and the luminescent areas along the root were counted (counting from the point where it had come into contact with CECT 21242). These luminescent spots correspond to the PBS (Figure 3A). A high-sensitivity CCD camera and Indigo software were used to take the luminescence photographs.

[0116] Plants treated with P. melinii CECT 21242 showed a significant increase in PBS compared to control plants not treated with CECT 21242. Specifically, approximately 1.5 times more PBS (Student's t, PcO.001 ) in plants treated with P. melinii CECT 21242 (Figure 3B). This increase occurred under both optimal phosphate conditions (300 pM P¡) and under excess (625 pM P¡) or lack of phosphate (5 pM P¡), 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 phosphate availability, precisely based on this greater capacity to produce a greater number of PBS.

[0117] Example 4.

[0118] The P. melinii strain CECT 21242 increases the growth of the root system and the biomass of the aerial part of A. thaliana plants grown in vitro under optimal temperature conditions (22 e C) or high temperature (32 e C) (Figure 4).

[0119] Surface-sterilized seeds of A. thaliana accession Col-0 were sown on Murashige and Skoog (MS) medium. 1 Z> modified (without sucrose or vitamins and with a phosphate concentration of 625 pM Pi), were stratified to 4 e C for 2 days and then kept in a controlled growth chamber in a long cycle (16 hours of light per day) at 23 e C. After 7 days the plants were transplanted to another plate with the same MS medium 1 Z> modified where the P. melinii strain CECT 21242 had been grown for 4 days under the same conditions as the plants. From that point on, the plants were maintained at 22 e C or 32 eC. In the latter case, the aerial part was at 32 e C, but the roots were maintained at a temperature gradient between 24 and 32 e 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 e C are subjected to thermal stress which is manifested by a lower growth of the root system and lower biomass of the aerial part.

[0120] Under both conditions, plants grown in the presence of CECT 21242 showed greater root growth (Figure 4A, C) and a significant increase in shoot biomass compared to untreated plants (75% increase for 22 e C and 45% for 32 e Student's t test (P<0.05) (Figure 4B, D), showing that CECT 21242 promotes the growth of plants grown under optimal conditions or under heat stress.

[0121] Example 5.

[0122] P. melinii strain CECT 21242 increases the growth of the main root and secondary roots of A. thaliana plants grown in vitro under conditions of high temperature and phosphate deficiency (20 pM P¡) in the culture medium (Figure 5).

[0123] Surface-sterilized seeds of A. thaliana accession Col-0 were sown on Murashige and Skoog (MS) medium. 1 Z> with phosphate deficiency (without sucrose or vitamins and with a low phosphate concentration of 20pM P¡), were stratified to 4 e C for 2 days and then kept in a controlled growth chamber in a long cycle (16 hours of light per day) at 23 e C. After 7 days the plants were transplanted to another plate with the same MS medium 1 Z> modified where the P. melinii strain CECT 21242 had been grown for 4 days under the same conditions as the plants. From that point on, the plants were maintained at 32e C (aerial part), but the roots were maintained at a temperature gradient between 24 and 32 e 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 e C are subjected to thermal stress which is manifested by a lower growth of the root system and lower biomass of the aerial part.

[0124] Plants grown in the presence of P. melinii CECT 21242 showed greater root growth than untreated plants, showing that treated plants become tolerant to the combination of both stresses (phosphorus deficiency and high temperature).

[0125] Example 6.

[0126] The P. melinii strain CECT 21242 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 lack or excess of phosphorus in the culture medium (Figure 6).

[0127] Surface-sterilized seeds of A. thaliana accession Col-0 were sown on Murashige and Skoog (MS) medium. 1 Modified Z> (without sucrose or vitamins and with a phosphate concentration of 625 pM and 20 pM Pi), were stratified to 4 e C for 2 days and then kept in a controlled growth chamber at 23 e C, in a long cycle (16 hours of daily light) with light input to the plant's root system. After 7 days, the plants were transplanted to another plate with the same MS medium. 1A modified Z> in which P. melinii strain CECT 21242 was grown for 4 days under the same conditions as the plants. The light input to the root produces stress because this organ is adapted to underground growth in darkness, and this is manifested by reduced plant growth.

[0128] Plants grown under this stress are more sensitive to excess (625 pM) or deficiency (20 pM) of phosphate (Pi) content in the medium due to the addition of stresses. 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, showing that plants treated with P. melinii CECT 21242 become tolerant to combined stress due to the supply of light to the root and the excess or low availability of phosphorus.

[0129] Example 7.

[0130] P. melinii strain CECT 21242 decreases the accumulation of free amino acids in plants subjected to salinity stress (Figure 7).

[0131] Surface-sterilized seeds of A. thaliana accession Col-0 were sown on Murashige and Skoog (MS) medium. 1 Z> modified without sucrose or vitamins, were stratified to 4 e C for 2 days and then kept in a controlled growth chamber at 23 e C, in a long cycle (16 hours of daily light) within the D-Root system that prevents light from reaching the plant's root system. After 5 days, the plants were transplanted to another plate with MS 1 / 2 without sucrose or vitamins and modified or not with 100 pM NaCl as a salinity stress condition, where the CECT 21242 strain had been grown for 4 days under the same conditions as the plants. Seven days after transplantation, the plants were harvested for extraction and determination of free amino acids as described in López-Hidalgo et al. 2021. Plant Cell Environ. 44: 1977–1986. https: / / doi.org / 10.11 11 / pce.14007.

[0132] Plants treated with CECT 21242 showed lower accumulation of free amino acids in response to 100 pM NaCl salt stress treatment than control plants, indicating decreased protein degradation in response to abiotic stress, as described in López-Hidalgo et al. 2021. Plant Cell Environ. 44: 1977–1986. https: / / doi.org / 10-11 11 / pee.14007. Example 8.

[0133] P. melinii strain CECT 21242 decreases the expression of genes related to the plant's abiotic stress response (Figure 8).

[0134] Surface-sterilized seeds of A. thaliana accession Col-0 were sown on Murashige and Skoog (MS) medium. 1 Z> without sucrose or vitamins under conditions of deficiency (20 |iM P¡) or optimal phosphate (300 |iM P¡), were stratified to 4 e C for 2 days and then kept in a controlled growth chamber at 23 e C, in a long cycle (16 hours of daily light) within the D-Root system that prevents light from reaching the plant's root system. After 5 days, the plants were transplanted to another plate with MS 1Z> (where the CECT 21242 strain had been grown for 4 days under the same conditions as the plants). Three days after transplantation, the plants were harvested for RNA extraction and subsequent sequencing. Differentially expressed genes (DEGs) were identified using the R package DESeq2 v.1 .40.1, defined as those genes with a p-value <0.05 and Iog2(fold-change) >0.5 and < -0.5. To identify genes related to abiotic stress, the “Abiotic Stress” gene ontology category of the Mapman 3.5.1 program was selected using the A. thaliana TRO genome annotation (June 2016). The comparison was performed using the variance-stabilizing transformation to the raw data matrix of gene counts and applying a Z-score standardization of the expression of each gene in the mentioned category.

[0135] Plants treated with CECT 21242 showed lower expression of abiotic stress response genes 3 days post-inoculation under both phosphate-deficient and optimal growth conditions, indicating lower stress (T-test, p<0.001). The results observed under both phosphate-deficient and optimal growth conditions indicate that treatment with CECT 21242 improved the overall abiotic stress status of these plants, even though they had not been subjected to any specific known stress (e.g., phosphate deficiency). Therefore, application of CECT 21242 improved plant growth in any type of abiotic stress.

[0136] Example 9.

[0137] P. melinii strain CECT 21242 increases root thickness, lateral root growth and total surface area occupied by the root system, and promotes the growth of barley plants grown under general nutrient deficiency conditions using germination paper as a support (Figure 9).

[0138] Barley seeds (Hordeum vulgare var. Rubiana) were germinated on moistened filter paper for 5 days and the resulting seedlings were immersed in a suspension of 10 5 spores / ml (or water for control) for 30 seconds. Treated seedlings were placed on germination paper within the adapted D-Root system and watered with a nutrient solution composed of 1 / 8 MS, conferring a general nutrient deficiency, for 7 days.

[0139] Plants treated with P. me / / n / 7 CECT 21242 showed greater root development, with thicker roots (Figure 9A), longer lateral roots (Figure 9A), and a greater total surface area occupied by the root system (32% increase; Figure 9B). This greater root development resulted in a significant increase in plant growth, measured as the weight of the aerial part of the plant (11% increase, Student's t-test P<0.05; Figure 9C), produced under conditions of general nutrient deficiency.

[0140] Example 10.

[0141] P. melinii strain CECT 21242 increases the growth of barley plants (Figure 10A and 10B) and corn grown in agricultural soil in a greenhouse (Figure 10C and 10D).

[0142] Barley (Hordeum vulgare var. Rubiana) and maize (Zea mays var. LG3490) 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 control) and the pots were kept in the greenhouse for 6 weeks.

[0143] Plants treated with P. melinii CECT 21242 showed greater height than untreated plants when measured at 3 weeks (increases of 15 and 16% for barley and corn, respectively, and statistically significant, P<0.05, for corn). Treated barley plants also showed greater leaf area than untreated plants, measured at 5 weeks (19%).

[0144] Example 1 1. P. melinii strain CECT 21242 increases the growth rate of maize plants grown in agricultural soil in a greenhouse (Figure 11 ).

[0145] Corn seeds (Zea mays var. LG3490) 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. Plants treated with P. melinii CECT 21242 showed a higher height growth rate than control plants, measured as the slope of the growth curve (Figure 1 1 ).

[0146] Example 12.

[0147] Treatment with P. melinii strain CECT 21242 increases root development (12A) and total length of the root system (12B) of tomato plants growing in peat with optimal fertilization conditions (T-test compared to control, *: p < 0.05).

[0148] Tomato seeds (Solanum lycopersicum var. Optima) were germinated on moistened filter paper for 3 days. The resulting seedlings were transplanted into containers called rhizotrons filled with fertilized peat under optimal growth conditions. A rhizotron is a rectangular prism-shaped container with at least one of the largest surfaces made of transparent material. This makes it possible to observe the root system of the plant grown in this type of container. Three days after transplanting, the plants were inoculated with 1 mL of a 10 7 spores / ml (or water for control). The plants were maintained in a growth chamber at 23e C with a light cycle of 16:8h of light and darkness respectively.

[0149] Plants treated with P. melinii CECT 21242 showed greater root development, measured by a greater total length of the root system (45% increase, Student's T P<0.05; Figure 12).

[0150] Example 13.

[0151] P. melinii strain CECT 21242 increases root growth of greenhouse-grown tomato plants in nutrient-poor blond peat fertilized with normal levels of nutrients except for phosphorus (Figure 13). Tomato (Solatium lycopersicum var. Moneymaker) seeds were germinated in coarse vermiculite for 15 days. The resulting seedlings were transplanted into 4x4cm pots of soil fertilized with 1 / 8 the recommended amount of phosphate (0.073 g / L single superphosphate). The remaining nutrients were added at the recommended rate (3 g / L NK White). After 3 days, the soil near the plant stem was inoculated with 1 mL of a 10 6 spores / ml of CECT 21242 (or water for the control called “Mock”) and the pots were kept in the greenhouse for 15 days.

[0152] Plants treated with CECT 21242 showed greater root mass measured in fresh weight than those not treated under phosphorus deficiency conditions (T-test, p<0.05).

[0153] Example 14.

[0154] The P. melinii strain CECT 21242 increases the fresh (Figure 14A) and dry (Figure 14B) weight of plants, the number of marketable first and second category tomato fruits (Figure 14C), the weight of marketable fruits (Figure 14D) and the total marketable yield (tons / hectare) obtained from the total harvest (Figure 14E) in field trials under optimal fertilization conditions and under phosphate stress conditions measured in untreated (control) and treated (CECT 21242) plants (ANOVA and Post-hoc Tukey p<0.15).

[0155] Field trials were designed with eight replicates per treatment. Treatments consisted of a combination of two levels of phosphorus availability (optimal and deficiency) and the application or absence of the fungus. The optimal phosphorus level was obtained by applying the recommended phosphorus fertilization dose for tomato, taking into account the previous amounts of phosphorus in the soil determined by chemical analyses prior to planting. Phosphorus deficiency was achieved by halving the phosphorus input through fertilization. Additionally, both conditions were fertilized with the recommended levels of nitrogen, potassium, calcium, and magnesium. The CECT 21242 strain of P. melinii was applied at a dose of 4 x 10 7spores / mL at 10 days after transplanting tomato seedlings (Solanum lycopersicum var. N-283) into the field. Control plants were treated with the same volume of water. The fresh and dry weights of plants collected 21 days post-inoculation were determined. The fruits of the plants were harvested and the number and weight of marketable first and second category fruits were determined. First category fruits were defined as marketable fruits with a diameter > 45 mm. Second category fruits were defined as marketable fruits with a diameter < 45 mm. The yield of marketable fruits at harvest was determined in tonnes per hectare of crop.

[0156] P. melinii CECT 21242 increased the fresh weight of plants collected at 21 days post-inoculation by 26% under optimal P¡ conditions and by 28% under P¡ deficiency conditions (Figure 14A) and the dry weight by 16% under optimal P¡ conditions and by 39% under P¡ deficiency conditions (Figure 14B). P. melinii CECT 21242 increased the number of marketable fruits by 37% under optimal P¡ conditions and by 34% under P¡ deficiency conditions (Figure 14C). P. melinii CECT 21242 increased the weight of marketable fruits by 27% under optimal P¡ conditions and by 16% under P¡ deficiency conditions (Figure 14D). P. melinii CECT 21242 increased marketable fruit yield by 27% under optimal P¡ conditions and by 16% under P¡ deficiency (Figure 14E).

[0157] Example 15

[0158] P. melinii strain CECT 21242 increases foliar total phosphorus content in tomato plants in field trials under optimal phosphate (P¡) fertilization conditions.

[0159] Field trials were designed with eight replicates per treatment. Plants were fertilized with the recommended phosphorus dose for tomato, taking into account the previous amounts of phosphorus in the soil determined by chemical analyses prior to planting. Additionally, plants were fertilized with the recommended levels of nitrogen, potassium, calcium, and magnesium. P. melinii strain CECT 21242 was applied at a dose of 4 x 10 7 spores / mL at 7 days after transplanting tomato seedlings (Solanum lycopersicum var. Malpika F1) in the field. Control plants were treated with the same volume of water. Foliar phosphorus content of plants collected 21 days post-inoculation was determined by ICP-OES (inductively coupled plasma atomic emission spectroscopy).

[0160] P. melinii increased phosphorus use efficiency, measured as total foliar phosphorus content, by 7% in plants grown under optimal P¡ conditions (Figure 15).

[0161] Example 16.

[0162] P. melinii strain CECT 21242 increases the partial productivity factor of phosphorus in tomato plants grown in field trials under P¡ deficiency conditions (Figure 16). Field trials were designed with eight replicates per treatment. Phosphorus deficiency was achieved by not fertilizing with phosphorus. Additionally, plants were fertilized with the recommended levels of nitrogen, potassium, calcium, and magnesium. P. melinii strain CECT 21242 was applied at a dose of 4 x 10 7spores / mL at 14 days after field transplantation of tomato (Solanum lycopersicum var. Globe Trotter) seedlings. Control plants were treated with the same volume of water. The partial productivity factor (PPF) of phosphorus was determined based on the recommendations described in technical specifications CEN / TS 17700-1 -2022 and CEN / TS 17700-2-2022 for the determination of nutritional efficiency for biostimulants, which indicate that the partial productivity factor index is calculated following the formula: PPF=Y / F, where Y is the crop yield in the cultivated area and F the amount of nutrient initially available in the soil.

[0163] P. melinii strain CECT 21242 increased phosphorus use efficiency, measured as phosphorus PFP, by 7% in plants grown under phosphate (Pi) deficiency conditions (Figure 16).

[0164] Example 17.

[0165] Testing of different Penicillum melinii strains on Solanum lycopersicum var. Moneymaker plants. The strains tested included:

[0166] - Strain CECT 21242;

[0167] Strain CBS 139142, isolated from South Africa, Stellenbosch in 2014 from soil;

[0168] CBS 218.30 strain, isolated in the USA in 1930 from a forest soil;

[0169] Strain CBS 285.65, isolated from England, Lancashire (Grange-over-Sands) in 1965 from Querqus sp. litter in an acid pH (4.5) field.

[0170] With the exception of strain CECT 21242, the strains tested came from the Westerdijk Fungal Biodiversity Institute and have been classified as Penicillium melinii by that institute.

[0171] Tomato seeds (Solanum lycopersicum var. Moneymaker) were germinated in coarse vermiculite for 15 days. The resulting seedlings were transplanted into 4x4cm pots filled with soil fertilized with 1 / 8 of the recommended amount of phosphate (0.073 g / L single superphosphate). The remaining nutrients were added in the amount recommended for tomato cultivation. After 3 days, the soil in the stem area of ​​the plant was inoculated with 1 mL of a 10 6 spores / ml of the strain to be tested (or water for the control called “Mock”) and the pots were kept in the greenhouse for 15 days.

[0172] As shown in Figure 17, plants treated with all P. melinii strains showed a significant increase in root fresh weight (Student's t test, p<0.05) and shoot fresh weight (Student's t test, p<0.05) compared to untreated control plants (Figures 17A and 17B, respectively).

[0173] Therefore, P. melinii strains CECT 21242, CBS 139142, CBS 218.30, and CBS 285.65 increase root and shoot growth in tomato plants. The same effect was observed for CECT 21242 as well as for other strains of the same species, P. melinii, so the characteristics of CECT 21242 can be generalized to the P. melinii species as a whole. Therefore, P. melinii increases root and shoot growth in tomato plants, and this increase occurs under conditions of low phosphorus availability. This demonstrates that plants treated with P. melinii show tolerance to phosphorus deficiency stress, as they are capable of producing greater plant tissue growth under these conditions.

[0174] Example 18.

[0175] Strains CECT 21242, CBS 139142, CBS 218.30 and CBS 285.65 of P. melinii, increase the capacity to form lateral roots measured as the number of zones along the main root with the capacity to form a lateral root (PBS) (Figure 18).

[0176] The experiment was performed on A. thaliana plants transformed with the synthetic DR5 promoter fused to luciferase (DR5::LUC), a marker for lateral root formation by indicating the areas along the main root capable of forming a lateral root called pre-branching sites (PBS).

[0177] Surface-sterilized seeds of A. thaliana accession Col-0 DR5::LUC were sown on Murashige and Skoog (MS) medium. 1 Z> modified (without sucrose or vitamins), were stratified to 4 e C for 2 days and then kept in a controlled growth chamber at 23 eC, in a long cycle (16 hours of daily light) within the D-Root system that prevents light from reaching the plant's root system. After 5 days, the plants were transplanted to another plate with Minimal Medium where the P. melinii strain to be tested had been grown for 4 days under the same conditions as the plants. Minimal Medium produces general nutrient deficiency stress in the plant, since it contains quantities of nutrients that are too low for optimal plant growth. The P. melinii strains tested were CECT 21242, CBS 139142, CBS 218.30 and CBS 285.65. Three days after transplanting, the plants were sprayed with the luciferase substrate (luciferin) and the areas along the root that emitted luminescence were counted (counting from the point where it had come into contact with the strain). These luminescent dots correspond to the PBS (Figure 18).A high-sensitivity CCD camera and Indigo software were used to capture luminescence images. Plants treated with P. melinii strains CECT 21242, CBS 139142, CBS 218.30, and CBS 285.65 showed a significant increase in the number of PBS (Student's t test, P<0.05) compared to untreated control plants. Furthermore, the increased PBS in plants treated with these strains demonstrates that these plants are becoming stress-tolerant, as they are capable of producing higher numbers of PBS under the general nutrient deficiency conditions imposed by the minimal culture medium.

[0178] The same effect was observed for both CECT 21242 and other strains of the same species, P. melinii, so the characteristics of CECT 21242 can be generalized to the P. melinii species as a whole. Therefore, P. melinii increases the plants' ability to form lateral roots and their tolerance to general nutrient deficiency stress.

[0179] Example 19.

[0180] Freeze-dried mycelium and spore extract of P. melinii strain CECT 21242 (mycelium) or culture filtrate of P. melinii strain CECT 21242 (filtrate) increase the length (Figure 19A) and number (Figure 19B) of lateral roots of A. thaliana plants grown under phosphate deprivation and root illumination.

[0181] Extracts of CECT 21242 were obtained after two weeks of culture in potato dextrose broth (PDB) growth medium. The 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 filtered medium, free of fungal tissue, 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 control culture medium extract (PDB Medium Control), the same procedure was performed, but with PDB medium in which the fungus had not been incubated. Each extract was applied to a plate containing Murashige and Skoog (MS) medium. 1Z> with phosphate deficiency (without sucrose or vitamins and with a low phosphate concentration of 20 pM P¡. Seeds of A. thaliana accession Col-0, surface sterilized and previously stratified at 4 e C for 2 days were sown on the culture plates where each treatment had been previously applied (mycelium and spore extract, filtrate, PDB control and sterile distilled water for the control) and then were kept in a controlled growth chamber in a long cycle (16 hours of light daily) at 23 e C for 14 days, after which the roots were measured.

[0182] Example 20.

[0183] Treatment with different types of CECT 21242 extracts increases fresh weight growth in plants under nutritional stress due to phosphate (Pi) deficiency.

[0184] For solid medium growth of the fungus, a plant substrate derived from bagasse, a waste product from the brewing industry, was used as a culture medium. Prior to fungal cultivation, the plant substrate was sterilized, pulverized by ultra-freezing in liquid nitrogen, ground in a mortar, and spread onto round Petri dishes supplemented with 2 mL of a 5% sucrose solution. Each plate was inoculated with five 6 pL drops of the CECT 21242 strain for growth in the plant substrate from which the CECT 21242 extract will originate. Control plates that were not inoculated with the fungus (called plant extract) were also used. The plates inoculated with CECT 21242 and the controls were incubated at room temperature for 27 days.

[0185] For extraction, both the fungus and the culture medium were ground with a mortar and pestle using liquid nitrogen to ultrafreeze and pulverize the sample. Subsequently, 50 mM Tris-HCl buffer, pH 6.8, was added in a 1:5.5 weight-to-volume ratio, and the mixture was stirred for 1 hour at 6.5 rpm. Three different extraction protocols were used on this mixture:

[0186] 1. Normal (aqueous extract adding Tris-HCI buffer and without additional treatment).

[0187] 2. Temperature: an extraction increasing the temperature to 50 e C for 15 min.

[0188] 3. Vacuum: consisting of two negative pressure pulses of -700 psi.

[0189] All treatments were then centrifuged at 4000 rpm for 30 minutes. The supernatant of each extract was filtered through a 0.45 pm filter to remove all solid components from the fungus and the culture medium.

[0190] To evaluate the effect of the extracts, surface-sterilized seeds of A. thaliana accession Col-0 were sown on Murashige and Skoog (MS) medium. 1 Z> modified to obtain phosphorus deficiency conditions (without sucrose or vitamins and 20 pM P¡), were stratified at 4 e C for 2 days and then kept in a controlled growth chamber at 23 e C, in a long cycle (16 hours of daily light) within the D-Root system that prevents light from reaching the plant's root system. After 7 days, the plants were transplanted to another plate with MS 1Z> supplemented with the extracts obtained from the different extraction methods (normal, temperature and vacuum). The plants were untreated (control), treated with plant extracts or with extracts of CECT 21242. The fresh weight of the plants and the total length of the root system were determined 14 days post-transfer. The results of the untreated plants (control), treated with plant extract and treated with the different types of CECT 21242 extract are shown in Figure 20A-D. Plants treated with the various methods to obtain CECT 21242 extracts are superior to plants treated with plant extracts and to untreated plants, in fresh weight (Figure 20A). Plants treated with the extract called “Normal” of the CECT 21242 strain showed a significant increase in root length and fresh weight of the shoot (ANOVA post- hoc Tukey, P <0.05) compared to untreated control plants or those treated only with the plant residue extract (Figures 20B, C and D).

[0191] Example 21.

[0192] The aqueous extract of P. melinii strain CECT 21242 increases the amount of barley stems.

[0193] Barley plants (Hordeum vulgare var. Rubiana) were grown in a greenhouse with complete fertilization (optimum, 0.58 g / L of single superphosphate) in nutrient-poor blond peat. 3 g / L of NK White fertilizer was also added to the peat. A completely randomized design was used with five replicates and one pot per replicate. The bagasse plant extract was applied as a control, or the extract of the CECT 21242 strain obtained using the procedure described in the previous example (aqueous extract in Tñs-HCl).

[0194] The number of stems was evaluated as a measure of the extract's effect. Plants treated with the extract of strain CECT 21242 showed a higher number of stems (Figure 21).

Claims

CLAIMS 1. Method for protecting plants from different types of abiotic stress and / or increasing the tolerance of plants to different types of abiotic stress, 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 strains, and / or filtrates of said species or strains.

2. Method according to claim 1, wherein the protection of the plants from different types of abiotic stress and / or the tolerance of the plants to different types of abiotic stress is determined by one or more of the following parameters: increase in the growth or development of the plant or of some of its parts under stress conditions, decrease in the expression of plant genes related to abiotic stress and / or decrease in the content of free amino acids in the plant.

3. Method according to any of claims 1 to 2, wherein the microorganism is the Penicillium melinii strain deposited under deposit number CECT 21242.

4. The method according to any of claims 1 to 3 wherein the plants are Angiosperms.

5. The method of claim 4 wherein the plants are tomato (Solanum lycopersicum), Arabidopsis thaliana, barley (Hordeum vulgare), corn (Zea mays) or wheat (Triticum spp.).

6. The method according to claim 5, wherein the plant is Solanum lycopersicum var. Moneymaker, or Solanum lycopersicum var. Optima, or Solanum lycopersicum var. N-283, or Solanum lycopersicum var. Malpika F1, or Solanum lycopersicum var. Globe Trotter, or Hordeum vulgare var. Rubiana or Zea mays var. LG3490.

7. The method according to any one of claims 1 to 6 wherein the plants are grown in vitro, on germination paper, in peat or in agricultural soil.

8. The method according to any one of claims 1 to 7, wherein the microorganism is in the form of spores, strands, mycelium or sclerotia.

9. The method according to any one of claims 1 to 8 wherein the composition is applied to the seeds of the plant.

10. The method according to any one of claims 1 to 9 wherein the composition is applied to the aerial parts of the plant.

11. The method according to any one of claims 1 to 10 wherein the composition is applied to the roots of the plant or other underground parts of the plant.

12. The method according to any of claims 1 to 11 wherein the composition is applied to the substrate for growing the plant.

13. The method according to any one of claims 1 to 12 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.

14. The method according to any one of claims 1 to 13 wherein the composition is a liquid, a solid, a paste or a gel.

15. The method according to any one of claims 1 to 14 wherein the composition is a powder, tablet, tablet, granule or emulsifiable concentrate.

16. The method according to any one of claims 1 to 15 wherein the composition is applied by spraying, spraying, immersion, irrigation or dusting.

17. Use of a microorganism of the species Penicillium melinii, and / or strains of said species, and / or extracts of said species or strains, and / or filtrates of said species or strains, to protect plants from different types of abiotic stress and / or obtain stress-tolerant plants, and / or increase the tolerance of plants subjected to different types of abiotic stress.

18. The use of claim 17 wherein the microorganism is the Penicillium melinii strain, deposited under deposit number CECT 21242.

19. The use of claims 17 to 18 wherein the stress conditions are one or more conditions selected from the group: absence of phosphorus, low availability or excess of phosphorus, light stress, heat stress, water stress, toxicity stress, salinity stress and nutritional deficiency.

20. Plant propagation material comprising a microorganism of the species Penicillium melinii, and / or strains of said species, and / or extracts of said species or strains, and / or filtrates of said species or strains.

21. Propagation material according to claim 20, wherein the microorganism is the CECT 21242 strain of the Penicillium melinii species.

22. Propagation material according to claims 20 to 21, selected from the group comprising seeds, seedlings, young plants, cuttings, bulbs and tubers.

23. Substrate for the cultivation of plants comprising a microorganism of the species Penicillium melinii, and / or strains of said species, and / or extracts of said species or strains, and / or filtrates of said species or strains.

24. Use of the substrate of claim 23 to obtain stress-tolerant plants and / or increase the tolerance of plants subjected to stress.

25. Strain of the microorganism Penicillium melinii, deposited under deposit number CECT 21242.

26. Method for promoting growth in plants and / or increasing the nutritional efficiency of plants both under optimal growth conditions and under stress conditions, characterized by comprising a step of contacting said plants with a composition comprising the CECT 21242 strain of the Penicillium melinii species, and / or extracts of said strain, and / or filtrates of said strain.

27. Method according to claim 26, wherein the growth in the plants is determined by one or more of the following parameters: number of “pre-branching sites” or PBS in the root, area occupied by the root, root weight, length of the shoot, weight of the shoot, number of stems, leaf area, number of fruits, and / or weight of the fruits and / or the total yield of all the plants that make up a crop area.

28. Method according to claim 26, wherein the nutritional efficiency of the plants is determined by the nutrient content in the plant tissues or by the increase in the partial productivity factor of a nutrient.

29. The method according to any of claims 26 to 28 wherein the plants are Angiosperms.

30. The method of claim 29 wherein the plants are tomato (Solatium lycopersicum), Arabidopsis thaliana, barley (Hordeum vulgare), corn (Zea mays) or wheat (Triticum spp.).

31. The method according to claim 30, wherein the plant is Solatium lycopersicum var. Moneymaker, or Solanum lycopersicum var. Optima, or Solanum lycopersicum var. N-283, or Solanum lycopersicum var. Malpika F1, or Solanum lycopersicum var. Globe Trotter, or Hordeum vulgare var. Rubiana or Zea mays var. LG3490.

32. The method according to any of claims 26 to 31 wherein the plants are grown in vitro, on germination paper, in peat or in agricultural soil.

33. The method according to any one of claims 26 to 32, wherein the microorganism is in the form of spores, strands, mycelium or sclerotia.

34. The method according to any of claims 26 to 33 wherein the composition is applied to the seeds of the plant.

35. The method according to any of claims 26 to 34 wherein the composition is applied to the aerial parts of the plant.

36. The method according to any one of claims 26 to 35 wherein the composition is applied to the roots of the plant or other underground parts of the plant.

37. The method according to any of claims 26 to 36 wherein the composition is applied to the substrate for growing the plant.

38. The method according to any one of claims 26 to 37 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.

39. The method according to any one of claims 26 to 38 wherein the composition is a liquid, a solid, a paste or a gel.

40. The method according to any of claims 26 to 39 wherein the composition is a powder, tablet, tablet, granule or emulsifiable concentrate.

41. The method according to any one of claims 26 to 40 wherein the composition is applied by spraying, spraying, immersion, irrigation or dusting.

42. Use of the CECT 21242 strain of the Penicillium melinii species, to promote plant growth and / or increase nutritional efficiency both under optimal growth conditions and under stress conditions.

43. Substrate for the cultivation of plants comprising the CECT 21242 strain of the Penicillium melinii species, and / or extracts of said strain, and / or filtrates of said strain.

44. Use of the substrate of claim 43 to increase plant growth, increase the nutritional efficiency of plants, obtain stress-tolerant plants and / or increase the tolerance of plants subjected to stress.

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