Field of invention

The present invention relates to a biologicai soil amendment for diminishing nutrient leakage into aqua system. The soil amendment comprises microbes that are changing plant root architecture via rupturing apical dominance of the plant roots. The invention relates to modifying plant structure such that the plant is capable of better use and retain nutrients from fertilizers. The invention relates to beneficial effects other than crop increase due to better use of nutrients, especially nitrogen and phosphorus. The invention relates to ecologically sustainable agriculture .

Background of the invention

The use of fertilizers is crucial in the modern agricultural practices to enhance the soil fertility and crop productivity. The soil has large quantities of nitrogen (N), phosphorus (P) and potassium (K) that are present in highly insoluble forms that can’t be absorbed by plants. In order to improve plant production fertilizers such as organic fertilizers made from wastes and manure and chemical synthetic fertilizers are commonly used. Today most of the fertilizers used in the field are synthetic chemicals that have to be frequently applied to the soil because only a portion is taken up by plants, and the rest is lost. This causes an environmental problem which is commonly seen as increased nutrient concentrations in agricultural waters, which mainly end up into aquatic systems causing eutrophication, which further causes other environmental problems such as lack of oxygen and death of fish.

Moreover, the predominant application and long-term use of excess amounts of fertilizers can cause other negative effects such as soil depletion, and also health problems to humans.

For a successful agricultural yield of crops, fanners have to pay attention to the requirements of each crop. Many fertilizers are employed with the goal of supplying the optimal quantity of nutrients to obtain the best yield. These fertilizers can add one or more plant essential nutrients which may be macronutrients or micronutrients. Macronutrients are those needed in large quantities like carbon (C), oxygen (O), hydrogen (H), nitrogen (N), phosphorous (P) and potassium (K), whereas micronutrients are vitamins and other minerals that are required in smaller concentrations. The most commonly used fertilizers include N, P and K. Some fertilizers have additionally micronutrients like zinc and other metals.

Usually, chemical fertilizers are added to the soil to provide nitrogen in form of nitrate and phosphorus in form of phosphate. During the last decades both nitrate and phosphate levels have increased in waters leaking from agricultural system. Eutrophication of aquatic systems due to modern agriculture is a serious problem all over the world. For this reason, there is a serious need for agricultural practices that would lead to diminished leakage of nutrients into the aquatic systems.

Modern agriculture is reliant on high inputs of water and nutrients and breeding of crops responsive to such high input levels. An important goal of plant breeding has been production of plant varieties that, would produce high yields even in suboptimal environments. The approach of improving plant productivity by breeding has led to situation where the most commonly cultivated food and feed plants are genetically tuned to their maximum performance when provided with water and fertilizers within certain limits. This means that the plant production can seldom be increased any further by new or alternative fertilizers, or by adding more fertilizers to the soil.

Meeting increasing food demand associated with global population growth requires novel strategies that, produce high yields while minimizing damage to the environment. To address this issue, this disclosure provides a soil amendment system, and use of a soil amendment that addresses the environmental issues of novel agriculture by diminishing leakage of nutrients into the agricultural waters, improving plant ability to retain the nutrients while not necessarily increasing crop yields which have already been genetically tuned to their maximum.

Plant growth promoting rhizobacteria, including species such as Azospirillum, Bacillus, Pseudomonas, and Providencia have been shown to have success in improving production of legumes as rhizosphere inoculants. Garcia et al. 2021 showed with soybean plants that nodulation rhizobium was improved along with increased coarse root development by Pseudomonas inoculation. Increased nodulation likely improved nitrogen fixation which would explain increased yield of the legumes.

It has been shown that addition of beneficial microorganisms that produce phytohormones, small molecules or volatile compounds can act directly or indirectly activating plant immunity or they can regulate plant growth, and production. (Ortiz-Castro et. al., 2009). Most of the research about microorganisms that can affect plant growth have shown an increase in root and shoot dry weight of different plants (Perez-Montano et. al. 2014). It has been shown that Azospirillum and Bacillus stimulate plant growth by the production of phytohormones like auxin and cytokinin and also by the production of volatile compounds which triggers hormonal signal networks in the plant. Further, different species from the Pseudomonas genus can antagonize with plant pathogens, improving plant health (Ortiz-Castro et. al., 2009). Only a few reports explaining the change in root architecture by microorganisms have been published, mainly using mycorrhiza and bacteria from the Bacillus genus (Gutjahr and Paskowski, 2013, Wang et. al., 2015). It has been shown that arbuscular mycorrhizal establishes a symbiotic relation with plants and induces a change in root architecture. For example, Glomus intraradices can colonize the roots of rice and induces an increase in crown root length and number of lateral roots (Gutjahr et. al., 2009). Although only a few studies of changes in root architecture induced by microorganism have been reported, little is known about the mechanism.

Here, a mechanism to changes in root architecture in non-leguminous plant is disclosed and as a result a solution to problems caused of nutrient leaking into aqua system from agriculture is provided.

Summary of the invention

This invention addresses the problem of nutrients leaking into aqua system from agriculture. The invention provides a new concept of using plant growth promoting bacteria in retaining nutrients in the plant biomass as opposed to leaking into the aqua system. The invention provides formulations that are capable of increasing plant uptake of nutrients even in optimal nutritional situation where increased nutrition intake no more results in increased crop production but to environmentally improved situation of preventing leakage of nutrients to end into aqua systems. The invention provides formulations that are capable of improving plant nutrient uptake in non optimal environmental conditions thereby preventing leakage of nutrients to end into aqua systems

This invention provides soil amendment system that is capable of diminishing leakage of nutrients, especially nitrogen and phosphorus from fertilized soils by altering root architecture of agricultural crops. The soil amendment system of this invention is capable of improving plant retainment of nutrients also in aeroponic or hydroponic cultures. The soil amendment system is capable of rupturing the apical dominance of plant root, resulting in changed root architecture, increased nutrient uptake and decreased leakage of nutrients into the aqua system

The soil amendment system comprises adding a formulation including a bacterial isolate of one or more bacterial species affecting plant root architecture. The bacterial species comprise preferably at least one Pseudomonas strain. According to a preferred embodiment soil amendment system comprises an isolate of bacterial strains extracted from roots of Deschampsia antarctica. According to a preferred embodiment the soil amendment system comprises an isolate of Pseudomonas species deposited with accession number ATC’C PTA- 122608; other preferable bacterial isolates from Deschampsia antrarctica roots include Pseudomonas antarctica strain deposited with accession number ATCC PTA 8990; Pseudomonas trivialis strain deposited with accession number ATCC PTA 8988, and Arthrobacteria sp. strain deposited with accession number ATCC PTA 8989.

The soil amendment system is capable of altering root architecture of several agriculturally important crops after just one treatment. The treatment may be applied together with an NPK- fertilizer or after the NPK-fertilizer. Treatment may be applied into the soil, or the seeds may be treated before sowing. The treatment may be added to grow medium of hydroponic or aeroponic cultures. The treatment may be provided together with a fungicide.

The soil amendment system of this invention reduces growth of the apical meristem of the plant’s roots and causes lateral relocation of auxin.

According to one aspect, this invention provides a novel sustainable biological soil amendment system based on the Pseudomonas spp bacteria for use of diminishing leakage of nutrients, especially nitrogen and phosphorus, into aqua systems.

It. is an object of this invention to use a bacterial formulation as a soil amendment wherein adding the formulation into the soil or growth medium in aeroponic or hydroponic culture with PKN -fertilizer increases uptake and retain of the nutrients by the plants and thereby decreasing leakage of nutrients into the aqua- system.

According to certain embodiments the bacterial formulation is used for non-leguminous plants to change the root architecture and retain nutrients thereby decreasing leakage of nutrients into the aqua system. According to certain embodiments the soil amendment comprising bacterial formulation reduces nitrogen and phosphate content of excess water not used by the agricultural plants, wherein the soil amendment is added into the soil or growth medium once before planting the plantlets or sowing the seeds.

It is an object of this invention to provide a method to reduce nutrient leakage into aqua systems from agricultural soils, the method comprising adding bacterial formulation into the soil before or together with a PKN fertilizer.

According to certain embodiments the method comprises adding a soil amendment comprising at least one bacterial isolate from Deschampsia antarctica rhizosphere into the soil or growth medium to increase uptake and retain of nitrogen and phosphorus in the plant foliage thereby diminishing nitrate and phosphate leakage into aqua systems.

It is an object of this invention to provide a method to decrease nitrogen and phosphate leakage into aqua systems by amending agricultural soil or plant growth media with a soil amendment comprising at least one bacteria strain changing root architecture by rupturing root apical dominance and thereby increasing uptake and retain of nitrogen and phosphorus in the plant foliage thereby diminishing nitrate and phosphate leakage into aqua systems.

It is an object of this invention to enable fertilization of agricultural plants by conventional NPK-fertilizers without increasing leakage of phosphate and nitrate from the fertilizer to leak into aqua systems by adding a soil amendment comprising at least one bacterial strain rupturing apical dominance of the root systems thereby changing root architecture and increasing uptake and retain of nitrogen and phosphorus in the plant foliage.

According to certain embodiments the soil amendment comprises bacterial isolates from Deschamps antarctica roots.

According to certain embodiment the soil amendment comprises one or more of Pseudomonas strains selected from the group consisting of Pseudomonas sp. that may be selected from the group consisting of Pseudomonas protegens, Pseudomonas saponiphila, Pseudomonas ficuserectae, Pseudomonas conge Pseudomonas tremae, Pseudomonas caricapayae, Pseudomonas madelii, Pseudomonas novoantarctica, Pseudomonas savastanoi, Pseudomonas syringae, Pseudomonas chlororaphis subsp. Pisciem, Pseudomonas cannabina, Pseudomonas marginalis. Pseudomonas simiae, Pseudomonas avellanae, Pseudomonas chlororaphis subsp. Aurantiaca, Pseudomonas c hlororaphis subsp. Chlororaphis, Pseudomonas extremaustralis, Pseudomonas kilonensis, Pseudomonas Uni, Pseudomonas Antarctica, Pseudomonas corrugate, Pseudomonas poae, Pseudomonas grimontii, Pseudomonas brassicacearum subsp., Neoaurantiaca, Pseudomonas meridian, Pseudomonas trivialis. Pseudomonas veronii, Pseudomonas lundensis, Pseudomonas salomonii, Pseudomonas rhodesiae, Pseudomonas arsenicoxydans, Pseudomonas thivervalensis, Pseudomonas deceptionensis, Pseudomonas palleroniana, Pseudomonas chlororaphis subsp. A ureofaclens, Pseudomonas costantinil, Pseudomonas iundd. Pseudomonas migulae, Pseudomonas oriental is. Pseudomonas extremor ientalis, Pseudomonas mediterranea, Pseudomonas brassicacearum subsp. Brassicacearum, Pseudomonas abietaniphila, Pseudomonas haetica, Pseudomonas brenneri, Pseudomonas psychrophila, Pseudomonas jessenil, Pseudomonas fragi, Pseudomonas tolaasii, Pseudomonas proteolytica, Pseudomonas taetrolens, Pseudomonas mohnii, Pseudomonas moorei, Pseudomonas moraviensis, Pseudomonas gessardii, Pseudomonas cichorii, Pseudomonas libanensis, Pseudomonas benzenivorans, Pseudomonas reinekei, Pseudomonas fluorescens, Pseudomonas agarici, Pseudomonas lutea, Pseudomonas mucidolens, Pseudomonas azotoformans, Pseudomonas viridiflava, Pseudomonas koreensis, Pseudomonas kuykendallii, Pseudomonas synxantha, Pseudomonas segetis, Pseudomonas marincola, Pseudomonas cedrina subsp. Cedrrina, pseudomonas graminis, Pseudomonas Vancouver enis, Pseudomonas cedrina subsp. Fulgida, Pseudomonas plecoglossicida, pseudomonas cuatrocinegasensis, Pseudomonas taiwanensis, Pseudomonas putida, Pseudomonas rhizosphaerae, Pseudomonas angiiilliseptica, Pseudomonas monteilii, Pseudomonas Juscovaginae, Pseudomonas mosselii, Pseudomonas taenensis, Pseudomonas asplenii, Pseudomonas entomophila, Pseudomonas cremoricolorata, Pseudomonas parafidva, Pseudomonas alcaliphila, Pseudomonas oleovorans subsp. Lubricantis, Pseudomonas borbori, Pseudomonas composti, Pseudomonas toyotomiensis, Pseudomonas batumici, Pseudomonas flavescens, Pseudomonas vranovensis, Pseudomonas punonensis, Pseudomonas balearica, Pseudomonas indoloxydans, Pseudomonas guineae, Pseudomonas japonica, Pseudomonas stutzeri, Pseudomonas seleniipraecipitans, Pseudomonas peli, Pseudomonas fulva, Pseudomonas argentinensis, Pseudomonas xanthomarina, Pseudomonas pohangensis, Pseudomonas oleovorans, Pseudomonas mendocina, Pseudomonas luteola, Pseudomonas straminea, Pseudomonas caeni, Pseudomonas aeruginosa, Pseudomonas tuomuei ensis. Pseudomonas azotgens, Pseudomonas indica, Pseudomonas azotgens, Pseudomonas indica, Pseudomonas oryzihabitans, Pseudomonas otitidis, Pseudomonas psychrotolerants, Pseudomonas zeshuii, Pseudomonas resinovorans, Pseudomonas oleovorans subsp. Oleovoraris, Pseudomonas thermotolerants, Pseudomonas bauzanensis, Pseudomonas duriflava, Pseudomonas pachastrellae, Pseudomonas citronellolis, Pseudomonas alcaligenes, Pseudomonas xinjiangensis, Pseudomonas delhiensis, Pseudomonas sabulinigri, Pseudomonas litoralis, Pseudomonas pelagia, Pseudomonas linyingensls, Pseudomonas knackmussii, Pseudomonas panipatensis, Pseudomonas nitroreducens, Pseudomonas nitrireducens, Pseudomonas jinjuensis, Pseudomonas pertucinogena, Pseudomonas halophile, Pseudomonas horeopolis, Pseudomonas geniculate, Pseudomonas be tel I, Pseudomonas hibiscicola, Pseudomonas pictorum, and Pseudomonas carboxydohydrogena.

According to certain embodiments the soil amendment comprises one or more Pseudomonas strains combined with kaolin and other ingredients to permit the strains to survive.

According to certain embodiments the soil amendment is applied as granular or powdery composition. According to certain embodiments it is applied as water solubilized liquid.

According to certain embodiments the soil amendment comprises one or more strains of Pseudomonas and/or Bacillus strains selected from the group of isolates deposited with accession numbers ATCC PTA- 122608, ATCC PTA 8990; ATCC PTA 8988, and ATCC PTA 8989

According to certain embodiments the soil amendment comprises at least Pseudomonas isolate ATCC PTA-122608 combined with kaolin (98% pure) and other ingredients (formula) to permit the survival of the strain.

It is an object of this invention to provide a method to decrease nutrient leakage into aeroponic cultivation water, by adding an amendment comprising at least one bacterial isolate from Deschampsia antarctica rhizosphere into the cultivation water or treating the seeds with the amendment.

Another utility presented by this patent is the reduction of the water footprint of the crops in which it is applied. Fundamentally due to the effect of increasing the root surface and absorption, which on one hand optimizes the use of water by the plant avoiding hydric stress and, on the other hand, optimizes the use of nutrients maintaining the level of productivity with less quantity of fertilizer. The latter influences a significant decrease in the impact of the gray water footprint of the crops in which it is applied, because by applying a smaller amount of fertilizers, the possibilities of contamination decrease drastically and consequently the gray water footprint will be much lower.

Short description of the drawings Fig. 1 shows the tomato root lengths of in vitro grown plants with treatments with different EcoPlus treatments.

Fig. 2 illustrates how treatment of tomato plants with EcoPlus (bacterial isolate in kaolin) reduces root apical meristem (RAM) size significantly as compared to control (kaolin (98% pure) only).

Fig. 3 A-E illustrates effects of Pseudomonas- inoculation to root growth of transgenic tomato primary roots. A and B Tomato meristematic zone marker line SIRPL11C::NTF grown on vertical plates for four days with 0 (mock), 10 ³ or 10 ⁶ CFU bacterial treatment. Scale bars represent 2 cm. M indicates the seedlings germinated during the 24h immediately prior to bacterial treatment and that were used in the experiment (n ≥ 6,p < 0.01). C. Root architecture (total length, surface area and volume and per diameter class) D . Total Length partition in cm.

Fig. 4 A-E illustrates how Pseudomonas- inoculation inhibits cell division in tomato primary roots while the quiescent center remains unaffected. A, Structure of a root tip with the meristem (blue) as well as quiescent center and initials (green) indicated. B, Tomato meristematic zone marker line SIRPL11C::NTF grown for four days with 0 (mock), 10 ³ or 10° CFU bacterial treatment. Green indicates NTF signal, red and yellow indicate autofluorescence, and scale bars represent 200 pm. C, Quantification of meristematic zone length based on the SIRPL11C::NTF marker expression (n ≥ 5, p < 0.01). D, Tomato quiescent center and initial marker line SlWOX5pro: :NTF grown for three days with 0 (mock) or 10 ⁶ CFU bacterial treatment. Green indicates NTF signal, red and yellow indicate autofluorescence, and scale bars represent 200 pm. E, Quantification of SlWOX5pro expression domain length shows no significant difference (w > 6).

Fig. 5. Rupture of apical dominance. Marker DR5-GFP signal (green) in the mature zone of the root. Red staining corresponds to FM4-64 labeling of the plasma membrane. Arrowheads highlight lateral root primordia. Detailed description of the invention

Definitions:

By EcoPlus it is meant here a formulation comprising one or more bacterial isolates from Deschampsia antarctica roots is called EcoPlus. By soil amendment as used here is meant a composition comprising one or more bacterial strains changing root architecture via disruption of apical dominance. Preferably the soil amendment comprises bacterial isolates from Deschampsia antarctica roots. More preferably the soil amendment comprises at least one Pseudomonas strain. The term ‘soil amendment’ is used for composition that is for use in soil, but also for in use in aeroponic or hydroponic cultures media. Soil amendment may include other ingredients, such as kaolin and/or indolic acetic acid.

Isolated psychrophilic bacteria that grow in the Antarctic frozen soil were isolated. The bacterial isolates comprised Pseudomonas strains. Altogether 70 isolates have been obtained. Exemplary' isolates have been deposited with accession numbers ATCC PTA-122608, ATCC PTA 8990; ATCC PTA 8988, ATCC PTA 8989.

Soil amendment formulations were provided by using the isolates. In preparation of the formulations, the isolates were grown Luria Bertani (LB) medium at 20°C in agitation at 250 rpm. Bacterial growth was controlled by temperature. At temperature above 30°C the bacteria die. Solid formulations of the bacterial strains were produced. Survival of the formulations w'ere tested and it was established that the bacteria survive at least 12 months in dry environment. Toxicity of the formulations were tested in rats and the formulations were found to be safe (oral, dermal an inhalation tests): LD50 as determined to be 2000mg/kg.

Biochemical characterization and enzyme profiling C utilization of isolate ATCC PTA-122608 was investigated by plating the isolate on a variety of media. Triple Sugar Iron agar (TSI), Simmons citrate agar, Motility-Indole-Ornithine medium (MIO) and Christensen urea agar were inoculated with a drop or stab from an 18 h LB culture of

Pseudomonas ATCC PTA-122608 and incubated at 18°C for 24 h. All media were manufactured by Difco (Becton Dickinson). Enzyme activities of Pseudomonas isolate ATCC PTA-122608 were determined using the API ZYM miniaturized test (Biomerieux) following the manufacturer’s instructions. The API strips were inoculated with 24 h-old cultures grown in LB broth and then incubated at 18°C for 24 h. The bacteria are Gram-negative rod, which has a non-femientative aerobic metabolism. The biochemical testing showed that this stains urease positive and use citrate as carbon source. Analyses of enzyme metabolism using Api ZYM assay, show that this strain has activity for Naphtol-AS-BI-phosphohidrolase, acid phosohatase, trypsin, alkaline phosphatase, esterase (C-4), esterase lipase (C-8), lipase (C-14) and leucine arylamidase.

A soil amendment formulation was prepared by mixing at least one of the isolates with kaolin. Typically, the mixing ratio is 1.100 v/v , but other ratios may also be used. The soil amendment formulation may additionally include auxin supplement e.g., in a small amount of IAA (Indol Acetic Acid) e.g. 0.1- 0.8 mg/1, preferably 0.5 mg/1 As an exemplary formulation in here we used ATC PTA-122608. In this disclosure the formulation comprising one or more bacterial isolates from Deschampsia antarctica roots is called EcoPlus.

In vitro and in vivo phosphate solubilization:

Laboratory and greenhouse experiments were performed to test whether the isolates solubilize non-bioavailable phosphate sources and alters P acquisition in tomato seedlings. A P -solubilization plate assay was set up using modified procedures outlined in Marra et al. (2015) and the National Botanical Research Institute Phosphate (NBRIP) growth media. The media was supplemented with a pH indicator and 1 g of non-bioavailable phosphate source. Ten plates were treated with the EcoPlus composition (bacterial isolates in kaolin) in quadruplicated 20 pl aliquots at 10 ⁸ CFU/ml and 10 plates were inoculated with 20 μl aliquots of kaolin (98% pure) only. Halos around the aliquots were examined three days after inoculation and the solubilization index was calculated as halo diameter (mm) / aliquot diameter (mm) (Marra et al., 2015). Shifts in media pH were also observed by noting the media color. Table 1 shows results of in vitro test with isolate ATC PTA-122608. Similar results were obtained with other isolates including ATCC PTA 8990; ATCC PTA 8988, ATCC PTA 8989. Table 1. Size of halo around aliquot of Ecoplus (bacterial isolate and kaolin (98% pure)) and control (kaolin (98% pure) only) on a P-containing medium.

The experiment shows that EcoPlus formulation is capable of solubilizing non bioavailable phosphate source, suggesting that the formulate when applied into the soil would increase available phosphate for use of plants.

Greenhouse experiments were performed to examine plant P acquisition from bioavailable and non-bioavailable soil phosphate sources. Tomato seeds (variety Haley 3115) were transplanted one week after germination iKin a non-P peat soil supplemented with CaHPO ₄ (bioavailable P source) and Ca3(PO4)2 (non-bioavailable P source) at four different P levels (0, 6.25, 12.5, and 25 ppm). Bacterial treatments were administered randomly one day after transplanting. All pots were arranged in a completely randomized design with ten replicates per treatment and seedlings were watered daily with DI water. Seedlings were harvested 21 days after transplanting. Rhizosphere soil was analyzed for pH and roots for rhizosphere acid phosphatase (APase) activity using as described in Zang et al. (2015). Root and shoot dry weight were recorded after drying at 68°C for 48h and shoot tissues were analyzed for total P using a Flow Injection Analyzer according A & L WESTERN AGRICULTURAL Laboratories. Ca, USA.

In another greenhouse experiment a fertilizer gradient was established by fertilizing plants daily with a nutrient solution diluted to 0.25x, ().5x, 0.75x, or lx. The lx solution contained NH4 ⁺ (6 ppm), NO3- (96 ppm), P (26 ppm), K (124 ppm), Ca (90 ppm), Mg (24 ppm), Su (16 ppm), Fe (1 .6 ppm). Ma (0.27 ppm), Br (0.5 ppm), Co (0.16 ppm), Zn (0.12 ppm) and Mo (0.016 ppm). Plants were inoculated either with the isolate formulated into an EcoPlus biofertilizer using a kaolin (98% pure) substrate and diluted to 10 ⁴ CFU/ml using DI water, kaolin (98% pure) alone (non-microbe control), or no solution (uninoculated control).

Plants were harvested at maturity (91 days after transplant) and root and shoot dry weight were recorded after drying at 68°C for 48h. Plant total carbon (C) and nitrogen (N) were measured using a Costech Elemental Analyzer according to the manufacturer’s recommendations. Fruits were harvested from each plant for determination of final harvestable yield. To analyze changes in root architecture in vivo, roots were scanned and analyzed via color analysis using WinRhizo software (Regent Instruments). Total root length and surface area were partitioned into three distinct diameter classes (fine root (d<0.5cm), intermediate root (0.5cm<d≤1cm), coarse root (d>1cm)). The experiment was repeated twice. Results are shown in Figure 4.

In vitro root architectural development assay

Root primary growth occurs through a combination of cell division and expansion largely occurring in two distinct developmental zones - the division or meristematic zone and the elongation zone (Beemster and Baskin, 1998). Furthermore, new cells are provided by the activity of the quiescent center which serves as a reservoir for the neighboring cells and which influences the meristematic potential of initial cells (vandenBerg et al., 1997). Stunting of root primary growth could be achieved by repressing the cell division or by repressing cell elongation. Division and elongation can be measured using the length of the meristematic and elongation zone, respectively.

To characterize the effect of ATCC PTA-122608 on the root meristem, we utilized the S. Lycopersicon cv. M82 meristematic zone marker line SIRPL11Cpro: :NTF (NUCLEAR TAGGING FUSION contains nuclear envelope tag WPP, GFP, and biotin ligase receptor peptide BLRP). First, we confirmed that the bacterial treatment (EcoPlus) had a repressive effect on primary root growth in plate grown marker line seedlings. Then, we looked at the effect of bacterial treatment on the expression of NTF driven by tomato promoter SIRPL11Cpro, which is active in the dividing cells of the root meristem (Ron et al., 2014). Both low (10- CFU) and high (10 ⁶ CFU) bacterial treatments (EcoPlus) reduced the length of the meristematic zone significantly (Fig. 3) and also reduced the expression level of the marker gene (not shown). The reduction in meristematic zone length and on the SIRPL1 ICpro expression domain indicates that EcoPlus likely inhibit cell division in the primary root, which leads to stunted primary root growth.

The stunting of the root meristem and inhibition of cell division could be regulated by a change to the quiescent center, which is an organizing center that maintains the stem cell potential of the surrounding cells. However, there was no change in the QC domain when we grew quiescent center and root initial specific marker line SlWOX5pro::NTF with different concentrations of the bacteria. This indicates that the chan oge in meristematic zone leng oth is likely not achieved by suppression of quiescent center function.

In vivo plant growth promotion

Analysis of root architecture in pot-grown plants revealed that the bacterial treatment significantly increased total root length and root surface area occupied by fine roots compared to non-inoculated plants (Figure 1). Inoculated plants (right) had significantly greater total root dry mass and R:S ratio. Notably, despite these changes to the root systems, no significant differences in aboveground biomass, harvestable yield, total C and N, or fertilizer use efficiency were detected.

While inoculation with ATCC PTA-122608 increased shoot biomass under P limitation in a 21 -day greenhouse experiment, inoculation did not affect shoot P and no increase in shoot biomass, harvestable yield, or plant C or N was seen in a. 91 -day greenhouse experiment. If solubilization of non-plant-available P was an important mechanism of growth promotion, plant P uptake and biomass should have been improved by inoculation in the non- bioavailable P treatment but not when the P source was bioavailable. Given that dry biomass was increased by inoculation across all P sources with no effect on plant P content, P solubilization does not appear to be the primary' mode of plant growth promotion by ATCC PTA- 122608.

In contrast, ATCC PTA-122608 appears to effectively regulate root architectural development both in vitro and in vivo. Inhibition of cell division in the meristematic zone restricts primary root elongation and proceeds through an unknown mechanism other than suppression of quiescent center function. In tomato, ATCC PTA-122608 increases total root length, root surface area, root biomass, and the proportion of fine roots. These alterations could facilitate resource acquisition since more surface area is available for nutrient uptake and fine roots are especially important in plastic responses to localized resources and stimulating microbial biogeochemical cycling (McCormack et al., 2015). However, root system architectural shifts did not appear to improve productivity over 91 days, as shoot biomass, harvestable yield, and plant C and N were unaffected across a fertilizer gradient. Increased investment belowground may have required increased allocation of resources away from the shoot, highlighting the importance of understanding tradeoffs inherent in plantmicrobe interactions under varied environmental conditions.

Due to the fact that the EcoPlus treatment caused changes in the root architecture and increased the nutrient content of the tomato leaves, but surprisingly did not improve the shoot biomass or harvestable yield, we decided to investigate the nutrient contents of tomato leaves and roots in aeroponic tomato culture and of the used water in the aeroponic tomato culture.

The plants were provided with 0.1g/l of Ultrasol® NPK-fertilizer (SQM AS, Chile) and part of the plants additionally received water solubilized Pseudomonas- isolate in concentration of 10 ⁵ cells/ml. After 30 days of treatment the uptake of primordial macronutrients (N, P and K) from leaves (L) and roots (R) was increased when we applied with both NPK-fertilizer and Pseudomonas ssp. isolate as compared with adding only NPK-fertilizer. This shows that the isolate improves uptake of the macronutrients provided in the NPK fertilizer. When testing the nutrient concentration in the water of the aeroponics after 30 days of cultivation it was clear that there was about 10% less residual N, P and K when NPK-fertilizer and bacterial inoculate was provided to the system as opposed to NPK fertilizer alone. This show's that the bacterial inoculate promotes a better nutrient uptake by the plant and diminishes leakage of the nutrients into the growth medium.

The results shows that the bacterial inoculate is an efficient soil amendment which increases uptake of nutrients via change of root architecture caused by rupture of apical dominance. Increased nutrient uptake is not necessarily causing increase of biomass, or crop yield as is shown here, but is an efficient way to diminish leakage of nutrients from the agricultural environment where NPK fertilizers are used. The soil amendment according to this disclosure thus provides a system NPK fertilizer scan be safely added to the soil without increasing leakage of nutrients into the aqua system. This invention provides thus means for sustainable agriculture by way of adding bacterial formulation, especially in form of EcoPlus (bacterial isolate in kaolin) to retain nutrients in the plant tissues.

Another utility presented by this patent is the reduction of the water footprint of the crops in which it is applied. Fundamentally due to the effect of increasing the root surface and absorption, which on one hand optimizes the use of water by the plant avoiding hydric stress and, on the other hand, optimizes the use of nutrients maintaining the level of productivity with less quantity of fertilizer. The latter influences a significant decrease in the impact of the gray water footprint of the crops in which it is applied, because by applying a smaller amount of fertilizers, the possibilities of contamination decrease drastically and consequently the gray water footprint will be much lower.

The invention is no described in light of non limiting examples.

EXAMPLE 1

Lateral root number in Arabidopsis thaliana seedlings increased when soil was treated with Pseudomonas ssp isolated from rhizosphere of Deschatnpsia antaretica

Bacterial isolate was obtained from rhizosphere of Deschampsiaantarctica. The isolate mainly comprised bacteria of genus Pseudomonas and Bacillus. 4-day old Arabidopsis thaliana seedlings grown in MS-agar were treated with different amount of bacterial (10 ⁴ and 10 ⁶ CFU/7ML) for 12 days. Higher lateral root number was observed when the seedlings were treated with the bacterial isolate. The size of the root apical meristem also showed differences (results not shown). Further, similar experiments were performed using Solarium Lycopersicon as model plant. Figure 1 shows example of tomato roots without EcoPlus amendment and with the amendment. Figure 2 shows the length of tomato roots when plants were grown in vitro, and the growth medium was supplemented with different levels of bacterial isolate (ATCC PTA-122608 ). Day 0 is the date of germination.

EXAMPLE 2

EcoPlus, a biological product to change the root architecture to increase the nutrient uptake by different crops

EcoPlus can colonize a plant and promote the root growth. When EcoPlus is co-cultivated with seeds of tomato, a change in the root architecture was observed, more specifically, an apical dominance break by the bacteria and a proteoid root growth.

To study the root structure, in vitro cultures of Arabidopsis thaliana seedlings with and without the product were performed, root length and lateral root number were measured at 5 and 12 days post inoculation (DPI). By treating Arabidopsis thaliana seedlings with 10 ⁴ and 10 ⁶ CFU/'ml of EcoPlus, shorter roots and more lateral roots are observed compared to the control (LB media added instead of the bacteria). Further, similar experiments were performed using Solatium Lycopersicon as model plant. In this case, we saw the same results as mentioned before (shorter roots and more lateral roots when the product is present (Figures 1, 2).

Two transgenic lines of Solamim Lycopersicon (SIRPL1 lCp:NTF and SIW0X5p:NTF) were used to study the root apical meristem (RAM) which is composed of stem cells that undergo several cell divisions to give rise to all types of cells in the root. Inside the RANI, a quiescent center is found that is a group of cells that surround the stem cells in their undifferentiated fate, this QC cells are mitotically inactive cells in the root tip (Cederholm et. al., 2012). The RAM size in SIRPL1 lCp:NTF 5. Lycopersicon is shorter when the product is present compared to the control plants. No remarkable difference in the QC size was observed when the bacteria is present using SIWOX5p:NTF 5. Lycopersicon (Table 2). This experiment was done twice. The reduction of the RAM was also observed in Arabidopsis thaliana.

Table 2. Apical meristem and quiescent center length in tomato plants with or without EcoPlus treatment MZ is meristem zone QC is quiescent center

Furthermore, the root structure was evaluated in Solamim Lycopersicon cultured at UC Davis’s greenhouses. Six-old tomato plants were grown in soil (1 gallon pots) using different treatments at greenhouses. At day 0 of the experiment, tomato plants were inoculated with 20 mg of EcoPlus dissolved in 200 ml of distillated water (di-water) while the control plants only diwater was added. After two days, 150 ml of fertilizer (using different amount: 100%, 50% and 25%) were applied to the plants during 1 month. Six treatments were applied (100%, 50% and 25% w/o and w/ bacteria, respectively) and 5 plants per treatment were used. After one month of treatment, the plants were harvested and root measurement was performed by using WinRhizo™ analysis program. The root architecture is very different comparing tomato plants treated with the same amount of fertilizer (25%) without and with EcoPlus, respectively, more lateral roots are observed in the plant treated with EcoPlus. The major differences in root architecture were found at low fertilizer level. Plants can change its root architecture when they faced against environmental stress like low ⁷ amount or low availability of nutrients. When EcoPlus is added, the root architecture is optimized, and plants can have a better nutrient uptake. EXAMPLE 3

EcoPlus-formuIatioo for the sustainable use of the soil

To analyze the sustainable use of the soil, we evaluated the nutrients uptake by the roots and leaves from tomato aeroponic culture. The plants were provided with 0. lg/1 of Ultrasol® NPK- fertilizer (SQM AS, Chile). Part of the plants additionally received water solubilized Pseudomonas- inoculant in concentration of 10 ⁵ cells/ml . After 30 days of treatment the uptake of primordial macronutrients (N, P and K) from leaves (L) and roots (R) was increased when we applied with both NPK -fertilizer and Pseudomonas ssp. isolate as compared with adding only NPK-fertilizer.

Table 3 below ⁷ illustrates the results of nutrient concentration in the tomato plants with or without the Pseudomonas -isolate. A considerable increase in the uptake of N, P and K and a decrease in Al are shown. Al is toxic for the plants causing negative effects in their growth.

Table 3 Furthermore, the quantity of the residual nutrients remained in the water that weren’t utilized by the plant was tested. In this case the same quantity of UltraSol -fertilizer and EcoPlus was added and the analysis were made of the water after 30 days of use. Table 4 below show the results: There is less residual near to a 10% of N, P and K when Ultrasol + EcoPlus is added than when Ultrasol is applied alone. In conclusion, EcoPlus promote a better nutrient uptake by the plant into 10 to 50% increase than the fertilizer alone, without contaminating the aquifers.

Table 4

EXAMPLE 4

Apical dominance break by EcoPlus EcoPlus can colonize a plant and promote the root growth. When EcoPlus was used with tomato plants, we observed a change in the root architecture.

Plants can establish symbiotic relations with several bacteria, for example the positive relation between EcoPlus and Deschanipsia amarctica is found in Antarctica. Also, a symbiotic relation was observed in the co-culture of EcoPlus with tomato plant, for example this plant can resist against abiotic stress like a deficit of water. This fact is promising for the use of less water in crops and in consequence a production costs savings for the agricultural companies.

Differentially expressed genes of EcoPlus during the response to root exudates were identified using a suppression subtractive hybridization analysis (SSH). The up- and down-regulated genes were mostly associated with processes related to root colonization by the bacteria, such as transport, metabolism, defense mechanisms, signal transduction, chemotaxis and motility, as well as stress responses and DNA replication, recombination and repair. EcoPlus induces a metabolic pathway in the plant like organic acid pathway to promote the break of the apical root and the appearance of proteoid root. This change in the architecture of roots promotes a better uptake of nutrients by the plant, mentioned before.