Biostimulant bioprotective composition comprising antarctic endophytes, which can be used in crops, in normal and drought conditions

Abstract

The present invention relates to agronomy, preferably to a biostimulant bioprotective composition comprising a mixture of microorganisms that can be used to increase the resistance of plant species cultivated under different types of stress, including water and heat stress, and under normal / standard irrigation conditions, wherein the plant species are selected from agroforestry species, native woodlands, ornamental plants and grasses, and, more preferably and additionally, no-till or transplanted agricultural crops. More preferably, the invention relates to a composition comprising a mixture of isolated Antarctic endophytic fungi selected from Penicillium brevicompactum, deposit number RGM 3549 of 17 January 2024 of the Chilean Collection of Microbial Genetic Resources (CChRGM) and Penicillium chrysogenum, deposit number RGM 3550 of 17 January 2024 of the CChRGM.

Description

[0001] BIOSTIMULANT-BIOPROTECTIVE COMPOSITION INCLUDING ANTARCTIC ENDOPHYTES, USEFUL IN CROPS, UNDER NORMAL AND DROUGHT CONDITIONS

[0002] TECHNICAL SECTOR

[0003] The present invention relates to agronomy, preferably to a biostimulant-bioprotective composition comprising a mixture of microorganisms useful for conferring resistance to crops subjected to cultivation under different types of stress, including water and heat stress, and under normal / standard irrigation conditions, where said plant species are selected from silvo-agricultural species, native forests, ornamental plants and grasses, and more preferably and additionally, agricultural crop plants either direct sowing or indirect sowing (transplant) such as vegetables selected from lettuce, spinach, paprika and chard, or fruit crops, under normal and drought conditions. More preferably, the present invention relates to a biostimulant-bioprotective composition comprising a mixture of endophytic fungi, useful for improving the capacity of plants to resist drought stress.Even more preferably, said endophytic fungi correspond to a mixture of isolated Antarctic endophytic fungi selected from Penicillium brevicompactum, deposit number RGM 3549 of January 17, 2024, of the Chilean Collection of Microbial Genetic Resources (CChRGM) and Penicillium chrysogenum, deposit number RGM 3550 of January 17, 2024, of the Chilean Collection of Microbial Genetic Resources (CChRGM).

[0004] STATE OF THE ART

[0005] Recent studies show that functional symbiosis, defined as "the permanent association between two or more distinct organisms, at least during part of their life cycle," is one of the most important and novel strategies for improving crop production or yield. Although symbiosis is an ancient biological phenomenon, known to be a ubiquitous and important aspect of life on Earth, most studies fail to consider the fact that all plants in natural ecosystems are symbiotic with microbial endophytes (bacteria, yeasts, and fungi), and that these endophytes can have considerable effects on plant nutritional status and yield in stressful environments. Recent research suggests that these positive interactions are especially important where abiotic stress is greatest, particularly those related to water scarcity or drought.Regarding the latter, the places on Earth with the most adverse environmental conditions for plant life correspond to the extreme zones of the Atacama Desert and its mountains, and the Antarctic continent, where survival conditions are limited by low temperatures, desiccation, abrasive winds, high radiation, and low availability of water and nutrients. However, even in this extreme environment, endophytes have been found capable of providing benefits to the vegetation native to these hostile environments. The latest published reviews highlight the benefits of using microorganisms in crops subjected to various types of stress, but to date, there is no information involving Antarctic endophytic fungi in the formulation of inoculants that can improve plants' ability to resist drought stress, acting as biostimulants and bioprotectants.

[0006] This research addresses the need for biostimulants and bioprotectants for plant species such as plants (whether agricultural crops or ornamental plants), trees (native, forest or fruit trees) or grasses under water stress conditions that allow the aforementioned species to grow and develop as if they were in normal conditions.

[0007] Thus, the present invention relates to the isolation and selection of a mixture of endophytes from the extreme Antarctic zone for use as biostimulants and bioprotectants for plant species, especially when they are under conditions of water stress. There are no solutions that allow plant species such as those mentioned above to tolerate stress due to drought and heat stress. The present invention offers an alternative biostimulant and bioprotectant to improve the capacity of plants to resist drought stress.

[0008] Among the patent documents it is possible to mention US20180177196A1 (Texas A&M University System) which discloses a synthetic composition that improves the pest resistance of a plant when it is heterologously arranged in an element of the plant, if compared to the pest resistance in the absence of said synthetic composition, comprising: a) a fungal endophyte comprising at least one endophyte of the genus PurpureocilHunr, and b) at least one carrier, where said fungal endophyte is in contact with the carrier. The carrier can be selected from alginic acid, carrageenan, dextrin, dextran, pelgel, polyethylene glycol, polyvinylpyrrolidone, methylcellulose, polyvinyl alcohol, gelatin or combinations thereof. The synthetic composition further comprises water, a detergent, an insecticide, a fungicide or combinations thereof. The fungal endophyte comprises fungal spores, and may be present at a concentration of around 10 2 , 10 3 , 104 , 10 5 , 10 6 , 10 7 , 10 8 o 10 9colony forming units (CFU) per gram or spores per gram and the plant element can be selected from a dicotyledonous plant such as soybean or cotton, a seed, a leaf tissue or a root tissue. The fungal endophyte has at least 97% sequence identity with SEQ ID No.:94. The pest can be selected from nematodes or hemipteran insects, in particular, the pest can be selected from Nezara viridula, Lygus hesperus or Aphis gossypi. Resistance is observed in one or more of the following characteristics: increased stand, improved survival, increased plant height, increased shoot biomass, increased root biomass, decreased disease presence, increased leaf area, decreased pest abundance, decreased pest biomass, increased yield, increased vigor or improved resistance to pathogenic bacteria, fungi or viruses.It also refers to a method of improving one or more plant phenotypes by inoculating a plant element with the composition described above.

[0009] CN116925920A (Shenyang Enzhi Research Institute Co ltd) describes the use of the fungus Fusarium graminearum EC220315, deposit number GDMCC No.62304 at the Guangdong Provincial Microbiological Bacterial Collection Center, cultivated and grown at 25°C and pH 6-8, to increase resistance to bacterial or fungal pests, particularly wheat scab. The composition optionally comprises additional components selected from one or more of an insecticide, a nematicide, an acahcide, a bactericide, a fungicide, a herbicide, a growth regulator, a disinfectant, a repellent, an attractant or a biological pesticide, wherein the ratio of Fusarium graminearum EC220315 cultures to additional components is 1:100-100:1. The Fusarium graminearum EC220315 culture is a sterile culture solution, fermentation broth, metabolite or compounds separated from the sterile culture, extract, among others.

[0010] CN106061243B (University of Guelph) describes a method that confers resistance to drought stress by improving the trait of a plant (vegetable lamb) and enhances its growth, which comprises contacting plant seed with dendritic branched spores from buds of C. uredinicola and Epicoccum nigrum, and effectively adjusts to the natural Alternaria endophyte for vegetable lamb.

[0011] US8101551 B2 (Adjuvants Plus Inc) describes a method for stimulating and providing an additive effect with rhizobia on the production of nitrogen-fixing nodules in vegetables and improving their growth, by applying a stabilized endophyte inoculant composition comprising vegetative or conidial phase of Clonostachys rosea in mixture with an agronomically acceptable stabilizing carrier, which can be selected from an emulsifier, which in turn can be selected from a biologically derived emulsifier, and which more particularly can be selected from cereal grain, or an extract or flour thereof. The composition is a water-based spray composition or a powder, and has a pH of 6 to 8 and is at a concentration of 10 6 to 10 12 spores / gram of composition. Inoculation benefits roots, cuttings, or transplants when applied alone or in conjunction with indole-3-butyl acid (IBA).

[0012] CN106399131 A (Flourishing bio tech ltd, Shandong) describes the use of an inoculum of Penicillium rubens, accession number CGMCC No.: 13189 of the Center for Microorganism Management Joint Committee on Plant Preservation, Bacterial and Microbial Diseases, specifically a fermented mycelial ethanolic extract, to promote culture growth or increase its yield, where the fermentation medium employed consists of potato extract (1000 ml), yeast extract (1.0 g), peptone (3.0 g), glucose (15.0 g) and agar (17.0 g), and the inoculum concentration is 5-15%, at a fermentation temperature of 20-30°C and cultures of 4-6 days. The crops are selected from peanut, cowpea and radish.

[0013] US10765084B2 (University College Dublin, National University of Ireland, Dublin, The Provost, Fellows, Foundation Scholars, & The other members of Board, of the College of the Holy & Undivided Trinity of Queen Elizabeth, Near Dublin) describes a seed coating composition for use in (a) increasing grain yield and dry sprout weight in a plant, comprising a mixture of fungal root endophytes isolated from a plant root and a carrier medium, or (b) increasing tolerance to the development of seed-borne fungal infections in germinated or non-germinated seeds, and comprising a mixture of fungal root endophytes isolated from a plant root sprouted from said seed and a carrier medium for application to the seed, wherein the endophyte is characterized by 100% sequence identity to the nuclear ribosomal internal transcribed spacer (nrlTS), and the endophyte is isolated from the plant Hordeum murinum,and where plants and seeds are under reduced nutrient conditions or stressed by drought or multiple stresses. The concentration of the fungal endophyte isolate is between 0.001% (w / v) and 1.0% (w / v). The carrier medium is selected from water, distilled water, sterilized water, an emulsified suspension, a moist powder, encapsulation of fungal endophyte spores in alginate beads, or a film coating. The seed may be coated, mixed, or encapsulated with the composition.

[0014] Therefore, in the state of the art, there is a need to provide a natural biostimulant-bioprotective composition, preferably a biostimulant-bioprotective composition comprising a mixture of Antarctic endophytes, useful for providing greater resistance to plant species subjected to cultivation under different types of stress, including water and thermal stress, and under normal / standard irrigation conditions.

[0015] BRIEF DESCRIPTION OF THE INVENTION

[0016] The present invention relates to a natural biostimulant-bioprotective composition, preferably a biostimulant-bioprotective composition based on a mixture of microorganisms, which confers greater resistance to plant species subjected to cultivation under different types of stress, including water and thermal stress, and under normal / standard irrigation conditions.

[0017] The invention relates to the isolation and selection of a mixture of endophytes obtained from native Antarctic plants, Colobanthus quitensis and Deschampsia antartica. The isolated endophytes were identified through molecular identification. The sequence of each endophyte was analyzed using MegaBLAST (Basic Local Alignment Search Tool) to determine the maximum identity percentage. The isolated strains were identified as Penicillium brevicom pactum, deposit number RGM 3549 of January 17, 2024, from the Chilean Collection of Microbial Genetic Resources (CChRGM), and Penicillium chrysogenum, deposit number RGM 3550 of January 17, 2024, from the Chilean Collection of Microbial Genetic Resources (CChRGM).

[0018] The aforementioned isolated strains were inoculated jointly into different plants, creating a symbiosis with the plant species. When water stress occurs, they allow the plants and their fruits to develop as if they were under normal irrigation conditions. Furthermore, their use allows water consumption to be reduced by up to 20% without affecting the production of the crops to which the endophyte mixture is applied. The plant species can be selected from silvo-agricultural species, native forests, ornamental plants and pastures, and more preferably and additionally, agricultural crop plants such as selected vegetables such as lettuce, spinach, bell peppers and chard, under normal and drought conditions. BRIEF DESCRIPTION OF THE FIGURES

[0019] Figure 1. Scheme of the inoculation treatment with Antarctic endophytes.

[0020] Figures 2A-2D. Growth of lettuce plants with and without endophyte inoculum. Fig. 2A) Inoculum and 100% water. Fig. 2B) Inoculum and 75% water. Fig. 2C) Without inoculum and 100% water. Fig. 2D) Without inoculum and 75% water. Inoculum corresponds to a mixture of Penicillium brevicompactum, accession number RGM3549, and Penicillium chrysogenum, accession number RGM3550, in a 1:1 ratio, at a concentration of 5000 spores / ml.

[0021] Figures 3A-3D. Survival of chard (Fig. 3A), spinach (Fig. 3B), lettuce (Fig. 3C) and bell pepper (Fig. 3D) with and without inoculum under normal irrigation conditions (Control) and under conditions of 25% less water (Drought).

[0022] Figures 4A-4D. Biomass generated by chard (Fig. 4A), spinach (Fig. 4B), lettuce (Fig. 4C) and bell pepper (Fig. 4D) with and without inoculum under normal irrigation conditions (Control) and under conditions of 25% less water (Drought).

[0023] Figures 5A-5D. Fiber generated by chard (Fig. 5A), spinach (Fig. 5B), lettuce (Fig. 5C) and bell pepper (Fig. 5D) with and without inoculum under normal irrigation conditions (Control) and under conditions of 25% less water (Drought).

[0024] Figures 6A-6D. Carbohydrates generated by chard (Fig. 6A), spinach (Fig. 6B), lettuce (Fig. 6C) and bell pepper (Fig. 6D) with and without inoculum under normal irrigation conditions (Control) and under conditions of 25% less water (Drought).

[0025] Figures 7A-7D. Proteins generated by chard (Fig. 7A), spinach (Fig. 7B), lettuce (Fig. 7C) and bell pepper (Fig. 7D) with and without inoculum under normal irrigation conditions (Control) and under conditions of 25% less water (Drought). DETAILED DESCRIPTION OF THE INVENTION

[0026] The present invention relates to a biostimulant-bioprotective composition based on a mixture of microorganisms useful for promoting resistance in plant species subjected to cultivation under different types of stress, including water and heat stress, and under normal / standard irrigation conditions, where said plant species are selected from silvo-agricultural species, native forests, ornamental plants and grasses and turf, and more preferably and additionally, agricultural cultivation plants under normal or drought conditions and where the water stress can be 80% or less of the normal irrigation water required for the cultivation of the plant species.

[0027] These native forests can be selected from Chilean native forests that include Alerce or Cahuén (Fitzroya Cupressoides), Algarrobo (Prosopis chilensis), Araucaria (Araucaria araucana), Bellota del norte (Beilschmiedia miersii), Boldo (Peumus boldus), Bollón (Kageneckia oblonga), Calafate (Berberís buxifolia), Canelo (Dhmys winteh), Chagual (Puya berteroniana), Chilco, Cypress of the Cordillera, Colihue (Chusquea culeou), Colliguay, Copihue (Lapageha rosea), Hawthorn (Acacia caven), Guayacán, Guindilla (Guindilla thnervis), Guindo Santo (Eucryphia glutinosa), Olivillo (Aextoxicon punctatum), Oak (Nothofagus obliqua), Coigüe (Nothofagus dombeyi), Hazelnut (Gomortega keule), Hazelnut (Gevuina avellana), and Eucryphia cordifolia, among others; and other native forests.

[0028] These ornamental plants can be selected from trees, shrubs, and garden species, both perennial, annual, and biennial, intended for cultivation in private gardens, public parks, or urban decoration; florist plants used as cut flowers; and indoor plants grown in pots for decoration in homes or public centers.

[0029] Such trees, shrubs and garden species can be selected from mimosa, cow's foot, Jupiter tree, crazy carob, azarollo, iron tree, Canada maple, Alpine laburnum, Junglas, silver willow, among others. Such florist plants can be selected from Azalea, Anthurium, Bromeliad, Bougainvillea, Calendula, Carnation, Chrysanthemum, Dahlia, Dandelion, Spathiphyllum, Lotus flower, Gardenia, Geranium, Gerbera, Jasmine, Kalanchoe, Lavender, Lily, Daisy, Orchid, Pansy, Petunia, Rose, Tulip, Verbena, among others.

[0030] Such indoor plants can be selected from ribbons, lilies, mother-in-law's tongue, jade tree, red anthurium, ficus, Adam's rib, aloe vera, among others.

[0031] These grasses and turf can be selected from alfalfa, clover, ryegrass, dactyl, oats, fescue, Bermuda grass, and bentgrass, among others.

[0032] These crop plants can be selected from agricultural crops. These agricultural crop plants can be selected from vegetables, cereals, legumes, oilseeds, fruit trees, among others. These agricultural crop plants are selected from agricultural crops, whether direct or indirect sowing (transplant), such as vegetables. Even more preferably, these direct sowing vegetables can be selected from carrots, corn, broad beans, peas, beans, garlic, among others. These indirect sowing vegetables can be selected from tomatoes, chard, lettuce, cabbage, cauliflower, onions, radishes, turnips, broccoli, spinach, bell peppers, among others. These cereal crops can be selected from rice, corn, oats, wheat, among others. These legume crops can be selected from beans, lentils, chickpeas, among others. These oilseed crops can be selected from rapeseed, sunflower, peanut, soybean, sesame, among others.These fruit crops can include table grapes, cherries, plums, blueberries, apples, avocados, pears, walnuts, almonds, among others.

[0033] More preferably, the present invention relates to a biostimulant-bioprotective composition comprising a mixture of endophytic fungi useful for promoting the ability of plant species to resist drought stress. Even more preferably, said endophytic fungi correspond to a mixture of isolated Antarctic endophytic fungi selected from Penicillium brevicom pactum, deposit number RGM 3549 of January 17, 2024, of the Chilean Collection of Microbial Genetic Resources (CChRGM) and Penicillium chrysogenum, deposit number RGM 3550 of January 17, 2024, of the Chilean Collection of Microbial Genetic Resources (CChRGM). Said plant species are as described above. Water stress comprises irrigation where the water supply to the plant is 80% or less compared to the water supply in normal irrigation for the cultivation of the plant.

[0034] The biostimulant-bioprotective composition of the present invention is used, which comprises a mixture of both endophytes (fungi, Penicillium brevicompactum, accession number RGM3549 and Penicillium chrysogenum, accession number RGM3550), preferably in a proportion in the range of 1:0.01 -0.01:1, even preferably a proportion of 1:1, and at a concentration in the range of 1 to 100,000 spores / ml, preferably a concentration of 5,000 spores / ml. The biostimulant-bioprotective composition is applied to the plant or one or more of its parts, in two stages, in a time range of between 7 and 20 days, preferably in a period of time of 14 days apart to ensure symbiosis.Before applying the first stage, the plant may be subjected to water stress, thereby ensuring that the symbiosis is realized to a greater degree. Water stress involves irrigation where the water supply to the plant is 80% or less compared to the water supply in normal irrigation for the plant's cultivation.

[0035] In the case of direct-seeded agricultural crop plants, the application of this biostimulant-bioprotectant composition should be carried out one week after the plant reaches the seedling stage, with the development of 3 to 4 leaves. For indirect-seeded or transplanted agricultural crop plants, the application of this biostimulant-bioprotectant composition should preferably be carried out on the same day as the transplant. Silvoagricultural management should be carried out normally, that is, using fertilizers, insecticides, and others.

[0036] This biostimulant-bioprotective composition is applied by direct irrigation to the plant, preferably to the stem of the plant, preferably applying 10 to 50 ml in the first stage, and applying an equal amount as a booster dose in the second stage, or by immersion of the roots of the plants or seedlings prior to transplanting.

[0037] This biostimulant-bioprotective composition is applied by direct irrigation to the plant, preferably to the stem, by sprinkling, spraying, drip irrigation, technical irrigation, nebulization, dispersion, spreading, channel irrigation, among others.

[0038] The present invention also relates to a method for promoting resistance in plant species subjected to cultivation under different types of stresses, including water and heat stress, and under normal / standard irrigation conditions, comprising: a) preparing a biostimulant-bioprotective composition comprising a mixture of endophytes selected from Penicillium brevicompactum, accession number RGM3549 and Penicillium chrysogenum, accession number RGM3550, preferably in a ratio in the range of 1:0.01-0.01:1, even more preferably, a ratio 1:1, and at a concentration in the range of 1 to 100,000 spores / ml, preferably a concentration of 5,000 spores / ml; and then b) applying the biostimulant-bioprotective composition prepared according to step a), on the plant or one or more parts thereof, by: b.1) direct irrigation, in a first stage, preferably, on the stem of the plant, and in a time range of between 7 and 20 days, preferably, in a time period of 14 days apart, applied as a second stage, once again by direct irrigation, and where before the first stage, the plant may have been subjected to water stress, and where the plant is a seedling; b.2) by direct irrigation, in a first stage, preferably, on the stem of the plant, after one week of the plant being in the seedling stage, with the development of 3 to 4 leaves, in a time range of between 7 and 20 days, preferably, in a time period of 14 days apart, and applied as a second stage, once again by direct irrigation, where before the first stage, the plant may have been subjected to water stress, where the plant is a sowing crop plant and where the plant is a seedling, or b.3) by direct irrigation, preferably at the stem of the plant, on the day of transplanting, where the plant is a transplanted crop, or by immersion of the roots of the plants or seedlings prior to transplanting. Where the crop plant species are as described above and the water stress involves irrigation where the water supply to the plant is 80% or less compared to the water supply in normal irrigation for the crop plant.

[0039] The present invention also relates to the use of a biostimulant-bioprotective composition comprising a mixture of endophytes selected from Penicillium brevicompactum, accession number RGM3549 and Penicillium chrysogenum, accession number RGM3550, in a ratio in the range of 1:0.01-0.01:1, preferably a ratio of 1:1, at a concentration in the range of 1 to 100,000 spores / ml, preferably a concentration of 5,000 spores / ml, to promote resistance in crop plant species subjected to cultivation under different types of stresses, including water and heat stress, and under normal / standard irrigation conditions, wherein said plant species are as described above and the water stress comprises irrigation where the water supply to the plant is 80% or less compared to the water supply in normal irrigation for the cultivation of the species. vegetables.

[0040] If irrigation needs to be reduced, it can be reduced by up to 20% during the season and production results will not be affected, as shown in the examples described below.

[0041] The application of the present biostimulant-bioprotective composition was tested on various plants, in greenhouses and in field trials. Following the treatment scheme shown in Figure 1 , lettuce, chard, spinach and bell pepper plants were inoculated. The images in Figures 2A-2D show the benefits of applying the present biostimulant-bioprotective composition comprising a mixture of the fungi Penicillium brevicompactum, accession number RGM3549 and Penicillium chrysogenum, accession number RGM3550), when growing plant species under drought conditions (25% less water than normal irrigation).

[0042] The results obtained (Figures 3A-3D and 4A-4D) clearly show the positive effect on plant development when applying the biostimulant-bioprotective composition of the present invention to chard, spinach, lettuce, and bell pepper. When the plants are subjected to normal irrigation conditions, both lettuce and chard are more likely to develop / grow when the present biostimulant-bioprotective composition has been applied to them. This positive effect is increased when the samples are subjected to stress due to drought as described above. Also, when the plants are inoculated with the present biostimulant-bioprotective composition, the amount of biomass produced by the plants is greater than when they are not inoculated, the biomass comprising one or more of roots, stems, leaves, shoots, among others.

[0043] When a proximal analysis of the main nutritional components (fiber, carbohydrates, proteins) of the plants inoculated with the biostimulant-bioprotective composition of the present invention and without inoculation (where it has not been applied), subjected to normal and drought conditions, it is observed that the presence of the present biostimulant-bioprotective composition (inoculum, endophytes) delivers a greater quantity of nutrients than those without the presence of the present biostimulant-bioprotective composition, a positive effect that is accentuated when the plants are subjected to drought conditions (see Figures 5A-5D, 6A-6D and 7A-7D). APPLICATION EXAMPLES

[0044] Example 1: Isolation of endophytes

[0045] Twenty-five plants of each of the aforementioned species (Colobanthus quitensis and Deschampsia antartica) were collected from King George Island, Antarctica (62°09'S; 50°28'W) during the summer. Roots were surface-sterilized by successive immersions of 1 minute in ethanol (70%) and 3 minutes in sodium hypochlorite (2%), followed by rinsing in distilled water for 2 minutes. Roots were placed on Petri dishes containing potato dextrose agar (PDA) supplemented with chloramphenicol at 100 µg ml / l. The plates with roots were incubated for 60 days at 18°C. Emerging fungi were transferred to potato dextrose agar (PDA). Growing hyphae were reinoculated onto new plates containing fresh potato dextrose agar (PDA) medium. The isolates were maintained in this routine until spore isolation was obtained. Individual colonies obtained were stored at 4°C.

[0046] Example 2: Identification of fungi

[0047] The fungi Penicillium brevicompactum, accession number RGM3549, and Penicillium chrysogenum, accession number RGM3550, were identified from individual colonies isolated according to Example 1. For molecular identification of the endophytes isolated from roots, the ITS regions (including ITS1 , ITS2 , and the intercalary 5.8S rRNA gene) and LSU (the 28S large subunit nuclear rRNA gene) were amplified. DNA was extracted from actively growing mycelia using the EZNA Fungal DNAMiniKit (Omega-Biotek). The ITS region was amplified using ITS5 (5'- GGAAGTAAAAGTCGTAACAAGG-3') and ITS4 (5'-

[0048] TCCTCCGCTTATTGATATGC-3') as forward and reverse primers, respectively. The LSU region was amplified using LR0R (5'- GTACCCGCTGAACTTAAGC-3') and LR06 (5'- CGCCAGTTCTGCTTACC-3') as forward and reverse primers, respectively. Each PCR reaction was carried out in a 15 pl volume containing 30-50 ng of DNA, of a tenfold diluted PCR buffer from a stock solution, containing 2 mM MgCl2, 0.1 pM of each dNTP, 0.5 pM of forward and reverse primers, and one unit (1 U) of Taq DNA polymerase. PCR amplifications were performed with an initial denaturation of 4 min at 94°C, followed by 35 cycles of 30 s at 94°C, 60 s at 50°C, and 60 s at 72°C, followed by a final step of 5 min at 72°C. After this, the PCR product was purified and both strands were sequenced using Macrogen sequencing service (Seoul, Korea). After sequencing, forward and reverse sequence fragments were edited using Geneious v5.4 software.The sequence of each isolated endophyte was analyzed with MegaBLAST (Basic Local Alignment Tool) (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi) to determine the maximum identity percentage and total scores with the sequences of that global database. Finally, the obtained sequences (ITS1 -5.8S-ITS2 and 28S) were assembled and deposited in the Gene-Bank database. The main results of maximum identity percentage and total scores are shown below: The first isolate accession number RGM3549 was identified as Penicillium brevicompactum, showing 100% similarity with P. brevicompactum accessions in both the ITS (e.g., KF156318.1 ; total score = 670) and LSU regions (e.g., JN938947.1 ; total score = 1579). While the second, accession number RGM3550, was identified as Penicillium chrysogenum since it showed 100% similarity with P.chrysogenum in both the ITS (e.g., KF5784432.1 ; total score = 627) and LSU (e.g., KF417576.1 ; total score = 1705) regions.

[0049] Example 3: Mushroom cultivation

[0050] The Penicillium brevicompactum strains, accession number RGM3549, and Penicillium chrysogenum, accession number RGM3550, are grown on PDA or sterilized rice in a controlled humidity environment at 80% and 20°C. After 5 days of growth, they are harvested. Example 4: Inoculum preparation

[0051] The present biostimulant-bioprotective composition of a mixture of selected endophytes of Penicillium brevicompactum, access number RGM3549 and Penicillium chrysogenum, access number RGM3550, or inoculum, is prepared by adding each of the two fungi, in a 1:1 ratio, at a concentration of 5,000 spores / ml.

[0052] Example 5: Plant Inoculation

[0053] The inoculum has been tested on various plant species in greenhouses and field trials. Following the treatment scheme shown in Figure 1, lettuce, chard, spinach, and bell pepper plants were inoculated.

[0054] 10 ml of the present biostimulant-bioprotective composition of the present invention is used as inoculant. Before the first inoculation, the plant was subjected to stress, that is, inoculation was carried out during transplantation; after 12 days, watering was stopped to generate drought stress; and after 2 more days, the booster inoculation was carried out.

[0055] Root and leaf characteristics were observed in a lettuce plant. Table 1 summarizes the crop comparison. See Figs. 2A-2D

[0056] Table 1 1The present biostimulant-bioprotector composition comprising a mixture of Penicillium brevicompactum, accession number RGM3549 and Penicillium chrysogenum, accession number RGM3550, in a 1:1 ratio and at a concentration of 5000 spores / ml.

[0057] In addition, the survival rate of chard, spinach, lettuce, and pepper crops was measured after 14 days of cultivation. See Figs. 3A-3D

[0058] Table 2

[0059] 2 The present biostimulant-bioprotector composition comprising a mixture of Penicillium brevicompactum, accession number RGM3549 and Penicillium chrysogenum, accession number RGM3550, in a 1:1 ratio and at a concentration of 5000 spores / ml, and 25% less irrigation than the control

[0060] Likewise, the biomass (grams) of the chard, spinach, lettuce, and pepper crops was measured after 60 days of cultivation. See Figs. 4A-4D. The biomass was determined by directly weighing the total mass of the plants (chard, spinach, and lettuce) on a scale (METTLER TOLEDO), removing the roots. In the case of peppers, the fruits from 10 plants subjected to each treatment were weighed and averaged. Table 3

[0061] 1 Control, cultivation without inoculum and normal irrigation

[0062] 2 The present biostimulant-bioprotector composition comprising a mixture of Penicillium brevicompactum, accession number RGM3549 and Penicillium chrysogenum, accession number RGM3550, in a 1:1 ratio and at a concentration of 5000 spores / ml, and 25% less irrigation than the control

[0063] Likewise, the amount of fiber (grams) of the chard, spinach, lettuce, and pepper crops was measured after 60 days of cultivation. See Figs. 5A-5D. The amount of crude fiber was measured using the AOAC method (1990). First, distilled water and the prepared solutions of 1.25% H2SO4 (MERCK) and 1.25% NaOH (MERCK) were heated. Approximately 4 g of sample were weighed into a 500 ml round-bottom flask. Subsequently, 250 ml of diluted H2SO4 and a few drops of octanol (MERCK) were added. The mixture was boiled under reflux for 30 minutes, after which a porcelain fragment was added to regulate the boiling point. The flask was removed from the hotplate, stopped boiling by adding 50 ml of cold water, and filtered through a Buchner funnel, where a kitasate connected to a vacuum pump was used. The residue was washed (5 times) with 100 ml of hot distilled water until the acidic reaction in the filtrate ceased.The residue was then quantitatively transferred back to the round-bottom flask, and 250 ml of the diluted NaOH solution was added. The mixture was boiled for 30 minutes, followed by 50 ml of cold water, which was then immediately filtered through a Buchner funnel using a kitasato connected to a vacuum pump. The residue was washed with hot water until the wash waters reached neutrality.

[0064] Finally, the residue was quantitatively transferred to a Petri dish, broken up if necessary, and dried in a KALSTEIN oven at 110 °C to constant weight. It was then allowed to cool in a desiccator and quickly weighed.

[0065] The crude fiber content of the sample was determined using the following equation:

[0066] % Crude Fiber = P2 x 100 Pi where:

[0067] P1: Weight (g) of the initial sample.

[0068] P2: Weight (g) of the residue, after drying.

[0069] Table 4

[0070] 1 Control, cultivation without inoculum and normal irrigation

[0071] 2 The present biostimulant-bioprotector composition comprising a mixture of Penicillium brevicompactum, accession number RGM3549 and Penicillium chrysogenum, accession number RGM3550, in a 1:1 ratio and at a concentration of 5000 spores / ml, and 25% less irrigation than the control. Additionally, the amount of carbohydrates (grams) of the chard, spinach, lettuce and pepper crops was measured after 60 days of cultivation. See Figs. 6A-6D. The amount of carbohydrates was determined using the AOAC method. The difference method was used to determine carbohydrates, using the following formula:

[0072] % HDC = 100 — (%Moisture + %Fiber + %Lipids + %C proteins + %Proteins) Table 5

[0073] 1 Control, cultivation without inoculum and normal irrigation

[0074] 2 The present biostimulant-bioprotector composition comprising a mixture of Penicillium brevicompactum, accession number RGM3549 and Penicillium chrysogenum, accession number RGM3550, in a 1:1 ratio and at a concentration of 5000 spores / ml, and 25% less irrigation than the control

[0075] Finally, the protein content (grams) of the chard, spinach, lettuce, and pepper crops was measured after 60 days of cultivation. See Figs. 7A-7D. The protein content was measured using AOAC 991.20 methodology. Approximately 2.5 g of homogenized sample was weighed on an analytical balance (CIENTEC brand, model ES / 22OA), which was then placed in a Kjeldahl digestion tube, and 5 glass beads, selenium catalyst, 1 g of hydrogen peroxide (MERCK), and 25 ml of concentrated sulfuric acid (MERCK) were added. The tube was then connected to the digestion trap (Buchi, 425) and to the heating mantle, where the temperature was raised proportionally until it reached its maximum level, avoiding abrupt bubbling. Once the contents inside the tube changed to a transparent color, it was left to boil for 15 to 20 minutes.Once the digestion was complete, the plate was turned off and the tube was allowed to cool without disconnecting the heating trap for 20 minutes to eliminate the toxic vapors generated by the reaction.

[0076] Once the mixture was cooled, the distillation process began. During the distillation process, 25 to 30 ml of distilled water and 1% phenolphthalein indicator were added to the Kjeldahl tube, which was then connected to the Kjeldahl distillation equipment's trumpet. Once connected, 32% sodium hydroxide (MERCK) (previously prepared) was slowly injected until the color changed (from clear to fuchsia).

[0077] The distillate was received in a flask containing 30 ml of 3% boric acid (MERCK), 4 to 5 drops of Tashiro indicator and 100 ml of distilled water, which was subsequently titrated with 0.1 N sulfuric acid until the color turned green to violet.

[0078] To determine the protein content, the percentage of nitrogen was first calculated and then the percentage of protein using the following equations:

[0079] % N-0.014 x N x VX100 % Protein-% N x6, 25 m

[0080] % Protein = % N x 6.25

[0081] Where:

[0082] V: 0.1 N H2SO4 flow rate - blank flow rate m: mass (g) of the sample.

[0083] N: normality of the reagent, 0.1 N

[0084] Factor: 6.25.

[0085] Table 6

[0086] 1 Control, cultivation without inoculum and normal irrigation

[0087] 2 The present biostimulant-bioprotector composition comprising a mixture of Penicillium brevicompactum, accession number RGM3549 and Penicillium chrysogenum, accession number RGM3550, in a 1:1 ratio and at a concentration of 5000 spores / ml, and 25% less irrigation than the control.

Claims

CLAIMS 1) Biostimulant-bioprotective composition to promote resistance in resistance in cultivated plant species subjected to different types of stresses, including water stress and heat stress, and preferably, water stress, and under normal / standard irrigation conditions, CHARACTERIZED in that it comprises a mixture of selected isolated Antarctic endophytic fungi of Penicillium brevicompactum, deposit number RGM 3549 of January 17, 2024, from the Chilean Collection of Microbial Genetic Resources (CChRGM) and Penicillium chrysogenum, deposit number RGM 3550 of January 17, 2024, from the Chilean Collection of Microbial Genetic Resources (CChRGM). 2) The biostimulant-bioprotective composition of claim 1 CHARACTERIZED in that the proportion of Penicillium brevicompactum, deposit number RGM 3549 of January 17, 2024, of the Chilean Collection of Microbial Genetic Resources (CChRGM) to Penicillium chrysogenum, deposit number RGM 3550 of January 17, 2024, of the Chilean Collection of Microbial Genetic Resources (CChRGM) is in the range 1:0-0:1 at a concentration in the range of 1 to 100,000 spores / ml. 3) The biostimulant-bioprotective composition of claim 1 CHARACTERIZED in that the proportion of Penicillium brevicompactum, deposit number RGM 3549 of January 17, 2024, of the Chilean Collection of Microbial Genetic Resources (CChRGM) to Penicillium chrysogenum, deposit number RGM 3550 of January 17, 2024, of the Chilean Collection of Microbial Genetic Resources (CChRGM) is 1:1 at a concentration of 5,000 spores / ml. 4) Method to promote resistance in plant species subjected to cultivation under different types of stress, including water and thermal stress, where water stress comprises irrigation where the water supply to the plant is 80% or less compared to the water supply in normal irrigation for the cultivation of the plant, and under normal / standard irrigation conditions, CHARACTERIZED in that it comprises: a. preparing a biostimulant-bioprotective composition comprising a mixture of endophytes selected from Penicillium brevicompactum, accession number RGM3549 and Penicillium chrysogenum, accession number RGM3550; and b. applying the biostimulant-bioprotective composition prepared according to step a), to the plant or one or more of its parts, by: b.1 ) direct irrigation, in a first stage, in step a), and in a time range of between 7 and 20 days, and applying it as a second stage, once again by direct irrigation, and where the plant is a seedling; b.2) by direct irrigation, in a first stage, after one week of the plant being in the seedling stage, with the development of 3 to 4 leaves, in a time range of between 7 and 20 days, and applying it as a second stage, once again by direct irrigation, where the plant is a sowing crop plant and where the plant is a seedling; or b.3) by direct irrigation, in a first stage, on the day on which the transplant is carried out, where the plant is a transplant crop plant, or by immersion of the roots of the plants or seedlings prior to transplanting. 5) The method of claim 4 CHARACTERIZED in that, in step a), the proportion of endophytes in the mixture, Penicillium brevicompactum, accession number RGM3549 and Penicillium chrysogenum, accession number RGM3550, is in the range of 1:0-0:1 and at a concentration in the range of 1 to 100,000 spores / ml. 6) The method of claim 5 CHARACTERIZED in that, in step a), the proportion of endophytes in the mixture, Penicillium brevicompactum, accession number RGM3549 and Penicillium chrysogenum, accession number RGM3550, is 1:1, and at a concentration of 5,000 spores / ml. 7) The method of claim 4 CHARACTERIZED in that, in step b.1) or in step b.2) or in step b.3), the biostimulant-bioprotective composition is applied to the stem of the plant. 8) The method of claim 4 CHARACTERIZED in that, in step b.1) or in step b.2), the second application of the biostimulant-bioprotective composition is carried out on day 14. 9)The method of claim 4 CHARACTERIZED in that, in step b.1) or in step b.2), before the first step, the plant is subjected to water stress. 10) Use of a biostimulant-bioprotective composition comprising a mixture of endophytes selected from Penicillium brevicompactum, access number RGM3549 and Penicillium chrysogenum, access number RGM3550, CHARACTERIZED in that it serves to promote resistance in crop plant species subjected to cultivation under different types of stress, including water and heat stress, and under normal / standard irrigation conditions, and where the water stress comprises irrigation where the water supply to the plant is 80% or less compared to the water supply in normal irrigation for the cultivation of the plant species, and under normal / standard irrigation conditions. 11) The use of claim 10 CHARACTERIZED in that the proportion, in the mixture of endophytes selected from, Penicillium brevicompactum, access number RGM3549 and Penicillium chrysogenum, access number RGM3550, is in the range of 1:0.01 - 0.01:1, at a concentration in the range of 1 to 100,000 spores / ml. 12) The use of claim 11 CHARACTERIZED in that the proportion, in the mixture of endophytes selected from, Penicillium brevicompactum, access number RGM3549 and Penicillium chrysogenum, access number RGM3550, is 1:1, at a concentration of 5,000 spores / ml. 13) The use of claim 10 CHARACTERIZED in that the crop plant species are selected from silvo-agrahic species, native forests, ornamental plants and grasses and turf, and additionally, agricultural crop plants under normal or drought conditions and where water stress can be 80% or less of the normal irrigation water required for the cultivation of the plant species.

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