Patent Application
20240389585
The present invention relates, in general terms, to plant-derived organic compounds and their use thereof to promote the growth of plants and biofilms.
As the world population is projected to reach 10 billion by 2050, food security has become one of the largest challenges of the 21st century. Agricultural productivity must be met sustainably despite the backdrop of climate change, land degradation, and increasingly unpredictable weather events. The use of agricultural microbials (or agro-microbials) and nature-based agrochemicals are now widely considered a promising strategy for sustainable agriculture.
Agro-microbials encompass crop-associated microbial communities that provide indispensable functions, including plant growth promotion, disease prevention, and nitrogen fixation. They also keep the soil fertile by breaking down organic matter, recycling nutrients, and creating humus to retain moisture. Rather than being free-floating or planktonic, a large percentage of soil microbes adopt the biofilm mode of life, where they are attached to a surface and embedded in a self-secreted polymeric matrix. This lifestyle confers various advantages to the microbial members, such as adhesion/cohesion capabilities and protection from environmental stressors. The diverse microbial community in biofilms may communicate with each other and share available resources.
Microbes can be 100-fold more abundant in vegetated compared to non-vegetated soils, and the majority colonise and form biofilms in the region around plant roots known as the rhizosphere. The rhizosphere microbiome can be distinct from the bulk soil microbiome, and is shaped by compounds released by the plants, including root exudates and volatile organic compounds (VOCs). Soluble root exudate components like coumarins, benzoxazinoids, salicylic acid, flavones, fumaric acid and citric acid establish a zone of rhizospheric influence that extends 2 to 10 millimetres from the root surface, to which soil microbes are drawn and within which they assemble into biofilms. These biofilms in turn provide beneficial feedback to the plant host, including nutritional provisioning through nitrogen fixation, direct protection against pathogens and indirect protection through induction of stress tolerance. The ability of root-derived compounds to influence biofilm assembly is largely unknown.
Accordingly, it would be desirable to overcome or ameliorate at least one of the above-described problems.
The present disclosure provides a method for promoting plant growth in a growth medium, the method comprising inducing biofilm growth in the growth medium by providing an oxylipin to the growth medium, wherein induction of biofilm growth promotes plant growth in the growth medium.
Disclosed herein is a method for inducing biofilm growth in a growth medium, the method comprising providing an oxylipin to the growth medium, wherein the root volatile organic compound induces biofilm growth in the growth medium.
Disclosed herein is a growth medium for promoting plant growth, wherein the growth medium comprises an oxylipin.
Disclosed herein is a kit for promoting plant growth, wherein the kit comprises a growth medium, wherein the growth medium comprises an oxylipin.
Disclosed herein is a method for isolating a microbe that is responsive to an oxylipin, the method comprising a) culturing a plurality of microbes in a growth medium comprising the oxylipin; and b) isolating microbes that are enriched in the growth medium comprising the oxylipin as compared to a growth medium that does not contain the oxylipin.
Embodiments of the present invention will now be described, by way of non-limiting example, with reference to the drawings in which:
The present specification teaches a method for promoting plant growth in a growth medium, the method comprising inducing biofilm growth in the growth medium by providing a root volatile organic compound to the growth medium, wherein induction of biofilm growth promotes plant growth in the growth medium.
Disclosed herein is a method for promoting plant growth in a growth medium, the method comprising inducing biofilm growth in the growth medium by providing an oxylipin to the growth medium, wherein induction of biofilm growth promotes plant growth in the growth medium.
As used herein the terms “microorganism” and “microbe” should be taken broadly. These terms are used interchangeably and include, but are not limited to, the two prokaryotic domains, Bacteria and Archaea, as well as eukaryotic fungi, algae and protists. Any reference to an identified taxonomic genus should be taken to include identified taxonomic species in that genus, as well as any novel and newly identified strains that may be classified under that genus.
The term “biofilm” refers to an assemblage of microorganisms embedded in an extracellular polymer matrix and attached to a surface. A biofilm may comprise a single species of microbe or a plurality of species of microbes. The present disclosure provides for biofilms that form in a plant growth medium. This is taken to include biofilms that form independent of a plant part in the growth medium, and also biofilms that form in or on a part of a plant in the growth medium. Microbes that form biofilms include, but are not limited to, Achromobacter sp., Actinomyces sp., Agreia sp., Alcaligenes sp., Arthrobacter sp., Azomonas sp., Azorhizobium sp., Azospirillum sp., Bacillus sp., Brevibacterium sp., Burkholderia sp., Caballeronia sp., Cellulomonas sp., Cladosporium sp., Derxia sp., Desulfovibrio sp., Exiguobacterium sp., Flavobacterium sp., Leuconostoc sp., Micrococcus sp., Nitrosocosmicus sp., Paenibacillus sp., Paraburkholderia sp., Pseudomonas sp., Rhizobium sp., Rhodococcus sp., Rhodopseudomonas sp., Serratia sp., Sphingomonas sp., Xanthobacter sp.
The term “plant” is used in its broadest sense to refer to a terrestrial member of the kingdom Plantae, and includes bryophytes, pteridophytes and gymnosperms and angiosperms. A plant referred to herein may be a seed, a spore or a juvenile or adult plant originating from a seed or spore. A plant includes, but is not limited to, any species of grass, sedge, rush, ornamental or decorative, crop or cereal, fodder or forage, fruit or vegetable, fruit plant or vegetable plant, flower or vine or shrub or tree, exotic plant or house plant.
As used herein, the terms “crop”, “crop plant”, “cultivated plant” or “cultivated crop” are used in their broadest sense. The term includes, but is not limited to, any species of plant consumed by humans or used as a feed for land animals or fish or marine animals, or used by humans, or viewed by humans (e.g., flowers) or any plant used in industry or commerce or education, such as vegetable crop plants, fruit crop plants, fodder crop plants, fibre crop plants, and turf grass plants.
As used herein, the terms “plant growth medium” and “growth medium” are used interchangeably, and refer to any solid medium that supports the growth of a plant or a biofilm. The growth medium may be natural or artificial including, but not limited to, soil, potting mixes, bark, vermiculite and tissue culture gels. Naturally-occurring growth media may also include sand, mud, clay, humus, regolith and rock. An artificial growth medium may be constructed to mimic the conditions of a naturally occurring medium. Artificial growth media can be made from one or more of any number and combination of materials including sand, minerals, glass, rock, water, metals, salts, nutrients and water. The growth media may be used alone or in combination with one or more other media. It may also be used with or without the addition of exogenous nutrients and physical support systems for roots and foliage. In some embodiments the plant growth medium is soil or a nutrient extract from soil. The plant growth medium may be sterile or it may include a plurality of native microbes.
As used herein the term “rhizosphere” is used to denote that segment of the soil that surrounds the roots of a plant and is influenced by them. A rhizosphere may include compounds released by plant roots and also the microorganisms present in the soil environment and in and on the roots of a plant.
A “root volatile organic compound (rVOC)” referred to herein is a molecule with a low molecular weight and a high vapour pressure that is released by plant roots into the rhizosphere. An rVOC has a molecular weight in the range of about 100 to about 500 Da and a vapour pressure greater than 10 Pa at 293.15 K. It may be present as a gas or dissolved in a liquid within the rhizosphere. An rVOC may be produced and released by other parts of a plant in addition to the roots, and may be collected from the roots or another plant part for the purposes of the methods herein. Roots produce a number of VOCs, non-limiting examples of which include terpenoids, benzenoids, phenylpropanoids, glucosinolates, and oxylipins. Without being bound by theory, RVOCs may have a biological effect on the microbiome and/or on biofilms in a growth medium, e.g., it may promote or inhibit the growth of certain microbes, promote or inhibit the formation of biofilms, or change the microbial composition of a soil microbiome or soil biofilm. These changes may in turn affect the growth of a plant in the growth medium.
In one embodiment, there is provided a method for promoting plant growth in soil, the method comprising inducing biofilm growth by providing a root volatile organic compound (such as an oxylipin) to the soil.
As used herein, the term “plant growth promotion” encompasses a wide range of improved plant properties, including, but not limited to, improved nitrogen fixation, improved root development, increased leaf area, increased plant yield, increased seed germination, increased photosynthesis, improved resistance to plant pathogens, increase in accumulated biomass of the plant, or a combination thereof. The plant pathogen may include one or a combination of insects, nematodes, plant pathogenic fungi, or plant pathogenic bacteria. For a cultivated plant, plant yield refers to the amount of harvestable plant material or plant-derived product, and is normally defined as the measurable produce of economic value of the cultivated plant. For crop plants, yield also means the amount of harvested material per acre or unit of production. Yield may be defined in terms of quantity or quality. The harvested material may vary from crop to crop, for example, it may be seeds, aboveground biomass, roots, fruits, plant fibres, any other part of the plant, or any plant-derived product which is of economic value. The term “yield” also encompasses yield potential, which is the maximum obtainable yield. Yield may be dependent on a number of yield components, which may be monitored by certain parameters. These parameters are well known to persons skilled in the art and vary from crop to crop. The term “yield” also encompasses harvest index, which is the ratio between the harvested biomass over the total amount of biomass. In some embodiments, the method described herein leads to an increase in leaf and root area in a plant compared to a control plant that was not provided with an oxylipin.
As used herein, the term “biofilm growth” refers to an increase in the biomass or biovolume of a biofilm, which may derive from, but is not limited to, the growth of one or more microbial strains already present in a biofilm, the incorporation of additional microbial strains into a biofilm, the incorporation of previously planktonic microbes into a biofilm, the production of more extracellular material within a biofilm, or a combination thereof. In some embodiments, the application of an oxylipin leads to a biofilm growth of at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, or at least 100%, as determined by an increase in biomass and/or biovolume of the biofilm.
In some embodiments, an rVOC used in the methods herein is selected from the group consisting of terpenoids, benzenoids, phenylpropanoids, glucosinolates, and oxylipins. In some embodiments an oxylipin is used in the methods herein.
An “oxylipin” of the present disclosure is a biologically active, oxygenated derivative of a polyunsaturated fatty acid, formed by oxidative metabolism of that fatty acid. Oxylipins are ubiquitous in animals, plants, algae, fungi and bacteria. In plants oxylipins may serve as signalling molecules regulating developmental processes like pollen formation or mediating responses to biotic and abiotic stresses.
In some embodiments, the oxylipin is a jasmonate or a derivative thereof. In some embodiments, the jasmonate is jasmonic acid, methyl jasmonate or dihydromethyl jasmonate, or a derivative thereof. In some embodiments the jasmonate is jasmonic acid, methyl jasmonate, dihydromethyl jasmonate, dihydro jasmonic acid, cis-jasmone, or a derivative thereof.
As used herein a “jasmonate” is a member of a family of oxylipins which is derived from or related to jasmonic acid. A jasmonate may be a natural or synthetic compound. Jasmonates include but are not limited to: jasmonic acid, methyl jasmonate (MeJA), dihydromethyl jasmonate (DHMJ), dihydro jasmonic acid, cis-jasmone, 7-iso-jasmonic acid, 9,10-dihydrojasmonic acid, 2,3-didehydrojasmonic acid, 3,4-didehydrojasmonic acid, 3,7-didehydrojasmonic acid, 4,5-didehydrojasmonic acid, 4,5-didehydro-7-isojasmonic acid, cucurbic acid, 6-epi-cucurbic acid lactones, 12-hydroxyjasmonic acid, 12-hydroxyjasmonic acid lactones, 11-hydroxyjasmonic acid, 8-hydroxyjasmonic acid, homo-jasmonic acid, dihomo-jasmonic acid, 11-hydroxy-dihomojasmonic acid, 8-hydroxy-dihomojasmonic acid, tuberonic acid, tuberonic acid-O-β-glucopyranoside, 5,6-didehydrojasmonic acid, 6,7-didehydrojasmonic acid, 7,8-didehydrojasmonic acid, dihydrojasmone, methylhydrojasmone, and jasmonic acid conjugated with amino acids. The compounds mentioned herein may contain a non-aromatic double bond and one or more asymmetric centres. Thus, they can occur as racemates and racemic mixtures, single enantiomers, individual diastereomers, diastereomeric mixtures, and cis- or trans-isomeric forms. All such isomeric forms are contemplated. For example, the jasmonate compound described herein includes all of any optical isomer that is based on the asymmetric carbon and is optically pure, any mixture of various optical isomers, or racemic form.
Methyl jasmonate (MeJA) and dihydromethyl jasmonate (DHMJ) are volatile jasmonates with known roles in plant communication in the aboveground parts of plants. MeJA has been applied exogenously to foliage to induce plant defensive responses and to maintain the post-harvest quality of fruits and vegetables. Disclosed herein is a role of MeJA and DHMJ in the belowground parts involving their effect on promoting biofilm growth in a plant growth medium.
The oxylipin may be provided to the growth medium as a solid, liquid or a gas. In many embodiments, the oxylipin will be applied in the form of an aqueous solution, but solid preparations, liquid suspensions, and preparations that allow the oxylipin to volatilise and expose the plant to oxylipin vapours may also be used. The oxylipin may be delivered in the form of emulsions, suspensions, solutions, powders, granules, pastes, aerosols and volatile formulations.
The oxylipin may be applied alone or in a formulation comprising other compounds. Some examples of other compounds that may be included in the formulation include wetting agents, adjuvants, emulsifiers, dispersants, spreaders, pastes, anchorage agents, coating agents, buffering agents, plant nutrients, and absorptive additives. The formulation may also include acids, bases, or other compounds that adjust or maintain the final pH of the formulation in order to increase solubility of certain compounds in the formulation or for other reasons. Those of skill in the art will recognise that a single ingredient may perform multiple functions, and may thus be classified or grouped in different ways. Particular examples of formulation ingredients include ionic, non-ionic, and zwitterionic surfactants, such as Triton® X-100, Triton® X-114, NP-40, Tween 20 (polysorbates) and sodium dodecyl sulfate; alcohols; and synthetic or natural oils, such as castor oil, canola (rapeseed) oil, and soybean oil. Citric acid may be used to acidify a formulation, and compounds such as dipotassium phosphate, calcium carbonate, and potassium silicate may be used to raise the pH.
The oxylipin preparation may be deposited or pumped into the growth medium or sprayed or fumigated or otherwise physically spread over the growth medium, by manual or mechanical means. The oxylipin preparation may be added in the vicinity of a plant or throughout the area of growth of a plant. The oxylipin may be applied once or repeatedly, depending on the formulation, the environmental conditions during and immediately after application, and the desired effect on biofilm and/or plant growth. A more dilute formulation may be used if repeated applications are to be performed.
In most embodiments the oxylipin is provided in an “effective amount” to promote plant growth. For the purposes of this disclosure, an effective amount of oxylipin is any amount of oxylipin that produces a quantifiable improvement in plant growth and/or a quantifiable increase in biofilm growth, compared to a control plant or a control biofilm which has not been provided with the oxylipin. It is understood by a skilled person that this effective concentration will not also induce any type of toxicity to the plant. In some embodiments, application of an effective amount of oxylipin leads to plant growth improvement that is an at least 5% increase, at least 10% increase, at least 25% increase, at least 50% increase, at least 75% increase, or at least a 100% increase in the property being measured. Thus, as non-limiting examples, the method according to this disclosure may produce an above-stated percentage increase in nitrogen fixation, or an above-stated increase in total root weight, or in leaf area or in plant product yield (e.g., an above-stated percentage increase in plant product weight), or an increased percentage of seeds that germinate, or rate of photosynthesis (e.g., determined by CO2 consumption) or accumulated biomass of the plant (e.g., determined by weight and/or height of the plant). The plant product is the item—usually but not necessarily—a food item produced by the plant.
In some embodiments, application of an effective amount an oxylipin leads to a biofilm growth of at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, or at least 100%, as determined by an increase in biomass and/or biovolume of the biofilm.
The effective amount of oxylipin will vary depending on plant and microbial species and plant cultivar, and will depend on the manner of application, the form in which the oxylipin is administered, and the environmental conditions around the growth medium and/or around the plant that may include, for instance, the microbiome in the growth medium. Thus, different concentrations and exposure times for any given formulation will vary according to the type of plant and variety, and the type of microbes present in the growth medium. The concentration will also vary depending on the growth stage of the plant.
The growth medium, plant, microorganism and oxylipin may be combined or exposed to one another in any appropriate order. In one embodiment, the plant, seed, seedling, cutting, propagule or the like is planted or sown into a growth medium which already contains one or more microbe or which has previously been inoculated with one or more microbe, and the oxylipin is applied to the growth medium containing plant and microbe. In another embodiment, a microbe is inoculated into the growth medium and the oxylipin is applied to the growth medium to allow biofilm formation before the plant, seed, seedling, cutting, propagule or the like is planted or sown into the growth medium. In yet another embodiment, the plant, seed, seedling, cutting, propagule or the like is first planted or sown into the growth medium, allowed to grow, and at a later time one or more microbes are applied to the growth medium followed by the application of the oxylipin.
In some embodiments, the oxylipin is provided as a liquid. The oxylipin may be provided as an undiluted liquid or in the form of a solution with any compatible solvent, including aqueous (water) solutions, alcohol (e.g., ethanol) solutions, or in combinations of solvents (e.g., water/ethanol). A “compatible solvent” refers to any solvent in which the oxylipin is at least slightly soluble and which is not phytotoxic in the amounts or concentrations used for oxylipin application.
In one embodiment the oxylipin is provided in a single application to the growth medium. The amount of oxylipin added may vary depending on the extent of biofilm formation and/or plant growth desired.
In some embodiments the oxylipin is provided as a diluted liquid at a concentration of about 1 nM to about 10 μM. In some embodiments, the oxylipin is added as a diluted liquid at a concentration of about 1 nM to about 25 nM, about 1 nM to about 20 nM, about 1 nM to about 15 nM, about 1 nM to about 10 nM, or about 1 nM to about 5 nM. In some embodiments, the oxylipin is added as a diluted liquid at a concentration of about 0.5 μM to about 10 UM, about 0.6 UM to about 7 UM, 0.7 UM to about 5 UM, 0.8 UM to about 3 UM, about 0.9 UM to about 2 μM, about 0.95 UM to about 1.5 μM, or about 1 μM.
In some embodiments the oxylipin is provided as an undiluted liquid at a concentration of about 5 μmol to about 50 μmol, about 5 μmol to about 45 μmol, about 5 μmol to about 40 μmol, about 5 μmol to about 35 μmol, about 5 μmol to about 30 μmol, about 5 μmol to about 25 μmol, about 5 μmol to about 20 μmol, about 5 μmol to about 15 μmol, about 5 μmol to about 10 μmol, about 5 μmol to about 9 μmol, about 5 μmol to about 8 μmol, about 5 μmol to about 7 μmol, about 5 μmol to about 6 μmol, or about 5 μmol.
In some embodiments, the method further comprises a step of enriching the soil with plant growth microbes.
In some embodiments the plant is a species selected from bryophyte, pteridophyte, gymnosperm, monocot, and dicot. In some embodiments, the plant is a cultivated monocot or dicot.
In some embodiments, the dicot is, by non-limiting example, one of the following: bean, pea, tomato, pepper, squash, alfalfa, almond, anise seed, apple, apricot, arracha, artichoke, avocado, bambara groundnut, beet, bergamot, black pepper, black wattle, blackberry, blueberry, bitter orange, bok-choi, Brazil nut, breadfruit, broccoli, broad bean, Brussels sprouts, buckwheat, cabbage, camelina, Chinese cabbage, cacao, cantaloupe, caraway seeds, cardoon, carob, carrot, cashew nuts, cassava, castor bean, cauliflower, celeriac, celery, cherry, chestnut, chickpea, chicory, chili pepper, chrysanthemum, cinnamon, citron, clementine, clove, clover, coffee, cola nut, colza, corn, cotton, cottonseed, cowpea, crambe, cranberry, cress, cucumber, currant, custard apple, drumstick tree, earth pea, eggplant, endive, fennel, fenugreek, fig, filbert, flax, geranium, gooseberry, gourd, grape, grapefruit, guava, hemp, hempseed, henna, hop, horse bean, horseradish, indigo, jasmine, Jerusalem artichoke, jute, kale, kapok, kenaf, kohlrabi, kumquat, lavender, lemon, lentil, lespedeza, lettuce, lime, liquorice, litchi, loquat, lupine, macadamia nut, mace, mandarin, mangel, mango, medlar, melon, mint, mulberry, mustard, nectarine, niger seed, nutmeg, okra, olive, opium, orange, papaya, parsnip, pea, peach, peanut, pear, pecan nut, persimmon, pigeon pea, pistachio nut, plantain, plum, pomegranate, pomelo, poppy seed, potato, sweet potato, prune, pumpkin, quebracho, quince, trees of the genus Cinchona, quinoa, radish, ramie, rapeseed, raspberry, rhea, rhubarb, rose, rubber, rutabaga, safflower, sainfoin, salsify, sapodilla, Satsuma, scorzonera, sesame, shea tree, soybean, spinach, squash, strawberry, sugar beet, sugarcane, sunflower, swede, sweet pepper, tangerine, tea, teff, tobacco, tomato, trefoil, tung tree, turnip, urena, vetch, walnut, watermelon, yerba mate, wintercress, shepherd's purse, garden cress, peppercress, watercress, pennycress, star anise, laurel, bay laurel, cassia, jamun, dill, tamarind, peppermint, oregano, rosemary, sage, soursop, pennywort, calophyllum, balsam pear, kukui nut, Tahitian chestnut, basil, huckleberry, hibiscus, passionfruit, star apple, sassafras, cactus, St. John's wort, loosestrife, hawthorn, cilantro, curry plant, kiwi, thyme, zucchini, ulluco, jicama, waterleaf, spiny monkey orange, yellow mombin, starfruit, amaranth, wasabi, Japanese pepper, yellow plum, mashua, Chinese toon, New Zealand spinach, bower spinach, ugu, tansy, chickweed, jocote, Malay apple, paracress, sowthistle, Chinese potato, horse parsley, hedge mustard, campion, agate, cassod tree, thistle, burnet, star gooseberry, saltwort, glasswort, sorrel, silver lace fern, collard greens, primrose, cowslip, purslane, knotgrass, terebinth, tree lettuce, wild betel, West African pepper, yerba santa, tarragon, parsley, chervil, land cress, burnet saxifrage, honeyherb, butterbur, shiso, water pepper, perilla, bitter bean, oca, kampong, Chinese celery, lemon basil, Thai basil, water mimosa, cicely, cabbage-tree, moringa, mauka, ostrich fern, rice paddy herb, yellow sawah lettuce, lovage, pepper grass, maca, bottle gourd, hyacinth bean, water spinach, catsear, fishwort, Okinawan spinach, lotus sweetjuice, gallant soldier, culantro, arugula, cardoon, caigua, mitsuba, chipilin, samphire, mampat, ebolo, ivy gourd, cabbage thistle, sea kale, chaya, huauzontle, Ethiopian mustard, magenta spreen, good king henry, epazole, lamb's quarters, centella plumed cockscomb, caper, rapini, napa cabbage, mizuna, Chinese savoy, kai-lan, mustard greens, Malabar spinach, chard, marshmallow, climbing wattle, China jute, paprika, annatto seed, spearmint, savory, marjoram, cumin, chamomile, lemon balm, allspice, bilberry, cherimoya, cloudberry, damson, pitaya, durian, elderberry, feijoa, jackfruit, jambul, jujube, physalis, purple mangosteen, rambutan, redcurrant, blackcurrant, salal berry, satsuma, ugli fruit, azuki bean, black bean, black-eyed pea, borlotti bean, common bean, green bean, kidney bean, lima bean, mung bean, navy bean, pinto bean, runner bean, mangetout, snap pea, broccoflower, calabrese, nettle, bell pepper, raddichio, daikon, white radish, skirret, tat soi, broccolini, black radish, burdock root, fava bean, broccoli raab, lablab, lupin, sterculia, velvet beans, winged beans, yam beans, mulga, ironweed, umbrella bush, tjuntjula, wakalpulka, witchetty bush, wiry wattle, chia, beech nut, candlenut, colocynth, mamoncillo, Maya nut, mongongo, ogbono nut, paradise nut, and cempedak.
In some embodiments, the dicot is from, by non-limiting example, one of the following families: Acanthaceae (acanthus), Aceraceae (maple), Achariaceae, Achatocarpaceae (achatocarpus), Actinidiaceae (Chinese gooseberry), Adoxaceae (moschatel), Aextoxicaceae, Aizoaceae (fig marigold), Akaniaceae, Alangiaceae, Alseuosmiaceae, Alzateaceae, Amaranthaceae (amaranth), Amborellaceae, Anacardiaceae (sumac), Ancistrocladaceae, Anisophylleaceae, Annonaceae (custard apple), Apiaceae (carrot), Apocynaceae (dogbane), Aquifoliaceae (holly), Araliaceae (ginseng), Aristolochiaceae (birthwort), Asclepiadaceae (milkweed), Asteraceae (aster), Austrobaileyaceae, Balanopaceae, Balanophoraceae (balanophora), Balsaminaceae (touch-me-not), Barbeyaceae, Barclayaceae, Basellaceae (basella), Bataceae (saltwort), Begoniaceae (begonia), Berberidaceae (barberry), Betulaceae (birch), Bignoniaceae (trumpet creeper), Bixaceae (lipstick tree), Bombacaceae (kapok tree), Boraginaceae (borage), Brassicaceae (mustard, also Cruciferae), Bretschneideraceae, Brunelliaceae (brunellia), Bruniaceae, Brunoniaceae, Buddlejaceae (butterfly bush), Burseraceae (frankincense), Buxaceae (boxwood), Byblidaceae, Cabombaceae (water shield), Cactaceae (cactus), Caesalpiniaceae, Callitrichaceae (water starwort), Calycanthaceae (strawberry shrub), Calyceraceae (calycera), Campanulaceae (bellflower), Canellaceae (canella), Cannabaceae (hemp), Capparaceae (caper), Caprifoliaceae (honeysuckle), Cardiopteridaceae, Caricaceae (papaya), Caryocaraceae (souari), Caryophyllaceae (pink), Casuarinaceae (she-oak), Cecropiaceae (cecropia), Celastraceae (bittersweet), Cephalotaceae, Ceratophyllaceae (hornwort), Cercidiphyllaceae (katsura tree), Chenopodiaceae (goosefoot), Chloranthaceae (chloranthus), Chrysobalanaceae (cocoa plum), Circaeasteraceae, Cistaceae (rockrose), Clethraceae (clethra), Clusiaceae (mangosteen, also Guttiferae), Cneoraceae, Columelliaceae, Combretaceae (Indian almond), Compositae (aster), Connaraceae (cannarus), Convolvulaceae (morning glory), Coriariaceae, Cornaceae (dogwood), Corynocarpaceae (karaka), Crassulaceae (stonecrop), Crossosomataceae (crossosoma), Crypteroniaceae, Cucurbitaceae (cucumber), Cunoniaceae (cunonia), Cuscutaceae (dodder), Cyrillaceae (cyrilla), Daphniphyllaceae, Datiscaceae (datisca), Davidsoniaceae, Degeneriaceae, Dialypetalanthaceae, Diapensiaceae (diapensia), Dichapetalaceae, Didiereaceae, Didymelaceae, Dilleniaceae (dillenia), Dioncophyllaceae, Dipentodontaceae, Dipsacaceae (teasel), Dipterocarpaceae (meranti), Donatiaceae, Droseraceae (sundew), Duckeodendraceae, Ebenaceae (ebony), Elaeagnaceae (oleaster), Elaeocarpaceae (elaeocarpus), Elatinaceae (waterwort), Empetraceae (crowberry), Epacridaceae (epacris), Eremolepidaceae (catkin-mistletoe), Ericaceae (heath), Erythroxylaceae (coca), Eucommiaceae, Eucryphiaceae, Euphorbiaceae (spurge), Eupomatiaceae, Eupteleaceae, Fabaceae (pea or legume), Fagaceae (beech), Flacourtiaceae (flacourtia), Fouquieriaceae (ocotillo), Frankeniaceae (frankenia), Fumariaceae (fumitory), Garryaceae (silk tassel), Geissolomataceae, Gentianaceae (gentian), Geraniaceae (geranium), Gesneriaceae (gesneriad), Globulariaceae, Gomortegaceae, Goodeniaceae (goodenia), Greyiaceae, Grossulariaceae (currant), Grubbiaceae, Gunneraceae (gunnera), Gyrostemonaceae, Haloragaceae (water milfoil), Hamamelidaceae (witch hazel), Hernandiaceae (hernandia), Himantandraceae, Hippocastanaceae (horse chestnut), Hippocrateaceae (hippocratea), Hippuridaceae (mare's tail), Hoplestigmataceae, Huaceae, Hugoniaceae, Humiriaceae, Hydnoraceae, Hydrangeaceae (hydrangea), Hydrophyllaceae (waterleaf), Hydrostachyaceae, Icacinaceae (icacina), Idiospermaceae, Illiciaceae (star anise), Ixonanthaceae, Juglandaceae (walnut), Julianiaceae, Krameriaceae (krameria), Lacistemataceae, Lamiaceae (mint, also Labiatae), Lardizabalaceae (lardizabala), Lauraceae (laurel), Lecythidaceae (brazil nut), Leeaceae, Leitneriaceae (corkwood), Lennoaceae (lennoa), Lentibulariaceae (bladderwort), Limnanthaceae (meadow foam), Linaceae (flax), Lissocarpaceae, Loasaceae (loasa), Loganiaceae (logania), Loranthaceae (showy mistletoe), Lythraceae (loosestrife), Magnoliaceae (magnolia), Malesherbiaceae, Malpighiaceae (barbados cherry), Malvaceae (mallow), Marcgraviaceae (shingle plant), Medusagynaceae, Medusandraceae, Melastomataceae (melastome), Meliaceae (mahogany), Melianthaceae, Mendonciaceae, Menispermaceae (moonseed), Menyanthaceae (buckbean), Mimosaceae, Misodendraceae, Mitrastemonaceae, Molluginaceae (carpetweed), Monimiaceae (monimia), Monotropaceae (Indian pipe), Moraceae (mulberry), Moringaceae (horseradish tree), Myoporaceae (myoporum), Myricaceae (bayberry), Myristicaceae (nutmeg), Myrothamnaceae, Myrsinaceae (myrsine), Myrtaceae (myrtle), Nelumbonaceae (lotus lily), Nepenthaceae (East Indian pitcherplant), Neuradaceae, Nolanaceae, Nothofagaceae, Nyctaginaceae (four-o'clock), Nymphaeaceae (water lily), Nyssaceae (sour gum), Ochnaceae (ochna), Olacaceae (olax), Oleaceae (olive), Oliniaceae, Onagraceae (evening primrose), Oncothecaceae, Opiliaceae, Orobanchaceae (broom rape), Oxalidaceae (wood sorrel), Paeoniaceae (peony), Pandaceae, Papaveraceae (poppy), Papilionaceae, Paracryphiaceae, Passifloraceae (passionflower), Pedaliaceae (sesame), Pellicieraceae, Penaeaceae, Pentaphragmataceae, Pentaphylacaceae, Peridiscaceae, Physenaceae, Phytolaccaceae (pokeweed), Piperaceae (pepper), Pittosporaceae (pittosporum), Plantaginaceae (plantain), Platanaceae (plane tree), Plumbaginaceae (leadwort), Podostemaceae (river weed), Polemoniaceae (phlox), Polygalaceae (milkwort), Polygonaceae (buckwheat), Portulacaceae (purslane), Primulaceae (primrose), Proteaceae (protea), Punicaceae (pomegranate), Pyrolaceae (shinleaf), Quiinaceae, Rafflesiaceae (rafflesia), Ranunculaceae (buttercup orranunculus), Resedaceae (mignonette), Retziaceae, Rhabdodendraceae, Rhamnaceae (buckthorn), Rhizophoraceae (red mangrove), Rhoipteleaceae, Rhynchocalycaceae, Rosaceae (rose), Rubiaceae (madder), Rutaceae (rue), Sabiaceae (sabia), Saccifoliaceae, Salicaceae (willow), Salvadoraceae, Santalaceae (sandalwood), Sapindaceae (soapberry), Sapotaceae (sapodilla), Sarcolaenaceae, Sargentodoxaceae, Sarraceniaceae (pitcher plant), Saururaceae (lizard's tail), Saxifragaceae (saxifrage), Schisandraceae (schisandra), Scrophulariaceae (figwort), Scyphostegiaceae, Simaroubaceae (quassia), Simmondsiaceae (jojoba), Scytopetalaceae, Solanaceae (potato), Sonneratiaceae (sonneratia), Sphaerosepalaceae, Sphenocleaceae (spenoclea), Stackhousiaceae (stackhousia), Stachyuraceae, Staphyleaceae (bladdernut), Sterculiaceae (cacao), Stylidiaceae, Styracaceae (storax), Surianaceae (suriana), Symplocaceae (sweetleaf), Tamaricaceae (tamarix), Tepuianthaceae, Tetracentraceae, Tetrameristaceae, Theaceae (tea), Theligonaceae, Theophrastaceae (theophrasta), Thymelaeaceae (mezereum), Ticodendraceae, Tiliaceae (linden), Tovariaceae, Trapaceae (water chestnut), Tremandraceae, Trigoniaceae, Trimeniaceae, Trochodendraceae, Tropaeolaceae (nasturtium), Turneraceae (turnera), Ulmaceae (elm), Urticaceae (nettle), Valerianaceae (valerian), Verbenaceae (verbena), Violaceae (violet), Viscaceae (Christmas mistletoe), Vitaceae (grape), Vochysiaceae, Winteraceae (wintera), Xanthophyllaceae, and Zygophyllaceae (creosote bush).
In some embodiments, the monocot is, by non-limiting example, one of the following: corn, wheat, oat, rice, barley, millet, banana, onion, garlic, asparagus, ryegrass, millet, fonio, raishan, nipa grass, turmeric, saffron, galangal, chive, cardamom, date palm, pineapple, shallot, leek, scallion, water chestnut, ramp, Job's tears, bamboo, ragi, spotless watermeal, arrowleaf elephant ear, Tahitian spinach, abaca, areca, bajra, betel nut, broom millet, broom sorghum, citronella, coconut, cocoyam, maize, dasheen, durra, durum wheat, edo, Pique, formio, ginger, orchard grass, esparto grass, Sudan grass, guinea corn, Manila hemp, henequen, hybrid maize, jowar, lemon grass, maguey, bulrush millet, finger millet, foxtail millet, Japanese millet, proso millet, New Zealand flax, oats, oil palm, palm palmyra, sago palm, redtop, sisal, sorghum, spelt wheat, sweet corn, sweet sorghum, taro, teff, timothy grass, triticale, vanilla, wheat, and yam.
In some embodiments, the monocot is from, by non-limiting example, one of the following families: Acoraceae (calamus), Agavaceae (century plant), Alismataceae (water plantain), Aloeaceae (aloe), Aponogetonaceae (cape pondweed), Araceae (arum), Arecaceae (palm), Bromeliaceae (bromeliad), Burmanniaceae (burmannia), Butomaceae (flowering rush), Cannaceae (canna), Centrolepidaceae, Commelinaceae (spiderwort), Corsiaceae, Costaceae (costus), Cyanastraceae, Cyclanthaceae (Panama hat), Cymodoceaceae (manatee grass), Cyperaceae (sedge), Dioscoreaceae (yam), Eriocaulaceae (pipewort), Flagellariaceae, Geosiridaceae, Haemodoraceae (bloodwort), Hanguanaceae (hanguana), Heliconiaceae (heliconia), Hydatellaceae, Hydrocharitaceae (tape grass), Iridaceae (iris), Joinvilleaceae (joinvillea), Juncaceae (rush), Juncaginaceae (arrow grass), Lemnaceae (duckweed), Liliaceae (lily), Limnocharitaceae (water poppy), Lowiaceae, Marantaceae (prayer plant), Mayacaceae (mayaca), Musaceae (banana), Najadaceae (water nymph), Orchidaceae (orchid), Pandanaceae (screw pine), Petrosaviaceae, Philydraceae (philydraceae), Poaceae (grass), Pontederiaceae (water hyacinth), Posidoniaceae (posidonia), Potamogetonaceae (pondweed), Rapateaceae, Restionaceae, Ruppiaceae (ditch grass), Scheuchzeriaceae (scheuchzeria), Smilacaceae (catbrier), Sparganiaceae (bur reed), Stemonaceae (stemona), Strelitziaceae, Taccaceae (tacca), Thurniaceae, Triuridaceae, Typhaceae (cattail), Velloziaceae, Xanthorrhoeaceae, Xyridaceae (yellow-eyed grass), Zannichelliaceae (horned pondweed), Zingiberaceae (ginger), and Zosteraceae (eelgrass).
Disclosed herein is also a method for inducing biofilm growth in a growth medium, the method comprising providing an oxylipin to the growth medium, wherein the oxylipin induces biofilm growth in the growth medium.
In some embodiments, the biofilm growth that is induced by an oxylipin is capable of promoting plant growth in the growth medium. The biofilm may have a plant-beneficial effect without growing on any parts of a plant. The biofilm may, for instance, grow in the vicinity of plant roots and produce growth-promoting VOCs that are absorbed by plant parts underground and/or aboveground.
In some embodiments the plant is introduced to the growth medium after the growth of the biofilm. In some embodiments, the plant may induce changes to biofilm composition, which may include, but is not limited to, changes to the number and diversity of microbes in the biofilm, the behaviour and/or metabolic activity of microbes in the biofilm, the amount of extracellular material in the biofilm, the biomass or biovolume of the biofilm. These plant-induced biofilm changes may result in beneficial effects on the plant.
In some embodiments, the biofilm is capable of sequestering one or more plant pathogens. Pathogenic microbes may include, but are not limited to, plant fungal pathogens, plant bacterial pathogens, Alternaria sp., Aspergillus sp., Botrytis sp., Cercospora sp., Claviceps sp., Erwinia sp., Fusarium sp., Glomerella sp., Macrophomina sp., Magnaorthe sp., Pantoea sp., Phoma sp., Phytophthora sp., Pythium sp., Ralstonia sp., Rhizoctonia sp., Tilletia sp., Ustilago sp., Xanthomonas sp. The biofilm may sequester plant pathogens preferentially over other microbes, including plant-beneficial microbes.
Disclosed herein is a growth medium for promoting plant growth, wherein the growth medium comprises an oxylipin.
Disclosed herein is a kit for promoting plant growth, wherein the kit comprises a growth medium, wherein the growth medium comprises an oxylipin.
Disclosed herein is a method for isolating a microbe that is responsive to a root volatile organic compound, the method comprising culturing a plurality of microbes in a growth medium comprising a root volatile organic compound, isolating a microbe that is enriched in the growth medium as compared to a growth medium that does not contain the root volatile organic compound.
In one embodiment, the method comprises identifying the isolated microbe. The isolated microbe may be identified by sequencing. This may be done by sequencing the 16S rRNA gene segments from genomic DNA isolated from the microbe.
In some embodiments, the isolated microbe is capable of promoting plant growth in a growth medium.
It will be appreciated that many further modifications and permutations of various aspects of the described embodiments are possible. Accordingly, the described aspects are intended to embrace all such alterations, modifications, and variations that fall within the spirit and scope of the appended claims.
As used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).
As used in this application, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “an agent” includes a plurality of agents, including mixtures thereof.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
Throughout this specification and the claims which follow, unless the context requires otherwise, the phrase “consisting essentially of”, and variations such as “consists essentially of” will be understood to indicate that the recited element(s) is/are essential i.e. necessary elements of the invention. The phrase allows for the presence of other non-recited elements which do not materially affect the characteristics of the invention but excludes additional unspecified elements which would affect the basic and novel characteristics of the method defined.
The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.
Certain embodiments of the invention will now be described with reference to the following examples which are intended for the purpose of illustration only and are not intended to limit the scope of the generality hereinbefore described.
Soil extract medium was prepared by autoclaving 70 g of JIFFY soil substrate in 1 liter of water. It was cooled down and was then filtered through a 0.22 μm Nalgene filtration unit. For preparing soil extract agar plates, 1% (w/v) agarose was added to the filtered media and the solution was autoclaved again before pouring into the plates. This is the default broth and agar medium for all the experiments in the manuscript unless stated otherwise.
5 g of JIFFY soil substrate was resuspended in 20 ml of PBS. This suspension was vortexed for 4 minutes and sonicated for 1 minute. It was then filtered through a strainer. The slurry that didn't pass through the strainer was again resuspended in 20 ml of PBS medium and subsequent steps were repeated twice. The filtrate was then centrifuged at 150G for 2 minutes to settle large soil particles and the supernatant was decanted into another tube. This supernatant was then centrifuged at 5500G for 5 minutes to obtain a bacterial pellet. This pellet was resuspended in 20 ml of soil extract medium. This suspension was referred to as “soil microbiota inoculum”. The final concentration in all inocula was around 1×108 bacteria per ml as quantified by Baclight bacterial counting kit (flow cytometry) or manual counting with a hemocytometer.
For the confocal imaging experiments, soil inoculum was enriched in soil extract media overnight at 37° C.
Arabidopsis insertional mutant lines were acquired from Arabidopsis Biological Research Centre at Ohio State University (details mentioned in key resource table) and segregated for homozygous lines wherever viable. In most cases, Arabidopsis thaliana (col-0) and mutant seedlings were grown in pots with Jiffy universal potting soil up to 12 days in a plant growth chamber with the following settings: 16 hours of light at 23° C. followed by 8 hours of darkness at 21° C. with 80% relative humidity. Tomato, tobacco, rice, and fern were grown similarly. For in vitro experiments, plants were grown in soil extract agar (preparation described below). Before germination, plants were surface sterilised with 50% Chlorox and stratified for two days at 4° C.
This system is a modification of the bipartite system (Ryu, C. M. et al., Proc. Natl. Acad. Sci. U.S.A 100, 4927-4932 (2003)) that is routinely used to study microbial VOCs. Circular Petri plates (90 mm diameter) were filled with MS media, and Arabidopsis seeds were grown on it (post-sterilisation) for 12 days. A square portion of MS media was cut out and a smaller Petri plate (35 mm diameter) with microbial inoculum (1 ml) was placed in it. There was sufficient headspace to allow for gaseous exchange. The lid was then tightly closed with parafilm to avoid the loss of VOCs. At particular time points, the smaller plates were taken out, and biofilm was quantified with crystal violet staining assay (as described below).
This system is an implementation of designs proposed in previous reviews (Delory, B. M. et al., Plant Soil 402, 1-26 (2016); Tholl, D. et al., Plant J. 45, 540-560, (2006)).
This system consists of an aerator/pump (to push air), 5 μm charcoal filter (to adsorb gaseous impurities), 0.22 μm filter (to trap microbial contamination), gas wash bottle (to moisturize the air), a source chamber (to host the source of volatiles), recipient chamber (to receive volatiles) and vacuum pump (to pull the air out) (
Charcoal filters (5 μm) and polytetrafluoroethylene (PTFE) filters (0.22 μm) were procured from Omega Scientific Pte Ltd, Singapore. The source chambers and receiving chambers were custom made by Million Fabricators, Singapore. Silicone tubings were used to connect all parts of the system. Airflow from the inlet (aerator) and outlet (vacuum) of the pot was measured using a mechanical flowmeter to be around 400 ml/min.
This method was used to get a proxy for biofilm biomass. Briefly, planktonic cells were discarded. 50 μL of 0.1% CV solution was added to the well very gently. The biofilm was stained for 10 minutes. The dye was removed gently. 100 μL of PBS was added to the well to wash off the excess CV. PBS was removed, and the wells were left to dry overnight. The next day, 200 ul of 1% SDS was added to each well and was resuspended vigorously with a pipette. After 20 minutes, 20 ul from the top suspension was removed and added to a new 96-well plate. The well was diluted with 180 ul of water and absorbance was checked at 595 nm on a spectrophotometer.
Volatile trapping and TD-GCMS
rVOCs and soil VOCs were trapped as described previously (Schulz-Bohm, K. et al., ISME J. 1-11 (2018)). Briefly, 2 Tenax cartridges were fitted into the side-arms of the glass pots in such a way that their opening is exposed towards the plant roots/soil. VOCs were sampled for 40 hours and immediately analysed by thermal desorption-gas chromatography-mass spectrometry.
Sample preparation and injection were performed using the fully automated Gerstel MPS-2 autosampler and Gerstel MAESTRO software. Volatile compounds were adsorbed on a Tenax TA tube. A thermal desorption unit (TDU) was used to thermally desorb the volatiles in splitless mode at 230° C. for 10 min. To ensure that the volatiles released from the TDU are quantitatively trapped, the cooled injection system-programmed temperature vaporiser (CIS-PTV) was used. The CIS was heated from 80° C. to 230° C. at the rate of 12° C./sec with the split valve closed during sample injection into the GC inlet. Analyses of volatile compounds were performed on an Agilent 7890B GC coupled to a 5977B quadruple mass spectrometer. Separation of compounds was performed on a DB-FFAP column (60 m×250 μm×0.25 μm, Agilent Technologies, Middleburg, OI, USA). Helium was used as the carrier gas at a flow rate of 1.9 ml/min and the solvent vent mode was used. The inlet temperature was 250° C. The oven program was as follows: initial temperature 50° C. held for 1 minute, then increased to 230° C. at the rate of 10° C./min and held for 20 minutes. The temperature of the ion source and transfer line was 250° C.
The mass spectrometer was in electron ionisation mode with an ionization energy of 70 eV, scan range of 40-300 m/z and solvent delay of 3.75 minutes. Analysis was performed in Single Ion Monitoring (SIM) mode by monitoring the following ions 83, 151.1, 224.1 with the dwell time of 150 ms. Mass Hunter Qualitative Analysis was used to extract and integrate peak spectra. Peak area of these ions was considered for the relative quantification of MeJA among different samples.
Soil microbiota inoculum was prepared as described in the section above. MeJA was added to the microbiota to achieve the desired concentration (0, 1, 5, 25 nM for nucleic acid imaging experiment, and 0 and 5 nM for matrix imaging experiment). 50 L of microbiota suspension was added to every well of Ibidi™ p-Slide 18 Well (Cat. No: 81816) that had a cover glass bottom. 50 UL of SYTO™9 (Thermo cat. no. S34854) solution (final concentration of 5 μM) was also added to all the wells. For matrix imaging, FilmTracer™ SYPRO™ Ruby Biofilm Matrix Stain (Thermo, cat. no. F10318) was added instead (ready to use, 1×concentration). For live imaging, Zeiss LSM 900 with Airyscan (Definite Focus 2) was used and images were acquired every 30 minutes for 24 hours with 65×oil objective at the NUS Centre for Bioimaging Science (CBIS). For both the dyes (separate experiments), a 488 nm laser was used.
Image analysis was performed using BiofilmQ software (Hartmann, A. et al., Plant Soil 312, 7-14 (2008)). Images were aligned along the Z-axis and along time. 2-class Otsu thresholding was used to detect the signal against the background. Sensitivity was set based on thresholding feedback. The rest of the settings were kept at default. Biofilm-related global properties were calculated and exported. We mainly focused on the 3D biovolume of our samples. Linear mixed-effects were used to model the biovolume where time, treatment, and their interaction were the fixed effects, and every sample was considered a random effect (Table 1 and Table 2). Following packages from R were used: nlme (Pinheiro, J. et al., R package version 3.1-153 (2021)), ggplot2 (Wickham, H., Wiley Interdiscip. Rev. Comput. Stat. 3, 180-185 (2011)).
Identification of rVOCs- and MeJA-Responder Strains
Using the push-pull airflow system, soil microbiota inoculum was exposed to four VOC treatments 1) soil VOCs; 2) WT Arabidopis rVOCs; 3) jmt Arabidopsis rVOCs; 4) jmt Arabidopsis rVOCs+MeJA. Biofilm and planktonic parts of the samples were collected from 28 wells after 16 and 24 hours and stored at −80° C. The collective sample from 28 wells was treated as a single experimental replicate. The whole experiment was repeated eight times.
Biofilm DNA Extraction and 16S rRNA Gene Amplicon Sequencing
Biofilm was scraped at specific timepoints and resuspended in PBS solution. DNA-RNA shield was added in 1:1 proportion and samples were stored at −80 C. Zymobiomics DNA miniprep kit was used to isolate DNA from the samples based on their protocol. 16S V4-V5 region was amplified using 515F-Y and 927R primers (Walters et al., mSystems 1 (2016)). 20 UL of reaction contained 2 μL of 10×DreamTaq buffer, 2 μL of 2 mM dNTP mix, 0.5 μL of each primer (10 UM), 0.5 μL of DreamTaq polymerase (5U/μL), 10 ng of template DNA, and molecular grade water to make up the volume. PCR conditions were as follows: initial denaturation at 95° C. for 3 minutes, 35 cycles of denaturation at 95° C. for 45 s, annealing at 50° C. for 45 s, extension at 68° C. for 90 s and final extension at 68° C. for 5 minutes. PCR products were purified with a Genejet PCR purification kit. Amplicon concentration was measured using Qubit DNA BR Kit and Qubit fluorometer. 16S amplicons were submitted for next-gen sequencing on Illumina MiSeq V3 Run (300 base pairs paired-end) at Singapore Centre for Life Science Engineering (SCELSE). Rarefaction analysis was performed to calculate appropriate depth for sequencing (
qPCR for 16S Copies (Bacterial Load)
To enumerate the 16S rRNA gene copy numbers, the primer pair 515F and 806R (Walters, W. et al., MSystems 1, (2016)) were used in qPCR to amplify the 16S gene using Applied Biosystem real-time PCR system. The PCR assay mixture consists of 10 ul of PowerUp™ SYBR™ Green Master Mix, 1 ul of each primer from 10 UM stock, 1 ul of DNA of extracted DNA from the microbial population, and 7 ul of sterile nuclease-free water. The PCR amplification program encompassed an initial denaturation step at 95° C. for 3 min followed by 40 three-step cycles at 95° C. for 30 s, at 52° C. for 30 s and 72° C. for 30 s. Plasmid with the fragments of 16S rRNA gene part amplified with same primer pair was taken as standard for creating standard curve with known copy number for absolute quantification. Pearson correlation was calculated for the qPCR-derived copy number and the DNA yield from all the samples (
Raw and demultiplexed sequencing data was analysed as follows (also described in a flowchart in
PICRUSt 2.0 pipeline was used to understand the predicted functions of the community. The differential functions were identified in the same way as identification of differential taxa (integration with qPCR bacterial load data with gene tables followed by Wilcoxon Rank Sum test with Bonferroni-Hochberg correction).
Effects of Complex Biofilms on Hosts from a Distance
The host benefit assay system of biofilms consists of two major parts (
For assaying the plant response with intact biofilm as depicted in “2B” of
Isolation, Biofilm Assay of Monoculture Strains, and their Effect on Plant Growth
Complex microbiota biofilm exposed to volatile MeJA was scraped off and resuspended in PBS. Through serial dilution, this inoculum was plated on soil extract agar, minimal media agar, and LB agar. Colonies with unique morphology were picked and streaked onto a fresh LB plate to acquire single colonies. The isolated strains were identified using Sanger sequencing of the PCR product with primers 27F and 11.
Their biofilm response to volatile MeJA was tested using “push-pull” setup (as described above for complex biofilms). Their response to soluble MeJA was tested by directly adding MeJA to the monoculture inoculum (final concentration of 5 nM). For both assays, biofilms were stained with crystal violet and quantified after 24 hours as explained in the biofilm staining protocol. The initial OD of the inoculum was 0.2.
To test the effect of isolated strains on the plant growth from a distance, bipartite assay was performed where 50 ul of 0.2 OD inoculum was smeared on part of the plate and 3-5 seedlings (four days old) were placed in the other part of the plate without spatial contact. Plant growth was monitored non-invasively using photography. Leaf area was quantified using an ImageJ macro as described in the previous section.
Plant Root VOCs Promote Biofilm Formation in the Soil Microbial Community
To understand the effects of plant VOCs on PGPR, a known model PGPR, Pseudomonas protegens Pf-5, was co-cultured with Arabidopsis seedlings in vitro with shared headspace without physical contact to ensure only gaseous interaction (
To test the effect of total plant VOCs on the soil microbiome community (
To further determine the universality of the observed phenomenon, induction of biofilms by rVOCs from different plant species spread across the plant kingdom (pteridophyte, monocots, and dicots) that are separated by at least 400 million years of evolution was tested. The dynamic push-pull system was chosen due to its advantage in testing rVOCs activity from soil-grown roots. In all cases, rVOCs from these diverse plant species promoted biofilm formation in soil microbiota (
Methyl jasmonate is a potent rVOC that signals biofilm promotion in the soil microbiome
To identify the class(s) of plant rVOCs responsible for triggering the biofilm formation in soil microbiome, a genetic approach was taken that involved screening biosynthetic mutant lines of Arabidopsis for the major known classes of plant VOCs. Of the 10 mutant lines belonging to the biosynthesis of 6 VOC classes namely terpenoids, benzenoids, phenylpropanoids, glucosinolates, and oxylipins (
Methyl jasmonate (MeJA) is known to be one of the major bioactive compounds in the oxylipin class of plant volatiles. Given the involvement of LOX1 and JMT genes in MeJA biosynthesis and the inability of their mutants to promote biofilms, we tested whether the plant roots release MeJA as a VOC. The presence of MeJA was detected in the rVOCs of Arabidopsis, using a polymer-packed cartridge followed by direct thermal desorption (
As MeJA can exist in both soluble and volatile forms, both forms were tested for their biofilm-promoting activity. To test the effect of volatile form, 5, 25, and 50 μmol of MeJA was spiked in the soil chamber of the dynamic system setup followed by quantification of the biofilm in the recipient chamber. The potency of MeJA in biofilm promotion is higher at low concentrations (5 μmol) and gradually declined in a dose-dependent manner (
To identify the potential functional group of MeJA for its biofilm-inducing property, several jasmonic acid analogues were screened for their biofilm-inducing capability in the soil microbiome. The analogues were used to study biofilm induction in both complex soil microbiomes (
Biofilm induction of the soil microbiome was tested with several concentrations of pure MeJA at the nanomolar level. It was confirmed that a concentration range from 500 nM of MeJA was effective to induce biofilms without being detrimental to plant growth.
Phylogenetically Diverse Taxa from the Soil Microbiota Biofilms Respond to rVOCs and MeJA
Three lines of evidence, namely 1) compromised ability to induce biofilms by jmt mutant; 2) release of volatile MeJA from plant roots; and 3) biofilm induction by pure MeJA, taken together indicate that root-derived volatile MeJA promoted biofilms in soil microbiota. This was further corroborated by the comparable biofilm induction by WT rVOCs and rVOCs from jmt mutant complemented with pure MeJA (imt+MeJA) around the rhizosphere (
Alpha diversity analysis revealed that biofilm phase hosted a significantly higher diversity as compared to the planktonic phase across all the samples (
To obtain absolute abundance of each ASV in the biofilm community, the bacterial load of each sample (quantified through through qPCR) was integrated with the microbiome profile. To identify the biofilm members responding to WT rVOCs, soil VOC-induced and WT rVOCs-induced biofilm communities were compared. Similarly, MeJA-responding biofilm members were identified by comparing jmt rVOCs-induced and jmt+MeJA-induced biofilm communities.
It was observed that the WT rVOCs induced a significant shift in the abundance of ˜8% (86 ASVs inclusive of 24 promoted and 62 repressed ASVs) of the biofilm community compared to those exposed to soil VOCs (
The responder communities, at both 16 and 24 hours, consisted mostly of repressed taxa as compared to the induced taxa (
Overall, rVOCs and MeJA dynamically induced subtle changes in phylogenetically diverse strains within the soil microbiome biofilms.
rVOCs-Induced Complex Biofilms Promote Plant Growth
Given the evolutionary conserved nature of the bioactivity of rVOCs on soil biofilms, it was hypothesised that there would be a reciprocal ecological role of these complex biofilms on plant growth possibly through volatile signals. In order to simulate the long-distance effect of MeJA-induced biofilms, their benefit to the host plants was tested from a distance. Functional in vitro assays of plant growth were conducted with planktonic and biofilm microbiota, as well as with intact complex biofilms (
Plant growth promotion by complex biofilms was next tested to investigate whether these traits can be recapitulated at an individual strain-level. Randomly-selected strains were cultured from MeJA-induced complex biofilms (
The ability of MeJA-induced biofilms to get invaded by a plant pathogen (Xanthomonas sp) and a plant-beneficial strain (P. protegens Pf-5) was tested. The tested pathogen Xanthomonas sp is closely related to wilt causing Xanthomonas campestris that affects a wide variety of cruciferous vegetables worldwide. MeJA-induced biofilms trapped a significantly higher number of plant pathogens (
Isolation of Plant Beneficial Isolates from Soil Biofilms Using MeJA
MeJA-responders were isolated to study their effects on plants. A novel workflow was developed for quick isolation of the MeJA responders through a combination of the push-pull system and a culturomics approach (
| Number | Date | Country | Kind |
|---|---|---|---|
| 10202114308S | Dec 2021 | SG | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/SG2022/050926 | 12/22/2022 | WO |
