WO2020214018A1 - Endophyte bacterial strains, mixture, product and method, for compensating microbiota and controlling rot in vegetables - Google Patents

Abstract

Isolated Bacillus safensis strain (C4), having the features of deposit CM-CNRG TB70; isolated Bacillus licheniformis strain (C71), having the features of deposit CM-CNRG TB72; and isolated Enterobacter ludwigii strain (C14), having the features of deposit CM-CNRG TB71. Said strains are characterised in that they exhibit compensatory activity of microbiota and bactericidal and fungicidal activity against rot-causing pathogens in a vegetable. Mixture and product, useful in agriculture, which comprises the three aforementioned strains and product which in turn comprises said mixture. A method for controlling compensation of microbiota and rot in vegetables, which comprises applying an effective amount of the mixture or product of the present invention to the vegetables that require it.

Description

ENDOPHYTIC BACTERIAL STRAINS, MIXTURE, PRODUCT AND METHOD, TO COMPENSATE THE MICROBIOTA AND CONTROL ROT IN

VEGETABLES

TECHNICAL FIELD OF THE INVENTION

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.

BACKGROUND OF THE INVENTION

It is currently known that bacteria and fungi have profound impacts on adaptation, stress and plant health (Lugtenberg & Kamilova, 2009; Partida-Martínez & Heil, 2011; Gaiero atal., 2013; Méndez atal., 2013; Philippot atal ., 2013; Panke-Buisse atal., 2015). Microorganisms have helped plants to withstand different types of stress, both abiotic and blotlic, among these are salinity, drought, nutrient limitation, attacks by herbivores and pathogens (Amold at al., 2003; Rodríguez at al., 2008 , 2009; Partida-Martínez & Heil, 2011; Hardoim atal., 2012).

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 Acidobactaría 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). Likewise, it was found that the rhizosphere of 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).

In the research by Desgarennes et al. In 2014, it is described that the bacterial communities in the leaves were predominantly the Acinatobacter and Bacillus genera, and in the root endosphere Stenotmphomonas and Agrobacterium, in contrast in wild agaves it was dominated by Actinosynnamatacaate, Promicromonospora and Rhizobialas. Regarding the season, they observed that bacteria such as Actinobacter, Bacillus, and aby-Protaobactaria mixtures predominate in A. taquilana and A. salmiana during the dry season. In the analysis of A. salmiana and A. taquilana, the phyla Proteobactaria was the most common in both. 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. tequilana became in a monoculture. 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. Endophytic bacteria can represent an alternative to improve growth and reduce diseases in plants. In the tissue of the A. tequilana plant, a population density of 3 million CFU / g of fresh plant tissue was found. (Martínez-Rodríguez 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). In 2014, Beltrán-García et al., Isolated a strain of S. tequilansis from A. tequilana seed, in which they verified that the bacterium, previously enriched with 15N, is capable of degrading nitrogen and transferring it to agave plants. Degradation via the production of hydrogen peroxide secreted by the plant occurs when there is a limitation of nutrients in the soil. So the plant chooses to degrade its own endophytes. This finding opens an important panorama that brings together the understanding of how plants manage their microbial communities in environments with limited organic matter, and thus be able to support the hypothesis that some plants under nutrient limitation can degrade and obtain nitrogen from their endophytic microorganisms ( Beltrán-García et al., 2014).

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 (Alegría 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. In grade 3 the plant frequently has leaves with black, watery spots and leaf curl up to two thirds. In 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).

In the northern area of Jalisco, the disease that occurs most frequently is bud rot, where Tepatltlán is one of the most affected municipalities and Atotonilco el Alto is the least affected municipality with 75% of healthy plants during 2016 (SENASICA, 2016). In recent years the surface area cultivated with A. tequilana has decreased in the state of Jalisco, due to the presence of the soft rot of the bud. Bud rot was first reported in 1989 (Jiménez-Hidalgo at al). This disease not only affects A. tequilana but also Oaxaca agaves, and has been classified as the most dangerous disease of this crop (Aquino-Bolaños at 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). In another Investigation, Erwinia cactidda, Pantoea agglomerans (synonymous with Erwinia agglomerans) and Pseudomonas sp. as causes of symptoms of soft rot of bud of A. tequilana, applying Koch's postulates, where P. agglomerans and E. cacticida contain pectinolytic enzymes, these being the two most pathogenic in the tests (Jimónez-Hidalgo et al., 2003 ).

In the state of the art there are already products based on endophytic microorganisms to be used as biological controllers of fungi and bacteria; For example, 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; For its part, 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; and 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 .

The consumption of A. tequilana for the production of tequila has decreased by more than 150 thousand tons in the last 6 years (CRT, 2016). Limitations in production are mainly associated with phytosanitary problems, together with the deficiency of an orderly and efficient plant production plan. The tequila industry in 2004 reached a record figure of 109 million liters in tequila exports (CRT, 2004); This exerted pressure on producers to generate the raw material necessary for demand. This causes an overproduction of agave, this increase causes a decrease in the price of the plant kilo, making the producers stop taking care of the properties. Consequently, phytosanitary problems increase, the plant is scarce and prices increase again inducing overproduction, so the acid starts again. Consequently, pests increase, becoming more resistant. Added to this, environmental factors linked to climate change and the indiscriminate use of herbicides and pesticides, causes soil erosion problems by eliminating more than 90% of the plant cover (organic matter) during seven years of cultivation.

The rotting of the bud causes agave to become scarce and consequently the production of tequila. In addition to the series of scientific reports on this disease, they suggest that not only P. carotovora is the cause, but also other microorganisms. Therefore, we think that the loss of the balance of native endophytic microorganisms (soil and plant microbiota) combined with environmental and / or nutritional factors could be an explanation for the development of the disease. Until now, this disease has not been eradicated, nor controlled in a viable way, much less understand exactly how bacteria are acquired and infect the plant.

An analysis carried out by our research group indicates that diseased plants have a decreased population of beneficial endophytic microorganisms in the order of 10 ² . On the other hand, healthy Agave tequilana plants have a number of endophytes between 10 ⁵ and 10® (Beltrán-García et al., 2015).

Preliminary data suggest that pesticides and herbicides influence the loss of beneficial bacteria, predisposing the agave to develop diseases or affect the production of conventional suckers. This consequently causes the microorganism-plant balance to be broken, causing an influential stress in the loss of conditions of adaptation to the environment and defense response to the colonization of phytopathogenic organisms. Therefore, we suggest that the maintenance of endophyte communities in balance is essential to maintain plant health and growth, their loss must alter in a physiological and molecular context the behavior of plants and a weak response to stress. We think that the study of microbial communities under conditions of bud rot and healthy plants will serve to establish whether there is a relationship between the different bacterial Phylas and the disease. That is to say, this analysis of the percentage of phyla present in different states of the plant will indicate that the modifications in some of the groups may influence the development of the phyla after a stress event.

In order to counteract the aforementioned drawbacks, 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.

The characteristic details of the present invention are shown in the following description of some of its preferred embodiments, examples and figures that are attached, but not limited to, where:

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

Proteobactaries found in healthy buds from A. tequilana L. Weber isolated by the maceration method.

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.

DETAILED DESCRIPTION OF THE INVENTION

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.

Isolation of endophytic microorganisms from plant tissues of A. tequilana L Weber

Collection of samples

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.

Procedure for handling samples to obtain endophytes

Once the plant samples were obtained and selected, they were processed by two different methods to obtain endophytic microorganisms: liquefying and maceration with mortar. The blending method using an industrial blender tends to make it a bit more aggressive due to mechanical cutting. The samples with damage from bud rot were processed by this method as they were softer and the samples with less damaged tissue were used with the mortar method due to their hardness, to compare the extraction efficiency where the tissues were previously sanitized with gold. Culture and isolation methods

After being extracted, all the samples were cultivated in EMB agar (Eosin Blue Metlleno) as a medium for the selection of gram negative bacteria, since it is believed that the cause of the soft bud rot disease is the Enterobacteriacaaa family, and trypticasein soy agar (CASOY) as a nutritive medium for fastidious bacteria, without inhibiting gram negative ones. These media were cultured and incubated under aerobic and anaerobic conditions.

A) Sample liquefaction method

1. 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.

2. 500 g of sample were weighed, which were washed with 3% gold and 3 washes with sterile double-distilled water, each for 10 min.

3. 1 L of sterile double-distilled water was used with the 500 g of samples in the sterile blender. It was liquefied for 5 min at medium speed, until obtaining a sample liquid. Subsequently, the content of the blender was filtered with a sterile handle, where the solid was frozen at -20 ºC, and the aqueous extract was used for the direct cultivation of microorganisms in the agaves.

4. The aqueous extract obtained from the previous filtration, 6 dilutions were made in 0.9% sterile saline solution in 10 mL vials. Each vial contained 9 mL of saline solution, where one mL of the extract was added to the first vial, it was vortexed and one mL was taken to add it to the second vial and yes successively until six dilutions (1x10®) were obtained.

5. Then 0.1 mL of each dilution was added to Petri dishes with the culture media, EMB and CASOY, with a micropipette. It was plated with a Drigalski spatula to spread the sample throughout the box until completely dry.

6. Once labeled, the Petri dishes were incubated at 36 ° C, for 24 and 48 h, respectively, under aerobic and anaerobic conditions. A control of each type of medium was left in which only sterile water from the last wash was plated. It was performed in duplicate of the boxes in anaerobiosls and aeroblosis for better control.

B) Method of maceration by mortar

1. 100 g of sample were weighed and placed in a 500 mL beaker with 250 mL of a 3% doro solution and stirred for 10 min, then 3 sterile water washes of 10 min.

2. After the third wash, the sample was dried on sterile paper towels for subsequent maceration in a sterile mortar. The extract was carefully filtered with sterile gauze.

3. The same procedure mentioned above was repeated for the dilution and plating in the different media, as well as aerobic and anaerobic incubation at 36ºC, for 24 and 48 h, respectively.

C) Anaerobiosis Conditions

To generate the anaerobic condition, 5 g of citric acid and 5 g of iron were weighed and mixed in a glass Petri dish. Water was added to cover the mixture, and then put it in the anaerobiosis chamber (GasPak) and closed it under vacuum, where the mixture reacted producing CO2. Strips were used to check anaerobiosis that changes from blue to white, which were left for 48 h.

Figure 1 shows the isolates of aerobic microorganisms from the liquefied of diseased buds, which were subjected to 24 h incubation at 36ºC. Where A) is a 6 dilution in EMB medium; and B) a 6 dilution in CASOY. identification of microorganisms.

To identify the microbial communities, the MALDI-TOF mass spectrometry technique was used as methods and in some cases the 16s gene sequencing.

A) 16s rRNA 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.

For the molecular identification of the bacterial strains, the first ones were used: rD1 5 'AAG GAG GTG ATC CAG CC 3' and fD1 5 'AGA GTT TGA TCC TGG CTC AG 3'. Gene segment amplification was done using the polymerase chain reaction under the following PCR conditions:

95 ° C - 2 mln, (35 cycles) 94 ° C - 1 min; 55 ° C - 1 min; 72 ° C - 2 mln, 72 ° C - 6 min. In some cases the following degenerate primers were used: 27 f 5'AGA GTT TGA TCC TGG CTC AG 3 'and 1492 RY 5' GGY TACCTT GTT ACG ACT T 3 ', with the following PCR conditions at: 94 ° C - 5 min, (32 cycles) 94 ° C - 30 sec; 53 ° C - 40 s; 72 ° C - 40 s, 72 ° C - 5 min. Both primers have been used to generate the amplification of a 1500 bp fragment of the 16s rRNA gene sequence (Benson et al., 1996; Thomas et al., 2008).

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). For the identification of endophytic bacteria, the 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). B) MALDI-TOF MS Mass Spectrometry Technique.

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.

2. Sufficient bacterial blomass was taken with a toothpick and resuspended in the water.

3. Subsequently 900 mL of pure ethanol were added and mixed. 4. The tube was centrifuged 2 times at 12000 rpm for 2 min, discarding the supernatant each time.

5. The pellet was allowed to dry at room temperature.

6. Once the pellet was dry, 70% formic acid and acetone were added.

7. It was mixed and centrifuged at 12000 rpm for 2 min.

8. 1 mL of the supernatant was taken and placed in the "target plate" of the equipment, it was left to dry at room temperature.

9. Subsequently, 1 mL of HCCA matrix was placed and allowed to dry.

10. 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.

Results

In summary, 152 isolates were obtained, of which 80 were different genera of microorganisms derived from all the samples studied (A. tequilana L. Weber plants with apparently damaged grade 3 and 4 buds, as well as from the root, sucker and healthy bud).

Comparison of endophytic bacteria extraction methods.

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. From dilution number 5, the difference between one strain and another was beginning to be noticed, as well as the isolation of these microorganisms. 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.

Likewise, 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.

On the other hand, 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.

In the root we found that by the maceration method, 22,000 CFU / g were obtained for aerobic CASOY, 28,000 for aerobic EMB, 13,000 for anaerobic CASOY and 21,000 for anaerobic EMB as shown in Table 1. It is highlighted that there was a development higher CFU found in aerobic and anaerobic EMB compared to CASOY. The types of cottons that were found were circular, irregular and punctate in shape for CASOY anaerobic and aerobic, with convex and umbilicated surfaces, as well as some creamy yellowish and translucent as shown in figure 12. The colonies of the aerobic EMB media and anaerobic were circular, irregular and rhizoid in shape with convex, umbilicated and raised surfaces, as well as rounded and filamentous edges.

Finally, 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.

Phyla and genera isolated in the telldos of the bud of different plants of A. teauilana L. Weber.

Next, the isolated genera and species of each of the bud samples used in this work will be described. In them, as a summary table, the isolates and the method by which they were identified either by mass spectrometry or sequencing of the rDNA amplification product will be listed.

A) Analysis of cultivable endophytic communities of damaged bud derived from plants with soft bud rot lesions.

This analysis shows photos of the tissues used for the extraction of endophytes, as well as photos of the isolates made in the different culture media, as well as the microorganisms that were isolated and identified either by MALDI-TOF or 16s rDNA. , separating into their corresponding Edges.

As described below, 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) Riña 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;

C) Plña split in half with a grade 3 rot; D) Sample obtained from plant B) for the extraction of endophytes by the mortar method. The isolates of aerobic and anaerobic microorganisms from the liquefied of diseased buds which were subjected to 24 and 48 h incubation at 36 ° C, respectively, are shown in figure 3. The medium CASOY (A) appears on the upper left side. with 10 ⁵ dilution under anaerobic conditions, and with 10® dilution (C) under aerobic conditions, and on the upper right side of figure 3 is the EMB medium (B) with 10 ⁵ dilution under anaerobic conditions, and with 106 dilution ( D) under aerobic conditions. 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. On the upper left side appears the CASOY medium (A) with dilution 104 under aerobic conditions, and with dilution 104 (C) under anaerobic conditions, and on the upper right side of the figure is the EMB medium (B) with dilution 104 in aerobic conditions, and with dilution 104 (D) under anaerobic conditions. 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 2. Description of the MALDI-TOF rating range.

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. In this Table 3 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. In the isolates made by the liquefaction method, 31 total isolates were found, of which 22 were genera and species without repeating. 3 microorganisms with more than one isolate were found, which were Alcaliganas faacalis with 7 different strains, Stanotrophomonas maltophila with 2 and Morganalla morganii 3. While, by the maceration method, 42 total isolates were found, of which 30 were without repeating genus and species. In 8 microorganisms more than one isolate were found, which were Achmmobactar spanius with 2 different strains, Enterobacter cancarogenus with 3, Enterobacter ludwigii with 2, Psaudomonas axtramoríantalis with 3, Acinatobactar guillouiaa with 3, Corynabacterium variabila with 2, and Eschar Alcafíganas with 3. coli with 2.

Table 3. Endophytic bacteria obtained from bud with symptoms of soft rot of a plant of A. taquilana L. Weber, separated by phylum, genus and species, number of isolates, MALDI-TOF MS score and sequence obtained from the Genebank by the method from 16s.

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. taquilana isolated by the two methods of liquefying and macerating the bud; where Gammaproteobacteria (Entarobactarias, Pseudomonas, efc.), Betaproteobacteria (Achromobactar, Burkhoidaria, etc.) and Alphaproteobactarias (Rhizobium, Bravundimonas etc.) were found. It should be noted the Gammaproteobacteria pnesenda with 72.50%, then the Betaproteobacteria with 17.50% and finally the Alphaproteobacteria with 10%.

B) Analysis of cultivable endophytic communities of suckers derived from plants with soft rot lesions of the bud.

In figure 7 in section A) a plant of A. taquilana L. Weber is observed with symptoms of soft rot of the bud in degree 3 of severity with its sucker joined by the rhizome, in indso B) the sample obtained is observed of the sucker in its washing with distilled water to eliminate the gold used in the previous steps for the elimination of microorganisms from the surface, which was macerated with mortar and pistil for the extraction of endophytic microorganisms.

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. Weber separated by phylum, genus and species, number of isolates, MALDI-TOF score and if it was identified by the 16s method or not, where It is worth highlighting the presence of Firmicutes with almost half of the isolates above Proteobacteria and Actinobacteria, which will be discussed later in Table 4, in which a description of the endophytic bacteria isolated from the maceration method is made. . In the isolates made by this method, 20 total isolates were obtained, of which 17 did not repeat genus and species. Curtobactarium sp, Mathyiobactarium radiotoiarans, Pantoea agglomerans, 2 strains of each were obtained.

Table 4. Endophytic bacteria obtained from the offspring of an A. tequilana plant

Weber, with symptoms of bud rot.

The percentage of culturable bacterial phyla where an increase in the percentage content of Firmicutes bacteria is observed with 47% with almost half of the isolates, while Proteobacteria with 35% and Actinobacteria with 18%, which is described in Figure 9. This is important because at that stage the plant still retains a majority in Firmicutes.

While figure 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

(Entembacteria and Stanotrophomonas), Betaproteobacteria (Burkholdaría) and Alphaproteobacterlas (Mathylobactarium). It is worth noting the presence of Gammaproteobacteria with 66.70% of the isolates, then Betaproteobacteria and Alphaproteobacteria with 16.70%.

C) Analysis of cultivable endophytic communities of the roots of plants with soft rot lesions of the bud.

In part A) of figure 11 the rhizome of an A. tequilana plant is observed with symptoms of soft rot of the bud in grade 3 severity; where A) Union rhizome between the mother plant of A. tequilana with symptoms of soft bud rot with its sucker, B) Sample extracted from the root that was used for the extraction of endophytes by the mortar method. In Section B) of figure 11, the sample obtained from the rhizome is observed, which was a piece of root before washing with gold and distilled water for the elimination of the microorganisms from the surface as described previously, this tissue was use for the extraction of endophytes by the mortar method.

The isolates of aerobic and anaerobic microorganisms from diseased plant suckers, which were subjected to 24 and 48 h of incubation at 36 ° C, respectively, as shown in figure 12. In the upper left side of the figure appears the CASOY medium (A) with a 10 ² dilution under aerobic conditions, and (C) with a 10 ² dilution under anaerobic conditions, and upper right side of the figure is the EMB medium (B) with dilution

10 ² under aerobic conditions, and (D) with KP dilution under anaerobic conditions.

The list of cultivable strains identified from the extraction of endophytes from the root of A. taquilana separated by phylum, genus and species, number of isolates, MALDI-TOF score and if it was identified by the 16s method or not, where it should be noted the presence of Protaobactarias with more than half of the isolates above Firmicutes and Actinobacteria is described in Table 5. In this table a description of the endophytic bacteria isolated from the maceration method is made. In the isolates made by this method, 20 total isolates were obtained, of which in 16 strains neither genus nor species was repeated. In 2 microorganisms more than one strain was found, as in the case of Stenotrophomonas sp and Rhizobium radiobactar where 3 isolates of each were obtained, other important genera were also found such as Psaudomonas syríngaa, Bacillus my ¥ ides, Rhizobium tropici, among others.

The percentage of cultivable bacterial phyla obtained in the total of root isolates where an increase in the percentage content of Proteobacteria is observed with 70% with more than half of the isolates of the root sample, while Firmicutes and Actinobacteria with only 12% each is described in figure 13. This is important because these microorganisms come from the mother plant that showed symptoms of soft rot of the bud with severity grade 3, which suggests that this same plant is going to transmit a certain percentage of microorganisms to the young.

The percentages of the different classes of Protaobactaria found in the rhizome of A. taquilana L. Weber with symptoms of soft rot isolated by the maceration method are described in figure 14. In which Gammaproteobacteria (Entarobactarias, Pseudomonas,

Stenotrophomonas), Betaproteobacteria (Achromobactar, Burkholdaria, Acidovorax among others) and Alphaproteobacterlas (Rhizobium and Ochrobactrum). It should be noted the presence of Gammaproteobacteria and Betaproteobacteria with 36.36% of the isolates, and last but close to the

Alphaproteobacteria with 27%.

Table 5. Endophytic bacteria obtained from the rhizome that unites the mother plant of A. tequilana L. Weber with the sucker.

D) Analysis of cultivable endophytic communities of healthy bud derived from healthy plant.

In 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. In 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.

The list of cultivable strains identified from the extraction of healthy bud endophiles of A. tequilana L. Weber separated by phylum, genus and species, number of isolates, MALDI-TOF score and if it was identified by the 16s method or not, where it is worth highlighting the presence of Firmicutes and Proteobacteria with very similar percentages 40 and 55% respectively, as seen in Table 6. In this table a description of the endophytic bacteria isolated from the maceration method is made. In the isolates made by this method, 37 total isolates were obtained, of which in 20 genus and species were not repeated. More than one strain was found in 7 isolates, as in Cellulosimicmbium cellulans, Enterobacter aerogenes, Enterobacter cancerogenus, Klebsiella oxytoca, Rahnella aquatilis, Pantoea agglomerans and Paenibacillus amylolyticus.

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.

Table 6. Endophytic bacteria obtained from healthy A. taquilana L bud

Weber.

10 Genebank Identification percentage for 16s according to Gene Bank (NCBI). ND: Not Determined. The percentages of the different classes of Proteobacteria found in the healthy bud of A. tequilana, isolated by the maceradón method are described in figure 18. In which Gammaproteobacteria (Enterobacteria, Acinetobacter among others), Betaproteobacteria (Burkholdería and Alcaligenes) were found . It is worth highlighting the presence of Gammaproteobacteria with 81.81% of the isolates, then Betaproteobacteria with 18.18%.

Discussion Isolates of endophytes

Isolation using the bud blending method

This method of extraction of microorganisms yielded interesting isolates such as Bacillus pumilus and Pantoea agglomeraos that have been described as responsible for the bud rot in A. tequilana L. Weber, which is important that they have been found in these isolates since it suggests that these bacterial genera are linked to this disease. Insulations using the mortar method.

Interesting microorganisms were found, such as Achromobacter spaniuses, Enterobacter cancerogenus, Pseudomona extremorientalis, Acinetobacter ursingii, Burkholdería xenovorans, Enterobacter aemgenes, Klebsiella pneumoniae, Bacillus safensis, Enterococcus casselifíavus, among others.

Comparison of the two isolation methods.

Comparing the two isolation methods that were used in this work to process the samples that were by the liquefied and macerated method, in which the CFU obtained were 21 times higher by the liquefied as observed in the results, this is due to that the blending method is more effective than the mash method in terms of isolating microorganisms as it is a more aggressive method in terms of cutting speed and tissue size obtained after the process.

Isolated endophytes.

Isolated endophytes of the plant with symptoms of soft rot of the bud.

What is interesting in the results obtained in the isolated plant microorganisms with symptoms of bud rot was that there were more isolates from the anaerobic than from the aerobic part in CASOY, which indicates a greater presence of microorganisms capable of growing in oxygen-deprived environments. It was also the tissue with the presence of more isolates where 73 microorganisms were found, of which in 1 1 more than one strain was found, which were mentioned in Table 4.

Regarding the bacterial phyla obtained, what is striking is the low content of Firmicutes (1 1%) and the high content of Proteobacteria (76%) since Firmicutes are related to beneficial or more stable microorganisms in the life of the plant, while the Proteobacteria tend to become a little more pathogenic and their percentage increases as soon as the plant begins to show symptoms of soft bud rot, from the seed, through the sucker, older plant and even getting sick.

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.

These results are interesting because it can help in the future to do an analysis of the A. tequilana plant and know how affected it may be, depending on the content of Proteobacteria in relation to Firmicutes and in turn the Gammaproteobacteria that it contains and how it can affect the plant by altering the balance of its microbiota.

Among the interesting microorganisms found in these samples were 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.

Isolated endophytes of plant-derived sucker with symptoms of soft bud rot

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%). This is interesting because the plant has about a year of life of its eight that will last until its use for the production of tequila, at that stage the plant still preserves mostly Firmicutes, this percentage tends to go down When the plant grows and generates stress, consequently, it becomes ill due to the fact that the content of Proteobacteria exceeds the Firmicutes and other phyla, until it reaches about 80% as seen in the diseased plant, which consequently causes a imbalance of microbial communities and subsequent plant disease.

In Protein bacteria, it is worth highlighting the presence of Gammaproteobacteria with 66.70% of the isolates, then 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.

Among the interesting microorganisms found in these samples were Pantoaa agglomarans, Paenibacillus amylotyücus, Entambactar cowan, Mathyfobactarium radiotolarans, Solibacillus silvestris, among others. 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.

It should be noted the presence of Gammaproteobacteria and Betaprateobacteria with 36.36% of the isolates, and last but close to the

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. In these isolates it is also worth mentioning the presence of Psaudomona syríngae, 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.

In these isolates of healthy plant there was a greater development of CFU found in CASOY compared to the EMB medium, which is interesting because usually the microorganisms that affect A. tequilana in the disease of bud rot are Gram negative due to their association with Prateobacteria.

The presence of Firmicutes and Prateobacteria were with very similar percentages 40 and 55% respectively, which gives a greater equity in terms of the distribution of populations of an apparently healthy plant, unlike A. tequilana with symptoms of bud rot, where If we compare the results obtained against the values for a healthy plant, the difference between Prateobacteria and Firmicutes was more than 50%, this is a sum importance since it is the key to this work, he as the loss of a certain edge of bacteria in this case Firmicutes, and the increase of another (Proteobacteria) causes such an imbalance in the plant microbiota that they cause disease in the plant.

It is worth highlighting the presence of Gammaproteobacteria with 81.81% of the isolates, then 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.

In these isolates it is worth highlighting the presence of Cellulosimicrobium cellulans, Rahnella aquatilises, Enterobacter cancerogenus, Bacillus litoralis, Lactobadllus plantarum, Alcaligenes faecalis, Burkholderia caribensis, Klebsiella pneumoniae, Pantoea agglomerans, the latter apparently being opportunistic, among others, Bacillus pumilus. The plant is healthy, they help it to stay healthy, but when the plant drops its defenses, these strains become pathogens, affecting the plant. Erwinia pyrifoliae was also isolated, which does not have a history of A. 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.

Table analysis of the isolates.

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.

It should be noted, as the amount of Firmicutes decreased during the life of the plant, where it starts with 55% in the seed, then 47% in the sucker and 40% in the healthy plant, to end with 1 1% in the diseased plant. On the contrary, in the case of Proteobacteria, it begins with 24% in seed, 35% in sucker, 55% in healthy plant and 76% in diseased plant. This change is due to the fact that Firmicutes are more related as beneficial agents in plants in terms of agronomic properties, as well as phytopathological controls and in some cases resistant to stress. The main objective sought in this thesis was to be able to determine the bacteria associated with bud rot in A. taqullana and its relationship with the loss of Firmicutes, as shown in Figure 19. This work can be compared with a study made in humans, which shows how the amounts of bacterial phylum in our body change. It was shown that as age passes and depending on our state of health, the predominance of different bacterial phyla. By increasing the amount of Proteobacteria, poor nutrition and diseases begin to be generated, on the contrary, by having a higher percentage of Flrmicutes the person presents a healthier state (Ottman et al., 2012). This opens the possibility of the production of biofertilizers based on Firmicutes to keep the plant in good condition, and avoid the imbalance of the microbiota. 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 amyfotytícus, Klabsialla pnaumoniaa, Pantoaa agglomerans and Stenotmphomonas maltophilia. Table 7. Origin of the microorganisms that coincided with the different samples isolated in A. taquilana Weber.

The 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.

Isolation of microorganisms with possible effects on soft bud rot. Three different microorganisms known to be the cause of bud rot were isolated in different plants, where the highest percentage was found in bad bud due to the amount of isolates found of these bacteria. As described in Table 9, two of these microorganisms were found in different isolated samples, as in the case of B. pumilus and P. agglomerans.

Table 9. Description of all the isolated bacteria which are known to cause bud rot disease in different plants.

Endophtho-pathogen-saprophyte relationship.

This relationship is very important, in this case, where a microorganism such as Bacillus pumilus and Pantoea agglomaranas, which are strong candidates for bud rot, are at the same time considered beneficial for the plant due to their agronomic conditions described by several authors.

This relationship allows us to say that bacteria, like any organism, see for their survival, being inside the plant they see the way to feed and grow, at the same time that their residues help the growth of the plant, in addition to being antagonists against other microorganisms possible pathogens for the plant, but at the moment that the Agave enters stress, by concentrating on the production of quiote, which is the new tissue lowering its defenses, causing the bacteria to take advantage, and with the help of pectinases, it generates an environment rich in simple sugars, and it is when they take advantage of different bacteria and take control of the quiote, rotting the heart of the plant, later leading to death. This is because the bacterium is always watching from its side, going from being a beneficial endophyte to a pathogen and saprophyte at the moment it begins to see decomposition of organic matter, which causes the increase of microorganisms, which could have been found in smaller quantities . All this leads us to understand how the proportions of Proteobacteria and Firmicutes can determine a great Importance when being in mutual balance.

Conclusion The conclusions that we can reach in this work that took a very long process in how at the beginning it was thought to identify Erwinia Carotovora (the first pathogen described for the soft rot disease of the bud) and fulfill the 4 postulates of Koch, then we decided Note that this microorganism was not found in the isolates, for which other microorganisms were searched as the cause of bud rot, which were found as in the case of P. agglomerans and B. pumilus, but saying that These microorganisms are the cause of everything that this disease implies. It is very difficult to ensure, although they have weapons (pectinolytic enzymes) to attack the plant, they also have very good agronomic and antagonistic properties against certain microorganisms.

These microorganisms, when found in healthy and diseased plants, lead us to the question: Why, if they are pathogens, were they found in healthy plants? Therefore, the isolation of many microorganisms was sought to make an evaluation of the percentages of bacterial phyla, as seen in the discussion. Throughout this work, the phylum that was related to plants with symptoms of bud rot of A. tequilana was Proteobacteria, while Firmicutes was related to a healthy plant (as shown in figure 19), the change that goes arising from the seed, sucker, healthy plant and diseased plant, the percentage of Firmicutes decreases and, on the contrary, that of Proteobacteria increases. This is caused by some external change to the plant either by some stress condition where the plant lowers its defenses that causes that symbiosis of harmony to be broken in the healthy plant and start the change in the microblota that causes the plant disease.

This relationship is believed to affect not only A. tequilana, but it can affect any type of plant, including humans. It was also found that, among the Classes of Proteobacteria, Gammaproteobacteria, due to the type of genera that it handles in relation to Betaproteobacteria and Alphaproteobacteria, tend to be more dangerous for the plant, which is interesting because an analysis of the plant can be done and According to the types of Proteobacteria, it could be diagnosed if one could become ill in case of stress causing an increase in Proteobacteria. As well as a proposal to prevent the plant from getting sick is to keep it nutritionally strong and add Firmicutes to it to always maintain the relationship of these according to the Proteobacteria, in good balance as seen in the healthy plant isolates.

Agronomic tests

In accordance with the results obtained and the literature, 8 bacterial strains were chosen (see Table 10) that promised to be useful in agronomy for disease control, favoring fertilization, balancing the microblot, etc .; and the following parameters were evaluated: production of slderophores, auxins, ACC deaminase, Inhibition of fusarium, nitrogen fixation, and solubilization of phosphates. These tests were carried out in three repetitions, in Laboratory 310 of Biotechnology of the Autonomous University of Guadalajara, located in Zapopan, Jalisco, Mexico, during the dates of March 1 to 24, 2018.

Table 10. Species of bacteria that were subjected to agronomic tests.

The detection was carried out by Chrome Azuroi S (CAS) according to Schym & Neilands (1997).

1. 60.5 mg of CAS were weighed in 50 mL of double distilled water.

2. 10 mL containing 1mM FeCb.6H20 and 10mM HC1 were mixed while stirring.

3. This mixture was added to 72.9 mg of CTAB (Hexadeditrimethylammonium Bromide or Cetyltrimethylammonium Bromide)

(C16H33) N (CH3) 3Br in 40 mL of H ₂ O and the medium was sterilized. Apart, a mixture of 750 mL of distilled water was sterilized with 15 g of bacteriological agar, 30.24 g of Pipes (buffer) and 12 g of a 50% w / w solution of NaOH. Once sterile, both solutions were mixed and shaken carefully to avoid foaming, and poured into Petri dishes. The bacteria were also grown separately in their ideal medium and were incubated for 24 h at 30ºC. Each strain was inoculated in triplicate by applying bacteria dots to each CAS box. It was left to incubate at 30 ° C for 6 days. Color changes from blue to orange, purple, or magenta (pink-purple) indicate siderophobic production. 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.

Note: Siderophore-producing endophytes serve two purposes:

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.

Auxin Evaluation

1. It was seeded in CASOY agar medium achieving an isolation, incubating 24 h at 30 ° C.

2. Subsequently, it was transferred to 15 mL of 50% CASOY broth, incubated under shaking for 24 h in 50 mL falcon tubes at 30 ° C.

3. After 24 h the bacterial suspension was centrifuged at 6,000 rpm for 7 min, the supernatant was discarded.

4. The pellet obtained was resuspended with 5 mL of CASOY broth at 50%, enriched with L-Tryptophan at 5mM.

5. The concentrated bacterial suspension was diluted with 50% CASOY broth enriched with L-Tryptophan ai 5 mM until reaching an absorbance of

0.5 to 500 nm.

6. It was distributed in three 2 mL microtubes with 1 mL of bacterial suspension, incubated for 24 h while shaking at 30 ° C.

Subsequently, the colorimetric analysis was continued for the semi-quantitative determination of the production of indoleacetic acid IM using the Salkowsky reagent, which consists of a 10 mM solution of Iron Chloride FeCl ₃ dissolved in 35% perchloric acid HCIO4. 1.35 g of FeCb.6H20 were weighed and dissolved in 10 mL of double distilled water; and 1 mL of this solution was added to 50 mL of Perchloric Acid HCLO * 35%. It was prepared at the time of the test, due to the high instability of the solution.

7. Following step 6, each microtube was centrifuged and the pellet was discarded, obtaining the supernatant.

8. The supernatant was reacted 1: 1 with Salkowsky's reagent for 40 min in the dark.

9. To quantify the production of IM, a calibration curve is performed, with a stock solution of IM at 1,000 ppm.

10. Relevant aliquots were taken from this stock solution to obtain 40, 30, 20, 15, 10, 5, and 0 mL / mL solutions.

11. Solutions were reacted with Salkowsky's reagent for 40 mln in the dark.

12. Reactions were read with a spectrophotometer at 533 nm in triplicate and saved as a calibration curve in the equipment for the

quantifications of MI production by microbial isolates.

The positive result gives a deep pink or red color, and the negative result gives a yellow color (Bric ef e /., 1991). Table 12 shows the results. Table 12. Results of the auxin evaluation of the strains of bacteria subjected to agronomic tests, at 18 days, for 1 h.

(+) ^■ positive control; (-) = »negative control. Evaluation of ACC deaminase activity:

The qualitative determination of the activity of the deaminase enzyme of the 1-aminocyclo ropane-1-carboxylic acid or also known as ACC deaminase is based on the method of Penrose and Gllck 2003. 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).

1. A 0.5 M solution of ACC (Sigma Aldrich) was prepared.

2. The solution was sterilized by 0.45 pm membrane filtration.

3. The filtrate was collected and stored at -20 ° C, aliquots of 300 mL are used for every 50 mL of DF salts medium described by Penrose and Gllck 2003.

Note: 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.

Note: 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.

4. 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.

5. Separately, 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 α-ketobutyrate and ammonia. The results are shown in Table 13.

Table 13. Results of the evaluation of the ACC deaminase activity of the bacterial strains subjected to agronomic tests.

Evaluation of bacterial inhibition against Fusarium: 1. Bacteria were grown on PDA potato dextrose agar medium for 16 h prior to inoculation of the fungus.

2. Before mounting the bacteria, it was necessary to draw the inoculation zone of each organism in the Petri dish to ensure the accuracy of the distance of the bacteria against the fungus. Maintaining a distance of 2 cm from the bacteria with respect to the fungus.

3. 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 .

4. After 16 h of bacterial growth, 2,500 Fusarium spores were inoculated, it is recommended to do it in triplicate.

5. They were grown for 8 days at 30 ° C.

6. At 8 days, the evaluation was made by taking the radius between the center of inoculation of the fungus and the last milestone close to the zone of bacterial inoculation. These measurements are taken with a vemier graduated in centimeters.

7. You have to put a blank with a box only with the fungus with 2,500 spores to be able to compare against the tests, this would be the DHT (Development of the Control Fungus).

This is the formula for calculating the evaluation of the inhibition activity of each bacterium:

100- (DHE * 100 / DHT) = PID

DHT = Control Fungus Development

DHE: Development of the Test Fungus

PID: Percentage of Inhibition in the development of the fungus.

The results are shown in the following Table 14. Table 14. Results of the evaluation, at 16 days, of the bacterial inhibition against fusarium, of the bacterial strains subjected to agronomic tests.

(+) = positive control; (-) = negative control.

As can be seen, from the evaluations of the agronomic tests to which the selected endophytic bacterial strains were subjected, the strains C4, C14 and C71, were the ones that presented the best behaviors, a summary can be seen in Table 15.

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.

These 3 selected strains were deposited in the National Center for Genetic Resources (CNRG), a depository institution recognized by the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the purposes of Patent Procedure; on March 19, 2019, to which the following registration numbers were assigned: the isolated strain of Bacillus safensis (C4) was assigned the CM-CNRG registry

ΊGB70, the isolated strain of Bacillus lichaniformis (C71) was given the registry CM-CNRG TB72, and the isolated strain of Entambactar ludwigii (C14) was given the registry CM-CNRG TB71. Therefore, these bacterial strains are one more object of the present invention.

Table 15. Agronomic characteristics of the chosen endophytic bacterial strains.

Therefore, another object of the present invention is a mixture of endophytic bacteria for the preparation of a useful product in agriculture, which comprises:

An isolated strain of Bacillus safénsls (C4) CM-CNRG TB70;

An isolated strain of Bacillus licheniformls (C71) CM-CNRG TB72; and An isolated strain of Entambactar ludwigii (C14) CM-CNRG TB71.

As already mentioned, 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.

One way to use the mixture of selected bacterial strains is that 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. Likewise, 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.

Therefore, 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. Where 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.

2- Incubated 24 h.

3- With a sterile swab, the bacteria from the broth were inoculated into a 60 x 15 mm Petri dish with CASOY agar.

4- We tried to leave a space of 0.5 cm between the inoculum and the edge of the box.

5- Incubate for 24 h.

6- Some “wafers ^* were prepared, pouring 3 mL of CASOY agar into 60 x 15 mm Petri dishes and leaving them in the refrigerator to facilitate their handling.

7- These wafers were placed on top of the Petri dish inoculated with the initial bacteria with the help of sterile forceps and a small pressure is exerted to eliminate air bubbles that could have been trapped.

8- 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.

9- They were left to grow for 24 h at 30 ° C, so that these bacteria could confront the lower bacteria.

10- After 24 h it was observed whether there was growth or not. When there is growth, they are considered compatible, otherwise not.

In figure 20 it can be seen that the strains the isolated strains of Batíllus safénsis (C4) CM-CNRG TB70; Bacillus lichaniformis (C71) CM-CNRG TB72 isolate; and the isolated strain of Entambactar ludwigii (C14) CM-CNRG TB71, are compatible with each other, since there was growth between them. Which means that, when combining them to make a mixture between them, there is no antagonism and, therefore, there is no loss in their beneficial effects. The Micrococus luteus strain was not considered because it was not compatible with the other bacterial strains.

It should be noted that the description, figures and tests described herein are included in order to illustrate the preferred embodiments of the present invention, so they should not be considered as a limitation for the scope of the invention; therefore, all those embodiments of such an invention that are obvious to a person skilled in the art are included. Bibliography

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Claims

1. An isolated strain of Bacillus safensis (C4), which has the characteristics of the CM-CNRG TB70 deposit.

2. An isolated strain of Bacillus lichaniformis (C71), which has the characteristics of the CM-CNRG TB72 deposit.

3. An isolated strain of Enterobacter ludwigii (C14), which has the characteristics of the CM-CNRG TB71 deposit,

4. The strains of the preceding claims, characterized in that they exhibit compensatory activity of the microbiota; and bactericidal and fungicidal activity against spoilage pathogens in a plant.

5. The strains of the preceding claim, where the plant is of the genus Agave.

6. The strains according to the preceding claim, where the plant of the genus Agave is of the species Agava taquilana L.

7. A mixture, characterized in that it comprises: i) an isolated strain of Bacillus safensis, according to claims 1;

ii) an isolated strain of Bacillus lichaniformis, according to claims 2; Y

iii) an isolated strain of Enterobacter ludwigii, according to claims 3.

8. The mixture of the preceding claim, wherein the strains exhibit compensatory activity of the microbiota, bactericidal and fungicidal activity, against pathogens that cause rot in a plant.

9. The mixture of the preceding claim, where the vegetable belongs to the genus Agave.

10. The mixture according to the preceding claim, wherein the plant of the genus Agave is of the species Agave tequi! Ana L.

The mixture of claim 7, wherein the strains are in equal amounts.

12. A product to compensate the microbiota, and control pathogens that cause rot in a plant, said product is characterized in that it comprises the mixture of claims 7 to 11.

13. A method to compensate the microbiota and control pathogens that cause rot in vegetables, characterized in that it comprises applying an effective amount of the mixture, in accordance with claims 7 to 11.

14. A method to compensate the microbiota and control pathogens that cause rot in vegetables, characterized in that it comprises applying an effective amount of the product according to the preceding claim.

15. The method of the preceding claim, where the elective amount is 1x10® CFU per mL, once a week, for 4 consecutive weeks, repeating this treatment every year.