DESCRIPTION

Technical field

The present invention refers to a new bacterial strain, a microbial consortium comprising said strain and a composition comprising the bacterial strain or the consortium eventually in combination with a biostimulant for plants. Moreover, the present invention refers to the use of the bacterial strain, the microbial consortium or the composition in agriculture, preferably to improve plant processes, preferably nutrient up-take, and therefore to enhance plant growth and/or development. Background art In the past years, the heavy use of fertilizers, agro-chemicals and pesticides, which marked the First Green Revolution, strongly improved crop productivity, without taking in account adverse effects on environment and human health (Serpil, 2012), such as decreased soil fertility and increased susceptibility of plants to pests and disease (Jen-Hshuan Chen, 2006). Nowadays, sustainable agriculture is considered as the new way to produce more food to meet the increasing demand for food of an ever-growing population, while counteracting the adverse effects of the climate change on crop productivity. Biofertilizers have been considered a good and eco-friendly solution for sustainable agriculture compared to chemical fertilizers and can significantly improve crop productivity, ameliorate nutrient up-take and make plants more tolerant to several biotic and abiotic stress in a sustainable manner (Deepak et al., 2014).

In this context, the solution disclosed herein is based on a novel bacterial strain, a bacterial consortium or a composition thereof capable of increasing soil solubilization of main macronutrients and/or micronutrients, preferably phosphorus, zinc, iron, nitrogen, potassium and combinations thereof. Therefore, the strain, consortium or composition thereof is useful to improve micro and/or macronutrient uptake in plants and to ameliorate plant biology, in particular, the growth and/or the development of plants, nutrient use efficiency. Overall, the strain, consortium or composition disclosed herein is useful as biostimulant for plants, preferably to enhance in plants nutrient uptake, nutrient efficiency, tolerance to abiotic stress, crop quality and combination thereof.

Brief description of drawings - Figure 1 shows the phylogenetic tree obtained from the alignment of the full 16 rRNA gene sequences, with the “Maximum Likelihood” statistical method, “Tamura- Nei” substitution model and “Complete Deletion” mode (No. of bootstrap replications: 1000).

A) The strain Bacillus pumilus VMC30/195 is related to the species B. pumilus as it shares 97% of sequence similarity with the 16S rRNA sequence of Bacillus pumilus SH-B9.

B) The strain Bacillus megaterium VMC30/195 is phylogenetically related to the species B. megaterium, as it shares 94,98% of sequence similarity with the 16S rRNA sequence of Bacillus megaterium CtST3.5.

- Figure 2 shows protein mass spectra (in the range from 2 to about 12 KDa, peaks of a spectrum m/z values with a given intensity) of VMC30/195 (A) and VMC30/196 (B). Each peak in the graphs represents a different protein expressed by the microorganism and the intensity of the peaks represents the concentration of that proteins within the microbial cell. Since proteins are a direct expression of genome and genome of each strains is unique, thus the proteomic profile of each strain is unique and can be used as fingerprint differentiation and classification.

- Figure 3 shows the P-solubilization index of Bacillus pumilus VMC30/195, Bacillus megaterium VMC30/196 (alone or combined as consortium) compared to Herbaspitillum seropedicae ATCC 35893.

- Figure 4 shows the Zinc Solubilization Efficiency (SE) of Bacillus pumilus VMC30/195, Bacillus megaterium VMC30/196 (alone or combined as consortium) compared to Herbaspitillum seropedicae ATCC 35893.

- Figure 5 shows siderophores production of Bacillus pumilus VMC30/195, Bacillus megaterium VMC30/196 (alone or combined as consortium) compared to

Herbaspitillum seropedicae ATCC 35893.

- Figure 6 shows the potassium mobilization of Bacillus pumilus VMC30/195, Bacillus megaterium VMC30/196 (alone or combined as consortium) compared to an internal control.

- Figure 7 shows IAA production of Bacillus pumilus VMC30/195 and Bacillus megaterium VMC30/196 compared to Azospirillum brasilense ATCC 29145.

- Figure 8 shows the nitrogen fixation of Bacillus pumilus VMC30/195, Bacillus megaterium VMC30/196 (alone or combined as consortium) compared to Azospirillum brasiliense ATCC 29145 and Herbaspirillum seropedicae ATCC 35893. Detailed description of preferred embodiments of the invention The present invention refers to a novel strain of bacteria isolated and characterized for the ability to be used in agriculture, preferably for the purpose of solubilizing macro and/or micro-nutrients that are essential for plant biology, preferably for plant growth and development. Moreover, the strain disclosed herein is useful as biostimulant for plants and is preferably used to increase nutrient use efficiency in plants and/or resistance to abiotic stress of plants and/or plant growth and/or quality. As used herein, “strain” refers to isolate or a group of isolates exhibiting phenotypic, physiologic, metabolic, and/or genotypic traits belonging to the same lineage and different/distinct from that of other strains belonging to the same species.

As used herein, “isolate” means a microorganism which has been removed from its original environment, including, but not limited to, soil, air, fresh water, sea water, algae, higher plants, seeds, roots, leaves, fruits, etc. Preferably, the “isolate” has to be “pure”, meaning that it does not comprise other microbial contaminants in the isolate, according to the isolation method reported in the present invention.

As used herein, “macronutrients or main nutrients” refer - in general - to any nutrient that plays an essential role in any key physical, physiological and biochemical process of plants, providing a healthy and balanced growth and development during all lifecycle of plants. Preferably, said nutrient is selected from: nitrogen, phosphorus, potassium (also known as NPK elements), calcium (Ca), magnesium (Mg), sulfur (S) and combinations thereof. Phosphorus (P) is one of the most indispensable macronutrients next to nitrogen for the growth and/or the development of plants. A greater part of soil phosphorus, around 95-99%, is present in insoluble form complexed with cations like iron, aluminum, and calcium, all of them being chemical forms of unavailable P that, therefore, cannot be utilized by the plants. The use of natural phosphate-bearing materials, such as rock phosphate (RP), as fertilizer for P- deficient soils has received due attention in recent years since substantial deposits of cheaper and low-grade RP are locally available in many countries of the world. However, the solubilization of natural phosphate-bearing materials, such as rock phosphate (RP), rarely occurs in nonacidic soils with a pH greater than 5.5 to 6.0. Conventionally, RP is chemically processed by reacting with sulfuric acid or phosphoric acid to produce partially acidulated RP. The process incurs high cost and makes the environmental health worse. A much cheaper and convenient alternative is reclamation of exhausted soil through use of P-solubilizing microorganisms that have opened the possibility for solubilization of RP in soils. In this scenario, soil microorganisms play a critical role in natural phosphorus cycle and recently microbial-based approaches have been proposed to improve the agronomic value of RP. Therefore, microbial-based products represent cheaper approaches compared to the higher cost of manufacturing phosphate fertilizer in industry and, at the same time, they avoid the environment pollution posed by a traditional chemical process. Deficiency or excess of the main nutrients have relevant consequences such as great imbalance of the crops, delay of the growth, leaves and/or flowers loss, reduced photosynthetic activity, with subsequent low fruit production and, overall, the crop productivity is heavily reduced. In some cases, pathological consequences, such as plant tissues and/or fruit necrosis, can also take place.

As used herein, “plant biology” refers to any physiological and/or pathological plant process, preferably selected from: growth and productivity of plants, comprising plant biomass (mainly N, but also S), plants metabolism and energy production, flowers, fruit and seeds development (mainly P, but also Ca and Mg), efficiency of the photosynthetic process, osmotic balance, and/or production and quality of the fruit (mainly K, but also Ca).

As used herein, “micronutrients” relate to the main nutrients absorbed in very small amounts grams per hectare (g/ha) compared with macronutrients (kg/ha). Preferably, said micronutrient is selected from: boron (B), copper (Cu), iron (Fe), manganese (Mn), molybdenum (Mo), zinc (Zn) and combinations thereof. Zinc is an essential micronutrient, which means it is essential for plant growth and development but is required in very small quantities. It is crucial to plant development, as it plays a significant part in a wide range of processes, such as enzymes activation that are responsible for the synthesis of certain proteins. It is used in the formation of chlorophyll and some carbohydrates, conversion of starches to sugars and its presence in plant tissue helps the plant to withstand cold temperatures. Zinc is essential in the growth hormone production and internode elongation. Zinc deficiency is probably the most common micronutrient deficiency in crops worldwide. Symptoms caused by zinc deficiency vary depending on the crop. Typically, they include one or some of the following: stunting-reduced height, interveinal chlorosis, brown spots on upper leaves and distorted leaves. The most common fertilizer sources of zinc are zinc chelates (contain approximately 14% zinc), zinc sulfate (25- 36% zinc) and zinc oxide (70-80% Zinc), where zinc sulfate is the most used source of zinc. The major problem of the application of these fertilizers is that in most of the cases only a small part of the total applied Zn is available to plants while remaining gets fixed into soil. Thus, an alternative solution is to use zinc solubilizing bacteria that are able to solubilize insoluble sources of zinc, preferably Zinc Oxide - ZnO, Zinc(ll) Carbonate -ZnCO ₃ , or Zinc(ll) Phosphate - Zn ₃ (PO ₄ ) ₂ · With reference to Zinc, it must be pointed out that Zinc solubilizer strains are rare to be isolated and consequently make available on the market for this activity. Therefore, the strain disclosed herein represents a valuable source to address Zinc solubilization need. Micronutrient deficiency affects crop yield and quality. While excess of some micronutrients, despite taking place in rare situation, can interfere with the uptake of other nutrients resulting in unbalanced plant development.

As used herein, “solubilization and/or mobilization of nutrients and/or micro - macronutrients” refers to the process allowing the conversion of the mineral forms of macro/micro-elements, which are present in the soil under unavailable forms for plants, into forms which can be dissolved in water and consequently taken up through their roots. Advantageously, the bacterial strains here disclosed can solubilize and mobilize, preferably in the soil, insoluble forms of macro/micro- nutrients, preferably starting from the inorganic forms of macro/micro-nutrients. In other words, the bacterial strains here disclosed are able to improve the solubilization and/or the dissolution and/or the mobilization of macro/micro-nutrients, preferably in the soil, and, consequently, they are able to improve the availability, preferably in the soil, of micro and/or macronutrients as defined above for plants.

As used herein, “plant growth” refers to the extension and/or expansion of plant tissues and/or organs, which bring to an increase of the vegetative biomass. An optimal plant growth results in balanced development of the whole plant and boosts processes of differentiation in flowers, fruits, and seeds.

The isolated bacterial strain disclosed herein is a member of the genus Bacillus, species pumilus characterized by a DNA, more preferably the DNA of 16S rRNA gene coding comprising SEQ ID NO: 1 or any sequence characterized by 80-99,9% of identity with SEQ ID NO: 1. 16S ribosomal RNA sequences have been used extensively in the classification and identification of Bacteria and Archaea. The comparison of the 16S rRNA gene sequence of an isolate against sequences of type strains of all prokaryotic species provides an accurate and convenient way to routinely classify and identify prokaryotes.

To amplify the taxonomic-specific genes reported above any pair of primers located in the DNA sequence of interest can be used. Preferably, the pair of primers are the one disclosed in the examples and listed in Table I as SEQ ID NO: 3 and 4.

The isolated bacterial strain disclosed herein has been deposited on February 21 ^st 2018 with the NCMR accession number MCC0530 and with IDA accession number MCC0195 (1 ^st July 2020) and the following name (denomination): Bacillus pumilus strain VMC30/195 (VMC30/195 from now on) at the International Depositary Authority: National Centre for Microbial Research, Pune, Maharashtra-411021 , India (hereinafter NCMR) according to the provisions of Budapest Treaty.

VMC30/195 is gram positive and is preferably characterized by rod-shaped cells. The colony - when the bacteria are grown on nutrient agar - shows a yellowish color and is characterized by a size having an average diameter of 2-10 millimeters, preferably 3-5 millimeters. Preferably, the colony’s shape of VMC30/195 is irregular. According to a preferred embodiment of the invention, VMC30/195 can grow at a temperature up to 50C.

According to a further preferred embodiment of the invention, VMC30/195 is able to use at least one of the carbon source selected from: glycerol, arabinose, ribose, galactose, glucose, fructose, mannose, mannitol, methyl-αDmannopyranoside, esculin, salicin, cellobiose, saccharose, trehalose, gentiobiose, tagatose, malic acid and any combination thereof.

According to a further preferred embodiment of the invention, VMC30/195 is positive for: citrate utilization; and/or acetoin production and/or gelatinase.

According to a further preferred embodiment of the invention, VMC30/195 is preferably resistant to at least one of the antibiotic agent, preferably present in a concentration ranging from 8-32 μg/ml and/or 15 μg/dish, selected from: ceftazidime, miokamycin, lincomycin and any combination therefore, and/or susceptible to at least one of the antibiotic agent, preferably present in a concentration ranging from 4 to 200 μg/ml and from 5 to 30 μg/disc and 300 U/disc, selected from: chloramphenicol, gentamycin, streptomycin, polymyxin B, netilmicin, tobramycin, amoxicillin+clavulanic acid, ampicillin, ampicillin+sulbactam, piperacillin, ceflacor, cefonidic, ceftriaxone, cefuroxime, ciprofloxacin, levofloxacin, pefloxacin, azithromycin, clarithromycin, erthromycin, roxitromycin, fosfomycin, rifampicin, co-trimoxazole, novobiocin and any combination thereof, and/or intermediately susceptible to cefixime preferably present in a concentration of 32 μg/ml.

According to a further preferred embodiment of the invention, VMC30/195 is characterized by a population doubling of 30-40 minutes.

The invention refers also to a consortium comprising VMC30/195 and at least one further bacterial strain. Preferably said further bacterial strain is the isolated strain member of the genus Bacillus, species megaterium characterized by a DNA, more preferably the DNA of 16S rRNA gene coding, comprising SEQ ID NO: 2 or any sequence characterized by 80-99,9% of identity with SEQ ID NO: 2.

Preferably said isolated strain member of the genus Bacillus, species megaterium has been deposited on February 21st 2018 at the NCMR with the accession number MCC0524 and with IDA accession number MCC0194 (1 ^st July 2020) and having the following name (denomination): Bacillus megaterium strain VMC30/196 (VMC 30/196 from now on).

VMC30/196 is gram positive and is preferably characterized by rod-shaped cells. The colony - when the bacteria are grown on nutrient agar - shows a peach color and is characterized by a size having an average diameter of 2-10 millimeters, preferably 5-8 millimeters. Preferably, the colony’s shape of VMC30/196 is circular. According to a preferred embodiment of the invention, VMC30/196 can grow at a temperature up to 40°C, preferably up to 50°C.

According to a further preferred embodiment of the invention, VMC30/196 is characterized by a population doubling of time 30-40 minutes.

In this context, “consortium” means a group of different species and/or strains of microorganisms with different metabolic activities, preferably the consortium is defined “artificial” when it involves a multi-population system that can contain a diverse range of microbial species and is adjustable to serve a variety of industrial and ecological interests. Preferably, in this context consortium means a combination of VMC30/195 and at least one more bacterial strain, preferably a combination of VMC30/195 and VMC30/196, eventually comprising further bacterial species or strains.

According to a preferred embodiment of the invention, VMC30/195 and/or VMC30/196 and/or the consortium can be used as living microorganisms and/or dead cells and/or killed cells, preferably killed by heating, and/or as spores.

Moreover, VMC30/195 and/or VMC30/196 and/or the consortium may be used as a fresh or frozen sample. Alternatively, the strain(s) is(are) used dry, lyophilized or in liquid suspension, or encapsulated in the form of spores and/or living cells. VMC30/195 and/or VMC30/196 and/or the consortium may be cultivated continuously or discontinuously in any medium useful to grow bacteria, in liquid or solid form. Preferably VMC30/195 and/or VMC30/196 and/or the consortium is(are) cultivated on or in a medium(liquid/solid) that preferably comprises nutrient agar, meat extract, peptone, sodium chloride and yeast extract. The temperature of the culturing process ranges between 25°C and 30°C. Moreover, the pH value of the medium ranges preferably between 5 and 8, more preferably the pH is around 7. Therefore, a further aspect of the invention refers to a medium (broth) obtained/obtainable by culturing VMC30/195, eventually as consortium preferably with VMC30/196.The medium may contain the bacteria (the cells), that is the whole culture medium, or the medium may be a cell free medium (without the cells). The cell free medium may be obtained preferably by centrifuging the whole culture medium by using the common standard procedure useful for this purpose, to obtain a cell-free medium or supernatant. Therefore, VMC30/195, eventually as consortium preferably with VMC30/196, may be used as culture medium (broth), preferably whole culture medium (comprising cells), or cell-free medium or supernatant (without cells). Alternatively, VMC30/195, eventually as consortium preferably with VMC30/196, is used as bacterial derivatives, preferably as lysate, as extract, preferably cell free extract, as fraction or as a metabolite(s) derived from said bacterium (a).

As used herein, culture medium means a solid, liquid or semi-solid nutritive matrix to support the growth of cells (prokaryotic or eukaryotic cells).

In this context, alternative names for culture medium are preferably the following: proliferating medium, expansion medium, growth medium, nutrient medium.

As used herein, “whole culture medium” refers to a solution and/or a suspension containing bacterial cells, spores and derivatives thereof, such as nutrients, metabolites or cellular debris. As used herein, “supernatant” refers to the liquid part of a culture broth free from the bacterial cells/spores and containing extracellular metabolites. As used herein, “lysate” means the solution comprising all the intracellular and extracellular material released from the lysis of the bacterial cells. As used herein, “extract” refers to a specific part of the culture broth, including or not living cells/spores. As used herein, “cell free extract” means a solution containing all the microbial metabolites, without the presence of living cells/spores. As used herein, “fraction” means a specific part of the whole culture medium, for example only the solution, only the cells or the like. As used herein, “metabolite” means one or more intermediate(s) or final product(s) of the bacterial metabolism.

A further aspect of the invention refers to mutants and/or edited strains derived/obtainable from VMC30/195. As used herein, “mutant” means any microorganism obtained/obtainable by direct mutation, selection, or genetic recombination of VMC30/195. Mutant strains may be obtained by using any methods known in the art for this purpose, such as mutant selection, chemical mutagenesis, genetic manipulation physical mutagenesis (radiation) and biological mutagenesis. As used herein, “edited” means preferably gene-edited strains wherein at least one gene of interest is edited/modified by using the common biological tools useful for this purpose, in particular biological tools based on the use of nucleases such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly-interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas).

According to a preferred embodiment of the invention, VVMC30/195, eventually as consortium preferably with VMC30/196, is(are) used in combination with further microorganisms, preferably selected from: bacteria, preferably PGPR (or rhizobacteria), yeasts, mycorrhizae, fungi, any derivatives as disclosed above and any combination thereof.

The PGPR of interest that may be used in combination with the strain(s) of the invention is preferably selected from: Aeromonas rivuli, Agromyces fucosus, Bacillus spp. Bacillus mycoides, Bacillus licheniformis, Bacillus subtilis, Bacillus megaterium, Bacillus pumilus, Bacillus safensis, Microbacterium sp., Nocardia globerula, Stenotrophomonas spp., Pseudomonas spp, Pseudomonas fluorescens, Pseudomonas fulva, Pseudoxanthomonas dajeonensis, Rhodococcus coprophilus, Sphingopyxis macrogoltabida, Streptomyces spp., Enterobacter spp., Azotobacter spp., Azospiriullum spp., Rhizobium spp., Herbaspirillum spp., Lactobaccillus spp., Lactobacillus acidophilus, Lactobacillus buchneri, Lactobacillus delbrueckii, Lactobacillus johnsonii, Lactobacillus murinus, Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus plantarum, Lactococcus tactis, and combinations thereof.

The yeasts of interest that may be used in combination with the strain(s) of the invention is preferably selected from: Candida spp., Candida tropicalis, Saccharomyces spp., Saccharomyces bay anus, Saccharomyces boulardii, Saccharomyces cerevisiae, Saccharomyces exiguous, Saccharomyces pastorianus, Saccharomyces pombe, and combinations thereof.

The mycorrhizae of interest that may be used in combination with the strain(s) of the invention is preferably selected from: Glomus spp., Rhizophagus spp., Septoglomus spp., Funneliformis spp., and combinations thereof.

The fungi of interest that may be used in combination with the strain(s) of the invention is preferably selected from: Trichoderma spp., Trichoderma atroviride, Trichoderma viride, Trichoderma afroharzianum, Paecilomyces spp., Beauveria bassiana., Metarhizium spp., Lecanicillium lecanii, Penicillium spp., Aspergillus spp., Conythyrium minitans, Pythium spp, and combinations thereof.

As said before VMC30/195 eventually as consortium preferably with VMC30/196 can be used as plant biostimulant. However, according to a preferred embodiment of the invention, VMC30/195 eventually as consortium preferably with VMC30/196 is(are) used in combination with an additional plant biostimulant (PB or PBS).

As used herein and according to the current definition of European Biostimulant Industry Council, “biostimulant” refers to a substance(s) and/or micro-organisms or a composition comprising said substance(s) and/or micro-organisms whose function when applied to plants or to the rhizosphere is to stimulate natural processes to enhance/benefit nutrient uptake, nutrient efficiency, tolerance to abiotic stress, and crop quality. Preferably, said plant biostimulant comprises an extract of algae and/or an extract of microalgae and/or an extract of plant and/or a humic acid and/or a fulvic acid, and/or plant and/or animal byproducts.

As used herein, algae refer to a functional group of organisms that carry out oxygenic photosynthesis and are not embryophytes. They include both bacterial (cyanobacteria) and eukaryotic organism. The term encompasses organisms that are photoautotrophic, heterotrophic, or mixotrophic, and are typically found in freshwater and marine systems. The term algae include macroalgae (such as seaweed) and/or microalgae. Preferably said algae are brown algae, more preferably said algae are selected from: Ascophyllum nodosum, Ecklonia maxima, Laminaria saccharina, Laminaria digitata, Fucus spiralis, Fucus serratus, F. vesicuiosus, Macrocystis spp., Pelvetia canaliculata, Himantalia elongata, Undaria pinnatifida, Sargassum spp, and combinations thereof. Ascophyllum nodosum is particularly preferred for the purposes of the present invention. As used herein, microalgae refer to any microscopic algae that are unicellular and simple multi-cellular microorganisms, including both prokaryotic microalgae, preferably, cyanobacteria (Chloroxybacteria), and eukaryotic microalgae, preferably green algae (Chlorophyta), red algae (Rhodophyta), or diatoms (Bacillariophyta). Preferably said microalgae are selected from: Spirulina, Scenedesmus, Nannochloropsis, Haematococcus, Chlorella, Phaeodactylum, Arthrospyra, Tetraselmis, Isochrysis, Synechocystis, Clamydomonas, Parietochloris, Desmodesmus, Neochloris, Dunaliella, Thalassiosira, Pavlova, Navicula, Chaetocerous, and combinations thereof.

As used herein, plant means any one of the vast number of organisms within the biological kingdom Plantae. Conventionally the term plant implies a taxon with characteristics of multicellularity, cell structure with walls containing cellulose, and organisms capable of photosynthesis. Preferably, they include a host of familiar organisms including trees, forbs, shrubs, grasses, vines, ferns, mosses and crop plants as vegetables, orchards and row crops. For the purpose of the present invention, the whole plant or part thereof may be used, preferably said part is selected from: leaves, roots, stems, fruits, flowers, seeds, seedlings, bark, berries, skins, and combinations thereof. Preferably, the plant to be used as extract to add to the strains herewith disclosed is selected from: beet, sugar cane, alfalfa, maize, brassica, halophytes, soya, wheat, yucca, quillaja, hop, coffee, citrus, olive, and combinations thereof.

Preferably said plants or algae or microalgae are extracted with common process, preferably said process comprising the following steps: (i) providing a sample of algae and/or a sample of microalgae and/or a sample of plants; and (ii) contacting said sample(s) with an aqueous solution comprising an extraction agent, in other words an extraction solution having an aqueous base.

As used herein, the extraction agent can be a base and/or an acid and/or an enzyme. These kind of extraction agents can be used in any combination or singly. For the purpose of the present invention: the base is preferable an inorganic base, preferably selected from: NaOH, KOH, Na ₂ CO ₃ , K ₂ CO ₃ , NH ₃ , salts thereof, and any combination thereof; the acid is preferably selected from: H ₂ SO ₄ , HNO ₃ , HCI, H ₃ PO ₄ , and various acids of organic nature preferably selected from: acetic acid, citric acid, formic acid, butyric acid and ascorbic acid, gluconic acid, and any combination thereof; and the enzyme is preferably selected from: papain, trypsin, amylase, pepsin, bromelain and specific enzymes that degrade organic polymers present in the algae, preferably alginases, and any combination thereof. The selection of the extracting agent to be used for the process depends upon the kind of algae/microalgae/plant to be extracted and/or the molecules/components to be extracted from them. Preferably, the temperature of the extraction process ranges between -20 and 120°C, more preferably between 20 and 100°C. Preferably, the extraction time ranges from a few minutes to several hours, more preferably between 30 minutes and 18 hours. Preferably, the extraction process is realised at atmospheric pressure or at a pressure up to 10 Bar, more preferably at a pressure ranging from 1 to 8 Bar. The extraction process may be followed by a further step of separating/removing the non-solubilised and/or non-extracted component when it is desirable using only the extract in the formulation of the biostimulant. The removing/separating step is preferably performed by decantation, filtration or centrifugation. Alternatively, a suspension comprising both the extracted component and the non-extracted component can be used.

Preferably, the concentration of the extract from algae and/or microalgae in the biostimulant ranges from 1 to 60%, preferably it ranges from 5 to 50%, more preferably from 10 to 20%, still more preferably around 15%. Preferably, the concentration of the plant extract in the biostimulant ranges from 1 to 60%, preferably it ranges from 5 to 50%, more preferably from 10 to 20%, still more preferably around 15%. Preferably, the humic acid is extracted from leonardite, lignite, sub-products of the digestion of urban bio-waste and biochar. The extraction process is performed in water, in alkali, in acidic medium, or by pyrolysis. Preferably, the concentration of the humic acid in the biostimulant ranges from 1 to 20%. Preferably, the fulvic acid is extracted from peat, lignite, leonardite, digestion of urban bio-waste, biochar and vegetable materials. The extraction process is performed indifferently in water, in alkali, in acidic medium, and by pyrolysis. Preferably, the concentration of the fulvic acid in the biostimulant ranges from 1 to 20%, preferably from 5 to 10%.

Preferably, when VMC30/195, eventually as consortium preferably with VMC30/196, is used in combination with the PBS as disclosed above, said PBS is present in a concentration ranging from 5 to 50%, preferably from 10 to 40%, more preferably from 15 to 25 %, preferably around 20%. VMC30/195, eventually as consortium preferably with VMC30/196, is used in a concentration ranging from 0.01 to 20%, preferably from 0.01 to 10%, more preferably from 0.05 to 5%, more preferably from 0.1 to 1%. Alternatively, VMC30/195, eventually as consortium preferably with VMC30/196, is used in a concentration ranging from 10 to 20%.

Alternatively, VMC30/195, eventually as consortium preferably with VMC30/196, is used in a concentration ranging from 10M to 10 ^Λ 12 UFC/g each, preferably from 10 ^Λ 6 to 10 ^Λ 9 UFC/g, more preferably around 10 ^Λ 8 UFC/g.

The PBS percentage refers to the sum of the percentage of each plant biostimulant component. In this context, the plant biostimulant component is preferably selected from: plant extracts, seaweed extracts, humic acid, fulvic acid, animal byproducts and combinations thereof. The percentage of the strain(s) refers to the sum of dried biomasses for each microorganism in 100 g of final composition.

According to a further preferred embodiment, VMC30/195, eventually as consortium preferably with VMC30/196, or the medium or the extract, preferably bacteria-free extract, or the supernatant or the lysate or the fraction or the metabolite as disclosed above is/are mixed with the plant biostimulant as disclosed above and/or with a non- microbial component. Said non-microbial component comprises one or more components present in the mixture, preferably in the composition, with the exclusion of the microbial component, that is VMC30/195, eventually as consortium preferably with VMC30/196, or the medium or the extract, preferably bacteria-free extract, or the supernatant or the lysate or the fraction or the metabolite. The non-microbial component comprises preferably the PBS as disclosed above and/or at least one further ingredient, preferably a mineral component. Preferably said non-microbial component is present in a concentration up to 99,99%, 99,95 %, 99,90%, 99%, 95%, 90%, 85%, or 80% , and/or the bacteria are used in a concentration up to 20%, 15%, 10%, 5%, 1%, 0,1% 0.05% or 0.01 .

According to a preferred embodiment of the invention, said at least one further ingredient or mineral component is selected from:

- A nitrogen source, preferably selected from: ammonium phosphates, ammonium nitrate, ammonium sulfate, ammonium thiosulfate, potassium thiosulfate, ammonia, urea, nitric acid, potassium nitrate, magnesium nitrate, calcium nitrate, sodium nitrate, protein hydrolisates of vegetal and animal origin, aminoacids, proteins, yeast lysate, manganese nitrate, zinc nitrate, slow release urea, preferably ureaformaldehyde, similar compounds and combinations thereof; and/or

- A phosphorus source, preferably selected from: ammonium phosphates, potassium phosphates, phosphoric acid, sodium phosphates, calcium phosphate, magnesium phosphate, rock phosphate preferably hydroxyapatite and fluoroapatite, phosphorous acid, sodium phosphite, potassium phosphite, calcium phosphite, magnesium phosphite, organic phosphorus compounds, preferably inositol- phosphate, sodium glycerophosphate, ATP, similar compound and combinations thereof; and/or

- A potassium source, preferably selected from: potassium acetate, potassium citrate, potassium sulfate preferably mixed salts of magnesium, potassium thiosulfate potassium phosphate, potassium phosphite, potassium carbonate, potassium chloride, potassium hydroxide, potassium nitrate, mixed salts of magnesium and potassium, potassium sorbate, potassium ascorbate, organic forms of potassium, and combinations thereof; and/or

- A magnesium and/or calcium source, preferably selected from: magnesium nitrate, magnesium sulfate, magnesium chloride, magnesium phosphate, magnesium phosphite, magnesium thiosulfate, magnesium hydroxide, magnesium oxide, mixed salts of potassium and magnesium, mixed salts of magnesium and calcium (dolomite), magnesium acetate, magnesium citrate, magnesium sorbate, and organic forms of magnesium, magnesium carbonate, magnesium formate, magnesium ascorbate, and combinations thereof; and/or

- A sulfur source, preferably selected from: sulfuric acid, sulfates, thiosulfate, sulfated aminoacids, and combinations thereof; and/or - Iron and/or manganese and/or zinc and/or copper source, preferably selected from: iron sulfate, iron oxide, iron hydroxide, iron chloride, iron carbonate, iron phosphate, iron nitrate, chelated iron with EDTA, DTPA, HEDTA, EDDHA, EDDHSA, EDDHCA, EDDHMA, HBED, EDDS; complexed iron with aminoacids, ligninsufonates, humic acids, fulvic acids, gluconic acid, heptagluconic acid, iron citrate, iron malate, iron tartrate, iron acetate, iron lactate, iron ascorbate, organic form of iron, and combinations thereof .

According to a preferred embodiment of the invention, VMC30/195 and/or VMC30/196 is(are) used in combination with further molecules, preferably proteins, protein hydrolisates, peptides, oligopeptides, peptidoglycans, low-molecular weight peptides, synthetic and natural occurring aminoacids; molasses, polysaccharides, lypopolysaccharides, monosaccharides, disaccharides, oligosaccharides, sulfated oligosaccharides, exopolysaccharides, chitosan. Other molecules that can be advantageously used in combination with VMC30/195 and/or VMC30/196 are selected from: stress protecting molecules, such as betaines, mannitol, and other polyols with similar effects, and hormones and hormone-like compounds, such as melatonin, auxins, auxin-like compounds, cytokinins, cytokinin-like compounds, gibberellins, gibberellin-like compounds, jasmonates, hormones precursors like polyamines spermine, spermidine, putrescine, and metabolism stimulating substances like vitamins. Other molecules that can be advantageously used in combinations with VMC30/195 and/or VMC 30/196 are selected from: nucleic acids, uronic acids and polymers thereof, glucuronic acids and polymers thereof, small organic acids, such as oxalic and succinic acids. Preferably, said small molecules may be a synthetic and/or naturally derived nucleic acid molecules containing multiple nucleotides, preferably being defined an oligonucleotide when the molecule is 18-25 nucleotides in length and polynucleotides when the molecule is 26 or more nucleotides. Preferably said oligonucleotides or polynucleotides or a mixture of both, include RNA or DNA or RNA/DNA hybrids or chemically modified oligonucleotides or polynucleotides or a mixture thereof.

A further aspect of the invention refers to a composition, preferably an agricultural composition, more preferably a plant biostimulant composition, comprising VMC30/195, eventually as consortium preferably with VMC30/196 as disclosed above, eventually in combination with the further components/ingredients disclosed above and a carrier, preferably an agricultural compatible carrier. Preferably, said further components/ingredients is the PBS and/or the non-microbial component and/or the further ingredients or mineral component as previously disclosed.

As used herein “agricultural compatible carrier” refers to any synthetic or natural, organic or inorganic, derived compound able to deliver the product in an active form in the site of action, preferably said carrier is selected from the categories of surfactants, thickeners, suspension agents, wetting agents, and combinations thereof.

As used herein, surfactant means any molecule able to modify the surface tension of the water and allowing the product to impact a wider area of the leave and/or root and/or fruit, or any other part of the plant. Preferably said surfactant is selected from: ionic, non-ionic, cationic surfactants, synthetic or naturally derived, preferably alkyl sulfonates, alkylarylsulfonates, ethoxylated alcohols, alkoxylated ethers, ethoxylated esters, alkylpolyglucosides, block copolymers, lignosulfonates, saponins, and the like.

As used herein, thickener means any molecule able to modify the rheology of any given composition in the sense of improving the viscosity and stabilize it. Preferably, said thickener is selected from: natural and synthetic gums, lignosulfonates, molasses and the like.

As used herein, suspension agent means any molecule able to surround insoluble particles avoiding settlement and allowing the creation of a stable suspension of insoluble. Preferably, said suspension agent is selected from: natural and synthetic colloids, clays, and their derivatives and the like.

As used herein, wetting agent means any molecule able to avoid fast water evaporation on a given surface and retain moisture for a long time. Preferably, said wetting agent is selected from: glycols, glycerin and their derivatives and the like. VMC30/195, preferably as consortium, preferably with VMC30/196, eventually in combination with the further components/ingredients disclosed above and/or the composition disclosed above is/are formulated as: solution, suspension, water- soluble concentrates, dispersable concentrates, emulsifiable concentrates, emulsions, suspensions, microemulsion, gel, microcapsules, granules, ultralow volume liquid, wetting powder, dustable powder, or seed coating or treatment formulations. As shown in the experimental part, VMC30/195 and/or VMC30/196 is(are) able to solubilize macronutrients and/or micronutrients as defined above. Preferably, the strain(s) is(are) able to solubilize macronutrients and/or micronutrients present in the soil and therefore the strain(s) allow(s) said solubilized macronutrients and/or micronutrients being more available for plants that consequently show an improved capability of micro and/or macronutrient’s uptake. In other words, thanks to the macronutrients and/or micronutrients solubilizing (mobilizing/dissolving) activity of the strain(s) of the invention, plants improve the up-take capability of said solubilized macronutrients and/or micronutrients, preferably from the soil, and show a better growth. In this regard, the experimental evidence has shown that VMC30/195 is good performer in Phosporous solubilization, and when used with VMC30/196 the consortium works even better. In addition, VMC30/195 has shown great potential in Zinc solubilization more than the strain of reference known to have this activity. As mentioned before Zinc solubilizers are very difficult to be isolated and therefore VMC30/195 represent an interesting emerging alternative of selected strain for this biological function in plants. In addition, VMC30/195 shows promising potential also in nitrogen fixation and in this regard, it works better compared to known nitrogen fixator strains.

Finally, when VMC30/195 is used in combination with VMC30/196, the consortium shows complete framework of micro-macronutrients solubilization activity because besides the above disclosed properties the consortium shows also potassium and iron solubilization/mobilization activity. Indeed, VMC30/196 is a good producer of siderophores and so it helps and increases iron solubilization and consequently plant iron uptake. Moreover, VMC30/196 is good potassium solubilized. Therefore, VMC30/196 can complement the panel of solubilizing/mobilizing activity toward micro-macronutrients of VMC30/195.

Moreover, VMC30/196 is also able to produce IAA. This molecule is known to help plant in the production of longer roots with an increased number of root hairs and root laterals which are involved in nutrient uptake (Datta and Basu, 2000). Moreover, IAA promotes cell elongation, inhibit or delay abscission of leaves and induce flowering and fruiting. Overall, IAA producing bacteria may improve the fitness of the plant-bacterium interaction, colonize plant roots better than other bacteria by increased root system, weaken plant defense mechanisms making colonization easier, loosen plant cell walls and as a result promotes an increasing amount of root exudation that provides additional nutrients to support the growth of rhizosphere bacteria and provides more number of active sites and access to colonization for other PGPRs (Etesami et al., 2015). Therefore, VMC30/196, preferably as consortium with VMC30/195, may be also used for increasing number of root hairs and/or root laterals; and/or promoting cell elongation, inhibiting or delaying abscission of leaves and inducing flowering and fruiting; and/or improving the fitness of the plant-bacterium interaction, colonizing plant roots by increasing the root system, and/or weakening plant defense mechanisms making colonization easier, and/or loosening plant cell walls, and/or promoting an increase in root exudation and so additional nutrients to support the growth of rhizosphere bacteria, and/or increasing active sites and accesses to colonization for other PGPRs.

Therefore, VMC30/195 and/or VMC30/196, eventually in combination with the further components/ingredients disclosed above and/or the composition disclosed above is/are preferably used to solubilize (mobilize/dissolve) or to improve the dissolution, preferably in the soil, of phosphate, preferably any inorganic source of phosphate, more preferably selected from: phosphate tricalcium phosphate (Ca ₃ (PO ₄ ) ₂ ), ferric phosphate (FePO ₄ ), and aluminum phosphate (AIPO ₄ ). In other words, insoluble forms of phosphorus, preferably contained into soils, are converted, through several mechanisms, preferably by organic acids production, chelation, ion-exchange reaction or polymeric substances formation, into soluble forms preferably H ₂ PO ₄ - and/or HPO ₄ ^2- that are easier to be taken up plants.

According to a further embodiment of the invention, VMC30/195 and/or VMC 30/196, eventually in combination with the further components/ingredients disclosed above and/or the composition disclosed above is(are) able to solubilize (mobilize/dissolve) zinc, preferably any inorganic source of zinc, more preferably selected from: Zinc Oxide (ZnO), Zinc(ll) Carbonate (ZnCO ₃ ), and Zinc(ll) Phosphate (Zn ₃ (PO ₄ ) ₂ ). Preferably, VMC30/195 and/or VMC 30/196, eventually, in combination with the further components/ingredients disclosed above and/or the composition disclosed above is(are) able to convert into soluble forms, existing as free Zn ²⁺ ions and Zn chelates, by the bacterial production of organic acids such as 5-ketogluconic acid and 2-ketogluconic acid. Indeed, gluconic acids and ketogluconates are sugar acids having multiple conformations, which chelate the metal cations coming from solubilization process.

According to a further embodiment of the invention, VMC30/195 and/or VMC30/196, eventually, in combination with the further components/ingredients disclosed above and/or the composition disclosed above is(are) able to produce siderophores. Preferably, VMC30/195 and/or VMC30/196, eventually in combination with the further components/ingredients disclosed above and/or the composition disclosed above is(are) able to chelate iron ions (Fe ³⁺ ), making iron more available for plants. As used herein, “siderophores” mean small, high-affinity iron-chelating compounds generally secreted by microorganisms such as bacteria, fungi and grasses. Siderophores are amongst the strongest soluble Fe3+ binding agents known. These compounds are small proteic molecules generally <1000 Da, although some siderophores are bigger. They are rapidly assembled through short, well-defined metabolic pathways. These molecules comprise lateral chains and functional groups that confer a strong affinity (usually with K _d >10 ³⁰ M ^-1 ) to coordinate with the ferric ion (Fe ³⁺ ). Typically, microbial siderophores belong to at least one of class of molecules preferably selected from: catecholates, hydroxamates, and a-carboxylates, depending on the chemical nature of their coordination sites with iron. Preferably, the strain(s) of the invention is able to produce hydroxamates type siderophores. These siderophores form iron chelates by the binding site that is mounted on an L-ornithine derivative. Through this mechanism iron ion (Fe ³⁺ ) is taken from insoluble forms and became available for plants. Iron privation in plants causes as main effect the reduction in photosynthetic activity and as secondary effect the reduction in fruit quality in terms of color, size, sugar content, fruit hardness and taste. Moreover, siderophores support plant growth also by inhibition of soil-borne plant pathogens. Indeed, the siderophores - produced in iron-limited conditions -sequester the less- available iron from the environment and inhibit pathogens by depriving iron. Therefore, according to a further embodiment of the invention, VMC30/195 and/or VMC30/196, eventually, in combination with the further components/ingredients disclosed above and/or the composition disclosed above can be used to improve the capability of plants to uptake iron preferably from soil.

According to a further embodiment of the invention, VMC30/195 and/or VMC30/196, eventually, in combination with the further components/ingredients disclosed above and/or the composition disclosed above is(are) able to mobilize K making it more available for plants. Potassium (K) is an essential macronutrient and plays an important role in the growth and metabolism of plants. The concentrations of soluble potassium in the soil are usually very low and more than 90% of potassium in the soil exists in the form of insoluble rocks and silicate minerals (e.g., biotite, feldspar, illite, muscovite, orthoclase, and mica), not available for plant uptake. A deficiency of potassium causes a delay in the root and plant growth, a small seed production and a lower yield. With the intensification of agriculture and the imbalanced fertilizer application, potassium deficiency is becoming one of the major constraints in crop production. This aspect highlights the importance to maximize the bioavailability of such mineral in the soils for sustaining crop production. Soil microbes can play a central role in the natural potassium cycle and therefore, the potassium solubilizing microorganism present in the soil, can dissolve silicate minerals and release K through the production of organic and inorganic acids, acidolysis, polysaccharides, complexolysis, chelation, and exchange reactions. Therefore, according to a further embodiment of the invention, VMC30/195 and/or VMC30/196, eventually, in combination with the further components/ingredients disclosed above and/or the composition disclosed above can dissolve potassium from insoluble K-bearing minerals and make it available to plant uptake.

According to a further embodiment of the invention, VMC30/195 and/or VMC30/196, eventually, in combination with the further components/ingredients disclosed above and/or the composition disclosed above is(are) able to produce IAA.

According to a further embodiment of the invention, VMC30/195 and/or VMC30/196, eventually, in combination with the further components/ingredients disclosed above and/or the composition disclosed above is(are) able to fix nitrogen. Preferably, VMC30/195 and/or VMC30/196, eventually in combination with the further components/ingredients disclosed above and/or the composition disclosed above is(are) able to convert atmospheric N ₂ into plant-utilizable forms by biological N ₂ fixation which changes nitrogen to ammonia by nitrogen fixing microorganisms using a complex enzyme system known as nitrogenase.

As already disclosed above, when used as consortium with at least a further strain, preferably with VMC30/196, VMC30/195 is used in the same amount (1 :1) of the further strain. Alternatively, the range between the amount of VMC30/195 and the additional strain, preferably VMC30/196, varies between 1 :1 and 1 :100, preferably 1 :3, 1 :5 or 1 :10.

According to a preferred embodiment, the strain or the consortium or the strain derivatives or the composition here disclosed is preferably applied into the soil, with any useful means or way, preferably through fertigation or irrigation systems and is preferably diluted in water, more preferably through overhead applications with appropriate volumes of water to let the solution spreading through the soil. In general, the application of the strain (VMC30/195) or the consortium of the strain with at least one additional bacterial strain, preferably VMC30/196, or the composition comprising the strain or the consortium is performed with at least one of the following practice: drip, overhead irrigation, seed treatment, soil application, foliar treatment, indoor farming, vertical farming and combination thereof.

According to a preferred embodiment, the application is performed all over the crop cycle, preferably during the early stages of crop development.

According to a preferred embodiment, the strain or the consortium or the strain derivatives or the composition here disclosed is preferably applied at a rate ranging between 100g-10.000 g/hectare, preferably between 2 and 10 kg/hectare preferably up to 5 times/crop cycle.

Indeed, when tested together, especially in presence of a plant biostimulant, the biological properties discussed above are particularly enhanced as well as the crop yield. In this regard, the synergism between the strains in the presence of the PBS may be due to their ability to degrade the PB’s components and to generate metabolites able to support their growth/activities.

According to a preferred embodiment, VMC30/195 and/or VMC30/196 when used as consortium is/are able to emphasize, preferably to synergize, plants responses to any biostimulant substance. Preferably, a significant yield improvement has been observed when VMC30/195 and/or VMC30/196 is(are) used with a biostimulant as defined above.

Therefore, VMC30/195 and/or VMC30/196, preferably in combination with one or more of the further components/ingredients disclosed above and/or the composition as disclose above is(are) particularly useful in agriculture, preferably to improve, preferably into soil, macronutrients and/or micronutrients solubilization (mobilization/dissolution/fixation), preferably phosphate and/or zinc and/or potassium solubilization/mobilization, and/or atmospheric nitrogen fixation and/or siderophores production, preferably iron availability and/or IAA production. Consequently, VMC30/195 and/or VMC 30/196, preferably in combination with one or more of the further components/ingredients disclosed above and/or the composition as disclose above is(are) particularly useful to improve macronutrients and/or micronutrients plant uptake, phosphate and/or zinc and/or iron and/or nitrogen uptake from plants. In view of these effects, VMC30/195 and/or VMC30/196, preferably in combination with one or more of the further components/ingredients disclosed above and/or the composition as disclose above is(are) useful to improve plant growth. Preferably, said further components/ingredients is the PBS as previously disclosed.

As used herein, plant means any one of the vast number of organisms within the biological kingdom Plantae. Conventionally the term plant implies a taxon with characteristics of multicellularity, cell structure with walls containing cellulose, and organisms capable of photosynthesis. Modern classification schemes are driven by somewhat rigid categorizations inherent in DNA and common ancestry. In general, these species are considered of limited motility and generally manufacture their own food. Preferably, plant includes a host of familiar organisms including trees, forbs, shrubs, grasses, vines, ferns, mosses and crop plants as vegetables, orchards and row crops. More preferably plant includes leaf vegetables, preferably selected from: Solanaceae, Brassicaceae, Liliaceae, and combinations thereof; and/or orchard plants, preferably selected from: fruit trees, grapes, olive trees, citrus trees, and combinations thereof; and/or row crops, preferably selected from: cereals, preferably corn, soybean, wheat, winter wheat, rice, barley, sugar cane, sugar beet, legumes preferably peas, beans, chickpeas but are not limited to: brassica, bulb vegetables, cereal grains, citrus, cotton, curcurbits, fruiting vegetables, leafy vegetables, legumes, oil seed crops, peanut, nut trees, cocoa tree, herbs, cannabis, pome fruit, root vegetables, tuber vegetables, corm vegetables, stone fruit, tobacco, palm trees, musae spp., strawberry and other berries, and various ornamentals.

All the sequences disclosed in the present invention are listed in the following Table and are further submitted as Sequence Listing. Any sequence having at least 80- 99% of identity with the sequences herewith should be considered part of the present disclosure.

Table

EXAMPLE

The present invention will be described in detail by means of the following examples. The following examples are for illustrative purposes and are not intended to limit the scope of the invention.

Isolation of Novel Bacterial Strains

The microbes have been isolated from rhizosphere soil samples coming from India.

A soil sample (10 grams) has been taken and diluted into 90 ml of distilled water. After homogenization, serial dilution up to 10 ^-5 have been done. One ml of each serial dilution has been smeared on a nutritive agar plate and incubated for 24 h at 30 °C. After incubation period, the most relevant colonies found on the highest dilution ( 10 ^-5 ), have been picked and plated again on nutrient agar plates, incubated for 24 h at 30 °C.

This process allowed isolate a pure colony from each soil sample said colony being later identified as the Bacillus pumilus and the Bacillus megaterium of the invention. In particular, Bacillus pumilus was isolated from Chickpea (Cicer arietinum) collected from Ramanthapur village (Sanga Reddy), while Bacillus megaterium was isolated from Paddy rhizoshere soil collected from Indra Karan village (Sangareddy). The isolated strains have been deposited in National Centre for Microbial Research (NCMR, Pune, India) on 21 ^st February 2008, with the following name: Bacillus pumilus VMC30/195 (VMC30/195) having the deposit number MCC 0530, and Bacillus megaterium VMC30/196 (VMC30/196 from now on) having the deposit number MCC 0524.

Identification and Characterization of Bacterial Strains

16S-rRNA Sequencing

The strains VMC30/195 and VMC30/196 were typed by direct sequencing of PCR- amplified 16S rDNA.

For the amplification the following primers were used:

The amplification protocol is the following.

The 16S rDNA sequences of the strains are set forth in the sequence Listing as indicated in Table I.

The Sanger sequencing was followed by alignment and phylogenetically analysis of the obtained data with the 16S rRNA sequences from the Type Strains.

According the analysis of the 16S rRNA sequence:

The strain Bacillus pumilus VMC 30/195 is related to the species B. pumilus as it shares 97% and 97,74% of sequence similarity with the 16S rRNA sequences of the corresponding Type Strains (Table II and Fig.1.A). Table II

The strain Bacillus megaterium VMC 30/196 is related to the species B. megaterium as it shares 94,98% and 94,98% of sequence similarity with the 16S rRNA sequence of the corresponding strains (Table III and Fig. 1 B).

Table III

This technique allows identification of microbes as intact cells or cell extracts. In this case, intact cells were tested by using a MicroflexTM MALDI-TOF (Matrix-assisted laser desorption ionization-time-of-flight) mass spectrometer (Bruker Daltonics, Leipzig, Germany). Initial manual/visual estimation of the mass spectra was performed using the FlexAnalysis 2.4 software (Bruker Daltonik GmbH, Germany). For automated data analysis, raw spectra were processed using the MALDI BioTyper 1.1 software (Bruker Daltonik GmbH, Germany) with default settings. The smoothing, normalization, baseline subtraction and peak picking was carried out by the software, thereby creating a list of the most significant peaks of a spectrum (m/z values with a given intensity). Samples were prepared according to manufacturers’ instructions.

Briefly, after 24 hours of cultivation on Nutrient Agar (NA) at 30°C, a single colony was transferred with a toothpick onto the MALDI steel target plates in triplicate. Spots were overlaid with 1 μI of a saturated solution of -cyano-4-hydroxycinnamic acid (Sigma-Aldrich) in organic solution (50% acetonitrile, 2.5% trifluoroacetic acid), air-dried within minutes at room temperature and directly screened. Spectra were recorded by Flex Control software (Bruker Daltonics, Bremen, Germany) in a linear positive mode at a laser frequency of 200 Hz in the range from 2 to 20 kDa. In order to assess the reproducibility of MALDI-TOF-MS identification, strains were tested in triplicate (analyses were performed on three different days and starting from different cultures). For each measurement, at least 300 individual spectra (30 laser shots at 10 different spot positions) were collected and averaged. External calibration was performed with the Bruker bacterial test standard (Bruker Daltonics, Bremen, Germany).

Figure 2 shows MALDI-TOF MS mass spectra of VMC30/195 (A) andVMC 10/196 (B), respectively. Each bar in the graphs represents a different protein expressed by the microbes and the intensity of the bars represents the concentration of the proteins within the microbial cell. Since proteins are a direct expression of genome and genome of each strains is unique, thus the proteomic profile of each strain is unique and can be used as fingerprint. The most and unique representative signal for each one of the strains are in the range from 2 to about 12 KDa using spectrum m/z 10 ^Λ 3.

Morphological and Physiological Characterization

The color and the colony morphology of the strains have been characterized by direct observation of the microbial strains after growing on Nutrient Agar (NA) plates. In particular, the bacterial strains have been cultured on NA plates for 24 h at 30 °C. After incubation period, bacterial colonies appear on the plates and color and morphology can be observed.

The motility and the colony size have been characterized by using optical microscope with 100X magnitude. In particular, the bacterial strains have been cultured on NA plates for 24 h at 30 °C. After incubation period, one bacterial colony has been picked and smeared into 1 ml water drop on a glass slide. The glass slide is then observed under optical microscope with 100x magnitude.

Gram staining is determined by using the Gram staining kit.

The growth of the bacterial strains at different temperatures has been evaluated by streaking the microorganisms on NA plates (in triplicates) and by incubating them at different temperatures (4, 40 and 50 °C). After 24 h of incubation, the appearance of colonies on the plates indicates the ability of the strain to grow at a specific temperature. The ability of the bacterial strains to grow at different pH values is determined by streaking the microorganisms on NA plates adjusted at different pH values (5.5, 6.5 and 9.5). Experiment was done in triplicate. After 24 h of incubation, the appearance of bacterial colonies on the plates indicates the ability to grow at a specific pH value. The salinity tolerance of the strains is determined by streaking the microorganisms on NA plates, modified by the addition of different amount of NaCI (8, 10, 12 and 14 %). The experiment is performed in triplicate. After 24 h of incubation, the appearance of colonies on the plates, indicates the ability of the strain to grow at a specific NaCI concentration, thus, to tolerate a specific salinity level. The results are given in Table IV.

Table IV

Growth (Fermentabilitv) of Strains for In-Vivo Tests

The biomass production process of the strains is divided into four steps that are: inoculum preparation, fermentation, biomass recovery and biomass drying. Recovery of microbial biomass can be done through several processes such as centrifugation, micro-filtration or ultra-filtration.

Drying process is preferably made by freeze-drying.

In vitro Assays

For the assays different strains were used as control. They were chosen according to the literature. Molina et al. (2018) reports Azospirillum brasilense ATCC 29145 as IAA producer and we used it as control in the indole-3-acetic acid production assay. Herbaspirillum seropedicae ATCC 35893 was used as positive control for several biostimulant activities reported in literature, inter alia, inorganic tricalcium phosphate ((Ca3(P04)2) solubilization (Estrada et al. (2012), Rodriguez et al.,2004), siderophores production (Rosconi et al., 2012; Tortora et al., 2011) and zinc solubilization according Gontijo et al. 2017. Both Azospirillum brasilense ATCC 29145 and Herbaspirillum seropedicae ATCC 35893 were used as positive control for Nitrogen fixation (Rosconi et al., 2012 and Zeffa et al., 2019).

Phosphate solubilization activity

The phosphate solubilization activity of the VMC30/195 and VMC30/196 was shown by using an in-vitro plate assay according to Pikovskaya method (Yasmin and Bano, 2011). Tricalcium phosphate (Ca ₃ (PO ₄ ) ₂ ) was used as inorganic source of phosphorus.

The seeding was done superficially using an aliquot (10 μI) of the bacterial suspension (10 ⁶ CFU/ml).

Seeded samples were incubated at 30 °C for 7 days and colonies with a clear halo on plate were considered positive for phosphate solubilization.

P-solubilization activity was measured as solubilization index (SI), according to Yasmin and Bano (2011) after 7th days and compared one to each other to individuate the best phosphorus solubilizing bacteria (PSB).

Solubilization index was determined as follows:

Bacterial strains have been tested in mixed cultures, by spotting on plate an aliquot of 5 μI of each bacterial suspension and following the methodology described above. Moreover, the phosphate solubilizing activity of the microbes was evaluated in liquid medium in order to measure the exact quantity of phosphorus liberated from inorganic form (Ca ₃ (PO ₄ ) ₂ ).

The samples tested were the same as reported above. The bacteria (2ml - 10 ⁶ CFU/ml) was inoculated in liquid Pikovskaya medium (pH 6.5) and incubated at 30°C for 5 days, shaking constantly at 120 rpm. Samples were then centrifugated at 10,000 rpm for 10 minutes. An aliquot of the supernatant was taken to measure the soluble phosphorus (P) and the final pH value of the medium. The amount of soluble phosphorus in the supernatant was measured by molybdenum-blue method using a spectrophotometer at a wavelength of 600 nm (King, 1932). The amount of soluble phosphorus was determined from the standard curve of KH ₂ PO ₄ . Herbaspirillum seropedicae ATCC 35893 have been used as specific controls.

The results are showed in Fig. 3A and B.

VMC30/195 showed ability to solubilize phosphate on Picovskaya’s plates with SI =1 ,20 after seven days of incubation at 30°C. On the contrary Bacillus megaterium VMC30/196 did not express this activity under the tested conditions. Values of solubilized P concentrations were recorded among the bacterial strains in 5 days of incubation. The highest mobilized phosphate value (497,67 mg/L) was recorded from VMC30/195 followed by Herbaspirillum seropedicae ATCC 35893 with 454,50 mg/L of soluble-P. VMC30/196 did not show this activity. Results are summarized in Table V.

Table V

Interestingly, when bacterial strains were tested in mixed cultures, the consortia of VMC30/195 and VMC30/196 showed an increase of the SI value (SI = 1 ,85) compared to the strains used alone where SI is Sl=1 ,20 for VMC30/195 and no activity for VMC30/196. Results are shown in Fig. 3B.

Therefore, the strain VMC30/195 keeps the P-solubilization activity even when is co- cultured with VMC30/196.

Zinc solubilization activity

Zinc-solubilizing capacity of the bacterial strains were assessed by a plate assay method using a modified Pikovskaya method (Ghevariya and Desai, 2014and zinc oxide (ZnO) as inorganic source of zinc.

The isolates were inoculated into agar medium containing 0.1% insoluble zinc (ZnO) and incubated at 30 °C for seven days. The seeding was done superficially using an aliquot (10 μI) of each bacterial suspension, with a concentration of 10 ⁶ CFU/ml. Zinc solubilization efficiency (SE) was calculated as described by Sharma et al., 2014. Bacterial strains have been tested as single cultures as well as mixed cultures, by spotting on plate an aliquot of 5 μI of each bacterial suspension. A highly significant (P= 0.05) variation of SE was recorded among the bacterial strains. The results are summarized in Fig. 4A and 4B and showed that VMC30/195 is better zinc solubilizer (SE=266,67) compared to the control Herbaspirillum seropedicae ATCC 35893 (SE=110,32). VMC 30/196 does not showed the zinc solubilization activity in the tested conditions. Apparently, at least in the tested condition, we found out that for zinc solubilization the consortium works not as well as the single strain. This could be due to secondary metabolites production by both that can affect medium pH, leading to higher values. Such aspect can decrease the efficacy of the solubilization and final result under these experimental conditions. In this regard, indeed, Khanghahi et al., 2018 reported the effect of pH on Zn-solubilizing ability of selected bacteria.

Siderophore’s production

The production of siderophores of the strain of the invention has been determined according to Schwyn and Neilands (1987). Briefly, 10 μI of each bacterial suspension with a concentration of 10 ⁶ CFU/ml was spotted in triplicate on Nutrient broth (NB) and plated were incubated at 28°C for 72 h. Ten ml of chrome azurol S (CAS) agar medium were poured over the plates. After 24 h the formation of an orange halo was considered as indicator of siderophore production. The halo was measured according to Omidvari et al. (2010). Moreover, bacterial strains have been tested as mixed cultures, by spotting on plate an aliquot of 5 μI of each bacterial suspension. The results are summarized in the Fig.5A and B and show that VMC30/196 works better than control (35 vs 19mm) while VMC30/195 did not show siderophore production activity in the tested conditions. Interestingly, the consortium of VMC 30/195 and VMC30/196 of works better that the strains alone. A possible explanation is because of the secreted molecules can be shared between cells; indeed, siderophore production is often considered a form of cooperation. Siderophores are public goods and their production typically represents a mutualistic form of cooperation, providing direct benefits to both the producers and the recipients. Evidence to support this concept, emerged from studies on siderophores, including pyoverdine and pyochelin from P. aeruginosa, ornibactin from B. cenocepacia and enterochelin from Escherichia coli.

Potassium mobilization

In order to determine the potassium-solubilizing capacity of the two bacterial strains used in the present invention, they were screened by a plate assay method using Alexandrov’s agar medium according to the method described by Parmar and Sindhu (2013). The potassium solubilizing activity was evaluated using mica powder (potassium aluminum silicate) as insoluble form of potassium. The isolates were inoculated into agar medium containing 0.5% insoluble potassium. The seeding was done superficially using an aliquot (10 μI) of each bacterial suspension, with a concentration of 1 ,00E+06 CFU/ml. Bacterial strains have been tested as single cultures as well as mixed cultures, by spotting on plate an aliquot of 5 μI of each bacterial suspension. The test organisms were inoculated on these media and incubated at 28 °C for three days. Colonies with a clear halo on plate were considered positive for potassium (K) mobilization.

K-mobilization activity was measured as mobilization index (Ml), according to Parmar and Sindhu (2013).

Mobilization index was determined as follows:

MI = Diameter of z one of clearance f Diameter of growth.

The results are summarized in Fig. 6A and B. Only VMC 30/196 shows good mobilization activity (Ml=2,06) under the tested conditions.

IAA measurement

To determine the IAA-producing capacity VMC 30/195 and VMC 30/196 were screened by a liquid assay method according to Sarker et al. (2013). 1 ,00E+06 CFU/ml or each strain was inoculated into nutrient broth containing 0,1% of tryptophan. Inoculated samples were incubated in shaking incubator at 28°C (100- 110 rpm) for 48 hours (for obtaining log phase). Uninoculated broth was used as negative control. After incubation, the IAA production was determined mixing 2 ml of each sample (previously spun at 13000 g x 10 minutes and filtered with 0,2 m filters) with 4 ml of 0 incubated in the dark at room temperature for 30 minutes. Finally, each sample has been analyzed by a spectrophotometer reading at 530 nm and value compared with IAA standard curve to record ppm of lAA.The results are summarized Fig. 7 and show that VMC 30/196 works better that the control used (ppm=24,88 vs 19,04 ppm). Culture broth not inoculated as negative control.

Nitrogen fixation

The nitrogen fixation capacity of the strains of the present invention have been assessed according to Okon et al. (1977) with minor changes. In particular, the bacteria were inoculated using an aliquot (10 μI) of each bacterial suspension, with a concentration of 10 ⁶ CFU/ml in a medium made up of in 1 I: D-L malic acid 5 g, KOH 4 g, K ₂ HPO ₄ 0.5 g, MgSO ₄ 0.2 g, NaCI 0.1 g, CaCl ₂ 0.02 g, bromotymol blu (BTB) (0.5% dissolved in 0.2 N KOH) 2 ml, trace element solution 1 ml (NaMo04 200 mg, MnSO ₄ 253 mg, H ₃ Bo ₃ 280 mg, CuSO ₄ 8 mg, ZnSO ₄ 24 mg in a final volume of 200 ml of distilled water), Fe EDTA (EDTA 22.8 g/l, 250 ml of KOH 1 N, FeCI ₃ 10 g) 4 ml, agar 15 g. The final pH was adjusted to 6.8. Fe EDTA solution was sterilized by filtration with 0.2 μm filters. Fe EDTA, BTB and the trace solution were added to the medium after cooling. After 24 hours of incubation at 30°C, the blue colored zone producing isolates were marked as nitrogen fixers in the solid culture conditions. The coloring zone was calculated by deducting the colony diameter from the coloring zone diameter, according to Gothwal et al. (2008). Bacterial strains have been tested as mixed cultures, by spotting on plate an aliquot of 5 μI of each bacterial suspension.

Results are summarized in Fig 8A and B and show that both strains are able of nitrogen fixation. In particular, VMC30/195 is the best performer working even better than the two control strains (34 mm vs 21 and 20.33 mm). VMC30/196 works too but with lower performance (18 mm of diameter). The consortium shows good performance too.

Plant growth promotion activity on Lettuce plants The plant growth promotion activity of the strains was tested in combination with a plant biostimulant (PBs). In particular, in vivo bioassays have been done on Lactuca sativa cv. Canasta plants. Canasta plantlets were obtained from a local nursery. Two-week-old plantlets were transplanted in 17 cm diameter plastic pots, 2 Liters volume each. 4 replications of eight pots/treatment were prepared, on a sandy substrate, in a glasshouse. The environmental conditions during the experimental period were 22-33°C, with a relative humidity ranging from 70-80%, under natural light conditions. Nutrients were directly added to the substrate during the crop cycle, by providing 40 Kg/ha of water-soluble nutrient mix containing NPK (13-40-13), every 10 days. The density was 10 plants/m2. T reatments conditions were:

• Untreated Control (UTC)

• VMC 30/195+VMC 30/196: two applications every 10 days at 5 Kg/ha rate;

• PBS: two applications every 10 days at 5 Kg/ha rate; • VMC 30/195+ VMC 30/196+ PBS: two applications every 10 days at 5 Kg/ha rate.

The irrigation was carried out considering the substrate moisture content and the amount of water was determined to maintain the 80% of substrate water availability. Lettuce plants were harvested when the plants reached the commercial maturity stage, after 40 days of cultivation. Plant fresh weight (g) was determined at harvest. Data were subjected to two-way ANOVA and SNK comparison test was used for evaluating the differences among means at p < 0,05. Different letters indicate statistical differences for p < 0,05. Foliar Zinc, Nitrogen, Phosphorous and Iron were assessed, only for UTC and complete prototype (VMC30/195+VMC30/196+PBS), at the end of the cycle by leaf analysis performed in a dedicated local laboratory.

The results show that VMC30/195+VMC30/196 promote plant growth and this activity is more enhanced in the presence of a plant biostimulant.

Mode of Action

To investigate the overall molecular effect of a composition comprising the 2 strains of the invention and PBS compared to plants treated only with PBS, a microarray- based transcriptomic analysis has been performed.

The samples tested are as follow: Arabidopsis thaliana (Ecotype Col-0) was used in this experiment. The seeds were washed and rehydrated with water for 15 minutes before sowing, to facilitate the germination. After washing, single seed was taken with tweezers and sown on each single well (50/50 of earth and perlite) of the "Arasystem trays". After that, the trays were transferred into a growth chamber with controlled conditions to allow optimal plant growth. The microarray analysis (SureScan, Agilent system) has been performed after 24h (early effect) and 72h (mid effect) from the application on 15- days old A. thaliana seedlings.

GO-Term enrichment was performed for interpreting sets of genes making use of the Gene Ontology system classification, in which genes are assigned to a set of predefined bins depending on their functional characteristics and cut-off selected (0,01).

The results show that the following GO-Groups are shared between PBS and the prototype: carbohydrates, transport, and response to starvation; these are most probably modulated by the PBS.

The GO-Groups correlated to the microbial component (the 2 strains of the invention) instead are the following: biosynthetic process, biotic response, hormone response and metabolic process.

Effect on Sweet Pepper

The plant growth promotion activity of the strains was tested in combination with PBS. In particular, greenhouse experiment has been done on sweet pepper plants using sub-optimal nutrition.

Sweet pepper plantlets were transplanted in 17 cm diameter plastic pots, 2 Liters volume each. 4 replications of eight pots/treatment (7 treatments x 12 blocks) were prepared, on a sandy substrate, in a glasshouse. The environmental conditions during the experimental period were 22-33°C, with a relative humidity ranging from 70-80%, under natural light conditions. Nutrients were directly added to the substrate during the crop cycle.

Treatments conditions were:

• Untreated Control (UTC)

• Prototype: three applications every 7 days at 5 Kg/ha rate, first application after transplant at 14 days.

• PBS: three applications every 7 days at 5 Kg/ha rate, first application after transplant at 14 days.

• Prototype: three applications every 7 days at 10 Kg/ha rate, first application after transplant at 14 days

• PBS: three applications every 7 days at 10 Kg/ha rate, first application after transplant at 14 days

• Internal Valagro control: three applications every 7 days at 10 Lt/ha rate, first application after transplant at 14 days Sweet pepper plants were harvested when the plants reached the commercial maturity stage. Plant biomass (g) and fruits (total weight in grams) was determined at harvest. Data were subjected to Duncan’s test and SNK comparison test was used for evaluating the differences among means at p < 0,05 and p<0, 10. Different letters indicate statistical differences for p < 0,05 and p<0,10.

Plant Biomass showed statistical significance (SNK and Duncan’s test p<0,05 and p<0, 10) for Prototype (5-10 kg/ha) vs UTC;

For Fruits weight, plants treated with product Prototype (5-10 kg/ha) produced more fruits, with a higher weight (g) in comparison with the UTC. Statistical significance (SNK and Duncan’s test p<0,05 and p<0, 10).

Field Trials

The prototype was assessed on Melon and Tomato crops under open field conditions in Rabi season at Maharashtra, India. The experiment was carried out using Split plot design under two different approaches, a] Reduction of Phosphorous content i.e- 30% reduction b] Remobilization of Phosphorous i.e no available Phosphorous. In both the crops and in both the approaches, untreated control (UTC) was used.

In both the crops, first dose of application was done after 5 days of transplantation of seedlings with second dose after 20 days of planting and 3 ^rd dose after 40 days of planting. Soil samples were collected from all the plots before and after application treatments i.e @ 0, 1 , 30 and 60 days after application to estimate the total viable count of microbial load.

Statistical analysis: LSD (a 0.05)

Under the conditions of standard NPK fertilizer application, the effect of the prototype on yield (mt/ha) was increased significantly in case of Tomato crops in 2 different experiments, whereas, in Melon significant difference in yield was observed in treated plot compared to UTC in one experiment. Moreover, regarding 2 different strategies tested, yield increase was significant in case of Tomato in both the strategies, indicating that prototype is effective when P is reduced to 30% or reduced completely.

Effect of prototype was significantly high in both the crops considering fruit weight in Melon and number of fruits in Tomato. Moreover, it is clear from the results below that the 2 strains of bacteria present in prototype survived and maintained stable viable cells in all the treated plots after the application of the prototype.