Compositions and Methods Comprising Endophytic Bacterium for Application to Grasses to Increase Plant Growth, Suppress Soil Borne Fungal Diseases, and Reduce Vigor of

Weedy Competitors

By James F. White, Jr.

Kurt P. Kowalski

Kathryn Kingsley

Field of the Invention

This invention relates to the fields of plant biology and endophytic bacteria. More specifically, the invention provides new strains of endophytic bacteria which provide beneficial features to the plant upon colonization of the same.

Background of the Invention

Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated by reference herein as though set forth in full.

Invasive Phragmites australis subsp. australis (Cav.) Trin., native to Eurasian wetlands, is an introduced grass in North America that displaces entire communities of native flora and fauna. This plant is highly competitive and generally out-competes other plants to produce large monospecific stands. It is increasingly becoming clear that microbes that form symbiotic associations with Phragmites play roles in increasing its invasive character (Clay et al., 2016). Soares et al. (2016a) recently demonstrated that endophytic bacteria isolated from tiller meristems were capable of increasing nitrogen assimilation into plants in greenhouse

experiments. In another study, Soares et al. (2016b) showed that fungal endophytes that enter into roots of Phragmites growing in saline soils may enhance salt tolerance in the host, enabling it to thrive in high saline soils. Ernst, Mendgen and Wirsel (2003) demonstrated that a seed- borne fungal endophyte in genus Stagonospora enhanced biomass accumulation in experiments in microcosms. It is thus evident that symbiotic microbes may impact the aggressiveness of Phragmites.

Summary of the Invention In accordance with the present invention, a method for increasing root and shoot growth of grass plant hosts is provided. An exemplary method comprises artificially inoculating the host grass with one or more biologically pure Pseudomonas spp.endophyte strains wherein said strains confer protection to the host grass. Preferably, said one or more Pseudomonas sp. is Sandy LB4 and West 9 used alone or in combination.

Surprisingly, the present inventors have discovered that the artificially inoculated host grass exhibits suppressed growth of soil borne fungal pathogens of said host grass relative to host grasses which have not been inoculated with said endophyte. Inoculated plants also exhibit increased resistance to disease and reduce competitiveness of distantly related competitor weeds of said grass plant host.

In a preferred embodiment, the host grass is a turf and forage grass selected from

Agrostis spp., Poa spp., Festuca spp., Lolium spp., Cynodon spp., Zoysia spp., Koleria spp., and Danthonia sp. The host grass can also be a food grass crop selected from Triticum aestivum, Oryza sativa, and Zea maydis. In a preferred embodiment, the host grass is a Pooideae grass.

Application methods include, without limitation, use of bacteria present in a seed ball or spraying the host plant with a liquid formulation comprising said bacteria.

In another aspect of the invention, a synthetic combination comprising at least one purified bacterial population in association with a seed of a turf and forage grass , wherein the purified bacterial population comprises a seed bacterial endophyte that is heterologous to the seed and selected from Sandy LB4 and or strain west 9, wherein the seed bacterial endophyte is present in the synthetic combination at a concentration of at least 10 ³ CFU/seed on the surface of a seed, and in an amount effective to increase root and shoot growth in a plant produced from the seed, as compared to a reference plant grown under the same conditions. In certain embodiments, the bacteria are lyophilized. Alternatively, they are encapsulated in an alginate bead

formulation. In yet another aspect, the invention provides a method of reducing undesirable weed growth in soils comprising turf and forage grasses , comprising drenching or treating the soil with one or more biologically pure Pseudomonas sp.endophyte strains wherein said strains reduce weed growth relative to weed growth observed in untreated soil, wherein said one or more Pseudomonas spp. is Sandy LB4 and/or West 9.

Brief Description of the Drawings

Figures 1A-1D. Fungus without bacteria (Fig. 1 A) Pseudomonas fluorescens Sandy LB4, (Fig. IB) Pseudomonas sp. West 9 (Fig. 1C) and Microbacterium oxydans B2 (Fig. ID) were co- cultured on LB A with soil fungi Sclerotinia homeocarpa, (the causal agent of Dollar spot disease inturfgrass) for 7 days. Formation of zones of inhibition are shown.

Figures 2A-2B. Fig. 2A. Cynodon dactylon (Bermuda grass) Seedlings growing on 0.7% agarose and stained with DAB for reactive oxygen. Fig. 2B. Bermuda grass (Cynodon dactylon) grass seedling root in agarose without bacteria showing absence of root hairs. Figures 3A-3B. Fig. 3 A. Bacterial strain West 9 colonizes the Bermuda grass seedling root-tip meristem and becomes intracellular in meristem cells then transmits to all parts of the seedling root and root hairs as the meristem cells divide. In these seedlings root hairs form very close to the root tip. West 9 restores root hair development in sterile seedlings. Root stained in agarose using DAB (diaminobenzidine) to visualize reactive oxygen (Η ₂ 0 ₂ ) production around bacteria. Fig. 3B. Root hairs (arrow) behind the root-tip are seen to stain brown internally due to production of reactive oxygen in the vicinity of the intracellular bacteria.

Figures 4A-4E. Fig. 4A. Early developing root hairs show internal presence of the spherical wall-less L-forms (arrows) of bacterial strain West 9. Roots were stained with DAB then counterstained with aniline blue stain. Fig.4B. Early developing root parenchyma cells also show chains of the spherical L-form bacteria. The root was stained with DAB then counterstained with aniline blue stain. Fig. 4C. Cells from the root meristem show internal presence of bacteria (arrows). Fig. 4D. The bacteria are degraded internally. They are seen to swell and become clear internally (arrows). Clusters of degraded bacteria are seen in these root parenchyma cells. Fig. 4E. More degrading bacteria (arrows) in root parenchyma cells. Bacterial L-forms swell as their internal contents vanish due to degradation.

Figures 5A-5B. Fig. 5 A. As the root continues to develop the bacteria within root hairs and root parenchyma cells are seen to disappear, apparently due to complete degradation by the root cells. Here several root hairs are seen to be free of internal bacteria (arrows). Bacteria that were intercellular are not degrade and remain as endophytes in the plant. Fig. 5B. In older parts of the root all root hairs are seen to be free of internal bacteria and reactive oxygen staining (brown coloration). In the parenchyma cells of the root axis the brown coloration also lessens due to degradation of the internal bacteria. Figures 6A- 6C. Fig.6A. Poa annua seedling root in agarose without bacteria showing absence of root hairs. Stained using DAB to visualize reactive oxygen. Fig. 6B. Poa annua seedling root bearing strain West 9, showing production of root hairs. Fig. 6C. Poa annua root hair showing internal bacteria (arrows).

Figures 7A-7B. Fig. 7A. Strain Sandy LB4 (also from Phragmites) is also a meristem colonizer. It is shown here in a Bermuda grass seedling root. Fig. 7B. The bacteria appear to persist longer in Bermuda grass seedling roots than strain West 9. It appears that the effect of Sandy LB4 in Bermuda grass seedlings is of longer duration than that of West 9.

Figures 8A-8C. Fig.8 A. Fig. 8 A. Rumex crispus (Curly dock). Fig.8B. Strain Sandy LB4 from Phragmites in root hair of Rumex crispus Root hair blockage may affect the absorptive function of root hairs. This may reduce the competitive ability of seedlings. Fig. 8C. Normal root hair function involves internal cytoplasmic streaming. Cytoplasmic streaming cessation in blocked hairs may reduce nutrient absorption.

Figures 9A-9B. Fig. 9A. Dandelion seedlings (3-wk-old) showing high mortality when colonized by Phragmites bacterium Sandy LB4. Fig. 9B. Dandelion seedlings from inhibition experiment in soil.

Detailed Description of the Invention

Phragmites australis is an invasive grass that decreases biodiversity and produces dense stands in North America. In the present invention, we tested the hypothesis that invasive Phragmites australis establishes mutualisms with endophytic microbes that increase its growth, improve its ability to survive stressful environments and resist pathogens, and contribute to its competitive capacity against other plant species. Ten bacterial strains were isolated from the surfaces of Phragmites seeds collected from natural stands at four sites in New Jersey. 80% of the isolates were identified to genus Pseudomonas. Using a surrogate test grass Cynodon dactylon in seedling assays we determined that two of the isolated strains Sandy LB4

{Pseudomonas fluorescens) and West 9 {Pseudomonas sp.) were endophytes that enter into plant meristems and become intracellular within cells of root tissues. These bacterial strains, along with an endophyte {Microbacterium oxydans B2) isolated from Phragmites shoot tissues, were found to increase plant growth using the surrogate grass host Poa annua. These strains were also found to slightly improve seed germination and increase seedling root branching in assays using Poa annua. Strains Sandy LB4 and West 9 were further shown to protect plants from damping off disease caused by fungal pathogen Fusarium oxysporum using Poa annua as the surrogate host plant in magenta box experiments. To evaluate whether Phragmites bacteria might play a role in inhibiting growth of competitor plant species we conducted experiments using dandelion {Taraxacum officionale) and curly dock (Rumex crispus). In these experiments we found that one of the endophytic strains, Sandy LB4, has the capacity to increase mortality in seedlings of both competitor species. Further, Sandy LB4 reduces the growth rates of both competitor plant species and increases their susceptibility to diseases. I. DEFINITIONS:

An "endophyte" or "endophytic microbe" is an organism that lives within a plant or is otherwise associated therewith. Endophytes can occupy the intracellular or intercellular spaces of plant tissue, including the leaves, stems, flowers, fruits, seeds, or roots. An endophyte can be either a bacterial or a fungal organism that can confer a beneficial property to a plant such as an increase in yield, biomass, resistance, or fitness in its host plant. As used herein, the term

"microbe" is sometimes used to describe an endophyte.

In some embodiments, a bacterial endophyte is a seed-origin bacterial endophyte. As used herein, a "seed-origin bacterial endophyte" refers to a population of bacteria associated with or derived from the seed of a grass plant. For example, a seed-origin bacterial endophyte can be found in mature, dry, undamaged (e.g., no cracks, visible fungal infection, or prematurely germinated) seeds. The bacteria can be associated with or derived from the surface of the seed; alternatively, or in addition, it can be associated with or derived from the interior seed compartment (e.g., of a surface-sterilized seed). In some cases, a seed-origin bacterial endophyte is capable of replicating within the plant tissue, for example, the interior of the seed. Also, in some cases, the seed-origin bacterial endophyte is capable of surviving desiccation.

Seed-origin means that the bacterial entity is obtained directly or indirectly from the seed surface or seed interior compartment or is obtainable from a seed surface or seed interior compartment. For example, a seed-origin bacterial entity can be obtained directly or indirectly from a seed surface or seed interior compartment when it is isolated, or isolated and purified, from a seed preparation; in some cases, the seed-origin bacterial entity which has been isolated, or isolated and purified, may be cultured under appropriate conditions to produce a purified bacterial population consisting essentially of a seed-origin bacterial endophyte. A seed-origin bacterial endophyte can be considered to be obtainable from a seed surface or seed interior compartment if the bacteria can be detected on or in, or isolated from, a seed surface or seed interior compartment of a plant.

The compositions provided herein are preferably stable. The seed-origin bacterial endophyte is optionally shelf stable, where at least 10% of the CFUs are viable after storage in desiccated form (i.e., moisture content of 30% or less) for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or greater than 10 weeks at 4° C. or at room temperature. Optionally, a shelf stable formulation is in a dry formulation, a powder formulation, or a lyophilized formulation. In some embodiments, the formulation is formulated to provide stability for the population of bacterial endophytes. In one embodiment, the formulation is substantially stable at temperatures between about 0° C. and about 50°C for at least about 1, 2, 3, 4, 5, or 6 days, or 1, 2, 3 or 4 weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months, or one or more years. In another embodiment, the formulation is substantially stable at temperatures between about 4°C. and about 37°C. for at least about 5, 10, 15, 20, 25, 30 or greater than 30 days.

An agricultural plant can be a monocotyledonous (i.e., an "agricultural grass plant") or a dicotyledonous plant typically used in agriculture. An agricultural grass plant includes, but is not limited to, maize (Zea mays), common wheat (Triticum aestivum), spelt (Triticum spelta), einkorn wheat (Triticum monococcum), emmer wheat (Triticum dicoccum), durum wheat (Triticum durum), Asian rice (Oryza sativa), African rice (Oryza glabaerreima), wild rice (Zizania aquatica, Zizania latifolia, Zizania palustris, Zizania texana), barley (Hordeum vulgare), Sorghum (Sorghum bicolor), Finger millet (Eleusine coracana), Proso millet (Panicum

miliaceum), Pearl millet (Pennisetum glaucum), Foxtail millet (Setaria italic), Oat (Avena sativa), Triticale (Triticosecale), rye (Secale cereal), Russian wild rye (Psathyrostachys juncea), bamboo (Bambuseae), grasses, including Agrostis spp., Poa spp., Festuca spp., Lolium spp., Cynodon spp., Zoysia spp., Koleria spp., Danthonia sp or sugarcane (e.g., Saccharum

arundinaceum, Saccharum barberi, Saccharum bengal ense, Saccharum edule, Saccharum munja, Saccharum officinarum, Saccharum procerum, Saccharum ravennae, Saccharum robustum, Saccharum sinense, or Saccharum spontaneum).

A "host plant" includes any plant, particularly an agricultural plant, which an endophytic microbe such as a seed-origin bacterial endophyte can colonize. As used herein, a microbe is said to "colonize" a plant or seed when it can be stably detected within the plant or seed over a period time, such as one or more days, weeks, months or years; in other words, a colonizing microbe is not transiently associated with the plant or seed. A preferred host plant is a cereal plant.

As used herein, a "reference agricultural plant" is an agricultural plant of the same species, strain, or cultivar to which a treatment, formulation, composition or endophyte preparation as described herein is not administered/contacted. Exemplary reference agricultural plants are described herein. A reference agricultural plant, therefore, is identical to the treated plant with the exception of the presence of the endophyte and can serve as a control for detecting the effects of the endophyte that is conferred to the plant.

"Biomass" means the total mass or weight (fresh or dry), at a given time, of a plant tissue, plant tissues, an entire plant, or population of plants. Biomass is usually given as weight per unit area. The term may also refer to all the plants or species in the community (community biomass).

A "bacterial network" means a plurality of endophyte entities (e.g., bacteria, fungi, or combinations thereof) co-localized in an environment, such as on or within a grass agricultural plant. Preferably, a bacterial network includes two or more types of endophyte entities that synergistically interact, such synergistic endophytic populations capable of providing a benefit to the agricultural seed, seedling, or plant derived thereby.

An "increased yield" can refer to any increase in biomass or seed or fruit weight, seed size, seed number per plant, seed number per unit area, bushels per acre, tons per acre, kilo per hectare, or carbohydrate yield. Typically, the particular characteristic is designated when referring to increased yield, e.g., increased grain yield or increased seed size.

A "transgenic plant" includes a plant or progeny plant of any subsequent generation derived therefrom, wherein the DNA of the plant or progeny thereof contains an exogenous DNA not naturally present in a non-transgenic plant of the same strain. The transgenic plant may additionally contain sequences that are native to the plant being transformed, but wherein the "exogenous" gene has been altered in order to alter the level or pattern of expression of the gene, for example, by use of one or more heterologous regulatory or other elements.

The terms "pathogen" and "pathogenic" in reference to a bacterium includes any such organism that is capable of causing or affecting a disease, disorder or condition of a host containing the organism.

As used herein, an "agricultural seed" is a seed used to grow a plant typically used in agriculture (an "agricultural plant"). The seed may be of a monocot or dicot plant, and may be planted for the production of an agricultural product, for example grain, food, fiber, etc. As used herein, an agricultural seed is a seed that is prepared for planting, for example, in farms for growing.

In some cases, the present invention contemplates the use of microbes that are

"compatible" with agricultural chemicals, for example, a fungicide, an anti -bacterial compound, or any other agent widely used in agricultural which has the effect of killing or otherwise interfering with optimal growth of microbes. As used herein, a microbe is "compatible" with an agricultural chemical when the microbe is modified, such as by genetic modification, e.g., contains a transgene that confers resistance to an herbicide, or is adapted to grow in, or otherwise survive, the concentration of the agricultural chemical used in agriculture. For example, a microbe disposed on the surface of a seed is compatible with the fungicide metalaxyl if it is able to survive the concentrations that are applied on the seed surface.

In some embodiments, an agriculturally compatible carrier can be used to formulate an agricultural formulation or other composition that includes a purified bacterial preparation. As used herein an "agriculturally compatible carrier" refers to any material, other than water, which can be added to a seed or a seedling without causing or having an adverse effect on the seed (e.g., reducing seed germination) or the plant that grows from the seed, or the like. As used herein, a "portion" of a plant refers to any part of the plant, and can include distinct tissues and/or organs, and is used interchangeably with the term "tissue" throughout.

A "population" of plants, as used herein, can refer to a plurality of plants that were subjected to the same inoculation methods described herein, or a plurality of plants that are progeny of a plant or group of plants that were subjected to the inoculation methods. In addition, a population of plants can be a group of plants that are grown from coated seeds. The plants within a population will typically be of the same species, and will also typically share a common genetic derivation.

A "reference environment" refers to the environment, treatment or condition of the plant in which a measurement is made. For example, production of a compound in a plant associated with a purified bacterial population (e.g., a seed-origin bacterial endophyte) can be measured in a reference environment of drought stress, and compared with the levels of the compound in a reference agricultural plant under the same conditions of drought stress. Alternatively, the levels of a compound in plant associated with a purified bacterial population (e.g., a seed-origin bacterial endophyte) and reference agricultural plant can be measured under identical conditions of no stress.

As used herein, a "colony-forming unit" ("CFU") is used as a measure of viable microorganisms in a sample. A CFU is an individual viable cell capable of forming on a solid medium a visible colony whose individual cells are derived by cell division from one parental cell.

II. Methods of the Invention

The invention relates to a process and method for the production and use of endophytes as plant inoculants products that provide unique inoculant feature/benefits for the promotion of plant vigor, health, growth and yield comprising bacteria isolated from Phragmites, e.g., Strain Sandy LB4 and strain West 9. Each of these new strains of bacteria have been deposited with the ATCC under the terms of the Budapest Treaty, under accession numbers and respectively. The invention also relates to a process and method for producing economically acceptable quantities of preparations of the aforementioned bacteria. The invention further relates to an endophyte product(s) produced by such processes and methods. The endophyte product(s) may comprise a solid substrate of, for example, certain cereals e.g. rye, which contain sufficient natural emulsifiers in the form of various proteins, lignans, to provide the bacteria with excellent natural dispersing/wetting/sticker agents that allows for rapid site occupation on/in plants. The product formulation allows/enables practical use and application of the product(s) to roots, stems, leaves, flowers, bulbs, etc of plants as a water-based sprayable formulations or as a dusts for other uses e.g. seed treatment, or as dusts for insect/mite vectors. The product when applied to seeds, roots, stems, leaves, flowers, wounds or cut surfaces of plants enables the endophyte to act as an inoculant within the tissues of plants. The product provides improved roots, leaf, stem and or vegetative bud (flowers) growth to plants and or enhances/improves the germination and emergence of seeds and also causes mortality in neighboring competitor species. The product provides for reduction of environmental or cultural stress to plants e.g. root loss due to trimming, pruning, cutting or other stresses. The product provides improved crop quality and faster development to marketability of the crop. The product provides for a reduction in the dependency on chemical pesticides for pest control e.g. control of Botrytis, Fusarium, Pythium, spp. and the like. The product can be used for the production of a variety of

greenhouse, horticultural and agronomic field crops. The composition of the invention can be a plant inoculant composition comprising the bacteria described above in admixture with an agrochemically acceptable diluent or carrier. The invention also relates to a method of enhancing growth, health vigor or yield of a plant which method comprises applying the plant inoculant composition of the invention to a plant or plant locus. The invention further relates to a method of combating a plant fungus which method comprises applying an antifungally effective amount of the composition.

Lyophilization Procedure

Freeze drying bacteria (lyophilization) is a very well established method for the archiving and long-term storage. Initial reports of freeze drying bacteria can be found in the middle of last century. The approaches used vary widely, but they all following the standard process associated with lyophilization, namely the freezing of the sample, application of a high vacuum, warming of the sample while under vacuum which causes water sublimation, driving off excess water through a drying phase, and finally sealing of the sample to prevent water uptake. This general process is used to preserve bacteria, fungi, yeasts, proteins, nucleic acids, and any other molecules which may be degraded due to the presence of water. Thus in one aspect of the invention, one or more of the endophytic bacteria will be applied to a plant or a plant part (such as seeds) as a lyophilized (freeze-dried) powder. In brief, the liquid culture will be: centrifuged, resuspended in a lyophilization medium which will optionally include cryoprotectants and biological- and/or chemical-oxygen scavengers, transferred to a shelf lyophilizer, lyophilized, and packaged for transport and storage.

In an alternative approach, one or more bacteria may be encapsulated in alginate beads enriched with humic acid as described by Young CC et al., Biotechnol Bioeng. 2006 Sep

5;95(l):76-83. Also see "Alginate beads as a storage, delivery and containment system for genetically modified PCB degrader and PCB biosensor derivatives of Pseudomonas fluorescens F113 B" by Power et al., Journal of Applied Microbiology 110, 1351-1358, 2011.

Other approaches include coating seeds with preparations comprising the endophytic bacteria of the invention. For example, Sandy LB4 can be incorporated into a carrier, which include without limitation, alginate (micro-bead formation), chitosan, carboxymethylcellulose-starch, clay, finely-ground peat mixed with calcium carbonate, methacrylic acid, bio-char and biogels. In certain embodiments, carrier and Sandy LB4 may be mixed with additives (including: adhesives, nutrients, surfactants and stabilizers).

The carrier and Sandy LB4 (with additives) can be applied to seeds of grass crops using a commercial seed dressing machine (e.g., MAYJOY High Speed Seeds Dressing Machine/Cora Seed Dresser).

Sandy LB4 may also be used as an additive to create seed balls. In this approach,clay mixed with freeze-dried preparation of Sandy LB4 and seeds (2-3) are added to the center of a small clay ball. The seed balls are then dried and stored for future use.

Sandy LB4 can also be applied to host plants in soil drenching aooroaches. For example, frreeze dried preparation of Sandy LB4 can be mixed with a liquid carrier (comprising water, buffers, plant nutrients, and microbial nutrients,). This liquid preparation of bacterium and carrier (with additives) may be applied to the soil around plant or seed or in the alternative be applied to soil and plants using a commercial sprayer.

The following materials and methods are provided to facilitate the practice of the present invention. Seed collection

Seeds of P. australis were collected in September of 2015 at four sites in New Jersey, including Sandy Hook National Park and towns of South River, West Windsor and Robbinsville.

Seed preparation Seeds were cleaned of associated awns, lemmas and paleas by rubbing seeds between a fine mesh screen and wooden block. Seeds were washed with agitation in sterile water then the water was discarded. Seeds were then washed a second time in 1-mL of sterile water after which the water was spread over the surface of agar plates containing trypticase soy agar (TSA), Luria Bertani Agar (LB A) and Yeast Extract Sucrose Agar (YESA) and plates incubated at room temperature for a 24-48 hr period. Colonies were removed and streaked to obtain pure cultures. A total of 12 isolates were obtained from P. australis seeds using this method.

Bacterial identification and characterization

Genomic DNA from bacteria was isolated using QIAGEN miniprep kits. Bacterial identifications were made by use of 16S rDNA sequences after methods employed by Lane (1991) (Tablel). For Sandy LB4 we used the recA gene to identify the bacterium to species.

Sequences were compared to GenBank accessions using BLASTn

Cultures on LB agar were also observed under ultraviolet light using an ultraviolet mineralogical lamp (UVGL-21, long wave UV-366nm, UVP, San Gabriel, California, USA). Phosphate solubilization was assessed by culturing bacteria on Pikovskayas agar, while protease testing was done by culturing in skim milk agar.

Endophytism determination

Two of the Phragmites seed bacteria (Pseudomonas fluorescens Sandy LB4 and

Pseudomonas sp. West 9) were selected along with control bacteria Microbacterium oxydans B2 (from Phragmites shoots) and Bosea thiooxidans TBN (from Japanese knotweed). These bacteria were screened to determine their capacity to become endophytic in grass roots. Bermuda grass (Cynodon dactylon) was used to evaluate endophytism because seeds could be surface sterilized and seedlings would readily grow in sterile agarose medium. Seeds without husks were surface disinfected by agitation in 4% NaOCl solution for 45 mins. Seeds were washed thoroughly by agitation in several washes of sterile water until chlorine odor could not be detected on seeds. Seeds were then placed onto 0.7% agarose in Petri dished. To inoculate seeds one drop of a water suspension of bacteria (at approximately 0.5 OD, 600nm) was placed onto each seed. For each treatment each plate contained 10-15 seeds, with 5 replicates per treatment. An axenic control was also done to confirm behavior of seedlings without bacteria. Plates were incubated at room temperature for 7-10 days.

Visualization of bacteria in/on seedling roots

To visualize bacteria in/on seedling roots, plates bearing seedlings were flooded and stained with a solution of 3, 3'-diaminobenzidine (DAB) using SIGMAFASTD D3, 3'- diaminobenzidine tablets from Sigma (White et al., 2014) and incubated at laboratory ambient temperature overnight for staining. Plates were then washed of excess DAB and the seedling roots examined through the reverse of the Petri plate using a compound light microscope.

Intracellular penetration of root cells was indicated by dark red or brown staining within root hairs and/or root parenchyma cells. Assessment of capacity for plant growth promotion

To assess plant growth promotion, Pseudomonas fluorescens Sandy LB4, Pseudomonas sp. West 9, Microbacterium oxydans B2 and Bosea thiooxidans TBN were inoculated onto previously surface disinfected (40 mins agitation in 4% NaOCl) seeds of Poa annua by soaking seeds in a suspension of bacteria (at approximately 0.5 OD, 600nm). Controls were not inoculated with bacteria. Seeds were planted in sterile soil in magenta boxes with approximately 15 plants/treatment and placed under florescent lights in the laboratory for 37 days. Plants were harvested by removal from soil and roots wiped of excess soil. Shoot and root lengths were then measured and recorded. Growth promotion experiments were also conducted using rice (cultivar Rex). In these experiments rice seeds were surface disinfected for 1 hour in 4% NaOCl solution, then washed in sterile water. To inoculate seeds, surface disinfected seeds were soaked for 1 hour with continuous agitation in a suspension of Pseudomonas fluorescens Sandy LB4 at approximately 0.5 OD, 600nm. Controls were surface disinfected seeds soaked in sterile water with agitation for 1 hour. 25 seeds for each treatment were planted in sterile soil in magenta boxes and incubated for 7 days under fluorescent lights at laboratory ambient temperature.

Seedlings were removed from soil and root and shoot lengths determined.

Seed germination and seedling root architecture

Experiments were conducted to evaluate Pseudomonas fluorescens Sandy LB4 effects of seed germination and early seedling root architecture. In these experiments seeds of Poa annua were surface disinfected for 45 mins as above then plated onto agarose media with without nutrients (Murashige and Skoogs basal medium) then monitored daily for 7 days for the parameters: percent germination, root development into agarose, root branching, and root and shoot lengths. Disease protection (co-culture experiments)

To assess potential for disease protection bacteria Pseudomonas fluorescens Sandy LB4, Pseudomonas sp. West 9 and Microbacterium oxydans B2 were co-cultured on LBA with soil fungi Sclerotinia homeocarpa or Fusarium oxysporum for 7 days. Formation of zones of inhibition were noted. Damping off disease control experiment

To evaluate whether the Phragmites bacteria could protect seedlings from disease an experiment was set up using the damping off pathogen Fusarium oxysporum. In this experiment surface disinfected seeds of Poa annua were inoculated by soaking in a suspension (at approximately 0.5 OD, 600nm) of strains Sandy LB4, West 9 or Microbacterium oxydans B2, then planted in magenta boxes containing previously sterilized soil that had been mixed with 10- mL of a conidial suspension of Fusarium oxysporum. Controls included treatment without bacterial or fungal treatments, and with fungus but without bacteria. Each treatment included 25 replicates. After 8 days and 15 days seedlings evident on the surface of the soil were counted (Table X). Also presence of fungal mycelium on the surface of soil was determined. To test for movement of the bacteria into soil a sterile probe was placed into soil between plants in each replicate, then the probe was streaked on a fresh Petri plant containing LB agar and incubated for 24 hours prior to examination. Effects of Phragmites bacteria on growth of competitor plants

Dandelion (Taraxacum officionalis) was used to screen Phragmites microbes for any antagonism leading to increased mortality in the dandelion seedlings. Here dandelion seeds were rubbed against a nylon screen to remove awns and associated fibers. Seeds were then surface disinfected for 30 mins in 0.4% NaOCl, then rinsed in five changes of sterile water to remove residual chlorine. Seeds were used in a series of experiments.

Competitor inhibition experiment 1 (Phragmites mixture 1)

Eleven seeds were placed onto the surface of agarose medium (0.7%) in Petri dishes. One drop of bacterial mixture 1 (including equal parts of Microbacterium oxydans B2, Psudomonas fluorescens Sandy LB4, and Pseudomonas sp. West 9 at approximately OD 0.5, 600nm) was applied to the surface of each seed (with 9 replica plates). Controls were moistened with sterile water only. Plates were then incubated at approximately 21°C for two weeks in alternating light/dark conditions (10-hours light/ 14-hours dark). Seedlings were then examined to determine whether they were alive (light green) or dead (brown). Competitor inhibition experiment 2 (Phragmites mixture 1 and 2 in soil)

In a second experiment dandelion seeds were cleaned of external fibers, surface disinfected as above and placed onto sterile soil in magenta boxes (10 seeds/treatment).

Treatments included bacterial mixture 1 (as indicated above) and bacterial mixture 2 (including strains: Pseudomonas sp. WY6, Pseudomonas sp. Sandy LB6-m, Pseudomonas sp. Sandy Y8, Pseudomonas sp. RoLB13w, Pantoea sp. Sandy Y9w). A control was not inoculated with Phragmites bacteria. To inoculate seeds with bacteria 2 mL of a bacterial suspension at approximately 0.5 OD, 600nm was mixed into soil in magenta boxes. Seeds of Poa annua were also pre-moistened with bacterial suspension or water (control) then placed into soil and boxes incubated for four weeks under alternating light/dark conditions (10-hours light/ 14-hours dark) and 21C. After four weeks seedlings were removed from soil, weighed, and root and shoot lengths determined (Table 4). Competitor inhibition experiment 3 (examination of individual bacterial strains)

A preliminary screen was conducted using individual strains of the bacteria included in mixture 1 (as indicated above). In this preliminary individual screen 14 disinfected dandelion seeds were placed on agarose media in Petri dishes. Seeds were inoculated as above with each bacterium, and control seeds were not inoculated. Three replica plates were made for each treatment and control. Plates were then incubated at approximately 21C for two weeks in alternating light/dark conditions (10-hours light/ 14-hours dark), after which mortality was assessed in each plate.

Competitor inhibition experiment 4 (P. fluorescens Sandy LB4) A larger experiment was conducted using strain Sandy LB4. In this experiment dandelion seeds were cleaned and surface disinfected as above, then placed on 0.7% agarose in 38 Petri dishes (15 seeds/dish). Seeds on nineteen of the plates were inoculated using a suspension of Sandy LB4 as above, and the other nineteen were not inoculated. Plates were incubated for two weeks under the conditions of the previous experiment, after which seedling mortality was assessed.

Competitor inhibition experiment 5 (effects on seedlings of curly dock (Rumex crispus))

Seeds of locally collected curly dock were cleaned of bracts and associated wings, then surface disinfected for 40 minutes in 4% NaOCl. Ten seeds were then placed onto each of five Petri plates containing 0.7% agarose. Each seed was inoculated with a drop of bacterial suspension (strain Sandy LB4). Five control replica plates were prepared where seeds were not inoculated. Plates were incubated at approximately 21C for one week in alternating light/dark conditions (10-hours light/14-hours dark) after which seedlings were stained using DAB and examined microscopically.

Competitor inhibition experiment 6 (curly dock in soil) A test was conducted in which 2 mL of individual bacteria (Sandy LB4, West 9, M. oxydans B2, or West 6-m) was mixed into previously sterilized soil in magenta boxes, then ten surface dis-infected seeds of curly dock were placed into the soil 2-3 mm beneath surface. A control treatment was not inoculated with bacteria. After two weeks, the number of seedlings emerging from the soil surface was determined.

The following example is provided to illustrate certain embodiments of the invention. It is not intended to limit the invention in any way. Example I

Host promotional properties of endophytic Pseudomonas spp. from seeds of invasive reed grass {Phragmites australis)

This invention involves application of a strain (Sandy LB4) of endophytic bacteria (belonging to genus Pseudomonas) to increase growth of turf and forage grasses (Agrostis spp., Poa spp., Festuca spp., Lolium spp., Cynodon spp., Zoysia spp., Koleria spp., Danthonia sp.) and food grass crops (Triticum aestivum, Oryza sativa, Zea maydis), and suppress soil borne fungal diseases (e.g., damping off disease), and reduce vigor of seedlings of distantly-related weeds includingTaraxacum officionale (dandelion) and curley dock (Rumex crispus). Sandy LB4 was obtained from seeds of the invasive grass Phragmites australis. Sandy LB4 colonizes root meristems and enters into the cells of roots. This endophyte is unique in entering cells of the plant where it elicits production of reactive oxygen. Elicitation of reactive oxygen likely increases the resistance of the grass host to oxidative stress by stimulating the plant to up- regulate oxidative stress resistance genes. Sandy LB4 increases the growth of grass seedlings resulting in seedlings with larger root systems and larger shoots compared to seedlings without the bacterium. Sandy LB4 also moves from the plant roots out into the soil and colonizes mycelium of soil fungi suppressing growth of fungal pathogens resulting in reduced seedling disease. Sandy LB4 colonizes seedlings of non-grass plants (dandelions and curly dock) and increases mortality of seedlings in laboratory experiments.

Application of the endophytic microbes described herein to grass crops (e.g., turfgrasses, wheat, rice, sorghum, etc.) improves crop growth and reduces soil borne diseases and weeds. Sandy LB4 may be used in soil additives or seed treatments to grow crops with reduced agrochemical inputs. Widespread application of Sandy LB4 commercially will enable decreased use of fertilizers, fungicides and herbicides on grass crops, resulting in reduced cost for expensive agrochemicals and reduced contamination of soil and water. Isolates

Ten bacterial isolates was obtained from the surfaces of Phragmites seeds. Of these, 8 were shown to belong in genus Pseudomonas based on 16S rDNA sequence data (Table 1). Two strains examined closely Pseudomonas sp. West 9 and Pseudomonas fluorescens Sandy LB4 were shown to become endophytic in the Bermuda grass seedlings (Fig. 1 and Fig. 2). These bacteria colonized root tip meristems and became intracellular in root cells, inducing formation of root hairs on seedling roots.

Table 1. Bacteria isolated from Phragmites australis seed surfaces

Collection Site Strain ID UV P ¹ Prot. ² Genus and Species

Sandy Hook Sandy LB 4 BF* Y Y P. fluorescens

Sandy Hook Sandy LB 6 LF Y — Pseudomonas sp.

Sandy Hook Sandy Y8 LF Y N Pseudomonas sp.

Robbinsville RoY12 F Y N Pantoea sp.

Robbinsville RoLB13w LF Y — Pseudomonas sp.

West Windsor WY9y NF — — Enterobacter sp.

West Windsor WY6 LF Y — Pseudomonas sp.

West Windsor West 9 LF Y N Pseudomonas sp.

West Windsor WY14 NF Y — Pseudomonas sp.

South River RiY3 NF Y ~ Pseudomonas sp.

*BF = bright fluorescence; Lf = light fluorescence; NF = no fluorescence observed.

Phosphorus solubilization, Y = yes;— = missing data.

² Protease production based on milk agar clearing; Y = yes; N = no;— = missing data.

Modes of colonizing Bermuda grass seedling roots

Strains Sandy LB4 and West 9 were both shown to enter root cells at the root meristem. Squash preparations of root tips showed presence of bacteria within meristematic cells. See Fig. 3A- 3B. In older root cells the spherical bacteria were seen to stain brown (positive indication of reactive oxygen) using the reactive oxygen stain DAB. Spherical bacterial L-forms were evident in root hairs and parenchyma cells (Fig. 4A -Fig. 4E) that comprised the main axis of the root. In older parts of the root the L-forms were seen to swell and the contents lacked protein as evident by failure to stain blue using the aniline blue counterstain. (Fig. 5). In still older root parts, including root hairs and parenchyma, the internal bacterial inclusions were no longer evident. Seedlings derived from seeds that had not been inoculated with either Sandy LB4 or West 9 did not show presence of bacterial L-forms within root cells.

Plant growth promotion capacity

Pseudomonas fluorescens Sandy LB4, Pseudomonas sp. West 9 and Microbacterium oxydans B2 were found to increase growth of Poa annua in the magenta box experiment. Roots and shoots of seedlings bearing these three bacteria were shown to be significantly longer than those without bacteria or inoculated with Bosea thiooxidans TBN (from Japanese knotweed; family Polygonaceae; Table 2). In the experiment where Sandy LB4 was applied to rice seeds, seedling shoot growth was increased 46% over seedlings without bacteria (Table 3). Although root lengths were not significantly different between inoculated and non-inoculated controls, roots of inoculated seedlings showed more adherent soil particles compared to non-inoculated seedling roots.

Table 2. Growth promotion of Poa annua in soil for 37 days (15 plants in each treatment) Treatment Shoot length (mm) ^* Root length (mm)

No bacteria 88.87 ± 23.53 ^a 17.27 ± 8.59 ^a

TBN 86.2 ± 32.35 ^a 13.73 ± 5.01 ^a

Sandy LB4 122.33 ± 27.38 23.8 ± 8.54

West 9 119.33 ± 20.43 27.33 ± 14.56

Microbacterium oxydans B2 113.73 ± 25.41 20.87 ± 6.79 ^a '

* Data is given as mean ± standard deviation. Means followed by the same letter within a column are not significantly different according to the Duncan multiple range test (p < 0.05).

Table 3. Rice seedling development with and without Pseudomonas fluorescens Sandy LB4 after 7-days-growth

Treatment Shoot length ± sd N samples Root length ± sd N samples

Sandy LB 4 52.957 ± 24.479* 47 58.596 ± 20.581 47

No bacteria 36.182 ± 25.971 44 52.045 ± 17.688 44

*Data given as means ± standard deviation. Shoot lengths are statistically different according to the student t test (p < 0.05). Root lengths were not statistically different. Seed germination rate and root architecture

In the germination and root growth study where we used axenic Poa annua to determine bacterial effects on seeds and seedlings, we found that the germination rate was only slightly increased in seeds bearing Sandy LB4. However, there was a slightly greater tendency for roots bearing the bacterium to branch compared to roots without the bacterium (Fig. 6). In the agarose medium roots bearing the bacterium were also seen to more frequently grow downward into the agarose medium than roots without the bacterium. It is notable that in seedlings grown on agarose containing Murashige and Skoogs nutrient base, the majority of the roots tended to grow downward into the medium (Table 4).

Table 4. Poa annua germination and growth with Phragmites bacterium Sandy LB4 over a 1-week period.

Production of fungal inhibitors

Inhibition zones were evident in co-culture experiments, where West 9 and Sandy LB4 were cultured with Sclerotinia homeocarpa and Fusarium oxysporum and seen to inhibit them in culture. Microbacterium oxydans B2 did not produce inhibitory substances in culture.

Damping off disease protection

In the experiment evaluating capacity of Phragmites bacteria to protect P. annua seedlings from damping off caused by Fusarium oxysporum, we showed that the least damping off was seen in the pathogen and bacterial free treatment with 23 seedlings surviving after 15 days. After 15 days Sandy LB4 showed the highest level of protection with 20 seedlings surviving; followed by West 9 with 16 seedlings surviving, Microbacterium oxydans B2 with 15 seedlings surviving, and the pathogen + no bacterium treatment with 12 seedlings surviving.

Effects of Phragmites bacteria on growth of seedlings of competitor species

In competitor inhibition experiment 1 we showed that a mixture of Phragmites bacteria (including Microbacterium oxydans B2, Pseudomonas sp. West 9 and Pseudomonas fluorescens Sandy LB4) increased mortality in dandelion seedlings compared to the non-inoculated control with the difference being statistically significant at the 5% level of significance in the Student's t-test. The bacterial mixture gave an 83.4 ± 15.7% mortality compared to the axenic control at 16.4 ± 22.8% mortality.

In competitor inhibition experiment 2 conducted in soil in magenta boxes we found that inoculation of soil with bacterial mixture 1 (including Sandy LB1, West 9 and Microbacterium oxydans B2) resulted seedlings that were significantly smaller in terms of weight, and shoot and root lengths than those of non-inoculated seedlings or those grown in soil containing Phragmites bacterial mixture 2 containing five other isolates (Table 5).

Table 5. Dandelion seedling growth inhibition in soil by Phragmites bacterial mixtures

Treatment Seedling mass Shoot length Root length

Wet weight (mg) (mm) (mm)

No bacteria 27.03 ± 9.54 ^*a 42.44 ± 12.6 ^a 40 ± 17.11 ^a

Mix 1 14.91 ± 8.71 23.88 ± 11.86 14.63 ± 5.21

Mix 2 26.36 ± 6.78 ^a 40.89 ± 10.71 ^a 43.33 ± 19.55 ^a *Data given as means ± standard deviation. Within columns means followed by the same letter are not statistically different according to the Duncan multiple range test (p < 0.05).

Identification of strains showing dandelion seedling inhibition

In competitor inhibition experiment 3 Pseudomonas fluorescens Sandy LB4,

Pseudomonas sp. West 9 and Microbacterium oxydans B2 were tested for their effect on mortality of dandelion seedlings on agarose (Fig. 9). In this test the most potent bacterium for increasing mortality of dandelion seedlings was Sandy LB4 (71%) > Microbacterium oxydans B2 (35%) > West 9 (31%) > no bacterium control (23%). On the basis of this test we concluded that Sandy LB4 was the most potent of the bacteria increasing mortality in dandelion seedlings and a larger study (Competitor inhibition experiment 4) was set up to gather statistical data for effect of Sandy LB4 on dandelion seedling mortality. In the larger study a statistically significant (p < 0.05 in student's t-test) increased mortality of 71.1 ± 15.3% was shown for Sandy LB4- inoculated seedlings compared to only 6.8 ± 7.4% for the non-inoculated control treatment. Effects of Pseudomonas fluorescein Sandy LB4 on curly dock (Rumex crispus) seedlings

In competitor inhibition experiment 5 we evaluated the colonization of seedlings of Rumex crispus by Sandy LB4. Here we found that Sandy LB4 actively colonized seedlings, inducing root hair development in the inoculated seedlings. Staining the seedlings using the reactive oxygen stain DAB and microscopic examination of roots showed that root hairs appeared to be occluded by large dark clusters of swollen degrading L-form bacteria (Fig. 8). In competitor inhibition test 6 in soil we showed that curly dock seedling emergence was: no bacterium treatment (7 seedlings) > West 9 (3 seedlings) > Microbacterium oxydans B2 (2 seedlings) > Sandy LB4 (0 seedlings).

Discussion Endophytic nature of seed-associated bacteria

While our examination was not extensive for all of the bacteria we isolated from

Phragmites seeds, we did closely examine Pseudomonas fluorescens Sandy LB4 and

Pseudomonas sp. West 9. In our in vitro assay using axenic Bermuda grass seedlings we showed that on germination of seeds both bacteria colonize the root meristem and enter into meristematic cells. In the process of becoming intracellular the bacteria lose their cell walls and their rod shapes to become spherical L-form bacteria. As the root meristem proliferates the intracellular bacteria become distributed in most or all cells that make up the root. Intracellular bacteria are visible early on in young differentiating root cells as small spherical aniline blue staining structures. The use of DAB to stain for Η ₂ 0 ₂ shows that the intracellular bacteria are generally surrounded by H ₂ 0 ₂ evident as red or brown staining. H ₂ 0 ₂ is likely produced by the host plant as a mechanism to control the intracellular bacterial endophytes. As tissues of the root, including root hairs become older the intracellular bacteria swell and their cytoplasmic contents disappear. Presence of H ₂ 0 ₂ around intracellular bacteria suggests that the disappearance of bacteria is due to their degradation by the host cells through oxidation. In more mature root tissues all evidence of the intracellular bacteria completely disappears from plant cells. Bacterial cells that do not enter the root tip cells do not appear to elicit a strong reactive oxygen response and may not be degraded; instead they likely remain as intercellular endophytes in the mature root tissues. While we did not examine shoot meristems for the presence of intracellular bacteria they could be present there as well since they somehow become deposited on the seeds.

Degradation of endophytic microbes as a nutritional source

Degradation of microbes in or on roots was proposed to be a mechanism of transfer of nutrients from microbes to plant (Paungfoo-Lonhienne et al., 2010; White et al., 2012; Beltran et al., 2015). Paungfoo-Lonhienne et al. (2014) denominated the microbe consumption process 'rhizophagy' because roots consumed microbes. For a mechanism like rhizophagy to provide substantial nutrients to plants the microbes must obtain their nutrients from outside roots, then enter roots and release their nutrients to the plant tissues. We have experimental evidence that in the case of the Pseudomonas endophytes of Phragmites the bacteria move from plant out into the soil and perhaps may return to recolonize the plant root meristem. If colonization of roots by the soil bacteria is occurring with any regularity a constant flow of nutrients from soil reserves to the plant may be occurring. In previous research we hypothesized that microbes may aid plants by scavenging organic or inorganic nitrogen (White et al., 2015; Soares et al., 2016a). However, all of the bacteria we isolated from Phragmites seeds showed the capacity to solubilize phosphate when grown on Pikovskayas agar. Further, Pseudomonas fluorescens and other fluorescent pseudomonads are known to secrete fluorescent siderophores called pyoverdines that actively bind iron (Visca, Imperi and Lamont, 2006). We confirmed that Sandy LB4 secreted a fluorescent pigment that fluoresced brightly in culture and that several other strains from

Phragmites seeds (Table 1) fluoresced lightly in culture when viewed under an ultraviolet light, suggesting that several of our isolates possess the siderophores. Sandy LB4 was also shown to produce a secreted protease (Table 1) that might enable it to degrade microbial proteins present around roots and thus acquire organic nitrogen (White et al., 2015). The capacity of the motile pseudomonads to actively move out into the soil and obtain iron and solubilize and absorb phosphorus and nitrogenous nutrients could enable the pseudomonads to provide critical nutrients to Phragmites to fuel its rapid growth. The endophytic pseudomonads could thus function as nutrient scavengers for their Phragmites seedlings (Zolg and Ottow, 1975). Whether pseudomonads are responsible for significant nutrients obtained by Phragmites will require additional experimentation to evaluate.

Intracellular endophytes as elicitors of reactive oxygen

Bacterial endophytes that internally colonize root meristems and increase occurrence of reactive oxygen in the young differentiating tissues of the root may trigger the host to express oxidative stress resistance features to make the host more resistant to the internal oxidations. In a study of fungal endophytes in grasses it has been shown that endophytes increase the oxidative stress resistance in the host (White and Torres, 2009; Hamilton and Bauerle, 2012; Hamilton et al., 2012). Growth promotion

Using Poa annua to test for growth promotion capacity of three Phragmites endophytes (P. fiuorescens Sandy LB4, Pseudomonas sp. West 9 and Microbacterium oxydans B2) and another bacterium, Bosea thiooxidans TBN from the distant family Polygonaceae, we found that the 3 Phragmites isolates increased growth of grass compared to the non-inoculated treatment (Table 2); while the bacterium Bosea thiooxidans TBN partially colonized grass seedlings and triggered root hair development but did not increase grass shoot or root growth in the soil experiment. We interpret that the Phragmites endophytes are compatible with the test grasses we used, but Bosea thioxidans TBN is only partially compatible with the grasses, incompletely colonizing root hairs and failing to increase growth of the grass host in experiments. In an experiment where we inoculated rice seeds with Sandy LB4, seedlings bearing the bacterium showed significantly more shoot growth compared to seedlings without the bacteria. Although roots were slightly longer in bacterial treated seedlings, they were not statistically different. It was notable that roots bearing bacteria showed greater adherence of soil particles to roots after extraction from the soil. This may be an indication that the presence of the bacterium may affect the physical relationship of the plant root to the soil matrix around roots. The bacterium may be increasing extraction of nutrients from soil, which could account for the increased growth of seedlings when the bacterium is present. Effects on seedling development

The effects of Sandy LB4 on Poa annua seedling growth were seen very early in development, including a very slightly increased germination rate and greater root branching (Table 4).

Suppression of soil borne disease

In co-culture experiments we demonstrated that Sandy LB4 and West 9 produced unknown antifungal substances that inhibited Sclerotinia homeocarpa and Fusarium oxysporum, while M. oxydans B2 did not appear to produce antifungal substances. In an experiment to assess protection from damping off disease caused by Fusarium oxysporum (Table 4) we showed that Sandy LB4, West 9 and M. oxydans B2 reduced damping off in seedlings when compared to controls inoculate with the pathogen; however Sandy LB4 was the most effective at reducing damping off. Microbacterium oxydans B2 was the least effective at reducing damping off disease.

In this same experiment we showed that the two pseudomonads originally inoculated onto seeds moved into the soil for some distance away from seedlings, suppressing mycelial growth in and on the surface of the soil. While M. oxydans B2 also moved out into the soil, it did not have any mycelial growth suppression effects and fungi grew readily over the surface of the soil in these treatments.

Some other Pseudomonas spp. that have been shown to be effective in biological control of diseases have been found to produce antifungal metabolites, including pyrrolnitrin, phenazines, pyoluteorin, hydrogen cyanide, etc... (Ligon et al., 2000). Whether any of our Phragmites pseumonads produce any of these antifungal metabolites has yet to be determined.

Endophyte inhibition of competitor species

Through a series of experiments using Phragmites bacteria to inoculate dandelion (Taraxacum officionale) and curly dock (Rumex crispus) we showed that the strain Sandy LB4, whether alone or in mixtures of bacteria, substantially increased mortality rates of dandelion seedlings (Table 5) and in general reduced growth and vigor in both weed species. One of the frequent features of Pseudomonas fluorescens is the production of hydrogen cyanide (HCN). HCN combines with cytochrome oxidase and prevents aerobic respiration. Perhaps differential susceptibility of the weeds and the grasses to HCN or other pseudomonad metabolite could be responsible for the inhibition of weed seedling growth and increased mortality. Differing sensitivity to HCN was previously suggested by Zeller, Brandle and Schmid (2007) to explain differing reactions of host and non-host species to Pseudomonas rhizobacteria. In this respect it is notable that many grasses secrete significant amounts of reactive oxygen (H ₂ 0 ₂ ) from roots (White et al., 2012). H ₂ 0 ₂ rapidly degrades HCN to form carbonate and ammonium ions (Yeddou et al., 2010). High production of hydrogen peroxide in grass roots could detoxify any HCN but also produce ammonia that could be absorbed and used to support plant growth. We did not see abundant H ₂ 0 ₂ production in the roots of seedlings of either dandelion or curly dock.

Another possible mechanism to explain differential responses of host and non-host seedlings was evident when seedling roots were stained using DAB to visualize bacteria in roots. In grass roots bacteria remained as small discrete spherical bodies, likely flowing in the cytoplasm with the normal cyclosis. However, the bacteria in the non-host species appeared to form large clusters that appeared to occlude the root hairs, likely blocking normal cyclosis in the root hairs. Whether differential sensitivity to HCN or physical occlusion of absorptive root hairs or other structural issues is responsible for the different responses of host grasses and non-host dandelion and curly dock seedlings to Sandy LB4 will require further experimentation.

Regardless of the mechanism of inhibition the presence of some endophytes in

Phragmites that show capability to inhibit competitor plants could provide a mechanism by which Phragmites excludes competitor species from its stands. The allelopathic exclusion of plants from Phragmites stands has been observed and previously attributed to secreted chemical inhibitors such as gallic acid (Weidenhamer et al., 2013) and phytotoxity of Phragmites residues (Uddin et al., 2014). It is possible that some of the allelopathic exclusion of competitors is a function of the growth promotional pseudomonads that are endophytic in plants and likely move out into soils colonizing and inhibiting competitor plant species that are not adapted to them. Conclusions

It is clear from our studies that Phragmites seeds carry pseudomonads that may stimulate growth and increase nutrients available to the grass, but may also inhibit soil borne fungi, suppress diseases and inhibit competitor weed species. The pseudomonad symbionts of

Phragmites appear to be diverse, multifunctional and likely increase the competitive capacity of the host.

References

Beltran-Garcia M, White JF, Prado FM, Prieto KR, Yamaguchi LF, Torres MS, ato MJ, Madeiros HG, Di Mascio P. 2014. Nitrogen acquisition In Agave tequilana from degradation of endophytic bacteria. Sci Report 4: 6938. DOI: 1.038/srep06938

Clay K, Shearin ZRC, Bourke KA, Bickford WA, Kowalski KP. 2016. Diversity of fungal endophytes in non-native Phragmites australis in the Great Lakes. Biological Invasions DOI 10.1007/sl0530-016-1137y.

Fischer MS, Rodriguez RJ. 2013. Fungal endophytes of invasive Phragmites australis populations vary in species composition and fungicide susceptibility. Symbiosis 61 : 55-62. Godlewski M, Adamczyk B, 2007. The ability of plants to secrete proteases by roots. Plant Physiol Biochem 45: 657-664.

Hamilton CE, Bauerle TL. 2012. A new currency for mutualism? Fungal endophytes alter antioxidant activity in hosts responding to drought. Fungal Divers 54: 39-49.

Hamilton CE, Gundel PE, Heiander M, Saikkonen K. 2012. Endophytic mediation of reactive oxygen species and antioxidant activity in plants: a review. Fungal Divers 54: 1 -10.

Kloepper JW, Ryu CM, Zhang S. 2004. Induced systemic resistance and promotion of plant growth by Bacillus spp. Phytopathology 94: 1259-1266.

Kowalski KP, Bacon C, Bickford W, Braun H, Clay K et al. 2015. Advancing the science of microbial symbiosis to support invasive species management: a case study on Phragmites in the great lakes. Fronteirs in Microbiology 6: 1-14.

Ligon JM, Hill DS, Hammer PE, Torkewitz NR, Hoffman D, Kempf H-J, van Pee K-H. 2000. Natural products with antifungal activity from Pseudomonas biocontrol bacteria. Pest

Management Science 56: 688-695.

Paungfoo-Lonhienne C, Rentsch D, Robatzek S, Webb R, Sagulenko E, Nasholm T, Schmidt S, Lonhienne T. 2010. Turning the table: plants consume microbes as a source of nutrients. PLOS One 5(7): el 1915.

Paungfoo-Lonhienne C, Schmidt S, Webb R, Lonhienne T. 2013. Rhizophagy— A New

Dimension of Plant-Microbe Interactions, in de Briujn FJ (Ed.) Molecular Microbial Ecology of the Rhizosphere . DOI: 10.1002/9781118297674.dll 15.

Rodriguez RK, Henson J, van Volkenburgh E, Hoy M, Wright L, Beckwith F, Kim Y-O, Redman RS. 2008. Stress tolerance in plants via habitat-adapted symbiosis. ISME J. 2(4): 404- 416.

Schulz B, Boyle C. 2006. What are endophytes? Pp 1-10, In Scultz B, Boyle C, Sieber T (ed) Microbial Root Endophytes, Springer- Verlag, Berlin.

Sharma SB, Sayyed RZ, Trivedi MH, Gobi TA. 2013. Phosphate solubilizing microbes:

sustainable approach for managing phosphorus deficiency in agricultural soils. SpringerPlus 2: 587.

Soares MA, Li HY, Kowalski KP, Bergen M, Torres MS, White JF. 2016a. Functional role of bacteria from invasive Phragmites australis in promotion of host growth. Microbial Ecology DOI: 10.1007/s00248-016-0793-x

Soares MA, Li HY, Kowalski KP, Bergen M, Torres MS, White JF. 2016b. Evaluation of the functional roles of fungal endophytes of Phragmites australis from high and low saline sites. Biological Invasions DOI 10.1007/sl0530-016-1160-z.

Stone JK, Bacon CW, White JW. 2000. An overview of endophytic microbes: endophytism defined. Pp 3-30, In: Bacon CW, White JF (ed) Microbial Endophytes Marcel -Dekker, New York.

Uddin MN, Robinson RW, Caridi D, Harun AY. 2014. Is phytotoxity of Phragmites australis residue influenced by decomposition condition, time and density? Marine and Freshwater Research 65: 505-516.

Visca P, Imperi F, Lamont IL. 2006. Pyoverdine siderophores: from biogenesis to

biosignificance. Trends in Microbiology 15: 22-30.

Weidenhamer JD, Li M, Allman J, Bergosh RG, Posner M. 2013. Evidence does not support a role for gallic acid in Phragmites australis invasion success. J Chem Ecol 39: 323-332.

White JF, Chen Q, Torres MS, Mattera R, Irizarry I, Tadych M, Bergen M. 2015. Collaboration between grass seedlings and rhizobacteria to scavenge organic nitrogen in soils. AoB Plants 01/2015. DOI: 10.1093/aobpla/plu093

White JF, Crawford H, Torres MS, Mattera R, Irizarry I, Bergen MS. 2012. A proposed mechanism for nitrogen acquisition by grass seedlings through oxidation of symbiotic bacteria. Symbiosis 57: 61-171. DOI: 10.1007/sl3199-012-01.

White JF, Torres MS. 2009. Is plant endophyte-mediated defensive mutualism the result of oxidative stress protection? Physiol Plant 138(4): 440-446. DOI: 10.1111/j.1399- 3054.2009.01332

White JF, Torres MS, Somu MP, Johnson H, Irizarry I, Chen Q, Zhang N, Walsh E, Tadych M, Bergen M. 2014. Hydrogen peroxide staining to visualize intracellular bacterial infections of seedling root cells. Microscopy Research and Technique. 05/2014;

DOI: 10.1002/jemt.22375

Yeddou AR, Nadjemi B, Halet F, Ould-Dris A, Capart R. 2010. Removal of cyanide in aqueous solution by oxidation with hydrogen peroxide in presence of activated carbon prepared from olive stones. Minerals Engineering 23 : 32-39.

Zeller S, Brandl H, Schmid B. 2007. Host-plant selectivity of rhizobacteria in crop/weed model system. Plos ONE 2(9): e846.DOL 10.1371/joumal.pone.0000846.

Zoig W, Ottow JCG (1975) Pseudomonas glathei sp. nov. a new nitrogen scavenging rod isolated from acid 1 at critic relicts in Germany. Z Allg Mikrobiol 15:287-299

While certain of the preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. Various modifications may be made thereto without departing from the scope and spirit of the present invention, as set forth in the following claims.