This invention relates to the fields of agriculture and propagation of plants under abiotic stress conditions. More specifically, the invention provides methods and microbial based compositions which facilitate improved plant growth and stress tolerance.
Several publications and patent documents are referenced throughout this application in order to more fully describe the state of the art to which this invention pertains. The disclosure of each of these publications and patent documents is incorporated by reference herein.
Pine barrens (pinelands) comprise a unique type of eco-system that is oligotrophic, and both drought- and fire-prone. Pine barrens occur throughout northeastern USA from New Jersey to Maine (Forman et al. 1998). Pines and oaks are the most common trees in pine barrens, while the understory is composed of grasses (Poaceae), sedges (Cyperaceae), blueberries and other members of heath family (Ericaceae). The largest and most uniform area of pine barrens in the United States is the 1.4 million acre pine barrens of New Jersey, where the soil is highly acidic, sandy and nutrient poor.
Dark septate endophytes (DSE) refer to a group of heterogeneous plant root-colonizing ascomycetes that produce melanized, septate hyphae. They have been isolated from over 110 plant families that grow in various environments (Knapp et al. 2012). The best studied DSE are the Phialocephala fortinii-Acephala applanata complex (PAC), a group of asexual fungi in Helotiales of Leotiomycetes (Wang et al. 2006). Fungi in PAC are characterized by darkly pigmented hyphae, and typically produce branched conidiophores, hyaline phialides with collarettes, and intracellular microsclerotia (Grünig et al. 2008a, 2008b; Yu et al. 2001). PAC are the common root associates of many tree species, specifically conifers in forests of the northern hemisphere (Grünig et al. 2008a, 2008b; Menkis 2004). Despite the global pervasiveness of DSE, their ecological roles, phylogenetic relationships and taxonomy remain poorly understood (Knapp et al. 2012; Mandyam and Jumpponen 2005). DSE fungal-plant interaction studies have yielded variable results, likely due to the use of differing experimental design strategies (Grünig et al. 2008b).
It is estimated that 30% of the world's total land area consists of acid soils, and 50% of the world's potential arable lands are acidic (Tuininga et al. 2004). In view of these adverse environmental conditions, improved methods to enhance growth of both edible and non-edible plants are needed.
In accordance with the present invention, a method for enhancing overall plant growth and resistance to adverse abiotic conditions comprising contacting a plant or seed therefrom with a composition comprising a biofertilizer comprising at least one endophytic fungi and optionally bacteria. In one aspect, the fungi is Acidomelania panicicola and the optional bacteria is from the Burkholderia genus. The method can include inoculating the seeds with the fungi and, or bacteria in agar or growth medium and placing seeds/agar composition in the soil. In another approach, after mixing the cultures, the seeds and cultures are subjected to drying to form a coating thereon. Vermiculite and rock phosphate may also be included in the composition to enhance plant growth and resistance to abiotic stress. The method can be applied to both monocots and dicots and can be used on plants which include without limitation, lettuce, corn, rice, soybeans, potatoes, barley, wheat, and carrots. In a particularly preferred embodiment, the plant is a turfgrass plant selected from a Ryegrass, Kentucky Bluegrass, Tall Fescue, Bermuda, St. Augustine or Zoysia plant or any other turfgrass plant.
In another aspect of the invention, a biofertilizer composition is provided. An exemplary biofertilizer includes an effective amount of A. panicicola and at least one agent or microorganism for promoting plant growth and resistance to abiotic stresses for use in the method described above. In a preferred embodiment, the composition contains A. panicicola and at least one Burkholderia species in equal concentrations. The composition may also contain a sun protecting product and a polysaccharide solution. The fungal strains may also be encapsulated in allignate beads.
In yet another aspect of the invention the fungus for use in the method is selected from the Barrenia genus. In a preferred embodiment, the fungi is Barrenia panicia. This composition may also comprise a bacteria selected from the Burkholderia genus.
The biofertilizer composition comprising at least A. panicicola and, or Barrenia and, optionally, other agents or microbial or fungal species are effective to enhance plant resistance to environmental stresses. Such agents may include gel formulations, agar, vermiculite, sun protectorants, rock phosphate, alginate, which when combined form an efficacious biofertilizer.
Drought and low nutrient stress typified early terrestrial environments when plant colonization of land occurred and was facilitated by root-symbiotic fungi (Stoyke et al. 1991). Beneficial endophytes encompass bacteria and fungi that have the ability to alleviate abiotic stresses in combination with plant growth promotion. Endophytes have been reported to enhance early root differentiation, improve drought and salinity tolerance and increased survival rate. These endophytes play critical roles in litter decomposition, nutrient absorption and cycling (Forman et al. 1998; Blackwell et al. 2011).
A group of new fungal species were discovered from switchgrass and other grass roots in the New Jersey Pine Barrens, which is a dry, highly acidic environment, low in nutrients (P, K, organic matter etc.), with high aluminum toxicity (von Uexkull et al. 1995). Herein we describe two new genuses, Acidomelania and Barrenia, discovered in pine barren switchgrass roots.
Barrenia was classified using multi-gene phylogenetic analyses, along with phenotypic and ecological characteristics. While the new species was isolated from roots of switchgrass and pitch pine in the acidic and oligotrophic New Jersey Pine Barrens, Barrenia likely has a wide distribution as its internal transcribed spacer (ITS) sequence has high similarity with a number of GenBank sequences obtained in various ecological studies. The majority of these similar ITS sequences were obtained from roots in plants growing in acidic, nutrient-poor environments, as well as from managed sugarcane plantations. Phylogenetic analyses of ITS, LSU and RPB1 sequence data strongly support that Barrenia is a monophyletic Clade in Helotiales, distinct from any known taxa. Barrenia is phylogenetically close to Acidomelania, Loramyces, Mollisia, and Phialocephala fortinii-Acephala applanata species complex (PAC), the dark septate endophytes. Barrenia can be distinguished from Loramyces and Mollisia by its association with living plant roots. While taxa in PAC also are root endophytes, they have complex phialid arrangements that appear to be lacking in Barrenia.
The present inventors have performed functional studies which demonstrate that application of biofertilizers comprising Acidomelania panicicola and Barrenia panicia significantly enhanced dense root hair growth in switchgrass. Acidomelania pancicola plant-fungal interactions with rice and lettuce seedlings under acidic and poor nutrient conditions also resulted in a significant promotion of root and shoot length.
In one aspect of the invention, a biofertilizer composition is prepared by inoculating seeds with fungi (e.g. Acidomelania panicicola or any fungus selected from the genus Barrenia) on agar or growth medium and placing seeds and agar in the soil. In another aspect of the invention, a biofertilizer composition is prepared by mixing fungi and bacterial cultures with seeds prior to placing seeds in the soil, the cultures optionally forming a coating around the seeds. In a third aspect of the invention, seeds are mixed with fungi and bacterial cultures and dried. In a fourth aspect of the invention, seeds are grown in fungal inoculated soil formulated with vermiculite and rock phosphate.
An endophyte is an endosymbiont, often a bacterium or fungus, that lives within a plant without causing apparent disease. Endophytes may enhance a plant's growth and improve the plant's ability to tolerate abiotic stresses such as drought or harsh soil conditions. In one embodiment an endophyte useful herein comprises the fungus, Acidomelania panicicola. In another embodiment, an endophyte comprises Barrenia panicia. Endophytes useful herein include the fungus Acidomelania panicicola in combination with certain bacteria selected from the bacterial species, Burkholderia. In yet another approach, the fungi Acidomelania panicicola and the fungi Barrenia panicia are used in combination to enhance plant growth under abiotic stress conditions.
The term “abiotic” includes non-living chemical and physical parts of the environment that affect ecosystems. An ecosystem's abiotic factors may be classified via “SWATS” (Soil, Water, Air, Temperature, Sunlight).
The term “biofertilizer” comprises at least one substance containing living microorganisms which, when applied to seed, plant surfaces, and/or soil, colonizes the rhizosphere or the interior of the plant and promotes growth by increasing the supply or availability of primary nutrients to the host plant. Biofertilizers can also comprise other agents which enhance the growth of the microorganisms present. Such agents include, without limitation, agar, gel, and minerals.
The term “crop” herein refers to any plant grown to be harvested or used for any economic purpose, including for example human foods, livestock fodder, fuel or pharmaceutical production.
The following materials and methods provided to facilitate the practice of the present invention.
Fungal Isolation
Poaceae grass roots were collected from three locations (N 40 12.00, W 74 30.00; N40 04.084, W74 26.696; and N 39 46.136, W 74 40.885) in New Jersey Pine Barrens in 2012 and 2013. Native pitch pine (Pinus rigida) roots were collected from two locations (N40 04.084, W74 26.696; and N 39 46.136, W 74 40.885) in New Jersey Pine Barrens in 2014 (Tables 1 and 2). Soil pH of the sampling locations ranged from 4.7 to 5.2. Root samples were rinsed thoroughly to remove soil from the surface, cut into 10-20 mm pieces then surface disinfected with sequential washes of 95% ethanol for 30 s, 0.5% NaOCl for 2 min and 70% ethanol for 2 min. After several rinses with sterile water and drying, the root samples were cut into 5 mm pieces and plated on acidified malt extract agar (AMEA, 1.5 ml 85% lactic acid per liter of 2% malt extract agar). Plates were incubated at room temperature with 12 h light and 12 h dark cycles. Fungal cultures were transferred to fresh AMEA and purified by sub-culturing from emergent hyphal tips.
Barrenia
panicia
Barrenia
panicia
Digitaria sp.,
virgatum, 5
Ochraceous Tawny, aerial
lacryma - jobi,
Barrenia
taeda E. Walsh &
rigida, 4 Jun.
Morphological Study and Growth Rate
Purified fungal isolates were grown on cellophane overlaid with 2% MEA (BD Difco, Maryland) and 2% water agar (WA). Cultures were incubated at 20° C. in the dark with three replicates. Colony diameter was measured after 20 days. The color names of colonies followed Ridgway (1912).
DNA Extraction, Amplification and Sequencing
Genomic DNA was extracted from fungal mycelium using the UltraClean Soil DNA isolation kit (MoBio, California) following the manufacturer's instructions. PCR was performed with Taq 2X Master Mix (New England BioLabs, Maine), following the manufacturer's instructions. PCR cycling conditions for the internal transcribed spacer (ITS) and the large subunit of ribosomal RNA genes (LSU) consisted of an initial denaturation step at 95° C. for 2 min, 35 cycles of 95° C. for 45 s, 54° C. for 45 s, 72° C. for 1.5 min, and a final extension at 72° C. for 5 min. For the largest subunit of RNA polymerase II (RPB1), the cycling conditions included an initial denaturation step at 95° C. for 2 min, 35° cycles of 95° C. for 60 s, 55° C. for 1.5 min, 72° C. for 2 min, and a final extension at 72° C. for 10 min. Primers used in this study are as follows: ITS1 and ITS4 for the ITS region (White et al. 1990), ITS1 and LR5 for the LSU locus (Rehner and Samuels 1995), and RPB1 Af (Hall and Stiller 1997) and RPB1 CrRev (Matheny et al. 2002) for the RPB1 gene. PCR products were purified with ExoSAP-IT (Affymetrix, California) and sequenced with the PCR primers by Genscript Inc. (Piscataway, N.J.).
Sequence Alignment and Phylogenetic Analyses
Six representative isolates of the new taxon (CM11m2, CM14P64, AL5m2, WSF1R37, WSF14P13, and WSF14P22) as well as other reference Leotiomycetes species (Table 3) were included in the phylogenetic analyses. The ITS dataset included sequences of the six new isolates from this study and 15 reference sequences of Helotiales. The LSU dataset included the six new sequences and 28 reference sequences of Helotiales and Rhytismatales. The three-gene (ITS, LSU and RPB1) alignment included the six new sequences and 12 reference sequences of. Sequences were aligned with MUSCLE (Edgar 2004). Maximum likelihood (ML) tree was generated with MEGA 6 (Tamura et al. 2013). Models with the lowest BIC scores (Bayesian Information Criterion) were considered to describe the substitution pattern the best. The best models for LSU, ITS and three-gene datasets were Tamura-3 parameter, Kimura 2-parameter, Kimura 2-parameter, respectively. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood approach, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites. Bootstrap was computed for 500 replications. All positions containing gaps and missing data were excluded from the analyses.
Acephala
Picea abies,
applanata
Acephala
Pinus
macrosclerotium
sylvestris, root
Acidomelania
Panicum
panicola
virgatum, root
Acidomelania
Schizachyrium
panicola
scoparium,
Barrenia
Digitaria sp.,
panicia
Barrenia
Coix lacrymajobi,
panicia
Barrenia
Panicum
panicia
virgatum, root
Barrenia taeda
Pinus rigida,
Barrenia taeda
Pinus rigida,
Barrenia taeda
Pinus rigida,
Botryotinia
fuckeliana
Bulgaria
inquinas
Chloroscypha
chloromela
Chlorovibressea
Collembolispora
aristata
Cudoniella
clavus
Dermea acerina
Acer rubrum
Fabrella tsugae
Hyaloscypha
aureliella
Hyaloscypha
vitreola
Lachnum
Alnus sp.,
virgineum
Lambertella
Aster
subsubrenispora
ageratoides
Leotia lubrica
Chrysolepis
chrysophyla
Loramyces
Equisetum
macrosporus
limosum
Microglossum
rufum
Mollista cinerea
Mollisia
Actinidia
dextrinospora
deliciosa cv.
Hayward
Monilinia laxa
Neobulgaria
lilacina
Neobulgaria
pura
Neofabrea
Malus sp.
malicorticis
Phialocephala
dimorphospora
Phialocephala
Pinus
fortinii
sylvestris, root
Phialocephala
Picea abies,
scopiformis
Spathularia
Tsuga
velutipes
Canadensis
Varicosporium
elodeae
Vibressea
Populus,
truncorum
Acephala sp.c
Cymbidium
insigne
Acephala sp.c
Phialocephala
Rhododendron,
a AFTOL = Assembling the Fungal Tree of Life project; ATCC = American Type Culture Collection, Manassas, Virginia, USA; CBS = Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands; ICMP = International Collection of Micro-organisms from Plants, Lincoln, New Zealand.
b Numbers in boldface indicating new sequences from this study.
c Taxon name was copied from GenBank. Phylogenetic analysis in this study indicated that they belong to Barrenia.
Plant-fungal Interaction Experiment
Fungal isolates WSF1R37, WSF14P22, and A. panicicola isolate 61R8 were used in the seedling inoculation experiment. Switchgrass (‘Kanlow’) seeds were surface disinfected as follows: 95% ethanol for 30 s, 0.5% NaOCl for 1 min, 70% ethanol for 1 min, rinsed with sterile distilled H2O and allowed to germinate in the dark at 25° C. for 3 days. Agargel (Sigma-Aldrich, USA) plates were made following manufacturer's instructions, and were cut in half, with one side removed. On the cut surface of an Agargel plate, three 10 mm×10 mm×5 mm plugs from a one-week old fungal culture grown on MEA were placed equidistance from one another. Germinated switchgrass seeds with visible radicle were then placed on the plugs. Sterile MEA plugs were used as negative control. Cultures were incubated at 25° C. under 12 hr light and dark cycle with nine replicates. Root length was measured 7 days after inoculation.
The following examples are provided to illustrate certain embodiments of the invention. They are not intended to limit the invention in any way.
A. Culture Morphology and Growth Rate
Isolate WSF1R37 produced dense Cinnamon Brown mycelium on MEA, and Ochraceous Tawny mycelium on WA. Colony diameter measurements for isolate WSF1R37 after 20 days were 75 mm on average on MEA with standard deviation (SD) of 2.6, and 47 mm on average on WA with SD of 2.6. Isolate WSF14P22 produced dense Cinnamon Brown mycelium on MEA, and Buckthorn Brown mycelium on WA. Colony diameter measurements for isolate WSF14P22 after 20 days were 28 mm on average on MEA with SD of 0.6, and 26 mm on average on WA with SD of 1.0.
B. Sequence Data and Phylogeny
There were 173 characters in the LSU alignment, 377 in ITS and 1291 in the three-gene alignment. Maximum likelihood trees based on LSU, ITS and three gene sequences are shown in
Based on the molecular phylogenetic analyses, morphological characters and their ecological features, a new genus and two new species have been identified. Barrenia differs from Loramyces by its association with living plant roots while Loramyces species are associated with submerged dead plants (Digby and Goos 1987; Ingold and Chapman 1952; Weston 1929). Taxa in the PAC are also root endophytes, but they exhibit complex phialid arrangements that appear to be lacking in Barrenia. Barrenia also differs from Mollisia because of its lack of phialide producing conidia. Moreover, Barrenia has 93% or less ITS sequence similarity to the above-mentioned close relatives or any other described species with accessible ITS sequences. The two Barrenia species differ from each other on host and growth rate. The pine associated B. taeda exhibited slower growth than the grass associated B. panicia on both WA and MEA. There is a 96% similarity in ITS sequences between B. panicia and B. taeda.
C. Plant-Fungal Interaction Experiment
Switchgrass seedlings inoculated with A. panicicola isolate 61R8 and B. panicia WSF1R37 produced dense root hairs all the way to the root apical meristem area, while the control seedlings only produced dense root hairs at the region of maturation of the root (
Our recent survey on fungi associated with grass roots uncovered a number of novel DSE in Leotiomycetes from the pine barrens ecosystem (Luo et al. 2014a, 2014b; Walsh et al. 2014). Leotiomycetes are morphologically and ecologically diverse and the phylogenetic relationships within this class are not well resolved due to lack of molecular data (Wang et al. 2006). Based on the multi-locus phylogenetic analyses, the new genus Barrenia described here belongs to Helotiales, which encompasses plant pathogens, saprobes and endophytes. The dark, septate hyphal morphology of Barrenia spp., their root-colonizing habit and phylogenetic closeness to PAC indicate that they likely are also DSE.
The best studied DSE is the PAC, specifically P. fortinii. However, the ecological functions of PAC and other DSE remain elusive. Host-fungal interaction experiments often yielded inconsistent results under various experimental conditions in different laboratories (Mandyam and Jumpponen 2005). This prompted us to examine the interaction between B. panicia, B. taeda, A. panicicola and switchgrass, which is the host of B. panicia and A. panicicola. Our inoculation results indicated that A. panicicola and B. panicia remarkably promoted the root hair growth in switchgrass. In switchgrass roots, B. panicia produced hyphopodium-like structures, which may perform penetration and nutrient exchange function between the fungus and the host plant (Delaux et al. 2013; Walker 1980). Barrenia taeda, originally isolated from pine roots, had negative effect on root elongation in switchgrass seedlings. These results corroborate Mandyam et al. (2010; 2012) that while DSE fungi have a broad host range, their effects and characteristics can be considered host specific.
The phylogenetic analysis in this study indicated that Barrenia is close to Acidomelania, Loramyces, Mollisia, and PAC. The phylogenetic proximity of Mollisia, Loramyces and PAC was also supported by Zijlstra et al. (2005) and Wang et al. (2006). Barrenia can be distinguished from Loramyces and Mollisia by its association with living plant roots. While taxa in PAC also are root endophytes, morphologically they can be distinguished from Barrenia. In addition, Barrenia has 93% or less ITS sequence similarities to the above-mentioned close relatives or any other described species with accessible ITS sequences. The family placement of Barrenia is not determined here because the Leotiomycetes phylogeny is poorly resolved and several families in this class likely are polyphyletic (Wang et al. 2006).
The six Barrenia isolates from New Jersey Pine Barrens were grouped into two well-supported clades. We delimited the two species based on the genealogical concordance phylogenetic species recognition (Taylor et al. 2000). The BLAST results in GenBank indicated that Barrenia might have a wide distribution. Sixteen ITS sequences in GenBank had 97-99% identity with that of B. panicia isolate WSF1R37, for example, GU973749 from sugarcane root in Brazil, HQ889709 from Cymbidium insigne root in China, and AY599235 from grass root in The Netherlands. Twelve ITS sequences in GenBank had 97-99% identities with that of B. taeda isolate WSF14P22, for example, JQ272328 from Rhododendron root in USA and KJ817299 from Vaccinium vitis-idaea in Inner Mongolia. The host plants of the matched sequences in GenBank are largely Ericaceae, terrestrial orchids, grasses and conifers, usually found in acidic and infertile soils (Keddy 2007). This distribution pattern was also found in Acidomelania panicicola, the other root associated fungus frequently isolated from the pine barrens (Walsh et al. 2014).
Additional experiments to uncover fungal-plant interactions included the inoculation of switchgrass seedlings with A. panicicola isolate 61R8 and B. panicia WSF1R37 produced dense root hairs all the way to the root apical meristem area, while the control seedlings only produced dense root hairs at the region of maturation of the root. In addition, the roots inoculated with A. panicicola isolate 61R8 and B. panicia WSF1R37 had a serpentine growth pattern, while the control roots were straight. The plant growth promotion effect of A. panicicola and B. panicia discovered in this study coupled with their distribution pattern indicate that these species may play a role in plant adaptation to acid, low nutrient soils.
In conclusion, we discovered a new genus and two species of root-colonizing fungi associated with plants living in an acidic, nutrient poor environment. The phylogenetic and taxonomic work and the plant-fungal interaction results reported here will aid future ecological and evolutionary studies on root-associated fungi.
A. Fungal Inoculation of Seeds on Agar
In this study, we performed functional studies that demonstrated that Acidomelania panicocola inoculation of seeds significantly increased root hair growth in switchgrass, rice and lettuce seedlings compared to the control.
To assess the effects of Acidomelania panicicola inoculation of switchgrass and rice seedlings on root hair abundance, fungus was grown on water agar under room temperature for 7 days. Seeds were germinated in sterile distilled water in a petri dish under room temperature in the dark for 7 days. Seedlings (roots down) were inserted in the 7 day-old fungal agar culture. Control seedlings were uninoculated but grown under the same conditions. Significant differences in root hair abundance were observed in inoculated seedlings when compared to negative, untreated controls (
To evaluate switchgrass seedling survival percent and root and shoot length, switchgrass seeds were next inoculated with Acidomelania panicicola or Fusarium oxysporum or uninoculated using the method described above and agar and seedlings were covered with top soil. 6 days post-inoculation, Acidomelania panicicola inoculated seedlings exhibited a significant increase in survival (
To assess the effects of Acidomelania panicicola inoculation on lettuce seed growth, seeds were inoculated and germinated as described above. Root length was assessed 4 days after inoculation. Increased root growth was observed for the inoculated lettuce seedlings (
B. Bacterial and Fungal Mixing and Inoculation of Seeds
Fungus (e.g. Acidomelania panicicola) is grown on water agar or other growth media under room temperature for 7 days. Bacterium (e.g. Burkholderia sp.) is cultured in Luria-Bertani broth (LB) overnight at 28° C. Seeds are mixed with the bacterial culture and the fungal cultures (ratio: 500 seeds: 10 mL overnight bacterial culture: 1 Petri dish 7 day old fungal culture) and placed on soil (e.g. Pine Barrens soil or other nutrient-poor soils). Seeds are then covered with top soil and grown under sufficient light.
C. Bacterial and Fungal Mixing, Inoculation and Drying of Seeds
Fungus (e.g. Acidomelania panicicola) is grown on water agar or other growth media under room temperature for 7 days. Bacteria (e.g. Burkholderia sp.) are grown in Luria-Bertani broth (LB) overnight at 28° C. Seeds are mixed with the bacterial culture and the fungal cultures (ratio: 500 seeds: 10 mL overnight bacterial culture: 1 Petri dish 7 day old fungal culture) and dried. Seeds are placed on soil (e.g. Pine Barrens soil or other nutrient-poor soils). Seeds are then covered with top soil and grown under sufficient light.
D. Bacterial and Fungal Mixing, Inoculation of Soil Formulated with Vermiculite and Rock Phosphate
Fungus (e.g. Acidomelania panicicola) is grown on water agar or other growth media under room temperature for 7 days. Bacterium (e.g. Burkholderia sp.) are grown in Luria-Bertani broth (LB) overnight at 28° C. Soil formulated with vermiculite and rock phosphate is inoculated with the fungal and bacterial cultures.
Roots were an early development in plant life evolving on land during the Devonian Period (416 to 360 million years ago; (von Uexkull et al. 1995)). The fossil record and molecular phylogenetic analysis suggest that from the outset, mycorrhizal fungi played a crucial role in facilitating plant invasion of land, which was dry and poor in nutrients at the time of colonization (Gensel et al. 2001). Such drought and low nutrient stress continue to challenge plants living in many extant habitats.
We describe herein a novel endophytic fungi, Acidomelania panicicola, for use alone, and optionally in the presence bacteria, which enter the root-interior and colonize the tissues of the plant, thereby effectively promoting plant growth and survival. Given that there are limited techniques—both time consuming and cost-intensive—to prevent adverse effects of abiotic stressors on plant growth, the present studies demonstrating that application of a biofertilizer comprising Acidomelania to seeds or seedlings results in increased seedling survival rate, root hair abundance, and root and shoot length will have great utility for promoting plant growth under adverse environmental conditions.
This example provides a liquid formulation of biofertilizer, where the formulation consists of two separate solutions that are combined before use as a seed coating.
For the first solution, the fungi are grown in a 1 L flask using an adequate medium and are concentrated by centrifugation in order to separate the solid. This solid is then suspended in a minimum amount of media. A sun protecting product, such as Congo red or green colorant can also be added to the media at 1% (w/v).
According to one preferred embodiment, A. panicicola only is used for the first solution in similar initial concentrations. In a second embodiment, the first solution contains a fungus from the genus Barrenia only (e.g. B. panicia). In another embodiment, the first solution is comprised of A. panicicola and at least one fungus from the genus Barrenia (e.g. B. panicia). In another embodiment, the first solution contains a mixture of A. panicicola and at least one bacteria from the genus Burkholderia. In another embodiment, a mixture of B. panicia and at least one bacteria from the genus Burkholderia is contained within the first solution.
For the second solution, a 1% (w/v) solution of a polysaccharide, such as guar gum, gelan gum, pectin, carboxymetil cellulose, agar-agar, xantan gum (or other food hydrocolloid) is prepared to be used as sticker. The two solutions are then mixed together to treat plant seeds as a coating. The seed should be dried before planting and it is preferable to wait at least two hours after application prior to planting.
This example provides a liquid formulation of a biofertilizer where the fungi and optionally bacteria are encapsulated and the fertilizer is in solid form. Alginate beads were prepared as follows: 1 ml of 30% glycerol was added to 1, 1.5 or 2% sodium alginate solution, depending on the alginate properties (M/G ratio) to obtain a final volume of 25 ml. Then, 250 ml of culture (obtained from a culture of A. panicicola only, a fungus from the genus Barrenia only (e.g. B. panicia), or a mixture of A. panicicola and Burkholderia, or a mixture of A. panicicola and Barrenia (e.g. B. panicia) was centrifuged, the cell pellet was washed with saline (0.85% NaCl, w/v) and suspended in 25 ml of alginate mixture and mixed thoroughly. This suspension was added drop wise into a pre-cooled sterile 1.5 or 2% (w/v) aqueous solution of CaCl2 under mild agitation to obtain the fungal-alginate beads. These beads were allowed to harden for 2-4 h at room temperature. Beads were collected by sieving and were washed several times with sterile water and stored at 4° C. In order to preserve the formulation, the fresh wet beads were frozen at −80° C. prior to lyophilization at −45° C. for 15 h. The lyophilized dry beads were stored in sterile glass bottles.
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.
This application is a § 371 of International Application No. PCT/US2015/048889, filed Sep. 8, 2015, which claims the benefit to U.S. Provisional Application No. 62/047,226, filed Sep. 8, 2014. The entire disclosure of each of the aforesaid applications is incorporated by reference in the present application.
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PCT/US2015/048889 | 9/8/2015 | WO | 00 |
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WO2016/040285 | 3/17/2016 | WO | A |
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20140342905 | Bullis | Nov 2014 | A1 |
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WO 2013090628 | Jun 2013 | WO |
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Number | Date | Country | |
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20180037517 A1 | Feb 2018 | US |
Number | Date | Country | |
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62047226 | Sep 2014 | US |