Patent Application
20210321617
The present disclosure generally relates systems and methods for an antipathogenic device, and in particular to a silicon nitride bioceramic which possesses antifungal properties against Plasmopara viticola pathogen having no toxicity to humans or adverse effects on the environment.
The application of agrochemicals is the most common method of preventing grapevine infections and improving harvest yields. However, powdery and downy mildew diseases, which can be caused by the oomycete Plasmopara viticola, require frequent applications of large quantities of antimycotic agents. Nearly two-thirds of all applied synthetic fungicides in the European Union are used to control these types of plant pathogens. Native to North America and accidentally introduced into Europe at the end of the last century, Plasmopara viticola can only be controlled through multiple weather-modulated annual applications. To minimize both health-risks and their environmental impact, only a limited number of fungicides at minimum concentrations are used, but both factors increase the risk that the pathogens will develop resistance. For these reasons, efforts to find alternatives to chemical treatments have garnered considerable attention, including the development of eco-friendly antifungal products. Some of the new concepts include microorganisms that confer systemic resistance to plant pathogens. Breeding resistance to infections via manipulation of host-pathogen interactions using population genetics is another favored technique. This is often done in parallel with chemical control. These latter approaches have their origins in transcriptomic and analytical methods. The search for alternatives to agrochemicals has become a priority of our modern society given the public's sensitivity to health, safety, and environmental issues.
Plasmopara viticola attacks all parts of the plant including leaves and young fruit. Assisted by temperate and humid weather, asexual sporangia release zoospores which eventually attach to and encyst into stomata to form a penetrating germ tube down to the substomatal cavity. The germ tube eventually transforms into an infection vesicle. A primary hypha emerges and quickly develops branches whose haustoria penetrate plant tissue to draw nutrients. After several days of infective incubation, the sporangiophores emerge to form new sporangia. A broad number of fungicides are applied during the growing season, but their type, amount, and timing depend on the nature of the disease and the variety of the grapevine. In most geographic areas, management of downy mildew requires several applications, starting very early in the growth cycle. The frequent use of fungicides, their high cost, and their long-term harm to the environment call for the development of more effective, long-lasting, and eco-friendly alternatives. Given the potential of these new concepts and substances, traditional fungicidal compounds should be restricted to conditions where they are truly needed.
It is with these observations in mind, among others, that various aspects of the present disclosure were conceived and developed.
Disclosed herein are methods for treating or preventing a pathogen in a plant, the method comprising contacting the plant with a composition comprising silicon nitride. The composition may comprise a slurry of silicon nitride particles and an aqueous solvent. In some embodiments, the solvent may include water. In some embodiments, the composition may comprise about 0.5 vol. % to about 20 vol. % of silicon nitride. The contacting step may include spraying, misting, or dipping. The plant may include an agriculture plant, a tree, or a vine. In some specific embodiments, the plant may be a grain, legume, tuber, grass, oilseed, vegetable, or fruit. In some other specific embodiments, the tree may be a fruit, landscape, or forest tree. In yet other specific embodiments, the vine may be a grapevine. In one example, the plant may be Vitis vinifera, including Cabernet Sauvignon, Cannonau, or Sultana. The pathogen may include a pathogen that causes a plant disease including downy mildew, powdery mildew, Botrytis rot, Fusarium rot, rust, Rhizoctonia rot, Clerotinia rot, or Sclerotium rot. The pathogen may be a fungus, such as Plasmopara viticola.
Other aspects and iterations of the invention are described more thoroughly below.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Reference characters indicate corresponding elements among the view of the drawings. The headings used in the figures do not limit the scope of the claims.
Various embodiments of the disclosure are discussed in detail below. While specific implementations are discussed, it should be understood that this is done for illustration purposes only. A person skilled in the relevant art will recognize that other components and configurations may be used without parting from the spirit and scope of the disclosure. Thus, the following description and drawings are illustrative and are not to be construed as limiting. Numerous specific details are described to provide a thorough understanding of the disclosure. However, in certain instances, well-known or conventional de-tails are not described in order to avoid obscuring the description.
The terms used in this specification generally have their ordinary meanings in the art, within the context of the disclosure, and in the specific context where each term is used. Alternative language and synonyms may be used for any one or more of the terms discussed herein, and no special significance should be placed upon whether or not a term is elaborated or discussed herein. In some cases, synonyms for certain terms are provided. A recital of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification including examples of any terms discussed herein is illustrative only, and is not intended to further limit the scope and meaning of the disclosure or of any example term. Likewise, the disclosure is not limited to various embodiments given in this specification.
Reference to “one embodiment” or “an embodiment” means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the disclosure. The appearances of the phrase “in one embodiment” in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments. Moreover, various features are described which may be exhibited by some embodiments and not by others.
As used herein, the terms “comprising,” “having,” and “including” are used in their open, non-limiting sense. The terms “a,” “an,” and “the” are understood to encompass the plural as well as the singular. Thus, the term “a mixture thereof” also relates to “mixtures thereof.”
As used herein, “about” refers to numeric values, including whole numbers, fractions, percentages, etc., whether or not explicitly indicated. The term “about” generally refers to a range of numerical values, for instance, ±0.5-1%, ±1-5% or ±5-10% of the recited value, that one would consider equivalent to the recited value, for example, having the same function or result.
As used herein, the term “silicon nitride” includes Si3N4, alpha- or beta-phase Si3N4, SiYAION, SiYON, SiAION, or combinations of these phases or materials.
The present disclosure relates to methods for treating or preventing a pathogen in a plant. The method includes contacting the plant with a composition comprising silicon nitride. In some embodiments, the contacting step may include spraying, misting, or dipping.
The surface of Si3N4 undergoes homolytic dissociation of Si—N covalent bonds in an aqueous environment. The release of nitrogen and silicon is the chemical origin of Si3N4's biological effectiveness. A hydrolysis process initiates the protonation of amino groups at the Si3N4 surface. The electrophilicity at the adjacent silicon sites increases, which in turn leads to the propensity of these sites to undergo nucleophilic attack by water. As water interacts with silicon sites, metastable pentacoordinated complexes first form, but then promptly decay with the liberation of ammonia/ammonium ions at a ratio that depends on the environmental pH. The pH-dependent elution of ammonia (NH3) or ammonium (NH4+) takes place together with the formation of silicon dioxide (SiO2) on the solid surface. Hydrolysis of this latter species produces orthosilicic acid, Si(OH)4. The basic chemical reactions of Si3N4 in water are as follows:
At room temperature, the fraction of NH3 varies with pH according to a sigmoidal dependence:
[NH3][H+]/[NH4+]=5.7×10−10 (4)
This indicates that the relative fraction of free NH3 at physiological pH is quite low (i.e., 1-2% of the overall amount of ammonium species eluted). However, at a pH of ˜8.3, it increases to ˜7%.
These reactions, Equations (5)-(11), represent a cascade of chemical events which includes free-electron release (Eq. (5)), splitting of water molecules (Eq. (6)), and the formation of radical oxygen anions and highly oxidative protonated species (Equations (7) and (8)). These latter species contribute to the dissociation of surface silanols (Equations. (9)-(11)), which in turn leads to the formation of additional oxygen radicals, i.e., (≡Si—O′) and (≡Si—O2′−. Free-electrons also oxidize ammonia (NH3) into hydroxylamine (NH2OH, i.e., ammonia monooxygenase) and its successive reaction with water to form nitrous acid HNO2 with the production of additional free-electrons and protons.
Equation (12) (i.e., ammonia monooxygenase) provides the free-electrons needed to catalyze NH3 oxidation, along with the formation of nitrous acid, additional free-electrons, and hydrogen protons. Equation (13) (i.e., hydroxylamine oxidoreductase) produces nitric oxide (NO), additional free-electrons, and hydrogen protons. The formation of additional NO and nitrite (NO2−) according to Eq. (14), together with oxygen radicals (O2*−) from Eq. (7) leads to the formation of peroxynitrite, ONOO−, as follows:
This ultimately leads to the formation of nitric oxide (NO) and peroxynitrite (OONO−) radicals. They are among the most lethal agents to pathogens. The formation of peroxynitrite has been experimentally confirmed in a recent study of the interaction of Si3N4 and Candida albicans using stimulated emission depletion microscopy and a specific fluorescent stain kit for nitrative stress sensing targeting peroxynitrite. Conversely, peroxynitrite is not toxic to plant cells and NO is a crucial signal in induction of plant resistance against pathogen infections, therefore exerting a positive indirect effect on plant expression of defense-related genes.
I. Composition
The composition of the present disclosure comprises silicon nitride.
In some embodiments, silicon nitride powder may be incorporated into compositions including, but not limited to slurries, suspensions, gels, sprays, or pastes. In at least one example, the composition may comprise a slurry of silicon nitride particles dispersed in a solvent. In some aspects, the solvent may be water. For example, silicon nitride particles may be mixed with water along with any appropriate dispersants and slurry stabilization agents, and thereafter applied by spraying the slurry onto various agricultural plants, fruit-trees, vines, grain crops, and the like. In at least one example, a silicon nitride slurry may be sprayed on fungi infected grape leaves.
In an example, the antipathogenic composition may be a slurry of silicon nitride powder and water. The silicon nitride powder may be present in the slurry in a concentration of about 0.1 vol. % to about 20 vol. %. In various embodiments, the slurry may include about 0.1 vol. %, 0.5 vol. %, 1 vol. %, 1.5 vol. %, 2 vol. %, 5 vol. %, 10 vol. %, 15 vol. %, or 20 vol. % silicon nitride.
The composition may include about 0.5 vol. % to about 20 vol. % silicon nitride. In some embodiments, the composition may include about 0.5 vol. %, 1 vol. %, 2 vol. %, 3 vol. %, 4 vol. %, 5 vol. %, 6 vol. %, 7 vol. %, 8 vol. %, 9 vol. %, 10 vol. %, 11, 12, 13, 14, 15, 16, 17, 18, 19 or about 20 vol. % silicon nitride. In some additional embodiments, the composition may include about 0.5 vol. % to about 3 vol. %, about 3 vol. % to about 6 vol. %, about 6 vol. % to about 9 vol. %, about 9 vol. % to about 12 vol. %, about 12 vol. % to about 15 vol. %, about 15 vol. % to about 18 vol. %, or about 18 vol. % to about 20 vol. % silicon nitride. In one exemplary embodiment, the composition includes about 1 vol. % to about 3 vol. % silicon nitride.
The silicon nitride powder may have a particle size of about 1 μm to about 5 μm. In at least one example, the silicon nitride powder may have a particle size of about 2 μm.
II. Pathogen
The method of the present disclosure may be used to treat or prevent many known pathogens in a plant. In some embodiments, the pathogen may cause one or more plant disease, including downy mildew, powdery mildew, Btrytis rot, Fusarium rot, rust, Rhizoctonia rot, Sclerotinia rot, Sclerotium rot, and other pathogenic plant diseases known in the art. In some additional embodiments, the pathogen may be a fungus, including Plasmopara viticola, Guignardia bidwellii, Uncinula necator, Botryotinia fuckelina, and other fungi known in the art.
III. Plant
The composition disclosed in Section I of the present disclosure may be applied to a plant, wherein the plant is an agriculture plant, a tree, or a vine. In some embodiments, the agriculture plant may include a grain, legume, tuber, grass, oilseed, vegetable, or fruit.
In some aspects, the grain may include teff, wheat, oats, rice, corn, barley, sorghum, rye, millet, triticale, amaranth, buckwheat, quinoa, bulgur, farro, freekeh, or other grains known in the art.
In some additional aspects, the legume may include peanuts, chickpeas, beans, peas, lentils, lupins, alfalfa, clover, mesquite, carob, soybeans, tamarind, and other legumes known in the art.
In yet additional aspects, the tuber may include beets, carrots, horseradish, parsnips, potatoes, radishes, sweet potatoes, turnips, rutabagas, taro, water chestnuts, yams, and other tubers known in the art.
In further additional aspects, the grass may include bamboo, marram grass, meadow-grass, reeds, ryegrass, sugarcane, and other grasses known in the art.
In still other aspects, the oilseed may include palm, soy, rapeseed, palm kernels, cottonseed, groundnut, olive, coconut, maize, sesame seed, linseed, safflower, sunflower, jatropha, camelina, cardoon, pennycress, and other oilseeds known in the art.
In still further aspects, the vegetable may include artichokes, asparagus, beetroot, broccoli, brussels sprouts, cabbage, carrots, cauliflower, celeriac, celery, fennel, garlic, ginger, kale, leeks, lettuce, parsnips, radishes, salad greens, shallots, spinach, spring onions, turmeric, turnips, watercress, and other vegetables known in the art.
In yet further aspects, the fruit may include apples, avocado, apricots, bananas, blackberries, blueberries, breadfruit, cantaloupe, cherries, clementines, coconut, cranberries, dates, figs, grapefruit, guava, honeydew melon, jackfruit, kiwi, kumquat, lemons, limes, mandarins, mangos, nectarines, oranges, papayas, passion fruit, peaches, pears, pineapples, plantains, plums, pomegranates, raspberries, rhubarb, strawberries, tangerines, watermelons, or any other fruit known in the art.
(b) Tree
In some embodiments, the tree may include a fruit tree, a landscape tree, or a forest tree.
In some aspects, the fruit tree may include almond trees, apple trees, apricot trees, avocado trees, cashew trees, cherry trees, coconut trees, fig trees, grapefruit trees, guava trees, jackfruit trees, lemon trees, lime trees, mango trees, olive trees, orange trees, peach trees, pear trees, pecan trees, plum trees, pomegranate trees, walnut trees, or any other trees known in the art.
In some additional aspects, the landscape tree may include magnolia trees, apple trees, dogwood trees, maple trees, maidenhair trees, katsura trees, spruce trees, arborvitae trees, birch trees, palm trees, cherry trees, holly trees, beech trees, and other landscape trees known in the art.
In yet additional aspects, the forest trees may include ash trees, birch trees, aspen trees, basswood trees, beech trees, cherry trees, chestnut trees, cottonwood trees, elm trees, fir trees, hickory trees, locust trees, maple trees, oak trees, pine trees, cedar trees, spruce trees, sycamore trees, willow trees, and other forest trees known in the art.
(c) Vine
In some embodiments, the vine may be a grapevine, watermelon vine, cucumber vine, ivy, creeper, hop, jasmine, or other vines known in the art. In some aspects, the vine is the grapevine Vitis vinifera. In some examples, the Vitis vinifera may include Cabernet Sauvignon, Cannonau, Sultana, Chardonnay, white Riesling, Pinot blanc, Pinot Gris, Gewurztraminer, Muscat Ottonel, Sauvignon blanc, Pinot noir, Pinot Meunier, Cabernet Franc, Merlot, Limberger, Gamay noir, Trollinger, Petite Verdot, Trebbiano Toscano, Garnacha, Syrah, Airen, Tempranillo, and other Vitis vinifera varieties known in the art.
IV. Methods
Further provided herein is a method of inactivating a pathogen by contacting the pathogen with a composition comprising silicon nitride. The pathogen may be a fungus or plant-based pathogen. The composition may be a slurry comprising silicon nitride particles and water.
In further embodiments, the method may include contacting the silicon nitride slurry with the surface of living agricultural plants, trees, grains, etc. infected with a plant-based pathogen. In an embodiment, infected leaves may be sprayed with an about 1 vol. % to about 40 vol % slurry of silicon nitride in water. The leaves may be exposed to the silicon nitride slurry for at least 1 minute, at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 5 hours, or at least 1 day.
In various examples, the infected area of leaves may be reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%. In an example, after 1 minute of exposure, the infected area of the leaves may be reduced by about 95%. It was surprisingly found that silicon nitride particles may be electrically attracted to and attach to the spores of the pathogen.
Downy mildew infections in grapevines are usually prevented by the intensive application of fungicides, including copper and sulfur (contact fungicides) or by synthetic broad-spectrum systemic fungicides such as benzimidazoles or triazoles. However, their use negatively impacts the environment and human health. Fungicide residues are long-term soil contaminants and non-negligible amounts of these compounds can be found in wine. While strict regulations attempt to minimize their harmful consequences, the situation calls for the development of alternative fungicidal strategies. These examples present the unprecedented case of a bioceramic, silicon nitride, which possesses antifungal properties against Plasmopara viticola, but no toxicity to humans or adverse effects on the environment. Raman spectroscopic assessments of living sporangia mechanistically showed that the nitrogen-chemistry of the bioceramic surface was responsible for inhibiting host infections.
These examples used silicon nitride (Si3N4), to knockdown Plasmopara viticola starting early in its infection cycle. The choice of this ceramic was based on its unique surface chemistry within an aqueous environment. It has antibacterial, antiviral, and antifungal properties, while still being friendly and supportive of eukaryotic cells. For these reasons, Si3N4 can be considered an environmentally friendly alternative for grapevine protection. In situ Raman spectroscopy was utilized to provide insight into the molecular mechanisms governing the pathogenicity of Plasmopara viticola on grapevine leaves and their inactivation by Si3N4. Raman spectroscopy is a non-invasive method that can be applied to living pathogens without markers, thus allowing time-lapse experiments to reveal their metabolic variations. The method monitors the structure of the pathogen and its evolution during chemical interactions with antipathogenic agents.
To show the effect of silicon nitride on the inactivation of agricultural fungi, Cabernet Sauvignon leaves were infected with Plasmopara viticola at a concentration of 3×104 spore sacs/ml. Treated Plasmopara viticola was exposed to a slurry of 1.5 vol. % silicon nitride for 1 minute.
The silicon nitride particles appear electrically attracted to and attach themselves to the spores of the pathogen, as seen in
Plasmopara viticola (P. viticola,) isolate harvested in a field in 2018 was axenically grown as described by Polesani et al., “General and species-specific transcriptional responses to downy mildew infection in a susceptible (Vitis vinifera) and a resistant (V. riparia) grapevine species,” BMC Genomics 11:117 (2010). To evaluate the possible phytotoxicity of Si3N4, treatments were performed using two different grape varieties, Cabernet Sauvignon and Cannonau. Cabernet Sauvignon leaves were taken from 3-year-old plants, while Cannonau leaves were obtained from young seedlings grown in a greenhouse under controlled conditions (16 hours light/8 hours dark, temperature range 18-28° C.).
Si3N4 powder with a particle size of about 2 μm was used. It was obtained by grinding sintered β-Si3N4 powder having a nominal composition of 90 wt. % α-Si3N4, 6 wt. % yttrium oxide (Y2O3), and 4 wt. % aluminum oxide (Al2O3). The constituents were sintered at ˜1700° C. for >3 h and hot-isostatically pressed at about 1600° C. for 2 h. After preparation, it was heat sterilized at 180° C. for 2 h before suspension in sterile distilled water.
For evaluation of preventive efficacy, three lots of five disks were cut from sterilized leaves for each grape variety. One lot was treated by full immersion in a 1.5 vol. % aqueous suspension of Si3N4 for 1 minute and inoculated with 40-μL of germinated sporangia suspension (3×104/mL) 24 h later (pre-treated samples). A second lot was exposed to sporangia combined with the 1.5 vol. % Si3N4 suspension. In this case, the Si3N4 granules remained in direct contact with the sporangia during germination (co-treated samples). The third lot was inoculated with P. viticola and served as an infection control group. All disks were incubated in a growth chamber at 21-24° C. with a day/night photoperiod of 16 hours and 8 hours, respectively, and monitored for 6 days until sporulation appeared on the controls.
To evaluate potential curative effects, three lots of six disks each were cut from sterilized grapevine leaves of the highly susceptible Sultana variety. All three lots were inoculated with P. viticola using 40 μL of sporangia suspension (3×104/ml) and incubated in a growing chamber at 21-24° C. with day/night photoperiods of 16/8 h to allow for the onset of infection. Droplets were removed 24 h later with the same procedure as discussed previously. Three days after the appearance of infection, two lots were treated by full immersion in a 1.5 vol. % aqueous suspension of Si3N4 for 1 minute. Then, one of the two lots was washed in distilled water for one minute to remove Si3N4 residue. The third lot was left untreated as a control group.
Microscopy Observation
Sporangia suspended in water or the 1.5 vol. % Si3N4 suspension (3×104 sporangia/mL) were observed under an epifluorescence microscope (excitation filter BP 340-380 nm; dichroic mirror 400 nm; suppression filter LP>430 nm) or stained with Fluorescein diacetate (FDA) and observed using a fluorescence microscope to check sporangia viability during a time course of 3 h. Observations were made in a cell counting Bürker chamber to calculate the percentage of viable sporangia in comparison to water-treated controls.
The pH of sterile double distilled water was measured with a pH-meter after the addition of 15 vol. % Si3N4 powder. Measurements were made while stirring at room temperature as a function of time for up to 800 s at intervals of 10 s until final pH stabilization. To check whether the pH trend was reproducible, the tested powder sample was separated by centrifugation (13×103 RPM for 3 min) and dried at 180° C. in air for 2 h. After cooling to room temperature, the powder was re-suspended at the same water concentration (i.e., 1.5 vol. %) for additional pH measurements. The procedure was repeated with the same powder for three subsequent cycles.
In situ Raman spectroscopy
In situ Raman spectra were collected on sporangia samples suspended in water solutions with and without Si3N4 powder. Raman spectra were obtained using a dedicated instrument operating in microprobe mode with a 50× optical lens. The spectroscope was equipped with a holographic notch filter which concurrently allowed high-efficiency and high-resolution spectral acquisitions. Excitation was made with a 785 nm laser source at a power of 15 mW. The Raman scattered light was monitored using a single monochromator connected with an air-cooled charge-coupled device (CCD) detector. The acquisition time of one spectrum was typically 60 s. The spectra for different sporangia samples were averaged over ˜-10 different collection locations. Raman spectra were deconvoluted into Gauss-Lorentz cross-product sub-band components using commercially available software (e.g. LabSpec 4.02). Spectral band assignments were made according to published literature.
pH analyses of Si3N4 powder in aqueous suspensions
The change in pH as a function of time for the 15 vol. % Si3N4 water-suspension is shown in
In situ microscopic monitoring of sporangia/Si3N4 granule interaction
A detailed observation of the interaction between the membrane of the sporangium and Si3N4 granules at a contact time of <1 min is shown in
Monitoring preventive and curative Si3N4 efficacy against P. viticola
The experimental results on leaf-disks from the two grapevine species, Cabernet Sauvignon and Cannonau, are shown in
The potential curative effect of Si3N4 was evaluated further by treating the highly susceptible Sultana variety 3-days post-infection. Since residual Si3N4 granules can remain in the stomata, this test investigated whether pathogenesis could be halted at a relatively advanced state of infection. In other words, this experiment sought to determine how Si3N4 affects the mycelium inside the leaf tissues or blocks the emission of new sporangia. It was observed that P. viticola had colonized the intercellular spaces and produced haustoria in mesophyll cells, yet without detectable sporulation. This normally would have occurred in 5-6 days. In this case, it appears that wet Si3N4 granules partially penetrated the stomata. The subsequent elution of nitrogen from the granules likely diffused into the intracellular space where P. viticola develops its hyphae. This inhibitory mechanism is like the antifungal effect of ammonium bicarbonate in which the bicarbonate anion supplies the alkalinity necessary to establish a sufficient concentration of NH3 to kill the pathogen.
In situ Raman spectroscopic monitoring of living sporangia
Raman spectra of P. viticola, collected after room-temperature immersion for 10 min in pure water and the suspension containing 1.5 vol. % Si3N4 powder are shown in
Spectra of sporangia exposed to pure water
Oomycetes have recently been re-classified in Stramenopiles according to an updated classification. Main structural characteristics include the presence of cellulose in the wall, mycolaminarine instead of glycogen as a carbon-based energy source, a conspicuous lack of chitin. Recent analyses of carbohydrate content in the oomycete Phytophthora parasiticia, closely related to P. viticola, revealed that the cell walls were completely devoid of chitin and consisted by ˜85% of β-glucans, about 40% of which was represented by cellulose. 1,3 β-glucans with low polymerization level, and 1,3,6 β-glucans were also present, together with lower fractions of glucuronic acid and mannan. Such detailed information is not available for P. viticola, but previous evidence indicate that this pathogen might slightly differ from other best-known organisms in the Peronosporales. Indeed, P. viticola can express at least two different chitin synthases, and chitin was detected on the surface of sporangia, sporangiophores, and hyphal cell walls during in planta growth.
When oospores are dormant (as in this case), the structure of the sporangia consists of large lipid globules distributed throughout the cytoplasm-filling the entire cell lumen. They serve as a storage material for oospore germination. Mitochondria reside in small interstices among the lipid globules. The globules (or vacuoles) are of different sizes and are contained into a relatively thin interspace. The overall external walls of the oomycete are complex and divided into two layers—the outer and the inner oospore walls (OOW and IOW, respectively). The OOW and IOW are separated from each other by a thin slightly undulating plasma membrane. The IOW mainly consists of β-1,3-linked glucans (˜80%; including chitin, a homopolymer formed with N-acetyl glucosamine), cellulose (˜10%), and proteins (divided into wall-associated enzymes and structural proteins). Glucans are the preponderant chemical species in the IOW structure. They contain fibrils of cellulosic nature oriented in straight parallel arrays along with minor fractions of mannose and glucosamine. Chitin has an important structural function since it contributes to the rigidity and strength of the wall. The OOW is mainly composed of mannans and proteins which link it to the inner wall with β-1,6-glucans, but it also contains lipids. The oogonial walls, a thicker fibrillar wall set on the external side and separated from the oospores by a periplasmic space, contain a relatively high amount of lipids and proteins. Lipids confer hydrophobicity to the structure, which is needed to keep the pathogen safe during dormancy. Negrel et al. recently searched for Plasmopara-specific metabolites and identified three types of atypical lipids—ceramides, and derivatives of arachidonic and eicosapentaenoic acids. These lipids were reported to exist in P. viticola from the very early stage of its development.
These structural features were observed in the Raman spectrum of
Signals related to nucleic acid were found from both phosphodiester and purine bonds. C′5-O—P—O—C′3 phosphodiester bond symmetric stretching in DNA (Band 25 at 782 cm−1) was the strongest signal detected in the low-frequency spectrum of P. viticola exposed to pure water (
Looking for peculiar signals from lipids usually present in cell membranes, a strong emission from phosphatidylserine in the studied spectral area was expected at about 734 cm−1. Band 21 at 731 cm−1 was observed in the spectrum for sporangia exposed to pure water (
Sterols are characterized by complex Raman spectra, which include clear low-frequency signals (cf. Table 1). However, an accurate screening revealed that none of these low frequency signals was free of overlapping signals from other membrane molecules. Sterols are essential components in modulating fluidity, permeability, and the integrity of the cell membrane. In contrast to true fungi, Peronosporales are unable to synthesize sterols, although they need them for both sexual and asexual reproduction. In Phytophthora, fitosteroles from the plant host are taken up and used without any further modification.
Regarding other lipid compounds, arachidonic acid is a well-known elicitor released by oomycetes in planta and recent findings indicate that ceramides and derivatives of arachidonic and eicosapentaenoic acid in P. viticola are produced during the very early stages of the infection process. Bands 32 (at 861 cm−1) and 35 (at 931 cm−1) were assigned to C—O vibrations in alpha-linolenic acid and C—H bending in arachidonic acid. The former band serves as a fingerprint of fatty acids peculiar to P. viticola, while the latter unfortunately overlaps with bands from glucose and histidine, (as described later). Fatty acids are commonly released into plants upon infection by oomycete pathogens. The strongest signal at low frequency of the glycerophospholipid lecithin (assigned to C—N stretching) could also contribute Band 20 (at 715 cm−1). Additional bands from lecithin appear at 764 and 827 cm−1 (Bands 24 and 29, respectively). These are attributed to O—P—O symmetric and antisymmetric stretching, respectively. An attempt to give complete labeling of the spectrum shown in
Spectra of sporangia exposed to 1.5 vol. % Si3N4 water suspension
Changes in the cellular structure P. viticola sporangia induced by the presence of Si3N4 in aqueous suspension are shown by the spectral variations between
Several bands of high or medium intensity disappeared or occurred only with significantly reduced intensity in the spectrum of sporangia in the Si3N4 suspension. They included: Bands 5 and 12 (at 535 and 623 cm−1, respectively) from adenine; Band 10 (at 594 cm−1) from cytosine; Band 25 (at 782 cm−1) from C′5-O—P—O—C′3 phosphodiester symmetric stretching in DNA; Band 31 (at 846 cm−1) from guanine; and, Band 33 (at 893 cm−1) from cellulose (possibly also contributed by chitin).
Three new bands appeared in the sporangia spectrum exposed to the Si3N4 suspension. They were: Band 11* (at 613 cm−1), Band 15* (at 654 cm−1), and Band 32* (at 872 cm−1). The origin of these Raman signals is due to chemical modifications of pre-existing molecules or from new chemical species produced by the sporangia in response to environmental stress (as discussed later).
Additional spectral variations in the presence of Si3N4 were: Band 2 from C—C backbone stretching in polysaccharides and Band 3 from D(+)-mannose (at 490 and 500 cm−1, respectively). These signals underwent an intensity-trend inversion, the former becoming more intense than the latter; and Band 9 from cellulose (at 583 cm−1) which also showed relatively high intensity. A similar trend was observed for Band 17 (at 681 cm−1), which was assigned to O═CN and CCO bending in ceramides, but also had contributions from the guanine ring. Band 23 (at 753 cm−1) representative of thymine, Band 35 from histidine (at 931 cm−1) and Band 36 from adenine (at 942 cm−1) experienced significant decreases in intensity.
The reasons for the bold spectral differences between sporangia exposed to pure water and the aqueous Si3N4-powder suspension was the result of chemical reactions occurring between sporangia and the Si3N4 granules.
The chemical interaction between P. viticola and Si3N4
By direct observation, this study confirmed the robust pH buffering of Si3N4 in an aqueous suspension and the release of gaseous ammonia (cf.
In
Interpretation of the Raman analyses
The main chemical reaction expected by ammonia on nucleic acid is hydrolysis. Nucleic acid is first decomposed into two dinucleotides, one containing adenine and uracil groups, while the other retains guanine and cytosine groups. Although the adenine-uracil dinucleotide is comparatively more stable than the guanine-cytosine, both decompose into mononucleotides at pH values >8. In the presence of NH3, adenine and guanine, and the phosphodiester bonds are deprotonated and strongly destabilized. At any alkaline pH, the hydrogen at N(3) in thymine is also removed due to the weak basicity of the nitrogen ring. Upon exposure to Si3N4, the most striking spectral variations were the disappearance of the two strongest signals, namely Band 25 and 31 (i.e., related to C′5-O—P—O—C′3 phosphodiester symmetric stretching in DNA and C4-N9-C8+N1-C2-N3 in-plane deformation of guanine rings, respectively). A significant decrease in intensity, if not the disappearance, of several bands related to adenine (Bands 5, 12, and 36) and cytosine (Bands 6, 10, 11, 19, and 26) was noted (cf.
Sonois et al. described the Raman behavior of several amino acids by both experiments and theoretical calculations. In the case of histidine, environments with increasing pH led to the appearance of new Raman bands at ˜613, 656, and 860 cm−1. These three bands correspond to the new bands detected in sporangia exposed to Si3N4 (cf.
In an attempt to strengthen the histidine interpretation for the newly formed Bands 11*, 15*, and 32*, the C—N stretching spectral area for the imidazole ring in the frequency range 1,050˜1,150 cm−1 was monitored. Different trends were observed when comparing Raman spectra collected on sporangia in pure water (pH=6.5) versus the Si3N4 suspension (pH=8.3, cf.,
A new strong Band appeared at ˜1075 cm−1. This is in addition to the band that was originally observed at ˜1090 cm−1 whose intensity appeared to be significantly weakened. These bands are related to stretching vibrations of the (C2-N3)+ and (C2-N3) bond configurations, respectively. The former configuration involves a stronger bond (i.e., due to N1-C2-N3 electron sharing). Its vibrational energy is greater and it appears at higher frequencies.
A similar trend was observed for the stretching bands in the pristine (C4-N3)+ and deprotonated (C4-N3) bond configurations, which appeared at ˜1125−1 (shoulder band) and ˜1118 cm−1 (pristine band), respectively. This trend can be explained using the same reasoning given in the preceding paragraph, even though the frequency shift toward a lower frequency is less pronounced than in the case of the preceding paragraph. This circumstance is related to the balance of bonding strength within the deprotonated ring. The C2-N3 bond is weaker than the C4-N3 bond because its neighboring double bond N1=C2 is stronger than the double bond C4=C5 next to C4-N3 (i.e., due to the higher electronegativity of N over C).
No significant shift or intensity variation, but only a slight broadening, was observed for the stretching band related to the (C2-N1(H))+ bond (at ˜1100 cm−1) when the ring configuration was deprotonated.
Note also that additional Raman analyses of the imidazole ring of histidine residues in the C═C and C═N spectral zone at ˜1600 cm−1 (not shown) provided features that were consistent with the results shown in
Histidine kinase proteins are present in most prokaryotic and eukaryotic organisms. They regulate several adaptive transcriptional responses to a variety of environmental factors. In oomycetes, functional analyses of histidine kinases are missing, while phosphorylation at histidine sites is a common metabolic response of fungi to osmotic stress. For example, in response to perceiving osmotic stress as a change in environmental conditions, conserved histidine residues are phosphorylated with a phosphate group from an adenosine triphosphate, which agrees with the reduction in adenosine Raman bands detected in
In the present study, this hypothesis is corroborated by the disappearance of the main signal for chitin (i.e., Band 33 at 893 cm−1) and by a significant reduction in the intensity of all other signals (i.e., Bands 3, 14, 15, and 19 at 500, 643, 649, and 710 cm−1, respectively) related to cellulose (and/or chitin) and other linear carbohydrates in the structure of the cell walls. Linear polymeric chains in cellulose are linked together by β-glycosidic bonds. These bonds are not affected by the alkaline pH levels induced by Si3N4, or by any direct interaction with NH3. On the other hand, hydrolytic enzymes can break down the glycosidic bonds of chitin and thereby alter the cell walls of phytopathogens. Given how the Raman experiments were conducted, the enzymatic reaction could only be intrinsic to sporangia themselves. It is known that phycomycetes are enzymatically capable of controlling the plasticity of their walls. Fungal walls are “softened” and must expand for bud emergence and subsequent growth. They are also remodeled during the formation of pseudohypha and spore walls with phenolic crosslinks. The walls' inner matrix of interlinked β-glucan and chitin provides tensile strength and rigidity. However, the wall composition can be remodeled in response to environmental changes through mitogen-activated protein kinase pathways. The cell walls' elasticity is modulated by rapid structural realignments, which enables pathogen survival to osmotic shock. As shown in
Turchini et al. measured a 50% decrease in chitin content for fungal cells grown in a high-osmolarity medium as compared to those grown in low-osmolarity, in agreement with previous data showing that the chitin synthase activity in fungi is higher for cells grown in a low- vs. a high-osmolarity media. These researchers interpreted the observed weakening of the fungal walls in high-osmolarity medium as a rescuing mechanism to enable membrane stretching and enhance the probability of maintaining cell integrity. Based on these studies, the disappearance of the main chitin Band 33 of the sporangia exposed to Si3N4 is an enzymatic fingerprint activated by the fungal cells in the attempt to resist osmotic stress. Finally, it should also be noted that premature germination, which was surprisingly observed after only 1 minute of contact with Si3N4 (
The effect of Si3N4 in comparison with other eco-friendly approaches
P. viticola enters the host leaf tissue through the stomata and remains in the substomatal air spaces where it slowly develops for 12-15 hours until forming the first haustorium. This initial period is considered the most critical in the overall infective process. Si3N4 particles could be preventively sprayed on grapevine leaves before the start of this period. Upon entering the stomata, they remain trapped inside for a long period (e.g., perhaps one season). During rain events, water will repeatedly activate the elution of ammonium moieties and a rapid rise in pH, thereby creating a hostile environment for the sporangia (
Three strategies have been pursued to replace the use of environmentally unfriendly agrochemicals: (i) the development of new eco-friendly antifungal products; (ii) the use of microorganisms for induction of systemic resistance against plant pathogens; and, (iii) the manipulation of host-pathogen interactions through the control of population genetics.
Effective eco-friendly molecules that can replace agrochemicals are polysaccharides. For example, oligosaccharide chitosan is an efficient promoter of plant defenses with the capacity of inducing an accumulation of molecules that inhibit the growth of parasites (i.e., phytoalexins, and potent antioxidants, such as trans- and cis-resveratrol and their derivatives). Chitosan also triggers the production of enzymatic molecules (e.g., chitinase and α-1,3-glucanase) in grape leaves which are capable of lysing pathogens, thereby significantly reducing the probability of downy mildew infections. Low-molecular-weight chitosan also possesses the ability to penetrate fungal conidia causing membrane disorganization and loss of cellular content. It interacts with external anionic components of the fungal plasma membrane which results in membrane rupture. Another polysaccharide capable of controlling Plasmopara viticola infections is the water-soluble β-1,3-glucan laminarin, which can be obtained from brown alga, Laminaria digitata. The origin of its antipathogenic effect resides in an efficient elicitation of defense responses in grapevine cells. However, despite the well-established antifungal efficiency of chitosan and laminarin, reports show undesired effects of these polysaccharides on the amino acid composition of must from grapevines, with alterations of nitrogen concentration in must, a key parameter in the final vine quality.
Vanillin and garlic extract have also been classified as eco-friendly antifungal substances. The former has aldehyde groups in its chemical structure, while in the latter includes the powerful antifungal activity of allicin, block lipids, proteins, and nucleic acid synthesis in fungal yeast. Nevertheless, the main disadvantage of these compounds is they readily react with water. Allicin, for example, promptly forms diallyl disulphide, a compound with less pronounced antimicrobial activity.
Other naturally derived compounds tested include hydrolyzed proteins, plant extracts, and inorganic salts. In this context, a recent promising strategy has been proposed based on the use of a selected aptamer peptide, specifically inhibiting P. viticola cellulose synthase 2, and therefore preventing infection with no adverse effects on non-target organisms.
Among inorganic salts, examples include mainly bicarbonates, phosphates, silicates, chlorides, and phosphites. Their activity has been mainly reported against powdery mildews of different crops including grapevine, while only sodium bicarbonate showed a limited efficacy against grapevine downy mildew. The development of silicon nitride-based phytosanitary products falls within this last category. In the present context, silicates deserve a particular mention: several soluble silicate salts possess direct and indirect activities against different fungal infections, acting by both stimulation of the plant's natural defense mechanisms and strengthening of plant cell walls. The production of SiO2 and Si(OH)4 from Si3N4 (as described in reactions (1)-(3)), may thus complement the direct action of ammonia on P. viticola sporangia and zoospores by inducing plant resistance, which can be at least partially responsible for the almost complete inhibition of the infection process observed in our experiments. Moreover, in comparison to soluble salts, which are readily washed off by rain, Si3N4 could provide more lasting protection through different elution cycles of ammonium moieties from the insoluble powder, and generation of reactive nitrogen species, in line with previous studies on human pathogens. In fact, upon treatment, Si3N4 particles may remain trapped inside the stomata (
From a more general viewpoint, the molecular mechanisms behind the pathogenesis of oomycete Plasmopara viticola are largely unknown. To trigger an infection, cytoplasmic and apoplastic effector proteins are secreted by oomycetes, which suppress immunity and enhance plant susceptibility. Effectors in sequenced oomycetes genomes have been found to rapidly evolve and to acquire new functions that counteract plant resistance genes and suppress plant RNA silencing mechanisms. Counteracting the action of effector proteins has been traditionally difficult because of the complexity involved with genome sequencing. It mandatorily requires multi-omics approaches. Utilizing comparative genomics, Brilli et al. recently reported the discovery of a missing metabolic feature in the Plasmopara viticola genome that could explain its biotrophic mode of life. They identified a protein effector triggering immunity in a resistant grapevine. A new method developed by German et al. for parallel analyses of RNA ends, which combines small RNA and genome-wide degradome sequencing, uncovered the complex network of small RNAs that target genes during infection. Accordingly, a new bi-directional RNA silencing strategy was suggested. Although pathometric techniques have recently reached high levels of sophistication, they have also unveiled the complexity of pathogen-host interactions. The studied system could only be coded and interpreted for a limited number of genes, (many gene-for-gene interactions were expected), even if only “resistant” and “susceptible” types of reaction were recognized.
Si3N4 exhibits an intriguing multi-mechanistic antipathogenic behavior with the potential of solving several of the shortcomings of the alternative approaches to environmentally friendly agrochemistry. The broad-spectrum antipathogenic effectiveness of Si3N4 is due to its nitrogen chemistry. Water acts as a trigger to release nitrogen leading to a cascade of reactions that result in lysis of the pathogen. The nitrogen species generated at the surface of Si3N4 alter the pathogens' proteins, induce nitrosative damage to DNA, and stimulate metabolic enzymes that modify the pathogen membrane structures. These findings are in line with previous studies on human pathogens. The multi-mechanistic lytic reactions occurring in the pathogen's cytoplasm due to the diffusion of NH3 across cell walls reduces the probability of mutations by the pathogen. Moreover, because Si3N4 is used as an implantable biomaterial, it is not toxic to eukaryotic cells. It contains only environmentally friendly elements, which are intrinsic to the earth's prehistory. Several plant species benefit from Si fertilization, particularly in alleviating biotic and abiotic stresses. Ammonium, which is generated by Si3N4 decomposition, is the primary inorganic species involved in the synthesis of organic nitrogen. Ammonium and nitrate ions in the soil are directly absorbed through root-specific transporters and effectively utilized. In the case of grapevines, NH4+ represents up to 80% of the total nitrogen before veraison while decreasing to 5-10% after maturation and even lower after must fermentation. Nitrogen eluted from Si3N4 may contribute to an improvement in berry quality and fermentation conditions. As a limiting factor, care should be taken to balance the quantities of nitrogen from fertilizers and Si3N4 because excess nitrogen may alter the production of phenolic compounds and the taste or quality of both grapes and wine. Several plant species benefit from Si fertilization, particularly in alleviating biotic and abiotic stresses.
These examples provide new insight into the effect of Si3N4 against grapevine infections by P. viticola. As an inorganic environmentally friendly agent, it has the potential to replace heavy metal agrochemicals and newer eco-friendly antipathogenic molecules. The use of Si3N4 is also consistent with current regulatory trends directed at reducing the use of heavy metals in viticulture. The unique chemistry of Si3N4 induces osmotic stress in sporangia and triggers abortion of their immature zoospores even at concentrations as low as 1.5 vol. %, which is in the molar range of concentration used for other inorganic salts in agriculture application, such as bicarbonates. Raman experiments provided important information on chemical mechanisms which included cleavage of phosphate deoxyribose backbone and disruption of guanine rings. Experiments on leaves from different grapevine species showed that Si3N4 was effective in severely reducing or blocking the infection process at very early stages, affecting sporangia germination and zoospores viability, as revealed by microscopic observations. The use of Si3N4 will be most beneficial for grapevines with a high nitrogen requirement, where copper-based formulations are detrimental not only to the environment but also to wine quality. Since Si3N4 only contains environmentally friendly elements, this ceramic will also be a suitable alternative to contact fungicides, which include toxic copper and sulfur elements. Si3N4 can be regarded as a promising biopesticide with multiple benefits in comparison to conventional synthetic products and technical advantages over other inorganic salts and may be a useful component in integrated disease management.
It should be understood from the foregoing that, while particular embodiments have been illustrated and described, various modifications can be made thereto without departing from the spirit and scope of the invention as will be apparent to those skilled in the art. Such changes and modifications are within the scope and teachings of this invention as defined in the claims appended hereto.
This application is a Continuation-in-Part of U.S. application Ser. No. 16/550,605, filed Aug. 26, 2019 that claims the benefit of U.S. Provisional Applications 62/727,724, filed Sep. 6, 2018 and 62/800,034, filed Feb. 1, 2019. This application also claims the benefit of U.S. Provisional Application No. 63/042,859, filed Jun. 23, 2020. The contents of all of which are entirely incorporated by reference herein.
| Number | Date | Country | |
|---|---|---|---|
| 62727724 | Sep 2018 | US | |
| 62800034 | Feb 2019 | US | |
| 63042859 | Jun 2020 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16550605 | Aug 2019 | US |
| Child | 17354120 | US |
