WO2018182555A2

WO2018182555A2 - Microorganisms which are effective for preventing plant's cold stress

Info

Publication number: WO2018182555A2
Application number: PCT/TR2017/050480
Authority: WO
Inventors: Metin Turan
Original assignee: Yedi̇tepe Sağlik Hi̇zmetleri̇ Anoni̇m Şi̇rketi̇
Priority date: 2016-10-05
Filing date: 2017-10-05
Publication date: 2018-10-04
Also published as: WO2018182555A3

Abstract

The present invention is directed to a microbial inoculant that is effective for preventing cold stress. In an important aspect of the invention, the microbial inoculant includes the Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans. The use of those as a microbial inoculant is effective for increasing the productivity of both nonlegume and legume plants as well as vegetable plants over a wide variety of soil types and climates. One important aspect of the invention is directed to biologically pure cultures of Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans. Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans, may be used as a microbial inoculant either alone or in combination with other agronomically beneficial microorganisms. Application of the microbial inoculant to the plant or the soil prevent cold stress, provides an increased level of soil nitrogen, improves mineral uptake into plants, stimulates plant growth through the production of plant growth regulators, and inhibits phytopathogenic microflora. In another important aspect, the present invention provides a method for growing Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans. In accordance with the method of the invention, Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans is inoculated into a plant extract medium and cells are growth under conditions effective for providing a cell density of about 108 to about 109 cfu/ml.

Description

MICROORGANISMS WHICH ARE EFFECTIVE FOR PREVENTING PLANT'S

COLD STRESS

FIELD OF THE INVENTION

The present invention relates to microorganisms which are effective for preventing plant's cold stress. More particularly, the invention provides a crop inoculant that includes Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans which is effective for significantly and consistently improving plant productivity over a wide range of soil and climate conditions.

BACKGROUND OF THE INVENTION

About two thirds of the world's landmass is annually subjected to temperatures below the freezing point and about half of it suffers from temperatures below - 20°C. It is therefore not surprising that the impacts of cold stress on plant life have been comprehensively studied and many attempts have been undertaken, to improve cold resistance in particular of important crop plants. However, progress in achieving frost hardiness of plants either by classical breeding or by gene transfer is difficult, due to the fact that cold resistance is not a quality conferred by the product of one gene, but has turned out as a syndrome, comprising many quite different traits of cell biology, such as fluidity of the biomembranes, synthesis and accumulation of low molecular weight and high molecular weight cryoprotectants, increase of the potential to cope with oxidative stress and others. The syndrome is even more complex because the various tissues of a plant are differently frost resistant, whereby meristematic cells are in general less frost hardy than mature tissues. Another phenomenon that complicates the investigation of cold as a plant stressor is the seasonal change of frost hardiness of many perennial plants of temperate and subarctic climates. Needles of the Central European Scots pine {Pinus sylvestris L.) are lethally damaged when exposed to - 10°C during the summer months, while in mid winter they survive exposure to - 80°C. Frost hardening and dehardening of a plant are extremely slow processes which cannot be studied like metabolic reactions and which require special methods of investigation. Maximum rates of frost hardening of Scots pine needles under laboratory conditions as well as in the natural environment were below - 1 °C per day, as will be shown later. Low temperature may impose stress on a plant in a two-fold manner: By the effects of low temperature alone, and by dehydration of the cells and tissues when cellular water freezes. Low temperatures above the freezing point are detrimental to many plants of the tropics and subtropics which cannot acclimatize to cold. This kind of damage has been termed 'chilling' and results primarily from loss of function of bio membranes connected with a decrease of their fluidity and an inactivation or at least deceleration of the membrane-bound ion pumps. Light energy which is absorbed independently of the temperature, produces oxidative stress, if metabolism cannot keep pace with the energization of the photosynthetic membranes. Freeze dehydration, on the other hand usually takes place at an unexpectedly high extent: More than 75% of the water of a frost-hardy evergreen leaf was frozen to ice that was deposited in the intercellular spaces. Whether, and to which extent a plant becomes damaged by exposure to low temperature depends on many factors, such as its developmental stage, the duration and severity of frost, the rates of cooling (and rewarming) and whether ice formation takes place intracellular^ or extracellularly in the intercellular spaces. Intracellular ice formation, by disintegration of the cellular membranes, is known to be inevitably lethal. The bilayer structure of the bio membranes depends on the hydrophobic interaction with the aqueous cellular phase which cannot be replaced by ice. An exception to this rule is the artificial vitrification, whereupon amorphous ice is formed due to an extremely rapid cooling (1 0x000 K x min^-1 ). Frost hardiness or sensitivity is a quality of each individual plant and is governed by its genetic potential as well as by environmental factors and therefore usually changes with time. As mentioned above frost hardening and dehardening are accomplished by thorough changes of a tissue's cell biology. Well known alterations affect the lipid composition of the bio membranes with respect to the maintenance of their fluidity, the synthesis and accumulation of compatible solutes, the synthesis of cold acclimation induced proteins, changes in the carbohydrate metabolism and the boosting of the radical scavenging potential of the cells. Upregulation of gene expression following exposure to cold has been reported in many studies, mainly with mono- and dicotyledonous.

Common traits between resistance to cold and to drought have been identified in particular with respect to intracellular signal transduction, which involves an increase of the cellular calcium level. While exposure to cold, salt or drought are possible signals triggering frost hardening of a short-lived annual herb that under natural conditions does not experience cold, perennial, and in particular evergreen plants must perceive signals triggering frost hardening prior to the incidence of the first frost event. Shortening of the photoperiod together with a decrease of the average temperatures have been identified as effective signals for frost hardening of. A prolonged exposure of already moderately frost tolerant plants to non-lethal subfreezing temperatures or a sequence of mild frost events induces further hardening to the final stage of extreme frost tolerance.

A number of microorganisms are known to have beneficial effects on cold stress. Among these are nitrogen fixing bacteria of the Rhizobium species, which are symbionts of leguminous species. Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans which are free living nitrogen fixing bacteria associated with the roots of grasses, are also now recognized for their plant growth promoting qualities. More specifically, certain strains of Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans have been shown to enhance accumulation of various minerals and prevent from freezing in wheat and soybean, increase dry weights of maize shoots, and increase dry weights of sorghum, pearl millet and napier grass. Inoculation of seeds or soil with beneficial microorganisms, including Azospinllum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans, for crop improvement has been practiced for a number of years. However, variable and inconsistent results have often been observed possibly due to loss of inoculant viability or variability of dosage due to changes in inoculant viability. Further, the use of specific types of microorganisms as crop inoculants has met with varying degrees of success most likely due to variables that include:

(1 ) the presence or absence of adequate micro- and macro-nutrients in the soil to support the propagation of the microorganisms;

(2) the amount of organic material in the soil available to hold nutrient and microbes in the soil and provide a suitable environment for microbial growth;

(3) the presence or absence of certain minerals or compounds required by the plant for proper uptake of the nutrients provided by microbial activity; and (4) variations in soil characteristics such as soil type, pH, temperature and moisture.

A number of Azospinllum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans, strains have been isolated from soil. These strains seem to have limited adaptive capabilities when introduced into other soil types in different climate. Further, these types of microorganisms were not very effective simulators of legume-rhizobia symbiosis as they tended to stimulate the growth of plant dry weight and nodule formation, but did not always provide an increase in nitrogenase activity.

SUMMARY OF THE INVENTION

The present invention is directed to a microbial inoculant that is effective for preventing cold stress. In an important aspect of the invention, the microbial inoculant includes the Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans. The use of those as a microbial inoculant is effective for increasing the productivity of both nonlegume and legume plants as well as vegetable plants over a wide variety of soil types and climates.

One important aspect of the invention is directed to biologically pure cultures of Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans. Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans, may be used as a microbial inoculant either alone or in combination with other agronomically beneficial microorganisms. Application of the microbial inoculant to the plant or the soil prevent cold stress, provides an increased level of soil nitrogen, improves mineral uptake into plants, stimulates plant growth through the production of plant growth regulators, and inhibits phytopathogenic microflora.

In another important aspect, the present invention provides a method for growing Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans. In accordance with the method of the invention, Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans is inoculated into a plant extract medium and cells are growth under conditions effective for providing a cell density of about 10⁸ to about 10⁹ cfu/ml.

DETAILED DESCRIPTION OF THE INVENTION Definitions The term "cold stress" or "frost tolerance" refers generally to any aspect subjected to temperatures below the freezing point and about half of it suffers from temperatures below - 20°C.

Thus, for purposes of the present invention, preventing cold stress refers broadly to improvements in yield of grain, fruit, flowers, or other plant parts harvested for various purposes, improvements in growth of plant parts, including stems, leaves and roots, improved resistance to disease, improved survivability in extreme climate, and similar improvements of the growth and development of plants. Examples of agronomically beneficial microorganisms include Bacillus, Lactocoecus, Pseudomonas, Rhizobia, phototrophic and cellulose degrading bacteria, Clostridium, Trichoderma and the like.

As used herein "microbial inoculant" or "inoculum" refers to a preparation that includes Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans. Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans refer to both the marked strain, i.e. having streptomycin resistance and unmarked strain.

As used herein "biologically pure" refers to a microbial inoculant were Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans, are the only agronomically beneficial strain added to the inoculum. The microbial inoculant may include other microorganisms that do not provide any agronomic benefit. As used herein an "agronomically beneficial strain" refers to microorganisms that are effective for increasing plant productivity. Microorganisms that provide agronomic benefit in addition to SAB include symbiotic and nonsymbiotic microorganisms which may be effective for making nutrients more bioavailable to plants, and microorganisms that inhibit phytopathogenic microflora and stimulate plant growth. Isolation of Organism

In this aspect of the invention, Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans was isolated from turfty-podzolic soil during March in Turkey. This type of soil is representative of a poor northern climate soil having low nutrient levels. While not intending to be bound by any theory, it is thought that Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans is better able to survive under poor soil conditions, and hence adapt to a wide variety of soil conditions, because it was initially isolated from a poor soil.

Soil samples were enriched using standard techniques by culture in medium containing about 1 % sodium lactate and about 0.1 % yeast extract. Pure cultures of Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans were isolated by passage of enriched preparations on solid agarlike media, such as for example, potato agar and beef extract with the addition of ammonium sulfate. The presence of Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans was confirmed by observing culture preparations under a phase contrast microscope. In an important aspect of the invention, Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans have the following morphological, physiological and biochemical characteristics.

Staining characteristics: gram negative Cell size: about 0.5 to 0.6 microns in diameter and about 1 to 1 .5 microns in length, slightly curved.

Morphology: morphology is dependent on the type of medium used for culturing. Vibrioid forms occur on solid and semisolid media and S-shapes occur in liquid culture. Growth on potato agar results in the formation of large, opaque, round, slimy pink colored colonies with a metallic luster. Growth on beef extract agar results in the formation of small round colonies which are round, opaque and white in color. In an important aspect of the invention, Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans, produce a pink carotenoid pigment, rodovibrin.

Motility: Movement is typically wavy and rotatory, and motility is provided by a single polar flagellum in liquid medium and by numerous lateral flagellum on solid medium. In an important aspect of the invention, the presence of flagellum provides Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans with a improved moving capacity and allows them applied as an inoculum to quickly reach the root systems of the plants.

Cyst formation: Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans., initially form cysts and involution forms (spheroplasts) when cultured for about 10 to 14 days on an enriched media such as potato agar, beef extract, the ability to form spheroplasts is lost, and spheroplast formation does not occur even where Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans are cultured on a poor media (for example, water with 0.5% calcium lactate). Growth of Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans

In an important aspect of the invention, Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans can be maintained and grown in a manner which is effective for maintaining the stability and consistency of the strain. Even with repeated subculturing, Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans, are stable in that the ability of those to prevent cold stress, fix nitrogen, produce plant growth regulators, and inhibit phytotoxic organisms does not decrease from its initial levels.

Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans can be grown and stably maintained on minimal mediums that include a salt of an organic acid and various macro and micronutrients. In an important aspect of the invention, Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans can be propagated with minimal mediums (such as water with about 0.5% Ca-lactate) and still maintain its ability to fix nitrogen.

In another aspect of the invention, Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans can tolerate or even grow under a wide variety of environmental conditions. Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans do not require additional growth factors, is chemoorganotrophic, and does not have fermentative ability. In another aspect of the invention, Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans can grow under anaerobic conditions. Under anaerobic conditions, Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans produce sugar as an terminal electron acceptor, ultimately resulting in the non-chilling.

The inoculated plant extract medium is then cultured at a time, temperature and aeration rate effective for providing a cell density having an optical density of at least about 3.0. In an important aspect of the invention, culture time will typically range from about 24 to about 48 hours, temperatures are maintained at about ambient temperature, and standard aeration rates are used such that O2 levels are not limiting.

Application of Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans

Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans may be applied by methods known in the art which include spraying, seed coating, applications with peat or planting materials. In the aspect of the invention Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans are firstly sprayed to plant in autumn. The inoculum thereafter may be applied twice to plant's components like stems, flowers and fruits in spring.

In the aspect of the invention where seed coating is utilized, coating may be affected by Humic Acid. In the aspect of the invention where Humic Acid is utilized, Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans may be cultured in fermentators to reach high population levels (i.e. 10⁸ -10⁹ cells/ml) and then added to pre-sterilized Humic Acid. The inoculum thereafter may be applied to seeds (by preparing a slurry containing the peat/bacteria mixture and gums or sugars to improve adhesion), by applying directly to soil (by dripping peat suspensions into planting furrows) or by mixing with other planting media, such as peat or Humic Acid.

In another aspect of the invention, Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans may used as an inoculum and applied either alone (monoculture) or in combination with other agronomically beneficial microorganisms. In a very important aspect of the invention, Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans are suspended with seeds to provide about 10⁸ to about 10⁹ cells/ml/seed. This suspension of seeds and microorganism is then planted into the soil. Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans are physiologically compatible with a wide range of bacteria and fungi, including for example, Bacillus, Pseudomonas, Rhizobia, phototrophic and cellulose degrading bacteria, Clostridium, Trichoderma, and the like.

In another aspect of the invention, Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans may be applied to plants or soil in combination with the application of boric acid. In this aspect of the invention, inoculation with Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans is effective for increasing plant productivity over treatments where boric acid is used without any crop inoculant. In another aspect of the invention, Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans may be applied to plants or soil in combination with the application of enzyme, 9- cis-epoxycarotenoid dioxygenase (NCED). In this aspect of the invention, inoculation with Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans is effective for increasing plant productivity over treatments where enzyme is used without any crop inoculant. In another aspect of the invention, Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans may be applied to plants or soil in combination with the application of abscisic acid. In this aspect of the invention, inoculation with Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans is effective for increasing plant productivity over treatments where abscisic acid is used without any crop inoculant.

Effect on Plant Growth and Soil Quality

In an important aspect of the invention, Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans act to prevent cold stress and to improve plant productivity and soil quality through nitrogen fixation, by improving uptake of minerals by plants, by stimulating plant growth, and by inhibiting a wide range of phytopathogenic microflora.

Prevention of cold stress: Cold stress prevention by Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans occur over a wide range of environmental conditions. For example, it occurs over a temperature range of from about -50° C. to about 40° C, with the optimal temperature being about -22° C. to about 0° C. Similar levels of cold stress prevention are observed in soil when Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans are applied to soil as a crop inoculant.

Improving mineral uptake: In another important aspect of the invention, Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans are able to produce amino acids which act to facilitate transport of nitrogen into plants. For example, Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans produce asparaginic acid and proline in levels effective for aiding the assimilation nitrogen by plants. Producing abscisic acid: In an important aspect of the invention, ability of Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans to stimulate plant growth is attributable at least in part to its ability to produce plant growth regulators, such as abscisic acid. In this aspect of the invention, Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans are effective for providing at least about 100 micrograms of abscisic acid per mg of protein. Inhibition of phytopathogenic microflora: In another important aspect of the invention, Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans are effective for inhibiting the growth of phytopathogenic microflora which commonly occur in the root zone and which tend to have a detrimental effect on plant productivity. As used herein, "inhibiting the growth of phytopathogenic microflora" refers to not only to inhibiting an increase in the biomass of the phytopathogenic microflora but also inhibiting any metabolic activities which may decrease plant productivity. Examples of phytopathic bacteria and fungi that Azospinllum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans. are known to inhibit the growth of include Fusarium oxysporum, Thelaviopsis basicola, Alternaria, Aspergillus flavus, Mucor fragilis, Penicillum and the like.

The following examples illustrate methods for carrying out the invention and should be understood to be illustrative of, but not limiting upon, the scope of the invention which is defined in the appended claims.

EXAMPLES Example 1

Characteristics of MKB

Azospirillum Brasilense, Bacillus Subtilis, Bacillus Megaterium, Lactocoecus spp. pseudomonas sp, pseudomonas putita, pseudomonas lurida, acinetobacter, paenibacillus sp, Burhholderia phytofinans are an aerobic microorganism possessing a mainly respiratory metabolism with O2 as a terminal electron acceptor. Additional biochemical characteristic were determined by standard methods known in the art. Additional characteristics are as follows: BETA-KSiLOSiDAZ BXYL (pozitif)

L-Lizin-ARiLAMiDAZ LysA (negatif)

BETA-GALAKTOS I DAZ BGAL (pozitif)

L-Prolidonil-ARiLAMiDAZ PyrA (pozitif)

ALFA-GALAKTOSIDAZ AGAL (pozitif)

Ala-Phe-Pro ARILAMIDAZ APPA (pozitif)

GLIKOJEN GLYG (negatif) myo-iNOSITOL INO (pozitif)

METiL-A-D-GLUKOPiRANOSiD asitle§mesi MdG (pozitif)

ELLMAN ELLM (pozitif)

MALTOTRIOZ MTE (negatif)

Glisin ARILAMIDAZ GlyA (pozitif)

D-MANITOL dMAN (pozitif)

PALATINOZ PLE (pozitif)

L-RAMNOZ IRHA (negatif)

BETA-GLIKOSiDAZ BGLU (pozitif)

BETA-MANNOSIDAZ BMAN (negatif)

FOSFORIL KOLIN PHC (pozitif)

PIRUVAT PVATE 0,0 mg (negatif)

ALFA-GLIKOSiDAZ AGLU (pozitif)

D-TAGATOZ dTAG (negatif)

D-TREHALOZ dTRE (pozitif)

INULIN INU (pozitif)

D-GLIKOZ dGLU (pozitif) oxidase +

catalase +

Anaerobic growth with nitrate

reduces NO3 to NO2 +

reduces NO2 to N2 O +

reduces N2 O to N2 +

Starch hydrolysis

Urease +

Liquification of gelatin

Indole formation from tryptophane - Growth in presence of 3% NaCI - Reaction of Voges-Proskauer

Reaction with Methy-Red -

Growth on milk with lacmus alkilining Mol % G + C of DNA 69% GC

Temperature range of nitrogen fixation and growth 10-40° C. Temperature optimum 22-30° C. pH range of nitrogen fixation and growth 5.5-8.5 pH optimum 6.5-7.2 Example 2

Growth Mediums

Azospinllum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans may be grown and maintained on the following mediums.

Semisolid Nitrogen Free Medium 1 sodium malate 5.0

MgSO₄ 7H₂ O 0.3

CaCI₂ 6H2 O 0.1

FeCI₂ 6H2 O 0.01

agar 4.0

H₃ BO₃ 5.0

MnSO₄ 4H₂ O 3.0

KJ 0.5

ZnSO₄ 7H₂ O 0.2

AI2 (SO₄)₃ I 2H2 O 0.3

PH 6.8-7.0

Semisolid Nitrogen Free Medium 2

MgSO₄ 7H₂ O 0.3

FeCI₂ 6H₂ O 0.01 glucose 1 .0

peptone 1 .0

Na lactate 0.5

Na acetate 1 .0

malate or Na succinate 0.5

yeast autolysate 0.5

agar-agar 15.0

Example 3

Industrial Scale Growth of Azospirillum brasi^'lense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans

Preparation of Plant Extract Medium

1 . Plants (seed and/or stems, but no roots) were added to water (200 g/l) and boiled for 20 minutes.

2. Solid material was separated from the mixture (by filtration) and the following components were added.

0.5% Na-malate

0.1 % (NH₄)2 SO₄ Culture Conditions

1 Azospirillum brasi^'lense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans were inoculated into the medium and grown with agitation and aeration at a temperature of 25° C. to 28° C. for 48 hours. 2. Cell densities reached an optical density of 3.0 to provide 600-700 mg of biomass.

Claims

1 . A biologically pure culture of Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans, respectively to prevent cold stress in plants.

2. A microbial inoculant is effective for application to a plant or to soil which comprises Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans, .

3. A microbial inoculant according to claim 2, wherein the microbial inoculant further comprises agronomically beneficial strains of bacteria in addition to Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans,

4. A method for preventing cold stress, the method comprising inoculating soil or plants with a microbial inoculant, wherein the microbial inoculant includes Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans,

5. A method for preventing cold stress according to claim 4, herein the microbial inoculant is applied as a flower, cereals, vegetables and fruit coating, spraying

6. A method for preventing cold stress according to claim 4, wherein the microbial inoculant is sprayed directly to plant.

7. A method for preventing cold stress according to claim 4, wherein the microbial inoculant is applied to plants or soil as a mixture of microorganisms and planting materials.

8. A method for preventing cold stress according to claim 4, wherein the microbial inoculant further comprises agronomical beneficial strains of bacteria in addition to preventing cold stress.

9. A method for preventing cold stress according to claim 4, wherein the microbial inoculant is applied to plants or soil in an amount effective for increasing the sugar content of the soil and plant.

10. A method for preventing cold stress according to claim 4, wherein the microbial inoculant is applied to plants or soil in combination with a fertilizer.

1 1 . A method for growing Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans, the method comprising:

^* Inoculating a plant extract medium with Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans,

^* Culturing the inoculated plant extract at a time, temperature and aeration rate effective for providing a cell density of at least about 10⁸ to about 10⁹ cfu (colony form unit)/ml (milliliter).

12. A method for growing Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida,

Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans, according to claim 1 1 , wherein the plant extract is a water extract of plant and includes a salt of an organic acid , amino acid and a nitrogen source.

13. A biologically pure culture of Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans,

14. A microbial inoculant is according to claim 2, wherein the microbial inoculant further comprises agronomically beneficial enzymes, 9-cis-epoxycarotenoid dioxygenase (NCED), super oxide dismutase (SOD), dehidrogenese, (DHG), catalase (CAT) and ascorbate peroxidase (APEX) in addition to Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans

15. A microbial inoculant is according to claim 2, wherein the microbial inoculant further comprises agronomically beneficial Humic Acid in addition to Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans

16. A microbial inoculant is according to claim 2, wherein the microbial inoculant further comprises agronomically beneficial boric acid in addition to Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans.

17. A microbial inoculant is according to claim 2, wherein the microbial inoculant further comprises agronomically beneficial colemanite ore in addition to Azospirillum brasilense,

Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans

18. A microbial inoculant is according to claim 2, wherein the microbial inoculant further comprises agronomically beneficial Abscisic Acid (ABA), indole acetic acid (IAA), Gibberellin in addition Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans

19. A microbial inoculant is according to claim 2, wherein the microbial inoculant further comprises glycoproteins such as AFP1 (Anti Freeze Protein 1 ) , AFP2 (Anti Freeze Protein 2), AFP3 (Anti Freeze Protein 3) and AFP4 (Anti Freeze Protein 4) in addition to Azospirillum Brasilense, Bacillus Subtilis, Bacillus Megaterium, Lactocoecus spp.

20. A microbial inoculant is according to claim 2, wherein the microbial inoculant further comprises sugar alcohols such as mannitol and sorbitol in addition to Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans

21 . A microbial inoculant is according to claim 2, wherein the microbial inoculant further comprises Potassium Iodine (Kl) and Silver Nitrate (AgN0₃) in addition to Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans

22. A microbial inoculant is according to claim 2, wherein the microbial inoculant further comprises cry proteins such as putrescine, cadaverine and spermidine in addition to Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans

23. A microbial inoculant is according to claim 2, wherein the microbial inoculant further comprises hormones such as auxin, indole acetic acid (IAA), gibberellin in addition to Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans

24. A microbial inoculant is according to claim 2, wherein the microbial inoculant further comprises amino acid such asalanine - ala - A, arginine - arg - R, asparagine - asn - N, aspartic acid - asp - D, cysteine - cys - C, glutamine - gin - Q, glutamic acid - glu - E, glycine - gly - G, histidine - his - H isoleucine - ile - I, leucine - leu - L, lysine - lys - K, methionine - met - M, phenylalanine - phe - F, proline - pro - P, serine - ser - S, threonine - thr - T, tryptophan - trp - W tyrosine - tyr - Y, valine - val - V, and organic acid such as formic acid, acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, oxalic acid, lactic acid, malic acid ,citric acid, benzoic acid, carbonic acid in addition to Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans

25. A microbial inoculant is according to claim 2, wherein the microbial inoculant further comprises liquid , powder, and pellet forms are used for %100 solubilization in water with the bacterias as Azospirillum brasilense, Bacillus subtilis, Bacillus megaterium, Lactocoecus spp., Pseudomonas sp, Pseudomonas putita, Pseudomonas lurida, Acinetobacter, Paenibacillus sp, Burkholderia phytofirmans.