WO2020214018A1 - Souches bactériennes endophytes, mélange, produit et méthode pour compenser le microbiote et lutter contre la pourriture dans des végétaux - Google Patents

Souches bactériennes endophytes, mélange, produit et méthode pour compenser le microbiote et lutter contre la pourriture dans des végétaux Download PDF

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WO2020214018A1
WO2020214018A1 PCT/MX2019/000040 MX2019000040W WO2020214018A1 WO 2020214018 A1 WO2020214018 A1 WO 2020214018A1 MX 2019000040 W MX2019000040 W MX 2019000040W WO 2020214018 A1 WO2020214018 A1 WO 2020214018A1
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plant
bud
rot
microorganisms
mixture
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Cristóbal FONSECA SEPÚLVEDA
Miguel Juan BELTRÁN GARCÍA
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Fonseca Sepulveda Cristobal
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor

Definitions

  • the present invention is related to the technical fields of Agriculture and Biotechnology, since it refers to endophytic bacterial strains, to a mixture, a product and a method, to compensate the microbiota and control the rotting disease in vegetables.
  • Microorganisms can be found in different parts of the plant such as episphere that includes the rhizosphere (10 mm next to the root) and the phyllosphere (surface of the leaves), the endosphere (which can be in the leaves or in the root) and in the soil attached to the plant.
  • Diazotrophic bacteria have been found associated with agaves, whose soils are characteristic for their low nitrogen content.
  • communities belonging to the Phylum Protaobactaria, Actinobacteria and Acidobactar ⁇ a are the dominant ones.
  • the communities of bacteria in the rhizosphere and phyllosphere are mainly influenced by the agave species, while in the endosphere they are affected by the season (Desgarennes atal., 2014).
  • A. taquilana contains communities of the order of Pseudomonadalas and Enterobactariales.
  • the communities of microorganisms associated with the two native agaves behave similar for biogeographic factors, suggesting that abiotic factors play an important role in the organization of the microbiome of A. tequilana (Coleman-Derr at al., 2015).
  • A. tequilana Of 65 bacteria, 51 were present in the two species, of which 18 bacteria are dlazotrophic, 10 in A. salmiana and 8 in A. tequilana. All members of A. taquilana were g-Proteobacterlas, 7 of which were Enterobacteriacaae and 1 Stenotmphomonas. The A. taquilana and A. salmiana plants share 78.5% of their bacteria. Soil-associated bacteria communities were affected by the season. It was suggested that the diversity of the bacteria may become higher in wild A. salmiana than in A. tequilana, since A. salmiana is found in natural environments coexisting with cacti and other plants and A.
  • Endophytic microorganisms are those that live within plant tissue without causing apparent damage. From the mother plant they can potentially be inherited to the suckers, remaining in the internal tissues despite the sterilization of the surface (Coleman-Derr at al., 2015). Micropropagation techniques in plants can reduce endophyte levels, which results in plants being less resistant to biotic or abiotic stresses and more dependent on the application of agrochemicals. Endophytic bacteria can protect the plant and provide nutrients that are difficult to obtain, since they can have properties such as nitrogen fixation, plant growth promotion (PGPB), auxin production, phosphate solubilization and may even have antifungal activity.
  • PGPB plant growth promotion
  • Endophytic bacteria can represent an alternative to improve growth and reduce diseases in plants.
  • tissue of the A. tequilana plant a population density of 3 million CFU / g of fresh plant tissue was found. (Mart ⁇ nez-Rodr ⁇ guez et al., 2014).
  • Badllus tequilansis is a Gram-positive, aerobic, spore-forming bacterium that was first isolated from a 2,000-year-old grave specimen located near Tequila, Jalisco, Mexico. When analyzing the 16S rRNA gene of this bacterium, it was found that it has a 99% similarity in its sequence with Badllus subtilltls, a bacterium that has been reported as endophyte.
  • Badllus tequilansis shows a yellow pigmentation in its growth, it is positive for Inulin and it is denitrifying (capable of reducing nitrate to nitrogen gas) (Gatson et al., 2006).
  • A. tequilana The reduction in the production of A. tequilana is mainly due to two diseases that can occur simultaneously during the year: a) soft rot of the bud caused by bacteria during the months of November-March and b) wilt caused by Fusarium oxysporum that affects the root during June-October, these diseases affect 30% of the plants (Alegr ⁇ a Anda González, 1998).
  • the disease of bud rot causes necrotic and watery lesions of the leaves, starting at the apical spine and progressing towards the bud, reaching the blunder and causing death. This disease is divided into four grades, which are visual scales.
  • Grade 2 is the plant with dry rot, usually at the tip of the bud, some watery black spots on the leaves and leaf curl up to a third.
  • grade 3 the plant frequently has leaves with black, watery spots and leaf curl up to two thirds.
  • grade 4 the bud rot reaches the apex of the blubber, presents a commonly fetid odor of the bud and the leaves are almost completely rolled (Gil Virgen, et al., 2009).
  • Soft rot or bud rot is characterized by the presence of a purple color and macerated tissues of the affected area of the plant and a foul odor (CRT, 2005). The lesions advance towards the center of the leaves, whose lamina can become wrinkled and have a purple color, the rot reaches the blunder until causing the death of the plant (Vélez-Gutiérrez, 1997).
  • This disease can appear in the suckers from the beginning of the plantation.
  • the spread of the disease from one plant to another is favored under conditions of rain combined with winds, especially when the temperature conditions are below 10 ° C. In these conditions, the movement of exudates from lesions to entry points in healthy plant leaves is facilitated (Avila-Miranda, 2011).
  • the agave is a plant with a high sugar content (20%), due to this, it is a good food for insects and microorganisms.
  • Badllus pumilus was recently described as a cause of soft rot of the bud of A. tequilana, where the presence of P. carotovora was also ruled out (Cen-Caamal, 2012).
  • patent document W02016057991 (A1) describes a method for the production of plant microbiomes comprising Flrmicutes, Actinobacteria and Proteobacteria, but there is no evidence of the isolation of strains of the microorganisms used in said procedure;
  • patent document US2018002244 (A1) describes a composition comprising strains of the same Badllus genus, solvents and a urease inhibitor, but there is no combination of other genera;
  • the patent document IN201637040339 (A) refers to an endophytic bacterium, specifically to an isolated strain of Badllus oryzicda YC7007 (KCCM 11275P), useful as a microbial agent as a fertilizer for the control of diseases caused by Pseudomonas glumae and other diseases in rice .
  • endophytic bacterial strains were isolated, from which 3 strains with better agronomic characteristics were selected. With these selected strains, a mixture useful in agriculture was developed; as well as a method was developed to control the compensation of the microbiota and the rot disease in vegetables.
  • Figure 1 illustrates the aspects of cultures by dilution method of aerobic microorganisms obtained from the liquefaction of the diseased bud of A. tequilana L. Weber, with 24 h of incubation at 36 ° C.
  • Figure 2 shows A. tequilana L. Weber plants used to obtain microorganisms associated with bud rot.
  • Figure 3 shows the number of colonies and their morphology of bacteria obtained from extracts of the diseased bud of A. tequilana L. Weber, by the blending method.
  • Figure 4 shows the number of colonies and their morphology of bacteria obtained from the extracts of the diseased bud of A. tequilana L. Weber, by the maceration method.
  • Figure 5 illustrates the percentages of the phyla of the cultivable bacteria obtained from the bud of A. tequilana L. Weber, with symptoms of soft rot.
  • Figure 6 shows the percentages of the different classes of Pmteobacteria found in the bud with soft rot symptoms of A. tequilana L. Weber isolated by the two methods used, bud blending and maceration.
  • Figure 7 illustrates an A. tequilana L. Weber plant with symptoms of soft bud rot in grade 3 severity, with its sucker.
  • Figure 8 shows the number of colonies and their bacterial morphology of the sucker derived from a plant with soft bud rot, of A. tequilana L. Weber.
  • Figure 9 shows the percentage of bacterial phyla of the shoot bud from a plant with symptoms of bud rot of A. tequilana L. Weber.
  • Figure 10 illustrates the percentages of the different types of Pmteobacteria found in the sucker bud from A. tequilana L. Weber, with symptoms of bud rot isolated by the maceradon method.
  • Figure 11 shows the rhizome of an A. tequilana L Weber plant with symptoms of soft bud rot in grade 3 severity.
  • Figure 12 illustrates the number of colonies and their morphology of root bacteria derived from an A tequilana L. Weber plant, with soft rot of the bud.
  • Figure 13 shows the percentage of bacterial root phyla from the plant rhizome with symptoms of bud rot of A. tequilana L. Weber.
  • Figure 14 illustrates the percentages of the different classes of Pmteobacteria found in the root of A. tequilana L. Weber, with symptoms of bud rot isolated by the maceration method.
  • Figure 15 illustrates the healthy bud of an A. tequilana L. Weber plant.
  • Figure 16 shows the number of colonies and their morphology of bacteria isolated from a healthy plant of A. tequilana L Weber.
  • Figure 17 shows the percentage of healthy bud bacterial phyla from A. tequilana L. Weber.
  • Figure 18 represents the percentages of the different classes of
  • Figure 19 illustrates the percentages of bacterial phyla found in different samples of Agave tequilana L. Weber, from the seed stage to the already developed plant stage.
  • Figure 20 illustrates a Bacillus safensis, Bacillus licheniformis, Enterobacter ludwigii, and Micrococus luteus compatibility test.
  • Microorganisms associated with the soft rot of the Agave tequilana L. Weber bud were identified, comparing them with the existing microbial communities in healthy plants, as well as suckers derived from the rhizome of diseased plants established in commercial plantations of up to four years. This in order to identify cultivable endophytic bacteria isolated from diseased plants with symptoms of soft rot of the bud; identify cultivable endophytic bacteria isolated from healthy plants; and analyze in the background of the identified microorganisms, the percentage of Proteobacteria, Firmicutes, Acti no bacteria and other bacteria in the analyzed plants.
  • Plants of A. tequilana L. Weber with symptoms of bud rot were studied, for which we proceeded to search in ranches in the Highlands of Jalisco, since they have been the regions that in recent years had more presence of this disease.
  • the samples were taken from two ranches located almost in the same geographic coordinates, in the ranch located in the municipality of Atotonilco el Alto at the following coordinates 20 ° 36'27.3 ,, N 102 ° 32'21.2 "W where samples of healthy and diseased agave were obtained, while at the other ranch located in the municipality of Arandas at the following coordinates 20 ° 43'14.1 "N 102 ° 25O1.2" W samples of diseased agave, sucker and root were obtained.
  • the two ranches present the same type of soil, a red clay soil, mainly due to the presence of iron, this type of soil it is considered as a soil rich in minerals.
  • EMB agar Eosin Blue Metlleno
  • ASOY trypticasein soy agar
  • the industrial blender was sterilized with 3% doro by immersion for 10 min and then with 70% alcohol immersed for another 10 min, followed of 2 washes with bidestiiada water. Once dry, the blender was placed in UV light for 15 min, inside the laminar flow hood.
  • the sample was dried on sterile paper towels for subsequent maceration in a sterile mortar.
  • the extract was carefully filtered with sterile gauze.
  • Figure 1 shows the isolates of aerobic microorganisms from the liquefied of diseased buds, which were subjected to 24 h incubation at 36oC.
  • A) is a 6 dilution in EMB medium; and B) a 6 dilution in CASOY. identification of microorganisms.
  • the MALDI-TOF mass spectrometry technique was used as methods and in some cases the 16s gene sequencing.
  • the strains were grown in CASOY medium for 24 h, incubated at 36oC, and the bacterial DNA was extracted from the isolated strains, with a DNA extraction kit (MOBIO) and following the methodology described in the extraction kit.
  • MOBIO DNA extraction kit
  • the amplification reaction was carried out in a total volume of 20 mL, composed of 1x PCR buffer, 1.5 mM MgCl2, 60 pM dNTPs, 0.2mL of the rD1 and fD1 primers; 27f and 1492 R-Y, 1.5 U of Taq DNA polymerase and 50 ng of genomic DMA.
  • the amplification product (20mL) was analyzed by electrophoresis on a 1.5% agarose gel, once the electrofbresis is complete, the gel already stained with Gelred 2mL, was visualized in a UV transluminator (Apollo).
  • PCR products were purified using the Zymo research kit and sequenced using the primers rD1 and 1492 R-Y. Sequence analysis was done with the Bioedit program. The comparison of the 16s RNA sequences was carried out through the Internet by depositing the sequences in the National Center for Biotechnology Information (NCBI) database and in the Ribosomal Database Project at the Michigan State University (RPD).
  • NCBI National Center for Biotechnology Information
  • RPD Ribosomal Database Project at the Michigan State University
  • the methodology used for the identification of bacteria by MALDI-TOF-MS was as follows: 1. 300 mL of ml of water were added to eppendorf tubes.
  • the samples were analyzed on a MALDI-TOF MS equipment with an LP 5-20KDa method, later they were analyzed with the MALDI-BIOTYPER software.
  • the highest number of CFUs was obtained when the plant material of the bud with symptoms of soft rot was liquefied. It was shown that the blending method is more effective than the maceration method, in terms of the isolation of microorganisms, as it is a more aggressive method, since the maceration method in the diseased bud sample turned out to be 21 times lower on average. the amount of CFU obtained, in comparison with the blending method, as shown in Table 1. In the case of the healthy bud, sucker and root, only the maceration method was used, because the tissue was very hard to do it in blender.
  • the amount of CFUs found in each culture medium and its form of Anaerobic and aerobic growth by the damaged bud liquefaction method was 116 * 000,000 for aerobic CASOY, 63.00000,000 for aerobic EMB, 29 * 000,000 for anaerobic CASOY and 10 * 800,000 for anaerobic EMB as shown in Table 1 , where it should be noted that there was more development in CASOY because it is not as selective a medium as EMB, as well as in the aerobic part, since most of the isolated microorganisms were growing with oxygen.
  • the types of colonies that were found were circular, irregular and some filamentous in CASOY anaerobic and aerobic, in general most of the colonies had a convex and flat surface as shown in figure 8.
  • the colonies of the aerobic and anaerobic EMB media were circular and filamentous in shape, with convex and flat surfaces, respectively, as well as rounded and filamentous edges.
  • the amount of CFU by the diseased bud maceration method was 2 * 790,000 for CASOY aerobic, 2 * 500,000 for EMB aerobic, 3 * 000,000 for CASOY aerobic and 2 * 040,000 for EMB anaerobic as shown in Table 1 , where it should be noted that there was more development in CASOY because it is not a culture medium as selective as EMB, the interesting thing is that more isolates were obtained under anaerobic than aerobic conditions, which indicates a greater presence of microorganisms capable of growing in environments with little or no oxygen.
  • the types of colonies that were found were circular, irregular and some filamentous in CASOY anaerobic and aerobic, with convex, flat and umbilicated surfaces, as well as some translucent as shown in figure 4.
  • the morphologies of the colonies obtained from the EMB media under aerobic and anaerobic conditions were circular and irregular with convex and umbilicated surfaces, as well as rounded and wavy edges.
  • the amount of CFU found in the sucker by the mashing method was 400 CFU / g for aerobic CASOY, 1,000 for aerobic EMB, 500 for anaerobic CASOY and 180 for anaerobic EMB as shown in ei Table 1.
  • the types of colonies that were isolated were circular and irregular in shape in the CASOY medium for anaerobic and aerobic conditions, with convex, flat and umbilicated surfaces, as well as some creamy yellowish as shown in figure 8.
  • the colonies of EMB media under aerobic and anaerobic conditions were circular, irregular, and punctate in shape with convex, umbilicated surface, as well as rounded, lobulated, and wavy edges.
  • the CFUs found using the mash method for healthy buds were 20,000 for aerobic CASOY, 300 for aerobic EMB, 50,000 for anaerobic CASOY and 100 for anaerobic EMB as shown in Table 1, where it should be noted that there was a greater development of CFUs found in CASOY aerobic and anaerobic compared to the colonies found when using the EMB medium.
  • the types of colonies found were circular, irregular, rhizoid and punctate for CASOY anaerobic and aerobic, with convex, raised and umbilicated surfaces, as well as some creamy white, others yellowish and translucent as shown in figure 16.
  • the Colonies on the aerobic and anaerobic EMB media were circular, irregular, and rhizoid in shape with convex and umbilicate surfaces, as well as lobed, wavy, and rounded edges. It is observed that the process by liquefying is more effective for obtaining microorganisms.
  • Table 1 Number of CFU obtained from the samples analyzed under the 2 treatments and incubation conditions.
  • figure 2 shows A. taquilana L. Weber plants with symptoms of soft rot of the bud with severity 4 and 3, which were used for the study; where A) Ri ⁇ a de A. taquilana L Weber split in half with a grade 4 rot; B) Sample obtained from plant presented in A) for the extraction of endophytes by the liquefaction method;
  • Figure 4 shows the isolates of aerobic and anaerobic microorganisms from the diseased bud maceration which were subjected to 24 and 48 h of incubation at 36oC, respectively.
  • CASOY medium A
  • EMB medium B
  • dilution 104 D
  • Table 2 The MALDI-TOF score values have a numerical value that represents the similarity of the protein profile of the sample against the MALDI-BIOTYPER BRUKER database, as described in Table 2.
  • Table 3 shows the list of identified strains, where it is worth highlighting the presence of Proteobacteria and the low presence of Firmicutes in the bud samples with rotting symptoms, which will be discussed further. ahead.
  • a description is made of the endophytic bacteria isolated from the extraction of sick A. taquilana with soft rot of the bud (grade 4) where the blending method was used and grade 3 where the maceration method was used.
  • the isolates made by the liquefaction method 31 total isolates were found, of which 22 were genera and species without repeating.
  • Figure 5 shows the percentage of the edges of the culturable bacteria obtained from the bud of A. taquilana L. Weber with symptoms of soft rot, where a decrease in the content of Firmicutes bacteria of only 11% is observed, the majority being the content of Proteobacteria with 76%, what calls attention is the low content of Firmicutes and the high content of Proteobacteria.
  • Figure 6 describes the percentages of the types of Proteobacteria found in the bud with symptoms of soft rot of A.
  • Gammaproteobacteria Entarobactarias, Pseudomonas, efc.
  • Betaproteobacteria Achromobactar, Burkhoidaria, etc.
  • Alphaproteobactarias Raritabium, Bravundimonas etc.
  • the isolates of aerobic and anaerobic microorganisms from diseased plant suckers are also shown (figure 8), which were subjected to 24 and 48 h incubation at 36 ° C, respectively.
  • On the upper left side appears the CASOY medium (A) without dilution under aerobic conditions, and (C) with 10 1 dilution under anaerobic conditions, and on the upper right side of the figure is the EMB medium (B) without diludon under conditions. aerobic, and (D) without dilution in anaerobic conditions.
  • the list of cultivable strains Identified from the extraction of endophytes from the sucker of A. tequilana L.
  • FIG 10 describes the percentages of the different classes of Proteobacteria found in the sucker bud with symptoms of soft rot of A. tequilana from the mother plant, isolated by the maceration method. In which Gammaproteobacteria were found
  • Betaproteobacteria (Entembacteria and Stanotrophomonas), Betaproteobacteria (Burkholdar ⁇ a) and Alphaproteobacterlas (Mathylobactarium). It is worth noting the presence of Gammaproteobacteria with 66.70% of the isolates, then Betaproteobacteria and Alphaproteobacteria with 16.70%.
  • Betaproteobacteria Achromobactar, Burkholdaria, Acidovorax among others
  • Alphaproteobacterlas Rosenobium and Ochrobactrum
  • part A) of figure 15 a blunder of A. tequilana L. Weber sana is observed; where part A) shows a bud of a plant A. tequilana L. Weber sana; he part B) illustrates a sample extracted from the healthy bud for endophyte extraction by the mortar method.
  • section B) of figure 15 the sample obtained from the healthy bud is observed in its second washing with sterile double distilled water after its previous disinfection with doro for the elimination of endophytic microorganisms as described above, this tissue was used for the extraction of endophytes by the mortar method.
  • the isolates of aerobic and anaerobic microorganisms from healthy buds, which were subjected to 24 and 48 h of incubation at 36 ° C, respectively, are shown in figure 16.
  • the CASOY medium (A) without dilution appears on the upper left side. under aerobic conditions, and (C) without dilution under anaerobic conditions, and on the upper right side of the figure is the EMB medium (B) without dilution under aerobic conditions, and (D) without dilution under anaerobic conditions.
  • Figure 17 describes the percentages of cultivable bacterial phyla where an increase in the percentage content of Firmicutes is observed with 40%, only 15% lower than that of Proteobacteria; because a balance was observed between the percentages of Firmicutes and Proteobacteria with 40 and 55% respectively; unlike the diseased A. tequilana L. Weber plants, where the difference of Proteobacteria against Firmicutes was 65%, which gives us a difference of 50% if we compare the ratio of Proteobacteria with Firmicutes from a healthy plant to a diseased plant. This is important because this relationship of bacterial phyla can be a very important point in the development of the soft rot disease of the bud in A. tequilana L Weber plants.
  • Achromobacter spaniuses Enterobacter cancerogenus
  • Pseudomona extremorientalis Acinetobacter ursingii
  • Burkholder ⁇ a xenovorans Enterobacter aemgenes
  • Klebsiella pneumoniae Bacillus safensis
  • Enterococcus casselif ⁇ avus among others.
  • Isolated endophytes of the plant with symptoms of soft rot of the bud Isolated endophytes of the plant with symptoms of soft rot of the bud.
  • Proteobacteria analysis clearly shows the presence of Gammaproteobacteria (72.5%) that tend to be microorganisms considered pathogenic in some plants and animals due to the genera of microorganisms found in these classes, which is expected by the tissue where these microorganisms were found. coming from diseased tissues, regarding Betaproteobacteria (17.50%) they tend to be chemolithotrophic or phototrophic bacteria, in some cases pathogenic and
  • Alphaproteobacteria (10%) tend to be rhizobia or phototrophs.
  • Paenibadllus amylolyticus which is known to contain pectinases but has not currently been found to cause bud rot in A. tequilana unlike Bacillus pumilus and Pantoea aggiomerans which have pectinases. and they are known as causing soft rot of bud.
  • the sucker bud was the tissue where fewer microorganisms were found compared to the other samples, this may be due to the fact that the extraction method was by maceration, coupled with the fact that it still does not present as much microbial load since it is a young plant and not It has been in the soil for so long unlike the other samples of adult plants, indicative that the mother plant (A. tequilana) has not passed as many microorganisms through the rhizome. 20 isolates of microorganisms were found, of which 3 were found more than one strain, mentioned in Table 6. Regarding the phyla of bacteria obtained mentioned, it is worth highlighting the presence of Firmicutes (47%) with almost half of the isolates above Proteobacteria (35%).
  • Betaprateobacteria and Alphaproteobacteria with 16.70%. This is interesting because these percentages found, where the presence of Gammaproteobacteria stands out, as was the case of the plants with symptoms of bud rot previously analyzed. This can give the guideline that this sucker in the future can suffer from bud rot when the plant is under stress conditions that can allow the growth of Gammaproteobacteria causing microbial imbalance.
  • Pantoaa agglomarans which is already known as a cause of soft bud rot, and is a microorganism that has been found in seed, sucker and diseased plant, suggests that it is part of the A. taqullana microblota that is transmitted from generation to generation.
  • Isolated endophytes from roots derived from plants with symptoms of soft bud rot In the root isolates, 20 isolates of microorganisms were found, of which 2 more than one strain was found. Almost the same percentage of Proteobacteria (70%) as that of the plant with symptoms of soft bud rot were found, it is interesting because there is a relationship between the microorganisms found in the mother plant and those of its root, which this can cause that this same plant is going to transmit a certain percentage of microorganisms to the youngster.
  • Alphaproteobacteria with 27.27% This is interesting because these percentages found are different from those found in diseased plants where the majority were Gammaproteobacteria, while here in the rhizome there was an equity between the 3 classes, and there was no imbalance unlike the diseased plant that were almost the same percentage of Prateobacteria. Causing in the future that within the plant this percentage begins to change during the life of the plant and its subsequent disease.
  • Psaudomona syr ⁇ ngae which is known in the literature to be the cause of cold stress in plants, better known as "frosts", this is of great importance because the main damage of bud rot in A.
  • tequilana is seen after edge time, where the low temperatures that cause cold stress arrive.
  • interesting microorganisms were also found such as Stenotrophomonas sp, Rhizobium radiobacter synonymous with Agrobacterium tumefaciens, Rhizobium tropici, Micrococus lutaus, Chryseobacterium gleum, Badllus mycoides, Achromobacter spanius, among others. Isolated endofltos of healthy bud derived from healthy plant.
  • Betaproteobacteria with 18.18%. This is interesting because these percentages found in healthy plants where the majority are Gammaproteobacteria, as in the case of diseased plants, can be presented as an idea that in the future this apparently healthy plant will become ill in a year or two in the case from some stress in its environment that has already been cold, nutritional, etc., which triggers this change in percentages in the microbiota, increasing the number of Proteobacteria or consequently reducing the number of Firmicutes.
  • tequilana nor does it contain pectinases, but does contain inulinases, which can make it an afflicted plant in a slightly more advanced stage of the disease of bud rot where they have a fundamental role, it is interesting to find it in a healthy plant perhaps as an opportunistic microorganism.
  • Figure 19 presents the most important part of this Research work, because it shows a summarized panorama of how the microblota of A. tequilana L. Weber changes according to life and state. plant health.
  • Table 7 is very important, especially because it includes microorganisms that appear in the three conditions studied. This means that they are natural endophytes and that the loss of some species, especially Firmicutes, suggests that this phylum is important for the development of symptoms, as is the case of Paenibaclllus amyfotyt ⁇ cus, Klabsialla pnaumoniaa, Pantoaa agglomerans and Stenotmphomonas maltophilia. Table 7. Origin of the microorganisms that coincided with the different samples isolated in A. taquilana Weber.
  • microorganisms of the different genera and species isolated in the samples used are compared one by one (Table 8), putting (+) where this microorganism was found and (-) where it was not found, this to have a broad picture of what type of microorganisms was found in what type of tissue and in some cases which microorganisms repeated in different types of tissues.
  • Table 8 Origin of the endophytic bacteria isolated in the different samples used from the mother plant of A. tequilana L Weber, with symptoms of soft bud rot.
  • Table 9 Description of all the isolated bacteria which are known to cause bud rot disease in different plants.
  • Table 10 Species of bacteria that were subjected to agronomic tests.
  • CTAB Heexadeditrimethylammonium Bromide or Cetyltrimethylammonium Bromide
  • Table 11 shows the results of the evaluation of siderophores. Table 11. Results of the evaluation of siderophores of the strains of bacteria subjected to agronomic tests.
  • Capture Fe (ll) generated by the oxidation of Fe (ll) in oxidant micro-niches of the plant and rhizosphere, increasing the local availability of iron or reducing the toxicity of Fe (ll) towards the plant by accumulation of sequestered metal in the interior of bacterial cells.
  • the pellet obtained was resuspended with 5 mL of CASOY broth at 50%, enriched with L-Tryptophan at 5mM.
  • the concentrated bacterial suspension was diluted with 50% CASOY broth enriched with L-Tryptophan ai 5 mM until reaching an absorbance of
  • each microtube was centrifuged and the pellet was discarded, obtaining the supernatant.
  • ACC deaminase improves plant nutrition by increasing the availability of ammonia in the rhizosphere and resistance to stress factors decreasing the concentration of ethylene (Esquivel-Cote et al., 2013).
  • the medium must be treated to determine the ACC deaminase activity, due to the instability of ACC against temperature and light.
  • the medium should be used as soon as possible to avoid degradation of the ACC.
  • the solution should be prepared by adding one compound at a time and adding the next one, until the previous one dissolves completely, the final solution should not have precipitates.
  • ACC was added to the DF medium at room temperature, it was homogenized by shaking and 15 mL were poured per Petri dish using a 5 mL micropipette.
  • the bacteria were grown in their ideal medium and incubated for 24 h at 30oC. Note: To inoculate the medium, it was necessary to give the bacterial cells a wash treatment with 0.9% saline solution to eliminate traces of the culture medium where they were cultured. 6. The bacteria were inoculated in the form of puncture in the medium and incubated for 3 days in the dark and without exceeding 35 ° C to avoid the inhibition of ACC deaminase. Several inoculations can be done in the same Petri dish dividing it into a grid. The result is favorable when observing the growth of the inoculated point in the ACC deaminase medium. In the medium, the only source of nitrogen is ACC, which induces the production of the ACC deaminase enzyme, which removes the amino group, leaving ⁇ -ketobutyrate and ammonia. The results are shown in Table 13.
  • the fungus was previously cultured on PDA for 7 days at room temperature, then the culture is light-stressed to form spores. Subsequently, a spore collection was carried out with sterile type 1 ultrapure water, the spores were counted in a Neubauer chamber and the pertinent dilutions were made to achieve a concentration of 5x10 5 .
  • PID Percentage of Inhibition in the development of the fungus.
  • the chosen bacterial strains can be used in agriculture, to prevent, control, etc., plant diseases, such as plant rot; and compensate the microbiota in diseased plants; the vegetables can be of the commonly denominated agaves. Another use of bacterial strains is that they can favor plant nutrition, due to the agronomic qualities described above.
  • Another object of the present invention is a mixture of endophytic bacteria for the preparation of a useful product in agriculture, which comprises:
  • these strains were isolated from tissues of the bud and bases of the leaves of the Agava taquilana Weber, so they can function as agents of prevention and control of bud rot and wilting in plants of the agavaceae family, at the same time it has functions of blofertlllzadón since it enhances the absorption of nutrients, stimulating the growth of the plant through nitrogen fixing activities. , solubilization of phosphates, production of siderophors, auxins, Amino Cidopropane Carboxilico deaminase (ACCd), and antifungal activities.
  • a bio-inoculant product based on Firmicutes and Procteobacteria can be formulated to compensate for the loss of microbiota in plants, such as agave plants, and more. specifically in Agave taquilana Weber obtained from the same plant to prevent the disease of bud rot. Therefore, this product is also one more object of the present invention.
  • this bio-inoculant product seeks to compensate for the loss of the microbiota in A. taquilana Weber, and other agave species that suffer from the disease called bud rot.
  • the Invendón also comprises a method to control the compensation of the microbiota and rot, in plants, by applying an effective amount of the mixture of bacteria of the selected strains, in accordance with the present invention, or a effective amount of the blo-inoculant product of the present invention, on the plants that require it.
  • a result of the method is when a concentration of 1x10 9 CFU per mL is applied, once a week, for 4 consecutive weeks, repeating this treatment every year.
  • Compatibility analysis between isolated microorganisms In order to observe the compatibility of the selected bacterial strains, they were subjected to a compatibility analysis, for which the procedure described below was followed. 1- The bacteria were cultured in 1 mL of CASOY broth.
  • the box was divided into 4 and in each area 10 mL of the 3 previously cultivated strains were inoculated in 1 mL of CASOY broth, leaving a free control space and a separate box as a control.
  • CVP crystal violet pectate
  • Erwinia pyrlfoliae an Erwinia species different from Erwinia amylovora, causes a necrotic disease of Asians pear three. France: Hallym University., 48, 514-520.

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Abstract

La présente invention concerne la souche isolée de Bacillus safensis (C4), qui a les caractéristiques du dépôt CM-CNRG TB70; la souche isolée de Bacillus licheniformis (C71), qui a les caractéristiques du dépôt CM-CNRG TB72; et la souche isolée de Enterobacter ludwigii (C14), qui a les caractéristiques du dépôt CM-CNRG TB71. Ces souches sont caractérisées en ce qu'elles présentent une activité de compensation du microbiote, une activité bactéricide et fongicide, contre des pathogènes provoquant la pourriture, dans un végétal. Le mélange et le produit, utiles en agriculture comprennent les trois souches mentionnées auparavant; et le produit comprend à son tour ledit mélange. L'invention concerne également une méthode pour contrôler la compensation du microbiote et la pourriture, dans des végétaux, qui consiste à appliquer une quantité efficace du mélange ou produit de la présente invention, sur les végétaux qui en ont besoin.
PCT/MX2019/000040 2019-04-17 2019-04-23 Souches bactériennes endophytes, mélange, produit et méthode pour compenser le microbiote et lutter contre la pourriture dans des végétaux WO2020214018A1 (fr)

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WO2023167579A1 (fr) * 2022-03-04 2023-09-07 Fonseca Sepulveda Cristobal Souches bactériennes endophytes, mélanges probiotiques, formulation et procédé, pour stimuler la croissance végétale

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