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THE INVENTORS OF AUX-ON GC, 
A ENDOPHYTE FUNGUS BACTERIAL BENEFICIAL FOLIAR

AUX-ON GC-Apply on corn and soybeans at post herbicide application. Before corn is 12″ tall and 2-3 trifoliate on soybeans. Product is compatible with most herbicides, always jar test on new herbicides. AUX-ON GC has a specific biological component that is designed to activate growth hormone production (GIBBERELLIN, CYTOKININ, AUXIN) and stress adaptation in the plant through foliar feeding. Stress Adaptations (Salt, heavy metals and drought) AUX-ON GC has surfactant in it already. 2 quarts per acre rate. 10 gallons per acre minimum of water. Cost is $28.50 per acre or $59.00 per gallon 25 bushel increase average on corn first year testing on over 1000 acres.. 10 bushel increase average on soybeans, only 100 acres tested.

Aux-on GC is only available through AFS, the Inventors: Aux-on GC is the only foliar biological on the market. All biologicals are usually put in furrow at planting or a seed treatment. We are beneficial fungus based with bacteria in the product. Most products only have beneficial bacteria. Aux-on GC has all components in the product. Surfactant, Bioactives, Biologicals, and translocation properties. Most products only have the biological. Example Pivot Bio. AFS has the only scientific data that we know of in the industry that proves Aux-on GC works from Biomakers. This is what it does, it turns on all 3 growth hormones (Auxin, Cytokine, Gibberellin) while also turning on stress adaption in the plant for (salt, drought and heavy metals).

FEBRUARY 2024 EDUCATION SERIES-EDUCATION IS POWER
UNDERSTANDING WHY WE HAVE ONE PRODUCT AND NOT HUNDREDS.
WHEN A SUPPLY HAS MANY PRODUCTS FOR THE SAME PURPOSE THEY ARE NOT SURE WHAT WORKS. 
WE DO, AUXON-GC 95% INOCULATION VS. 25-30%. HERE IS WHY.

Abstract The plant endosphere contains a diverse group of microbial communities. There is general consensus that these microbial communities make significant contributions to plant health. Both recently adopted genomic approaches and classical microbiology techniques continue to develop the science of plant-microbe interactions. Endophytes are microbial symbionts residing within the plant for the majority of their life cycle without any detrimental impact to the host plant. The use of these natural symbionts offers an opportunity to maximize crop productivity while reducing the environmental impacts of agriculture. Endophytes promote plant growth through nitrogen fixation, phytohormone production, nutrient acquisition, and by conferring tolerance to abiotic and biotic stresses. Colonization by endophytes is crucial for providing these benefits to the host plant. Endophytic colonization refers to the entry, growth and multiplication of endophyte populations within the host plant. Lately, plant microbiome research has gained considerable attention but the mechanism allowing plants to recruit endophytes is largely unknown. This review summarizes currently available knowledge about endophytic colonization by bacteria in various plant species, and specifically discusses the colonization of maize plants by Populus endophytes. Keywords: bacterial endophytes, colonization, microscopy, Populus endophytes Go to: 1. Introduction The term “endophyte” is derived from the Greek words “endon” meaning within, and “phyton” meaning plant. Previously, endophytes were defined as microorganisms such as bacteria and fungi that inhabit the plant endosphere during all or part of their life cycle without causing any apparent harm to the host plant [1,2]. However, the definition of endophytes has been revised multiple times by different authors [1,3,4]. More recently, Hardoim et al. [4] defined endophytes as microbes including bacteria, archaea, fungi, and protists that colonize the plant interior regardless of the outcome of the association. Conventionally, endophytes were isolated from surface sterilized plant tissue and cultivated in nutrient rich medium. In recent years, many endophytes have been identified through culture-independent approaches such as sequencing of the 16S rRNA gene, the internal transcribed spacer regions, ITS1 and ITS2, or through whole genome sequencing of endophyte communities [5,6,7,8]. Bacterial endophytes that are beneficial to plant growth and development are the focus of this review. They are found across many phyla, including the Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes [4,9,10,11]. Increased biomass and height in inoculated plants have been reported as a result of colonization by many endophytic genera such as Azoarcus, Burkholderia, Gluconobacter, Klebsiella, Pantoea, Herbaspirillum, Rahnella, and Pseudomonas [12,13,14,15,16,17,18,19]. Common characteristics of endophytes include the ability to synthesize plant hormones such as indole-3-acetic acid, solubilize phosphate, secrete siderophores, and confer plant tolerance to biotic and abiotic stresses [20,21,22,23]. Additionally, some bacterial endophytes carry genes necessary for biological nitrogen fixation (BNF), potentially enabling them to convert dinitrogen gas (N2) into usable forms of nitrogen such as ammonium and nitrate within the host plant [24,25]. Symbiotic N-fixation by rhizobia in legume plants or Frankia in actinorhizal plants, respectively, has been an active area of research for decades. However, the discovery of N-fixing bacterial endophytes in the non-nodulating plants such as sugarcane during the late 1980′s has expanded the area of BNF research [26,27]. Bacterial endophytes in several genera such as Azoarcus, Burkholderia, Gluconobacter, Herbaspirillum, Klebsiella, Pantoea, and Rahnella were found in many different plants, facilitating the growth of the host plant in nutrient-poor conditions [10,22,28,29]. N-fixation involves reducing the triple bonds of N2 molecules, which requires substantial amounts of energy. Given this energy demand, free-living N-fixers likely have relatively limited applications in agriculture compared to plant-associated N-fixers, which can overcome the energy requirement of N-fixation by deriving energy from the host plant [30]. Bacterial endophytes reside in the internal plant tissues which may be a favorable environment for N-fixation that minimizes competition with other microbes in the rhizosphere as well as possibly providing a microaerobic environment that is necessary for nitrogenase activity [31,32,33]. The use of bacterial endophytes in agriculture has immense potential to reduce the environmental impacts caused by chemical fertilizers, especially N fertilizers. Several studies have shown that a significant portion of N used in agriculture is lost to the environment. It is estimated that only 17 Tg N of every 100 Tg N used in global agriculture is utilized [34,35,36]. The N lost from farmlands eventually accumulates in lakes, rivers or marine systems causing excessive growth of algae, which has serious impacts on aquatic ecosystems. Greater atmospheric N, in the form of ammonia or ammonium, also coincides with areas of eutrophication in the downwind regions of farmland. Elevated concentrations of N in the form of ammonium, nitrate or nitric acid vapors in the atmosphere can reduce air quality, reduce visibility and impact plant growth [37,38]. In addition, microorganisms convert excess ammonium or nitrate in the soil into nitrous oxide, which is a potent greenhouse gas. The use of natural symbionts such as bacterial endophytes could reduce the need for fertilizer inputs in the growth of crop plants and potentially lead to making farming more environmentally sustainable in the future. Bacterial endophyte strains promote plant growth by synthesizing phytohormones including indole-3-acetic acid (IAA), cytokinins and gibberellins or through regulating internal hormone levels in the plant body [4,39,40]. IAA produced by endophytes within plants increases the number of lateral and adventitious roots, facilitating access to nutrients, and improving root exudation, offering more resources for soil microbes to interact with roots [40,41]. Growth enhancement by increasing plant height and/or biomass has been reported in many studies when plants were inoculated with bacterial endophytes capable of producing IAA [39,42,43,44,45]. Furthermore, bacterial endophytes secrete siderophores and solubilize phosphorus in soil while initiating the symbiotic interactions with host plants [4,41]. Siderophores are organic compounds secreted by microorganisms and plants in iron limited conditions enabling them to chelate iron from the environment for microbial and plant cells to uptake [4,46]. Similarly, phosphorus-solubilizing bacteria can solubilize immobile phosphorus in soil, which is potentially available for plants to absorb, an important trait for plant growth promotion [47,48,49,50]. Many recent reviews highlighted the mechanisms and importance of phosphorus solubilizing microorganisms in agriculture [4,51]. Bacterial endophytes can confer resistance or tolerance to the host plant from biotic and abiotic stresses by releasing antimicrobial compounds, producing siderophores, competing for space and nutrients, and modulating the plant resistance response [39,52,53]. Some bacterial strains can relieve plant stress by blocking the pathway of ethylene synthesis in plants. These bacteria utilize 1-aminocyclopropane-1-carboxylate deaminase, which helps to reduce ethylene concentrations accumulated in response to different stresses in plants, otherwise lethal to plant health [54]. Endophytic strains of Bacillus, Burkholderia, Enterobacter, Pseudomonas, and Serratia were found to be effective in suppressing the growth of pathogenic microorganisms in in vivo and in vitro conditions [53,55,56,57]. Moreover, endophyte strains in the genera Bacillus, Enterobacter, Pseudomonas, Azotobacter, Arthrobacter, Streptomyces, and Isoptericola were successful in alleviating drought, heat, and salt stress in different crop plants. More importantly, symbiotic plants with these endophytes were not only capable of relieving the stress but also significantly increased plant biomass and height [58,59,60,61,62]. However, the mechanisms used by bacterial endophytes to mitigate abiotic stress remain unclear. Go to: 2. Recruitment of Bacterial Endophytes by Host Plants The rhizosphere is defined as the soil-root interface where complex interactions take place between the plant and surrounding soil microorganisms [9,63]. It has been reported that plants can release significant amounts of photosynthates or exudates from its roots, which influence microbial communities in the rhizosphere. Root exudates including organic acids, amino acids, and proteins may be involved in recruiting bacterial endophytes from the rhizosphere [9,64,65]. Root exudates likely contain substrates that initiate early communication between host plants and bacterial endophytes, and consequently steer the colonization process. For example, evidence of the involvement of oxalate in the recruitment of the beneficial bacterial strain Burkholderia phytofirmans PsJNby host plants has been reported [66]. In this study, a Burkholderia phytofirmans strain defective in oxalate utilization was used to inoculate lupine and maize plants that secrete moderate and low levels of oxalate, respectively. The mutant was observed in significantly less numbers in both maize and lupine plants 3 days after inoculation as compared to the wild type strain. Interestingly, inoculation with both wildtype and mutant strains resulted in significant differences in colonization by the two strains in lupine but not in maize. Oxalate was also observed in Brachypodium root exudates, and high numbers of Proteobacteria were detected in the Brachypodium rhizosphere [64]. Moreover, bacterial quorum sensing compounds are likely involved in communication with the plant root and the subsequent colonization process. The importance of these compounds in the colonization and growth promotion of plants by endophytes is supported by a recent study that showed that a quorum sensing mutant of Bukholderia phytofirmans PsJN could no longer efficiently colonize Arabidopsis thaliana and did not promote its growth [67]. Plants are likely directly involved in quorum sensing as well, given that some plant extracts have been shown to have quorum quenching capabilities which could protect them against pathogens and some quorum sensing molecules have been shown to have direct plant growth promoting effects [68]. Additionally, several endophytes of Populus deltoides were found to have LuxR homologs hypothesized to be involved in responding to plant derived compounds [69]. This study also found that many of the surveyed endophyte genomes contained LuxR-LuxI type quorum sensing gene pairs pointing to their importance in the endophytic lifestyle. The importance of quorum sensing compounds for plant-microbe interactions has been reviewed in detail by Hartmann et al. [70]. The native soil composition and host plant genotype are also considered important in the recruitment of bacterial endophytes by the host plant. A detailed study of root endophytes of Arabidopsis plants grown in different soils concluded that soil type likely influences the composition of the bacterial endophyte community found in the host roots. This indicates that different soil types may be inhabited by variable bacterial populations that serve as the initial inocula [9]. In addition, Wagner et al. [71] showed that bacterial communities (epiphytic and endophytic) in Boechera stricta, a perennial wild mustard plant, are highly similar in both leaves and roots supporting the hypothesis that the communities are recruited from the soil. This study also showed that environmental conditions such as soil nutrition, moisture, temperature, and host genotype and age have a direct influence on root and leaf bacterial communities. Diverse bacterial communities were reported in grass species Dactylis glomerata, Festuca rubra, and Lolium perenne under different management regimes, such as fertilizer application and mowing frequencies, indicating that agronomic operations may influence bacterial endophyte recruitment in cultivated plants. Interestingly, in these grasses, the functional profile of the bacterial communities was not correlated with changes in community composition at the species-level, suggesting that selection of endophytes by the plant may be functionally driven rather than driven by phylogeny [11]. Furthermore, direct influence of crop genotype and N fertilizer application on the diversity of N-fixing (diazotrophic) endophytes was detected in maize and rice plants [72,73]. A detailed study of the root microbiome of Arabidopsis showed that only a narrow subset of rhizosphere communities was able to colonize and establish in the root endosphere [74]. Overall, molecular mechanisms by which plants select specific bacterial endophytes over others remain largely unknown [66,70]. Go to: 3. Attachment of Bacterial Endophytes to the Host Plant Surface The attachment or adhesion of bacterial cells to the plant surface is considered the first step of the colonization process. Bacteria in the vicinity of the plant roots most likely swim towards the roots, using chemotactic affinities for root exudates. This is followed by attachment to the root surface, which is likely important in getting access to potential entry sites at lateral root emergence areas or other openings caused by wounds or mechanical injuries. The exopolysaccharides (EPS) synthesized by bacterial cells may facilitate the attachment of bacterial cells onto the root surface and may be important in the early stages of endophytic colonization. The EPS produced by endophytic bacterium Gluconacetobacter diazotrophicus Pal5 was reported as an essential factor for rice root surface attachment and colonization [75]. A recent colonization study in rice plants using G. diazotrophicus Pal5 showed that bacterial cells were shielded from oxidative damage by exopolysaccharides, which may be crucial for colonization. Additionally, free radical concentrations in planta were decreased by the application of EPS. Colonization was reduced in an EPS knockout strain of G. diazotrophicus. Interestingly, this reduction in colonization was rescued by the addition of EPS produced by the wild type strain [76]. In another study, Balsanelli et al. analyzed the mutant strains of Herbaspirillum seropedicae that are deficient in EPS production and concluded that EPS is not required for plant colonization, which could potentially point to a variation in the genes required for colonization across different endophyte species [77]. The biology of bacterial EPS including its synthesis, chemistry and functions were reviewed elsewhere [78]. Bacterial structures such as flagella, fimbriae or cell surface polysaccharides are also likely involved in the attachment of bacteria to the plant surface. While studying colonization of maize plants by endophyte H. seropedicae, Balsanelli et al. reported that bacterial lipopolysaccharide (LPS) is necessary for attachment and subsequent endophytic colonization of plant roots [79]. Later, it was also demonstrated that binding of N-acetyl glucosamine of LPS with maize root lectins is required for bacterial attachment and subsequent colonization inside the roots [80]. Bacterial adherence and colonization of the root interior likely happen in close succession given how quickly colonization is observed in roots after inoculation with bacterial endophytes [81,82,83]. The process of adherence of Rhizobia on legume roots, plant pathogenic bacteria on plant leaf or root surfaces, and Agrobacterium on roots of the host plant has been thoroughly studied in the past [84,85,86]. However, the mechanisms by which bacterial endophytes attach on plant surfaces remain relatively unexplored [87]. Go to: 4. Entry of Bacterial Endophytes into the Host Plant Bacterial endophytes initially attach to the root surface also called rhizoplane, and explore the potential entry sites to access the internal plant tissues. Openings in the roots where root hairs or lateral roots emerge, as well as stomata, wounds and hydathodes in the shoots are considered the main entry points that endophytes use to enter the host plant [4]. Endophytic bacteria likely utilize these natural discontinuities in the plant body to access the internal plant tissues. Moreover, some bacterial endophytes may modify the plant cell wall by secreting cell wall cellulolytic enzymes such as cellulases, xylanases, pectinases, and endoglucanases, which facilitate bacterial entry and spread within the plant tissues [81,88,89]. One study supported this hypothesis by observing that the frequency of entry of an endoglucanase mutant of Azoarcus sp. BH72 into rice roots was decreased as compared to the wild type strain and the mutant was unable to spread to the aerial plant parts [88]. Many colonization studies suggested that natural cracks at the lateral root emergence site are the most common entry sites for endophytic bacteria [4,14,81]. Furthermore, some bacteria use root apex and root hairs as entry points followed by endophytic colonization in root cortex and vascular tissues [90,91]. Go to: 5. Bacterial Niches inside the Host Plant Bacterial endophytes most often occupy intercellular spaces in the plant, most likely because these areas have an abundance of carbohydrates, amino acids, and inorganic nutrients [4,12,27]. They likely exclusively colonize the intercellular spaces of various plant parts including roots, leaves, stems, flowers, and seeds [14,18,81,92,93,94]. Colonization can be localized at the tissue level or systemically throughout the plant body. In the early stages of endophytic colonization, endophytes are first observed in root hairs, and subsequently in the root cortex [83,90,95]. Inoculated Burkholderia sp. strain PsJN was observed in cortical cells, endodermis, and xylem vessels, and colonization was especially strong at primary and secondary roots and at the base of lateral roots and root tips. Interestingly, in this study, both intracellular and intercellular colonization was observed [81]. In maize plants, bacterial endophytic colonization was stronger in the lower stem compared to the stem closer to the shoot apex [96]. The mobility of bacterial cells accompanied by the synthesis of cellulolytic enzyme may help endophytes to spread to aerial plant parts including leaves and stems [12,25,81]. In leaves, bacterial endophytes have been observed in the intercellular spaces of mesophyll, and xylem tissues and substomatal areas. Using green fluorescent protein (GFP) labeling and β-glucuronidase (GUS) staining, Burkholderia sp. strain PsJN was observed in xylem and substomatal chambers of inoculated leaves of grape vine plants. Interestingly, bacterial cells leaving through the stomatal aperture were also observed in grapevine leaves [81]. The demand for nitrogen in the production of rubisco and other photosynthetic enzymes may suggest an important role for BNF by bacterial endophytes in the leaves. For example, studies have shown that diazotrophic endophytes Klebsiella variicola colonized the mesophyll cells of sugarcane leaves; Herbaspirillum sp. colonized young leaves and shoots of wild rice; Herbaspirillum seropedicae Z67 colonized leaf vein, mesophyll cells, and substomatal cavities of rice leaves; and Serratia marcescens colonized the leaf sheaths and leaf aerenchyma of rice plants [12,97,98,99]. Niches of indigenous bacterial endophytes in different sections of grapevine leaf pieces were found by florescence in situ hybridization (FISH) and confocal laser scanning microscopy. Bacterial microcolonies were observed in leaf veins, trichomes, and cut sections of leaf pieces. Colonization was strong in various layers of the leaf tissue [100]. One relatively new area of research that remains poorly studied is intracellular colonization of plant cells by endophytes. Endophytes are known to typically colonize the intercellular spaces of plants but several examples of intracellular colonization of plants by bacteria have been reported recently [101]. These examples include the presence of intracellular bacteria in shoot-tips of banana, shoot meristem of Scotch pine, seedling roots of switchgrass and in micro propagated peach palm [102,103,104,105]. While this area of research is relatively new and unexplored, several hypotheses exist as to the potential colonization pathway intracellular endophytes use. Root hairs offer a logical point of entry for these endophytes as many cases of intracellular plant-microbe interactions begin with colonization of the microbe through intracellular access to root hairs. This is the case in the very well-studied legume-rhizobium symbioses and is one method reported to be used by some endophytes [90,106]. The role of each symbiotic partner in intracellular colonization remains unclear. Endophytes may be capable of gaining access to the intracellular space directly by secreting cell wall degrading enzymes or through a phenomenon known as rhizophagy [104,107]. Rhizophagy is a recently observed process in which roots of certain plants actively bring microbes in the soil into their cells, possibly in order to digest them and acquire essential nutrients from them [108]. The advantages to this peculiar kind of endophytic colonization remain unclear. One possible hypothesis is linked to the observation that intracellular colonization by endophytes is associated with a bombardment of the colonizing endophytes by intracellular hydrogen peroxide. This allowed the authors to use a hydrogen peroxide stain to detect the intracellular bacteria but also points to a potential advantage of this interaction for the plant [104]. Briefly, increasing intracellular reactive oxygen species (ROS) concentrations in the plant could acclimate the plant to ROS stress, which could increase its tolerance to stressors linked to ROS stress such as drought, heat and salt stress [109]. Survival in the intracellular environment is likely a specific adaptation of the endophytes to this environment and could provide the endophyte with a niche with low competition. The specificity of this adaptation is supported by a change of shape of the intracellular endophytes of switchgrass to an L-form lacking a cell wall as well as the fact that many of these endophytes are not currently culturable [102,104]. While this phenomenon seems widespread, the difficulty of culturing intracellular endophytes makes them very difficult to study [102]. Classic microbiology methods relying on culturing the endophytes, including fluorescent tagging, may be difficult to implement in the study of these intracellular endophytes. It is possible that a stronger reliance on next generation sequencing, metagenomics and FISH may be necessary to further study the life cycle and ecology of these endophytes. Go to: 6. Bacterial Genes Involved in Plant Colonization The production of ROS, mainly superoxide (O2−), hydrogen peroxide (H2O2), and hydroxyl radicals (OH−), are well understood as being an important immediate plant defense response in plant-microbe interactions [110,111,112]. During the plant-endophyte interaction, ROS-detoxification occurs early on, after entry of the endophyte into the plant. During the early stages of rice root colonization, endophytic diazotrophic bacterium Gluconacetobacter diazotrophicus expressed ROS-deactivating genes such as superoxide dismutase (SOD) and glutathione reductase (GR) in greater amounts. Furthermore, SOD and GR mutants of G. diazotrophicus could not colonize rice roots supporting the hypothesis that ROS-deactivating genes are important during the initial stages of colonization [113]. In addition, a gum gene cluster, gumD, in G. diazotrophicus, involved in EPS production, was shown to be required for biofilm formation and plant colonization. A genomic survey using comparative genomics of endophyte strains hypothesized that many genes involved in biofilm production, adhesion, and motility contribute to plant colonization and the endophytic life style within the host plant [6,114,115,116,117]. The metabolic adaptations required for root attachment, modification of the plant cell wall and life in the microaerobic environment within the plant was reported in the endophyte strain Herbaspirillum seropedicae. Increased gene expression of genes linked to N-fixation, auxin production and ABC transporters during interaction with the host plant was reported in this strain [87]. Many genes involved in bacterial chemotaxis and secretion systems were found in bacterial groups colonizing the Brachypodium rhizosphere and may be expressed during the colonization of roots by these endophytes [64]. Go to: 7. Colonization Cycle of Bacterial Endophytes in the Host Plant Bacterial endophytes are capable of colonizing different seed parts including the embryo. These endophytes likely mobilize and grow in the developing seedlings during germination and early seedling growth [118,119,120]. As seedlings emerge and plant growth begins, interactions between the roots and the soil microbiome commence. Plant exudates fuel microbial activities in the rhizosphere, which facilitate the attachment and entry of bacteria into the plant roots. Eventually, certain endophytes initiate colonization of tissues beyond the roots such as the stems and leaves, and ultimately throughout the plant endosphere. The colonization pattern and growth promoting characteristics of bacterial endophytes in different plant species are presented in Table 1. Some bacterial endophytes also colonize flowers and seeds, and most likely get transferred vertically from the maternal endophyte community into the offspring [93,120]. Additionally, a recent study showed that endophytes could colonize corresponding seeds after the flowers were inoculated. Moreover, endophytes passed on to seeds resumed endophytic activity after the seeds were planted [93,120,121].