ES2976115A1 - METHOD TO PROTECT AGAINST STRESS AND INCREASE THE GROWTH OF PLANTS (Machine-translation by Google Translate, not legally binding) - Google Patents

Abstract

Method to protect plants from different types of stress and/or increase the number of lateral roots in plants, based on the use of a microorganism of the species Penicillium melinii, and specific strains of it. The promotion of plant growth and development takes place both in optimal growth conditions and in conditions of different types of stress. (Machine-translation by Google Translate, not legally binding)

Description

DESCRIPTION

METHOD TO PROTECT AGAINST STRESS AND INCREASE GROWTH

OF THE PLANTS

FIELD OF INVENTION

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.

BACKGROUND OF THE INVENTION

In the agricultural sector there is enormous interest in increasing plant growth, minimizing the use of chemical fertilizers and with greater sustainability. This interest is particularly important when it comes to improving tolerance or alleviating the stress produced in plants when they are not growing under optimal 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 the efficient absorption of water and nutrients by the plant, especially in the early stages of development, especially under stress conditions such as drought, salinity and extreme temperatures. Improving plant root development can therefore be a key factor in improving plant tolerance to stresses produced 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 corn 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 main root of corn (Jia, X. et al., 2018, J Exp Bot. 69(20). 4961-4970. Doi: 10.1093/jxb/ery252).

Plants in nature establish symbiotic associations with microorganisms called mutualists that confer benefits in their growth, survival and multiplication. These microorganisms can be isolated and sometimes used to improve crop yield. For example, colonization of rapeseed roots by Serendipita vermifera, Alternaria altemata or Leptosphaeria biglobosa, significantly increases root and shoot 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.BI0C0NTR0L.2014.02.018). This improved growth may be due to an improvement in the plant's efficiency in uptake or assimilation of nutrients. For example, Aspergillus niger and Trichoderma harzianus produce ammonium in wheat roots 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 may be the increase in plant tolerance to stresses such as salinity or toxicity of the medium or extreme nutrient shortage. For example, T. virens increases the number of lateral roots of Arabidopsis, making the plant more tolerant to salinity (Contreras-Cornejo, H. Aet al., 2009, Plant Physiol. 149, 1579-1592. doi: 10.1104/PP.108.130369). As these examples illustrate, different microorganisms may have different mechanisms of action that serve to improve in particular ways different 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, to alleviate the stress produced in those conditions and restore its productivity. The modes of action of microorganisms to improve plant stress tolerance may include increasing the development of the root system.

Mohamed Tarroumet al. (2021), Plants, 10, 784, https://doi.org/10.3390/plants10040784, shows a set of six fungal strains of different species isolated from the rhizosphere of a halophyte herb, Aeluropus littoralis, and analyzes their growth-promoting activities on 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 (CFF) were added at 0.5 NS (nutrient solution) in a closed hydroponic system, the highest effects on tobacco seedlings (shoot and root dry weight, leaf number, and root length) were observed when adding CFF from strains A5.1 and A8, presumably belonging to the species Byssochlamys spectabilis and Penicillium melinii, based on the similarity of a single genetic marker (ITS), which is not completely determinant in taxonomic identification, with the sequences of these species obtained from the NCBI Genebank database. It was found that CFF from all strains, when added at 0.5 NS, can substitute part of the chemical inputs, with a 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 substances related to indoleacetic acid (auxins), a growth-related hormone, by fungi. In addition, the authors show that the expression of the Tryp1 and YUCCA6-like genes, involved in the biosynthesis of the phytohormone auxin, is also induced in treated tobacco plants. With these results, this work proposes the use of the A8 strain of P. meliniium and its filtrates to replace part of the chemical fertilization necessary for optimal plant growth, but not as a stress reliever. The only root parameters measured were those related to growth (weight or length), but no other developmental parameters related to the formation of lateral roots were analyzed. This effect was not measured under stress conditions either. In this regard, the publication shows that high temperature (40°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.

WO2014046553A1 (BIOCONSORTIA INC) discloses a method for selecting microorganisms capable of imparting beneficial properties to plants. In example 4, the use of this method is described 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. In addition, 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.

BRIEF DESCRIPTION OF THE FIGURES.

Figure 1. Increased root growth in plants treated with CECT 21242 compared to untreated plants (control), under nutritional stress due to lack of inorganic phosphorus (Pi).

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 related genera to determine the taxonomic position of the 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 the strain CECT 21242 groups with the sequences of other strains of the species Penicillium melinii. Figure 3. Relative increase in branching initiation sites (or their English term “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 roots of DR5::LUC plants inoculated with different fungi (C).

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

Figure 5. Extract of CECT 21242 (mycelium) or culture filtrate of CECT 21242 (filtrate) increases the length (Figure 5A) and number of lateral roots (Figure 5B) of Arabidopsis thaliana plants grown under phosphate deficiency and with light stress due to illumination on the root.

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 pM).

Figure 7. Increased root growth (Figure 7A and C) and shoot biomass (Figure B and D) in Arabidopsis thaliana plants treated with CECT 21242 and grown at 22 °C or 32 °C.

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.

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

Figure 10. 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.

DESCRIPTION OF THE INVENTION

The present invention has identified, through the screening of 10 strains of different fungal species, the strain CECT 21242 of the species Penicillum melini which, surprisingly, increases the plant's capacity for lateral root development and serves as a plant stress reliever. The increase in root development occurs under stress conditions due to lack or excess of phosphorus, light stress on the root, or thermal stress, or combining several of these stresses. In addition, the application of extracts or filtrates of the fungus also increased the length and number of lateral roots of the plant under stress conditions. Therefore, the data indicate that plants treated with Penicillium melini or its extracts or filtrates acquire tolerance to different types of stress. Furthermore, plants treated with the Penicillium melinii strain CECT 21242 increased root development and aerial part growth both under stress conditions and under optimal conditions in in vitro culture or by growing the plants in agricultural soil.

The species identification 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 encoding 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 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).

Nothing in the state of the art indicated 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 indicate an increase in the biosynthesis of this hormone, both by the fungus and by the treated plant.

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 (WO2014046553A1), as well as the negative role played by different types of stress on the growth of said microorganism (Mohamed Tarroumet al. (2021).

A first aspect of the present invention is a method of protecting or relieving plants from different 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.

Hereinafter this is the method of the invention, and the term “microorganism of the invention” refers to the microorganism of the species Penicillium melinii and/or the strain CECT 21242 of said species.

Increased lateral root formation in the plant is understood as a greater length of the root system, comprising the main root and lateral roots, and/or a greater number of zones capable of forming a lateral root (branching initiation sites or their English term “pre-branching sites” (PBS)) along the main root.

Stress is understood as the set of processes that the plant undergoes when growing in unsuitable environmental conditions and which manifests itself in a reduction in growth compared to what it would have achieved under optimal conditions. An example of stress is the extreme lack of nutrients, such as phosphorus. Optimal conditions are those that allow adequate plant growth, as any expert in the field can appreciate.

A second aspect of the present invention consists in 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 conditions of different types of stress. The use can be made of the microorganism itself, or of extracts of said microorganism, and/or filtrates thereof.

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

A third aspect of the present invention consists of 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).

A fourth aspect of the present invention is a substrate for plant cultivation 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.

A fifth aspect of the present invention consists in the use of the substrate comprising a microorganism of the species Penicillium melinii, and/or strains of said species, in particular the 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 subjected to stress.

In this specification, “plant growing substrate” is understood as 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, humus or peat or mixtures thereof. Examples of synthetic substrates include, but are not limited to, Murashige and Skoog (MS), Hoagland or any other nutrient media comprising plant nutrients, in liquid form for use in hydroponic culture or solidified with gelling substances such as agar, for use in in vitro culture. Other examples are materials for use in hydroponic culture, such as germination paper, rock wool or coconut fiber.

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.

In this specification, “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.

The propagation material may be of different origin. 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.

The present invention can be applied to any type of plant, preferably gymnosperms, angiosperms, monocotyledons and dicotyledons. Some preferred, but not limiting, examples of plants are Arabidopsis, and in particular Arabidopsis thaliana, barley and corn.

The promotion of plant growth can occur both under different types of stress conditions and under optimal growth conditions, as well as in different artificial or natural substrates, including, but not limited to, hydroponic and in vitro culture. In the present invention, the plant can be a natural or transgenic or genetically edited plant.

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.

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 lack or excess of phosphorus, light stress, heat stress or combining several of these stresses, as well as under optimal growth conditions. The data indicate that plants treated with the Penicillium melinii strain CECT 21242 acquire tolerance to stresses. Furthermore, plants treated with the Penicillium melinii strain CECT 21242 increase the growth rate and the absolute growth of the aerial part (biomass or length) under all these conditions in in vitro culture, in germination paper, or by growing the plants in agricultural soil.

Although the examples of the present invention have been carried out with the CECT 21242 strain of Penicillium melinii, the present system is universal and can be extended to any other type of Penicillium melinii strain.

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

Phosphorus deficiency refers to phosphate concentrations of less than 25 pM in in vitro culture, which cause significantly less plant growth than under optimal conditions. Optimal conditions are those that allow adequate plant growth, as can be appreciated by any expert in the field. Specifically, for A. thaliana grown in vitro, the optimal phosphate conditions are 300 pM.

Excess phosphorus refers to phosphate concentrations greater than optimal, which produce less plant growth than under optimal conditions.

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.

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

Toxicity stress is the presence of substances that are harmful to the plant, producing different types of osmotic, physiological or metabolic disruption, which lead 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 and can be 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.

Thermal stress is understood as a temperature that is lower or higher than the optimum for plant growth and that produces an inhibition of its development or less growth in the aerial part and/or in the root system. The optimum conditions are those that allow adequate growth of the plant, as can be appreciated by any expert in the field. Specifically, for A. thaliana grown in vitro, the optimum temperature conditions are 22°C.

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 use water and nutrients, resulting in reduced growth of the plant with respect to optimal conditions and in many cases producing different types of physiological or metabolic disruption that can even lead to the death of cells and tissues of the plant, or of the entire plant.

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, cobs, seeds, bulbs, tubers, roots and seedlings. The application of the composition to the plant can be carried out at any stage, and as an 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 cultivation in a seedbed, or at the time of transplanting seedlings, or at the time of cutting 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.

Therefore, one embodiment is the method of the invention, wherein said composition is applied to the seeds of said plant.

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

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.

The method of the invention includes treatment by spraying or atomizing whole plants or any parts thereof with a suitable dilution of the composition according to the invention, or dipping whole plants or any parts thereof in such a 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 may also be mixed with the irrigation liquid. The method of the invention also includes treatment by means of agar fragments with mycelium which are brought into contact with a part of the plant, whether root, stem or leaves or even on the surface of the soil close to the roots of the crop.

Another embodiment is the method of the invention, wherein said 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, nonionic surfactants, cationic surfactants, amphoteric surfactants, water-soluble polymers, polysaccharides, preservatives, coloring agents, thickening agents, and/or stabilizing agents.

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

Another embodiment is the method of the invention, wherein said composition is a powder, tablet, tablet, granule or emulsifiable concentrate.

Another embodiment is the method of the invention, wherein said composition is applied by spraying, atomizing, dipping, irrigating or dusting.

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 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.

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

The culture medium can be left at rest or 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.

Another embodiment is the method of the invention, where said microorganism is in the form of spores, hyphae, mycelium or sclerotia.

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

The substrate is preferably treated so that the microorganism of the invention is cultured therein before the substrate is used for plant cultivation. Examples of treatment of the substrate include perfusion of a liquid into the substrate (by irrigation, injection or drip), spraying, dusting or direct mixing with the substrate. The method of the invention also comprises the treatment of a hydroponic medium, for cultivation in hydroponics. 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.

The substrate for plant cultivation may comprise the spore, 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.

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

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

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: 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 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.

Another embodiment is the method of the invention, where filtrates of the microorganism are applied. In the present specification, “filtrate” is understood as a liquid culture medium obtained from the culture of the microorganism of the invention. It is possible to obtain a liquid culture medium, 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 (then 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 different methods known to the person 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 min and then remove the solid fungal material by centrifugation.

Preferably, the filtrate is obtained by filtration of 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 hyphae 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.

The following are a number of examples which 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.

PREFERRED MODES OF EMBODIMENT.

Example 1. Screening for fungi that increase plant root development.

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

2013. Fungal Diversity 60, 71-89). These plants were devoid of 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°C). The fragments were periodically inspected by isolating the emerging mycelium on petri dishes with potato dextrose agar (PDA) medium containing 200 mg/L chloramphenicol. Among the isolates obtained, a screening was performed to find fungi that were beneficial by producing an increase in the length of the root system in plants subjected to phosphorus deficiency stress.

Surface-sterilized seeds of A. thaliana accession Col-0 were sown on Murashige and Skoog (MS)/modified medium (without sucrose or vitamins and with an optimal phosphate concentration of 300 pM or a deficiency of 5 pM), stratified at 4 °C for 2 days and then kept in a controlled growth chamber at 23 °C, in a long cycle (16 hours of light per day) 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/modified medium 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 fungus was growing.

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 5 pM, both in length of the main root 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 lack of inorganic phosphorus. Other fungi produced significant reductions in the length of the main root or root system or did not produce significant changes.

Table 1.

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).

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

Example 2. Taxonomic identification of strain CECT 21242.

The morphology of strain CECT 21242 indicated that the strain belongs to the Penicillium genus. To determine the species, the methodology of Houbrake et al., 2020 (Studies in Mycology 95: 5-169) was followed, obtaining the partial sequences of genes encoding the beta-tubulin proteins (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 Penicillium genus and other 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 groups in a cluster with all strains of the speciesP. 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, The Netherlands) and are 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).

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

Surface-sterilized seeds of A. thaliana accession Col-0 DR5:LUC were sown on Murashige and Skoog (MS)/modified medium (without sucrose or vitamins and with a phosphorus concentration of 625 gM, 300 gM or 5 gM), stratified at 4 °C for 2 days and then maintained in a controlled growth chamber at 23 °C, in a long cycle (16 hours of light per day) within the D-Root system that prevents light from reaching the root system of the plant. After 7 days, the plants were transplanted to another plate with the same MS/modified medium where P. melinii strain CECT 21242 had been grown for 4 days under the same conditions as the plants. At 4 days after transplantation, plants were sprayed with luciferase (luciferin) substrate and the areas along the root that emitted luminescence were counted (counting from the point where it had come into contact with CECT 21242). These luminescent points correspond to PBS (Figure 3A). A high-sensitivity CCD camera and Indigo software were used to take luminescence photos. 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, P < 0.001) in plants treated with P. melinii CECT 21242 (Figure 3B). This increase occurs both under optimal conditions of inorganic phosphorus (300 gM) and excess (625 gM) or nutritional deficiency of phosphorus (5 gM), which implies 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 based on this greater capacity to produce a greater amount of PBS.

Example 4.

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 on the root and lack or excess of inorganic phosphorus in the culture medium (Figure 4).

Surface-sterilized seeds of A. thaliana accession Col-0 were sown on Murashige and Skoog (MS)/modified medium (without sucrose or vitamins and with a phosphorus concentration of 625 pM and 20 pM), stratified at 4 °C for 2 days and then kept in a controlled growth chamber at 23 °C, in 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 MS/modified medium where the P. melinii strain CECT 21242 had been grown for 4 days under the same conditions as the plants. Light supplied to the root produces stress because this organ is adapted to underground growth in the dark, and is manifested by reduced plant growth.

Plants grown under this stress are more sensitive to excess (625 pM) or deficiency (21 pM) of inorganic phosphorus (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 both in 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 light supply to the root and the low availability of inorganic phosphorus.

Example 5.

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

Extracts of CECT 21242 were obtained after being cultured for two weeks in potato dextrose broth (PDB) growth medium. 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 freeze-dried and suspended in sterile distilled water. This mixture was freeze-dried, suspended in sterile water and filtered through 0.22 ^m. For the culture medium extract control (PDB control), the same procedure was performed, but with PDB medium in which the fungus had not been incubated. Each extract was applied on a plate with Murashige and Skoog (MS) medium / phosphate deficiency (without sucrose or vitamins and with a low phosphorus concentration of 20 ^M). Surface-sterilized seeds of A. thaliana accession Col-0, previously stratified at 4 °C for 2 days, were sown on the culture plates where each extract had previously been applied (mycelium, filtrate, PDB control and sterile distilled water for the control) and then kept in a controlled growth chamber in a long cycle (16 hours of light per day) at 23 °C for 14 days, after which the roots were measured.

Example 6.

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 high temperature and lack of inorganic phosphorus (20^M) in the culture medium (Figure 6).

Surface-sterilized seeds of A. thaliana accession Col-0 were sown on Murashige and Skoog (MS)/phosphate-deficient medium (without sucrose or vitamins and with a low phosphorus concentration of 20^M), stratified at 4 °C for 2 days and then maintained in a controlled growth chamber in a long cycle (16 hours of light per day) at 23 °C. After 7 days, the plants were transplanted to another plate with the same MS/modified medium where P. melinii strain CECT 21242 had been grown for 4 days under the same conditions as the plants. From that moment on, the plants were kept at 32 °C (aerial part), but the roots were kept 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/10.10167j.xplc.2022.100514). Plants grown at 32 °C are subjected to thermal stress manifested by reduced root growth and lower biomass of the aerial part.

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 (inorganic phosphorus deficiency and high temperature).

Example 7.

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

Surface-sterilized seeds of A. thaliana accession Col-0 were sown on Murashige and Skoog (MS)/modified medium (without sucrose or vitamins and with a phosphorus concentration of 625 pM), stratified at 4 °C for 2 days and then maintained in a controlled growth chamber in a long cycle (16 hours of light per day) at 23 °C. After 7 days, the plants were transplanted to another plate with the same MS/modified medium where P. melinii strain CECT 21242 had been grown for 4 days under the same conditions as the plants. From that moment on, the plants were maintained at 22 °C or 32 °C. In the latter case, the aerial part was 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 manifested by lower root system growth and lower aerial part biomass.

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

Example 8

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

Barley seeds were germinated on moistened filter paper for 5 days and the resulting seedlings were dipped in a suspension of 105 spores/ml (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.

Plants treated with P. melinii CECT 21242 showed increased 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 increased root development resulted in a significant increase in plant growth, measured as the weight of the aboveground part of the plant (11% increase, Student's t-test P<0.05; Figure 8C).

Example 9.

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

Barley and maize seeds were germinated on moistened filter paper for 5 days and the resulting seedlings were transplanted into 15 cm diameter pots with 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 greater height than untreated plants measured 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 greater leaf area than untreated plants measured at 5 weeks (19%).

Example 10.

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

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 height growth rate than control plants, as measured by the slope of the growth curve (Figure 10).

Claims (24)

1. Method for protecting plants from different 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. 2. Method according to claim 1, wherein the microorganism is the Penicillium melini strain deposited with deposit number CECT 21242. 3. The method according to claims 1 and 2 wherein the plants are Angiosperms. 4. The method of claim 3 wherein the plants are Arabidopsis thaliana, corn or barley. 5. The method according to any of claims 1 to 4 wherein the plants are grown in vitro, in germination paper or in agricultural soil. 6. The method according to any one of claims 1 to 5, wherein the microorganism is in the form of spores, hyphae, mycelium or sclerotia. 7. The method according to any of claims 1 to 6 wherein the composition is applied to the seeds of the plant. 8. The method according to any of claims 1 to 6 wherein the composition is applied to the aerial parts of the plant. 9. The method according to any one of claims 1 to 6 wherein the composition is applied to the roots of the plant or to other underground parts of the plant. 10. The method according to any of claims 1 to 6 wherein the composition is applied to the substrate for growing the plant. 11. The method according to any one of claims 1 to 10 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, nonionic surfactants, cationic surfactants, amphoteric surfactants, water-soluble polymers, polysaccharides, preservatives, coloring agents, thickening agents, and/or stabilizing agents. 12. The method according to any one of claims 1 to 11 wherein the composition is a liquid, a solid, a paste or a gel. 13. The method according to any of claims 1 to 12 wherein the composition is an emulsifiable powder, tablet, tablet, granulate or concentrate. 14. The method according to any one of claims 1 to 13 wherein the composition is applied by spraying, atomizing, dipping, irrigating or dusting. 15. 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, to promote plant growth, both under optimal growth conditions and under conditions of different types of stress. 16. The use of claim 15 wherein the microorganism is the strain of Penicillium melinii, deposited under deposit number CECT 21242. 17. The use of claims 15 and 16 where the stress conditions are one or more conditions selected from the group: absence of phosphorus, low availability or excess of phosphorus, light stress, thermal stress, water stress, toxicity stress, nutritional deficiency and biotic stress. 18. Strain of the microorganism Penicillium melinii, deposited under deposit number CECT 21242. 19. 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 strain, and/or filtrates of said species or strain. 20. Substrate according to claim 19, wherein the microorganism is strain CECT 21242 of the species Penicillium melinii and/or extracts of said microorganism and/or filtrates of said microorganism. 21. Use of the substrate of claims 19 and 20 to obtain stress-tolerant plants and/or increase the growth of plants subjected to stress. 22. Plant propagation material comprising a microorganism of the species Penicillium melinii, and/or strains of said species, and/or extracts of said strain, and/or filtrates of said strain. 23. Propagation material according to claim 22, wherein the microorganism is strain CECT 21242 of the species Penicillium melinii. 24. Propagation material according to claim 23, selected from the group comprising seeds, seedlings, young plants, cuttings, bulbs and tubers.