Plant pathogen defense stimulants

Abstract

The present invention relates to the use of cellobionic acid or its pharmaceutically acceptable salt as a plant pathogen defense elicitor and methods of using cellobionic acid or its pharmaceutically acceptable salt as a plant pathogen defense elicitor. Also provided are botanical pharmaceutical compositions comprising cellobionic acid or its pharmaceutically acceptable salt and uses thereof. In certain preferred embodiments, the composition may further comprise other oxidized cellodextrins or may comprise oxidized cellodextrins and natural cellodextrins. In certain preferred embodiments, the composition may be produced by decomposing cellulose with one or more lytic polysaccharide monooxygenases (LPMOs).

Description

Technical Field

[0001] The present invention is broadly in the field of phytodrug compounds and compositions, and in particular relates to stimulators of natural plant defenses (also commonly referred to as plant immune system activator molecules, or in layman's terms "bio-pesticides"), a class of environmentally friendly molecules that prevent the infection and proliferation of microbial pathogens on plants, and their uses. Background Art

[0002] Plant cell walls are dynamic structures composed primarily of high-molecular-weight polysaccharides: cellulose, hemicellulose, and pectin. This complex mixture confers structural integrity and physical protection to plant cells. Plant pathogens, including pathogenic fungi and bacteria, use a variety of strategies to colonize plants and obtain nutrients from their hosts.

[0003] For example, in order to overcome the cell wall, plant pathogens can use a heterogeneous enzyme consortium known as cell wall degrading enzymes (CWDEs) (Kubicek et al. Plant cell wall-degrading enzymes and their secretion in plant-pathogenic fungi. Annu Rev Phytopathol. 2014, vol. 52, 427-51). Although multiple cellulolytic enzymes are needed to break down cellulose into glucose, it has recently been discovered that the Lytic Polysaccharide Monooxygenase (LPMO) family may be the initial participants in this process, paving the way for other hydrolases. LPMOs are copper enzymes that catalyze the oxidative cleavage of glycosidic bonds in recalcitrant polysaccharides (including cellulose and chitin) and are widely distributed in bacteria and fungi. According to the Carbohydrate Active Enzymes database (www.cazy.org), LPMOs are currently classified into seven "auxiliary active" (AA) families (AA9, AA10, AA11, AA13, AA14, AA15, and AA16). LPMO-driven oxidative cleavage of glucans can occur at the C1 or C4 position of the pyranose ring, thus producing a mixture of native and oxidized (C1-oxidized, C4-oxidized, and / or C1 and C4 dual-oxidized) oligosaccharides of varying degrees of polymerization.

[0004] Plant cells have at least two partially overlapping defense layers, which are defined as pattern triggered immunity (PTI) and effector triggered immunity (ETI), which constitute the so-called plant immune system (Jones and Dangl.The plant immune system.Nature.2006, vol.444 (7117), 323-9).ETI confers narrow strain-specific resistance because it is initiated after cytoplasmic resistance genes (R genes) recognize toxic effectors (avirulence proteins). This typically results in a strong site-specific accumulation of reactive oxygen species (ROS), leading to apoptosis. In contrast, PTI provides broad-spectrum protection. Evolutionarily conserved pathogen-associated molecular-patterns (PAMPs) are sensed by plants through a large number of plasma membrane-anchored pattern recognition receptors (PRRs) (Albert et al. Surface Sensor Systems in Plant Immunity. Plant Physiol. 2020, vol. 182(4), 1582-1596). For example, flagellin (FLG22) and chitin (two well-characterized PAMPs) are sensed by leucine-rich repeat receptor-like kinases (LRR-RLKs), such as FLS2 and CERK1, respectively. In addition, plants can trigger PTI by recognizing damage-associated molecular patterns (DAMPs) (Boller and Felix. Arenaissance of elicitors: perception of microbe-associated molecular patterns and danger signals by pattern-recognition receptors. Annu Rev Plant Biol. 2009, vol. 60, 379-406; Jones and Dangl. 2006, supra). DAMPs can include certain cell wall-derived molecules and de novo synthesized stress-related peptides.In particular, pectin fragments and oligogalacturonides (OG) are known to be important DAMPs recognized by cell wall-associated kinase (WAK) receptors (Kohorn et al. Cell Wall-Associated Kinases and Pectin Perception. J Exp Bot. 2016, vol. 67(2), 489-94; Ferrari et al. Oligogalacturonides: plant damage-associated molecular patterns and regulators of growth and development. Front. Plant Sci. 2013, vol. 4, 49). Common signaling events that underlie PTI include Ca. 2+ PTI activates the mitogen-activated protein kinase (MAPK) cascade, activates the apoplast reactive oxygen species (ROS) accumulation, and extensive transcriptional reprogramming involving transcription factors (Yu et al. From Chaos to Harmony: Responses and Signaling upon Microbial Pattern Recognition. Annu Rev Phytopathol. 2017, vol. 55, 109-137). Along with the transcriptional response during PTI, plants produce different signaling hormones, such as salicylic acid (SA), jasmonic acid (JA), and ethylene (ET), and callose deposition occurs on the cell wall a few hours after PTI activation.

[0005] Compounds that produce such a defense response when perceived by plants are often referred to as plant pathogen defense elicitors, plant elicitors, or simply elicitors. Previously, the elicitor properties of oligogalacturonosides (OGs), a class of DAMPs derived from pectin hydrolysis catalyzed by polygalacturonase (PG), have been studied (Ferrari et al. 2013, supra). Only recently, other cell wall-derived compounds, in particular cellobiose, xyloglucan and non-branched β-1,3-glucan, have been identified as DAMPs (Souza et al. Cellulose-derived oligomers act as damage-associated molecular patterns and trigger defense-like responses. Plant Physiol. 2017, vol. 173, 2383-2398; Claverie et al. The Cell Wall-Derived Xyloglucan Is a New DAMP Triggering Plant Immunity in Vitis vinifera and Arabidopsis thaliana. Front. Plant Sci. 2018, vol. 9, 1725; Melida, et al. Non-branched β-1,3-glucan oligosaccharides trigger immune responses in Arabidopsis. Plant J. 2018, vol. 93(1), 34-49).

[0006] The agricultural industry is engaged in a relentless battle against plant pathogens, a struggle exacerbated by the changing environment caused by climate change, striving to avoid significant annual economic losses and uncertainty in the food supply chain. While only a few chemicals are commonly used as insecticides or fungicides, even this reserve is dwindling due to the emergence of biotic resistance in plant pathogens and the adverse effects of some of these chemical compounds on human health. Therefore, there is an urgent need for new plant protection compounds and compositions. Summary of the Invention

[0007] As demonstrated in the experimental section describing certain illustrative embodiments of the invention, the inventors have shown for the first time that cellobionic acids act as potent plant defense elicitors. Furthermore, the inventors have shown that compositions comprising oxidized cellodextrins, as well as compositions comprising both natural and oxidized cellodextrins, such as, in particular, compositions comprising one or more natural cellodextrins in combination with one or more C1 oxidized cellodextrins, one or more C4 oxidized cellodextrins, and / or one or more C1 and C4 oxidized cellodextrins (i.e., doubly oxidized cellodextrins), exhibit advantageous plant elicitor potential. In certain instances, the inventors have shown potent plant elicitor activity of mixtures of natural and oxidized cellodextrins produced by the breakdown of cellulose with one or more lytic polysaccharide monooxygenases (LPMOs).

[0008] Thus, one aspect of the present invention provides the use of cellobionic acid or a plant pharmaceutically acceptable salt thereof as a plant pathogen defense stimulant. A related aspect relates to a method for activating a plant's defense against plant pathogens, comprising contacting the plant with an effective amount of cellobionic acid or a plant pharmaceutically acceptable salt thereof. On the other hand, a botanical pharmaceutical composition is provided, comprising cellobionic acid or a plant pharmaceutically acceptable salt thereof, and a plant pharmaceutically acceptable carrier. In a particularly preferred embodiment, the carrier may comprise an anionic surfactant, a nonionic surfactant, an amphoteric surfactant and / or a cationic surfactant. Thus, a botanical pharmaceutical composition is particularly provided, comprising cellobionic acid or a plant pharmaceutically acceptable salt thereof, and a plant pharmaceutically acceptable carrier, wherein the plant pharmaceutical carrier comprises or is selected from one or more anionic surfactants, nonionic surfactants, amphoteric surfactants or cationic surfactants, or a combination thereof.

[0009] Another aspect of the present invention provides for the use of (i) one or more cellodextrins (i.e., natural or non-oxidized cellodextrins) and (ii) one or more C1 oxidized cellodextrins (aldonic acids) or their phytologically acceptable salts, one or more C4 oxidized cellodextrins (4-ketouledose or geminal diols), and / or one or more C1 and C4 oxidized cellodextrins (4-keto-oligosaccharide-1-aldonic acids) or their phytologically acceptable salts, e.g., in compositions comprising these components as plant pathogen defense elicitors. A related aspect relates to a method for activating a plant's defense against plant pathogens, comprising contacting the plant with an effective amount of the aforementioned components (i) and (ii). Another aspect provides a botanical pharmaceutical composition comprising the aforementioned components (i) and (ii) and a botanical pharmaceutically acceptable carrier, e.g., a botanical pharmaceutically acceptable carrier comprising one or more anionic, nonionic, amphoteric, or cationic surfactants, or a combination thereof.

[0010] Another aspect of the present invention provides the use of one or more C4 oxidized cellodextrins (i.e., 4-ketoidoses or geminal diols) as plant pathogen defense elicitors. A related aspect relates to a method for activating a plant's defense against plant pathogens, comprising contacting the plant with an effective amount of one or more C4 oxidized cellodextrins. Another aspect provides a botanical pharmaceutical composition comprising one or more C4 oxidized cellodextrins and a botanical pharmaceutically acceptable carrier, such as a botanical pharmaceutically acceptable carrier comprising one or more anionic, nonionic, amphoteric, or cationic surfactants, or combinations thereof.

[0011] Another aspect of the present invention provides the use of one or more C1 and C4 oxidized (i.e., doubly oxidized) cellodextrins (i.e., 4-keto-oligosaccharide-1-aldonic acid), or their phytologically acceptable salts, as plant pathogen defense elicitors. A related aspect relates to a method for activating a plant's defense against plant pathogens, comprising contacting the plant with an effective amount of one or more C1 and C4 oxidized cellodextrins, or their phytologically acceptable salts. Another aspect provides a botanical pharmaceutical composition comprising one or more C1 and C4 oxidized cellodextrins, or their phytologically acceptable salts, and a botanical pharmaceutically acceptable carrier, such as a botanical pharmaceutically acceptable carrier comprising one or more anionic, nonionic, amphoteric, or cationic surfactants, or combinations thereof.

[0012] Also provided are methods and uses of the phytopharmaceutical compositions for eliciting plant protection from pathogens.

[0013] These and other aspects and preferred embodiments of the present invention are described in the following sections and in the appended claims.The subject matter of the appended claims is hereby expressly incorporated into this specification. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 (A) LPMO gene expression during Arabidopsis-B. cinerea interaction. 5 spores ml -1) Five-week-old Arabidopsis plants were drop-treated, and LPMO gene expression was assessed 48 hours after fungal infection; B) Sequence-based comparison of the biochemically and structurally characterized BcAA9 and AA9. A phylogenetic tree was constructed based solely on the alignment of the catalytic modules. The oxidation region specificity is shown for each characterized LPMO (C1, blue; C4, green; C1 / C4, red). AA9 from Botrytis cinerea is marked with an arrow. C) Structural comparison between TtAA9E and BCIN_12g03920 was constructed using the Swiss-Model server and the TtAA9E crystal structure obtained from the Protein Data Bank (PDB: 3EII). The structural model is shown as a ribbon representation, and the copper ions are shown as blue spheres. (D) HPAEC-PAD analysis of products produced by TtAA9E using PASC 0.5% (w / v) as a cellulosic substrate. Glc2: Cellobiose, Glc3: Cellotriose, Glc4: Cellotetraose, Glc5: Cellopentaose, GlcGlc1A: Cellobiosonic acid, Glc2Glc1A: Cellotrisonic acid, Glc3Glc1A: Cellotetrasonic acid, Glc4Glc1A: Cellopentasonic acid, Glc5Glc1A: Cellohexasonic acid, Glc6Glc1A. Celloheptonic acid, Glc7Glc1A: Cellooctosonic acid.

[0015] Figure 2 Figure 2 shows the expression of FRK1 and WRKY18 genes in Arabidopsis thaliana in response to different concentrations of AA9_COS. The expression of FRK1 and WRKY18 genes was evaluated in 14-day-old Arabidopsis plants 1 hour after treatment with increasing concentrations of AA9_COS. Different letters correspond to significantly different expression levels (p < 0.05) according to the Mann-Whitney test.

[0016] Figure 3 Figure 2 shows analysis of early transcriptome changes in Arabidopsis seedlings treated with LPMO product (AA9_COS) and cellobiose. (a) Differentially expressed genes 1 hour after treatment with 100 μM cellodextrin AA9_COS and cellobiose. (b) Principal component analysis of differentially expressed genes (DEGs) induced by AA9_COS, cellobiose, and mock treatment. Two principal components (PC1 and PC2) were used to visualize the overall gene expression similarity between samples, capturing 39% and 19% of the total variation, respectively. (c and d) The results were obtained using the PC1 and PC2 relative to the negative logarithm of the DEGs. 10Volcano plots were generated by plotting gene expression ratios (log2 fold changes) against transformed t-test p values. Data points represent gene expression modulated by AA9_COS (c) or cellobiose (d) treatment, respectively.

[0017] Figure 4 Genes induced by the LPMO product AA9_COS are shown to be associated with pathogen defense. Gene ontology (GO) term enrichment analysis of biological processes associated with the 668 significantly upregulated genes revealed enrichment for plant defense responses. GO analysis was performed using g:Profiler software (Benjamini-Hochberg FDR < 0.05) and the EnrichmentMap function of Cytoscape. Each node represents a pathway, edges represent genes that overlap between nodes, and circle size represents the number of genes included.

[0018] Figure 5 Comparison between transcriptome changes induced by AA9_COS (COS) or by cellobiose (CB) treatment is shown. (a) Hierarchical cluster analysis of gene expression patterns showing significant expression changes (p-value ≤ 0.05 and log2 fold change (log2FC) ≥ 1.5) compared to mock 1 hour after AA9_COS or CB treatment in 14-day-old Col-0 plants. Clusters are divided into upregulated (left panel, except for cluster 5, which showed opposite expression patterns between treatments: upregulated by AA9_COS and downregulated by cellobiose) or downregulated (right panel). (b) Proportion of defense response-related genes with relative GO terms evaluated from each cluster. (c) Heat map of selected defense genes belonging to clusters I, II, and III. (d) Venn diagram showing specific and shared upregulated and up- and downregulated genes induced by AA9_COS and CB treatment. (e) Gene ontology enrichment analysis using PSGEA showed that defense-related genes were upregulated by both AA9_COS and cellobiose treatments. -log2(FDR) indicates the significance of gene ontology enrichment.

[0019] Figure 6 Shown are the effects of AA9_COS and the single components cellobionic acid ("Bionic") or cellotrionic acid ("Trionic") on the activation of three marker genes (CYB81F2, RBOHD, and WRKY30).

[0020] Figure 7Correlation between microarray and qPCR data is shown. For a subset of 10 differentially expressed genes in Arabidopsis, the fold changes in gene expression determined by qPCR and by cDNA microarray are plotted. 2 and FDR indicated good correlation between real-time PCR and microarray results.

[0021] Figure 8 AA9_COS does not trigger oxidative burst. Representative images of leaves stained with DAB (a) or NBT (b) from 4-5 week-old Arabidopsis wild-type plants 24 hours after treatment are provided. (c) Luminol-based assays for hydrogen peroxide detection show that neither AA9_COS nor cellobiose treatment triggers detectable oxidative burst formation in Arabidopsis plants. The positive control H2O2 curve is marked. Overlapping curves representing different treatments are marked with "Other." MV (methyl viologen) and NaCl (sodium chloride) were used as positive controls.

[0022] Figure 9 The results show that AA9_COS induces resistance to Botrytis cinerea. 5 spores / mL) were spotted on detached leaves from 5-week-old Arabidopsis plants. (a) Photos were taken three days after infection, and the lesion area was determined using ImageJ (b). (c) Three days after inoculation, in-plant growth of Botrytis cinerea was determined by qPCR using housekeeping genes specific to Arabidopsis thaliana (AtSKII) and Botrytis cinerea (BcTUB). (d) Callose was determined in 5-week-old plants 6 and 24 hours after treatment. (e) Representative photos 24 hours after the indicated treatments, with bars representing 500 μm.

[0023] Figure 10 Figure 2 shows that treatment with cellodextrin triggers camalexin signaling in Arabidopsis. (a) Expression of genes involved in camalexin signaling and biosynthesis was evaluated in 14-day-old Arabidopsis plants 1 hour and 24 hours after treatment with 100 μM AA9_COS or 100 μM cellobiose. Median values ​​are plotted in boxes, with data generated from two independent biological replicates. Different letters correspond to significantly different expression levels according to the Mann-Whitney test (significance set to p < 0.05), (b) proposed model for AA9_COS regulation of camalexin biosynthesis and signaling, (c) camalexin quantification, and (d) early immunohistochemical quantification or Western blot analysis of MPK3 / 6 phosphorylation after the specified treatment.

[0024] Figure 11Shown is gene expression of selected marker genes in SIF2 knockout mutant plants ("SIF2") 1 hour after treatment with 100 μM AA9_COS cellodextrin ("CD"). Wild type ("Col-0") and mock ("Mock") controls are included.

[0025] Figure 12 Shown are the plant elicitor activities of different concentrations of AA9_COS in tomato.

[0026] Figure 13 Shown are the plant elicitor activities of cellobiose vs. AA9_COS in tomato.

[0027] Figure 14 The plant elicitor activity of natural cellodextrins, and several combinations of C1 oxidized, C4 oxidized, and optionally C1 and C4 oxidized (double oxidized) cellodextrins is shown.

[0028] Figure 15 Ethylene emanation is shown.

[0029] Figure 16 Plant hormone quantification is shown. (Top) Salicylic acid (SA) and jasmonic acid (JA) content was quantified by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS / MS). Three pools (n=3) of fifteen leaves from three five-week-old Arabidopsis plants were treated with 100 μM AA9_COS or mock, and the leaves were collected, pooled, and immediately frozen in liquid nitrogen 24 hours after treatment. JA and SA were extracted and quantified for each independent pool. Bars represent the mean and standard deviation of the three pools, and asterisks indicate significant differences (one-way ANOVA test). (Bottom) Ethylene was measured in a time course analysis using square dishes containing 100 14-day-old seedlings each. Bars represent the mean and standard error of eight independent square dishes (n=8). Asterisks indicate significant differences (one-way ANOVA test). DW: dry weight. FW: fresh weight. DETAILED DESCRIPTION

[0030] As used herein, unmodified nouns mean one or more unless the context clearly indicates otherwise.

[0031] As used herein, the term "comprising" is synonymous with "including" or "containing" and is inclusive or open-ended and does not exclude additional, unrecited members, elements, or method steps. The term also encompasses "consisting of" and "consisting essentially of," which have recognized meanings in patent terminology.

[0032] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the corresponding ranges, as well as the recited endpoints. This applies to numerical ranges regardless of whether they are introduced by the phrase "from to ," the phrase "between and ," or another phrase.

[0033] As used herein, the term "about" or "approximately" when referring to a measurable value, such as a parameter, amount, duration, etc., is intended to encompass the specified value and variations relative to the specified value, such as variations of + / -10% or less, preferably + / -5% or less, more preferably + / -1% or less, and still more preferably + / -0.1% or less relative to the specified value, within the range that such variations are suitable for use in the disclosed invention. It should be understood that the value to which the modifier "about" or "approximately" refers is also specifically and preferably disclosed itself.

[0034] While the term "one or more" or "at least one" (e.g., one or more members or at least one member of a group of members) is self-explanatory, by way of further illustration, the term specifically encompasses reference to any one of the members, or to any two or more of the members, such as any ≥3, ≥4, ≥5, ≥6, or ≥7 of the members, and up to all of the members. In another example, "one or more" or "at least one" may refer to 1, 2, 3, 4, 5, 6, 7, or more.

[0035] The discussion of the background to the invention is included to explain the context of the invention. This should not be taken as an admission that any of the material referred to was published, known or part of the common general knowledge in any country as at the priority date of any claim.

[0036] Throughout this disclosure, various publications, patents, and disclosed patent specifications are cited by reference. All documents cited in this specification are hereby incorporated by reference in their entirety. In particular, the teachings or portions of such documents specifically mentioned herein are incorporated by reference.

[0037] Unless otherwise specified, all terms (including technical and scientific terms) used to disclose the present invention have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. By way of further guidance, term definitions are included to better understand the teachings of the present invention. Unless otherwise specified, when specific terms are defined in conjunction with specific aspects of the present invention or specific embodiments of the present invention, such connotations or meanings are intended to apply throughout this specification, i.e., also in the context of other aspects or embodiments of the present invention.

[0038] In the following paragraphs, different aspects or embodiments of the present invention are defined in more detail. Unless expressly indicated to the contrary, each aspect or embodiment so defined may be combined with any other aspect or embodiment. In particular, any feature indicated as preferred or advantageous may be combined with any other one or more features indicated as preferred or advantageous.

[0039] Throughout this specification, references to "one embodiment" or "an embodiment" mean that the described particular features, structures, or characteristics associated with the embodiment are included in at least one embodiment of the present invention. Thus, the phrases "in one embodiment" or "in an embodiment" appearing in multiple places throughout this specification are not necessarily all but may refer to the same embodiment. Furthermore, in one or more embodiments, as will become apparent to those skilled in the art from this disclosure, the particular features, structures, or characteristics may be combined in any suitable manner. Furthermore, while some embodiments described herein include some features but do not include other features included in other embodiments, as will be appreciated by those skilled in the art, combinations of features from different embodiments are intended to be within the scope of the present invention and to form different embodiments. For example, in the appended claims, any of the claimed embodiments may be used in any combination.

[0040] Aspects of the present invention relate to the use of:

[0041] Cellobionic acid or a plant pharmaceutically acceptable salt thereof;

[0042] Compositions comprising (i) one or more cellodextrins (i.e., natural or non-oxidized cellodextrins) and (ii) one or more C1 oxidized cellodextrins or their plant-pharmaceutically acceptable salts, one or more C4 oxidized cellodextrins, and / or one or more C1 and C4 oxidized cellodextrins or their plant-pharmaceutically acceptable salts;

[0043] one or more C4 oxidized cellodextrins; or

[0044] One or more C1 and C4 oxidized cellodextrins or their pharmaceutically acceptable salts.

[0045] Also provided are corresponding methods for activating plant defenses against plant pathogens, comprising contacting the plant with an effective amount of such a substance or composition, as well as phytopharmaceutical compositions comprising said substance and a phyto-pharmaceutical carrier. Such methods can also be considered as methods for obtaining plants resistant to pathogens.

[0046] Cellobionic acid is a disaccharide composed of β-D-glucosyl and D-gluconic acid groups linked together by a (1→4) bond. Chemical names include 4-O-β-D-pyranosyl-D-gluconic acid, 4-O-β-D-glucosyl-D-gluconic acid, β-D-glucosyl-(1→4)-D-gluconic acid, and maltobionic acid. Therefore, this compound is a disaccharide and a carbohydrate acid, showing the molecular formula C 12 H 22 O 12 and can be represented by any of the representative structural formulas (I) or (II):

[0047]

[0048] Therefore, cellobiose acid can also be considered as the C1-oxidized form of cellobiose, or the aldonic acid counterpart of cellobiose. Cellobiose is a disaccharide composed of two glucose units linked together by a β(1→4) bond, namely β-D-glucopyranosyl(1→4)D-glucopyranose, with the molecular formula C 12 H 22 O 11 and can be represented by either of the representative structural formulas (III) or (IV):

[0049]

[0050] The term "cellodextrin" refers to any one of two or more low molecular weight carbohydrates composed of two or more β (1 → 4) linked glucose monomers or a group or mixture thereof. Thus, cellodextrins may be referred to as linear β-1,4-D-glucose oligosaccharides. Cellodextrins can typically be produced by the hydrolysis of cellulose, for example, in particular by cellulase hydrolysis. Thus, for example, cellobiose is a cellodextrin composed of two β (1 → 4) linked glucose monomers, while cellotriose, cellotetraose and cellopentaose are cellodextrins composed of three, four and five β (1 → 4) linked glucose monomers, respectively. The number of linked glucose monomers in the cellodextrin, i.e., the degree of polymerization (DP) of the cellodextrin, can be variable, for example, but not limited to, from 2 to 20, or from 2 to 15, or from 2 to 10, such as, for example, 2, 3, 4, 5, 6, 7, 8, 9 or 10. For example, a population or mixture of cellodextrins can include cellodextrins with various degrees of polymerization, such as cellodextrins with a DP of 2 to 20, or 2 to 15, or 2 to 10, or 2 to 8. By way of example, and not limitation, a population or mixture of cellodextrins can have a number average or weight average degree of polymerization of 2 to 10, such as 3 to 7, for example, about 3, about 4, about 5, about 6, or about 7.

[0051] Thus, the term "cellodextrin," when used herein without further qualification, specifically refers to natural cellodextrins or a population or mixture of natural cellodextrins. "Natural" in this context means that neither the free anomeric carbon atom at position 1 (C1) of the glucose moiety at the reducing end of the cellodextrin nor the carbon at position 4 (C4) of the glucose moiety at the non-reducing end of the cellodextrin has been further oxidized. In other words, in natural cellodextrins, the glucose moiety at the reducing end of the cellodextrin can be represented as follows (Formula V),

[0052]

[0053] And the glucose portion of the non-reducing end of cellodextrin can be represented as follows (Formula VI):

[0054]

[0055] The dashed line indicates the point of attachment (via -O-) to the rest of the cellodextrin molecule.

[0056] Reference to "oxidized cellodextrin" encompasses cellodextrins in which at least one carbon atom is further oxidized, such as, in particular, a carbon atom bearing a hydroxyl (-OH) group is further oxidized to a carbon atom bearing an oxygen (=O) group or two hydroxyl (-OH) groups, or a hemiacetal carbon is further oxidized to a carboxyl (-COOH) group. In particular, oxidized cellodextrins encompass C1 oxidized cellodextrin (aldonic acid), C4 oxidized cellodextrin (4-ketaldose or geminal diol), and C1 and C4 oxidized cellodextrins (dioxidized cellodextrin, 4-keto-oligosaccharide-1-aldonic acid).

[0057] In C1 oxidized cellodextrins, the anomeric carbon atom at position 1 (C1) of the glucose moiety at the reducing end of the cellodextrin is oxidized to a carboxyl group, providing a gluconic acid moiety that can be represented as follows (Formula VII):

[0058]

[0059] The dotted line represents the point of attachment to the remainder of the C1 oxidized cellodextrin molecule (via an -O- linkage). Non-limiting examples of C1 oxidized cellodextrins include linear oligomers of the following structure: (D-glucosyl-(1→4)) n (1→4)-D-gluconic acid, where n is the degree of polymerization minus 1, for example, where n is 1, 2, 3, 4, 5, or 6. Non-limiting examples of C1 oxidized cellodextrins include cellobicoic acid (DP=2), cellotrioic acid (DP=3), cellotetraic acid (DP=4), cellopentacoic acid (DP=5), and the like, for example, DP=6, 7, 8, 9, or 10.

[0060] In C4 oxidized cellodextrins, the carbon atom at position 4 (C4) of the glucose moiety at the non-reducing end of the cellodextrin is present as a keto (-C(=O)-) or geminal diol (-C(-OH)2-) group, providing a moiety that can be represented as follows (Formulas VIII and IX):

[0061]

[0062] The dotted line represents the point of attachment to the remainder of the C4 oxidized cellodextrin molecule (via an -O- linkage). Non-limiting examples of C4 oxidized cellodextrins include linear oligomers of the following structure: 4-dehydro-β-D-glucosyl-(1→4)(β-D-glucosyl-(1→4)) n , where n is the degree of polymerization minus 1, for example, where n is 1, 2, 3, 4, 5, or 6. Non-limiting examples of C4 oxidized cellodextrins include 4-ketobiose (DP=2), 4-ketotriose (DP=3), 4-ketotetraose (DP=4), 4-ketopentaose (DP=5), and the like, for example, DP=6, 7, 8, 9, or 10.

[0063] In C1 and C4 oxidized (i.e., doubly oxidized) cellodextrins, both of the above situations apply. Non-limiting examples of C1 and C4 oxidized cellodextrins include linear oligomers of the following structure: 4-dehydro-β-D-glucosyl-(1→4)(β-D-glucosyl-(1→4)) m (1→4)-D-gluconic acid, where m is the degree of polymerization minus 2, for example, where m is 0, 1, 2, 3, 4, or 5. Such molecules can also be represented as 4-keto-disaccharide-1-aldonate (DP=2), 4-keto-triose-1-aldonate (DP=3), 4-keto-tetraose-1-aldonate (DP=4), 4-keto-pentaose-1-aldonate (DP=5), 4-keto-hexose-1-aldonate (DP=6), etc., for example, DP=7, 8, 9, or 10.

[0064] In C1-oxidized, C4-oxidized, and C1 and C4-oxidized cellodextrins as contemplated herein, the oxidation state of carbon atoms other than the C4 carbon of the non-reducing 4-ketoglucose moiety and / or the C1 carbon of the gluconic acid moiety is preferably the same as that in native cyclodextrins.

[0065] Reference to any compound contemplated herein (e.g., cellobionic acid or natural or oxidized cellodextrin) encompasses a given compound and any phytologically acceptable form of such a compound, such as any addition salt, hydrate, or solvate of the compound. The term "phytologically acceptable" as used herein, particularly in connection with a form of the compound such as a salt, hydrate, solvate, and in connection with a carrier (e.g., a support, a solvent, etc.), is consistent with the art and means that the compound is harmless to the recipient plant, e.g., does not produce any adverse effects when applied to a plant or plant organ, and, where applicable, is compatible with any other ingredients of the phytological pharmaceutical composition. Phytologically acceptable acid and base addition salts are meant to encompass the phytologically active non-toxic acid and base addition salt forms that the compound is able to form. Such salts can be conveniently obtained by treating the base form of the compound with an appropriate acid. Suitable acids include, for example, inorganic acids, such as hydrohalic acids, for example, hydrochloric acid or hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, or organic acids, such as, for example, acetic acid, propionic acid, ascorbic acid, gallic acid, glycolic acid, lactic acid, pyruvic acid, malonic acid, succinic acid (i.e., succinic acid), maleic acid, fumaric acid, dehydroascorbic acid, malic acid, tartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cycloamine, salicylic acid, p-aminosalicylic acid, pamoic acid, coumaric acid, ferulic acid, caffeic acid, sinapinic acid, cinnamic acid, resveratrol, catechin, 3-hydroxy-anthranilic acid, L-DOPA, melanin, eumelanin, lignin, vanillic acid, vanillin, etc. Conversely, the salt form can be converted into the free base form by treatment with an appropriate base. Compounds containing acidic protons (e.g., cellobionic acid, aldonic acid) can also be converted into their non-toxic metal or amine addition salt forms by treatment with suitable organic and inorganic bases. Suitable basic salt forms include, for example, ammonium salts, alkali metal and alkaline earth metal salts, such as lithium, sodium, potassium, magnesium, calcium salts, etc., aluminum salts, zinc salts, salts with organic bases, such as primary, secondary and tertiary aliphatic and aromatic amines, such as methylamine, ethylamine, propylamine, isopropylamine, tetrabutylamine isomers, dimethylamine, diethylamine, diethanolamine, dipropylamine, diisopropylamine, di-n-butylamine, pyrrolidine, piperidine, morpholine, trimethylamine, triethylamine, tripropylamine, quinuclidine, pyridine, quinoline and isoquinoline; benzathine, N-methyl-D-glucamine, hydrazine-amine salts and salts with amino acids (e.g., arginine, lysine, etc.). Conversely, salt forms can be converted into free acid forms by treatment with acid. The term solvate comprises hydrates and solvent addition forms, and salts thereof, which the compounds are able to form. Examples of such forms are, for example, hydrates, alcoholates, and the like.

[0066] Any compound expected herein can be a part of a composition.The term "composition" generally refers to a thing consisting of two or more components, and more particularly specifically refers to a mixture or blend of two or more materials (such as elements, molecules, substances, biomolecules or microbial materials), and reaction products and decomposition products formed by the material of the composition. For example, a composition can include any compound taught herein in combination with one or more other compounds or substances (whether it is one or more other compounds taught herein or one or more other compounds or substances). For example, a composition can be obtained by combining (such as mixing) a compound taught herein with one or more other compounds or substances. For example, a composition can be obtained by decomposing starting materials such as cellulose into a mixture of multiple decomposition products. In certain embodiments, the present composition can be configured as a plant drug composition. A plant drug composition generally includes one or more active ingredients (chemical and / or biologically active substances having one or more beneficial effects on plant health) and one or more plant pharmaceutically acceptable carriers. The compositions generally used herein can be liquid, semi-solid (e.g., gel), solid or volatile or vapor-based, and can include solutions or dispersions, such as suspensions, emulsions, oil-in-water emulsions, water-in-oil emulsions, gelled aqueous solutions or dispersions, solutions containing volatile organic solvents, etc. Examples of solid forms include, but are not limited to, powders, granules, pellets, water-dispersible powders, water-dispersible granules, or water-dispersible pellets. The compositions can be formulated as concentrates that are diluted prior to use, such as, for example, soluble concentrates, emulsifiable concentrates, liquid concentrates, etc.

[0067] As used herein, the term "carrier" is intended to include in a broad sense any and all solvents, diluents, fillers, buffers for pH control, dispersants, solubilizers, surfactants, wetting agents, emulsifiers, viscosity-increasing agents, thickeners, binders, preservatives, antioxidants, epidermolytic molecules, natural or regenerated minerals, and the like, and combinations thereof. Such materials should be non-toxic to plants and should not interfere with the activity of the active ingredients.

[0068] A preferred example of a plant-based pharmaceutically acceptable solvent is water, and thus, the compositions taught herein may comprise water, i.e., may be aqueous solutions or dispersions. Other examples of suitable solvents include, but are not limited to, aromatic hydrocarbons, such as, for example, a xylene mixture or a substituted naphthalene; phthalates, such as, for example, dibutyl phthalate or dioctyl phthalate; aliphatic hydrocarbons, such as, for example, cyclohexane or paraffin; alcohols and glycols and their ethers and esters, such as, for example, ethanol, ethylene glycol, ethylene glycol monomethyl ether or monoethyl ether; ketones, such as, for example, cyclohexanone; highly polar solvents, such as, for example, N-methyl-2-pyrrolidone, dimethyl sulfoxide or dimethylformamide; vegetable oils or epoxidized vegetable oils, such as, for example, epoxidized coconut oil or soybean oil; and water. In a particular aspect, the solvent is a volatile solvent, such as methanol and ethanol.

[0069] Some non-limiting examples of solid carriers include, but are not limited to, natural mineral fillers such as, for example, calcite, talc, kaolin, montmorillonite or attapulgite; highly dispersed silicic acid or highly dispersed absorbent polymers; pumice, broken brick, sepiolite or bentonite; calcite or sand; dolomite or powdered plant residues.

[0070] In certain embodiments, the composition may include one or more surfactants, such as anionic surfactants, nonionic surfactants, amphoteric surfactants, or cationic surfactants, or combinations thereof, such as, but not limited to, Triton X-100 (C 14 H 22 O(C2H4O)n, nonionic surfactants having a hydrophilic polyethylene oxide chain (e.g., an average of 9.5 ethylene oxide units) and an aromatic hydrocarbon hydrophobic group 4-(1,1,3,3-tetramethylbutyl)-phenyl); polysorbate-type nonionic surfactants, such as polyoxyethylene (20) sorbitan monolaurate (Tween-20), polyoxyethylene (20) sorbitan monopalmitate (Tween-40); and / or nonionic organosiloxane surfactants, such as L-77 (3-(2-methoxyethoxy)propyl-methyl-bis(trimethylsiloxy)silane).

[0071] In certain embodiments, cellobionic acid or a salt thereof can be included in a composition that also includes one or more cellodextrins; one or more C1 oxidized cellodextrins or plant-pharmaceutically acceptable salts thereof; one or more C4 oxidized cellodextrins; and / or one or more C1 and C4 oxidized cellodextrins or plant-pharmaceutically acceptable salts thereof.

[0072] In certain embodiments, one or more C4 oxidized cellodextrins can be included in a composition that also includes one or more cellodextrins; one or more C1 oxidized cellodextrins or plant pharmaceutically acceptable salts thereof; and / or one or more C1 and C4 oxidized cellodextrins or plant pharmaceutically acceptable salts thereof.

[0073] In certain embodiments, one or more C1 and C4 oxidized cellodextrins or salts thereof may be included in a composition that also includes one or more cellodextrins; one or more C1 oxidized cellodextrins or plant pharmaceutically acceptable salts thereof; and / or one or more C4 oxidized cellodextrins.

[0074] In certain illustrative embodiments, the composition may include the components described in the following table, where '+' indicates presence, '-' indicates absence, 'A' indicates cellobionic acid or a plant pharmaceutically acceptable salt thereof, 'B' indicates one or more natural cellodextrins, 'C' indicates one or more C1 oxidized cellodextrins other than cellobionic acid or a plant pharmaceutically acceptable salt thereof, 'D' indicates one or more C4 oxidized cellodextrins, and 'E' indicates one or more C1 and C4 oxidized cellodextrins or plant pharmaceutically acceptable salts:

[0075] Composition A B C D E 1 + - - - - 2 + + - - - 3 + - + - - 4 + + + - - 5 + + + + - 6 + + + + + 28 + - - + - 29 + + - + - 30 + + + - + 31 + - + + - 32 + - + + - 33 + - - + + 34 + + + + + 35 + - - - + 36 + - + + +

[0076] In certain illustrative embodiments, the composition may include the components described in the following table, where '+' indicates presence, '-' indicates absence, 'A' indicates one or more natural cellodextrins, 'B' indicates one or more C1 oxidized cellodextrins or plant pharmaceutically acceptable salts thereof, 'C' indicates one or more C4 oxidized cellodextrins, and 'D' indicates one or more C1 and C4 oxidized cellodextrins or plant pharmaceutically acceptable salts thereof:

[0077] Composition A B C D 7 + + - - 8 + - + - 9 + - - + 10 + + + - 11 + + - + 12 + - + +

[0078] Composition A B C D 13 + + + +

[0079] In certain illustrative embodiments, the composition may include the components described in the following table, where '+' indicates presence, '-' indicates absence, 'A' indicates one or more C4 oxidized cellodextrins, 'B' indicates one or more natural cellodextrins, 'C' indicates one or more C1 oxidized cellodextrins or their plant pharmaceutically acceptable salts, and 'D' indicates one or more C1 and C4 oxidized cellodextrins or their plant pharmaceutically acceptable salts:

[0080] Composition A B C D 14 + + - - 15 + - + - 16 + - - + 17 + + + - 18 + + - + 19 + - + + 20 + + + +

[0081] In certain illustrative embodiments, the composition may include the components described in the following table, where '+' indicates presence, '-' indicates absence, 'A' indicates one or more C1 and C4 oxidized cellodextrins or their plant pharmaceutically acceptable salts, 'B' indicates one or more natural cellodextrins, 'C' indicates one or more C1 oxidized cellodextrins or their plant pharmaceutically acceptable salts, and 'D' indicates one or more C4 oxidized cellodextrins:

[0082] Composition A B C D 21 + + - - 22 + - + - 23 + - - + 24 + + + - 25 + + - + 26 + - + + 27 + + + +

[0083] In certain preferred embodiments, the degree of polymerization (DP) of any of the cellodextrins, C1 oxidized cellodextrins, C4 oxidized cellodextrins, and C1 and C4 oxidized cellodextrins taught and used herein may each independently be from 2 to 20, e.g., from 2 to 15, or from 2 to 10, or from 2 to 8, or from 2 to 5. In certain preferred embodiments, the number average or weight average degree of polymerization of a population or mixture of cellodextrins, C1 oxidized cellodextrins, C4 oxidized cellodextrins, and C1 and C4 oxidized cellodextrins taught and used herein may each independently be from 2 to 10, e.g., from 3 to 7, such as, for example, about 3, about 4, about 5, about 6, or about 7.

[0084] In certain embodiments, the concentration of cellobionic acid or a salt thereof (e.g., in the (phytodrug) composition taught herein) may be 0.10 μM to 1000 μM, such as 0.10 μM to 900 μM, 0.10 μM to 800 μM, 0.10 μM to 700 μM, or 0.10 μM to 600 μM, preferably 0.10 μM to 500 μM, such as 0.10 μM to 400 μM. M, or 0.10 μM to 300 μM, more preferably 0.10 μM to 200 μM, even more preferably 0.10 μM to 100 μM, for example about 0.10 μM, about 0.50 μM, about 1.0 μM, about 5.0 μM, about 10 μM, about 20 μM, about 30 μM, about 40 μM, about 50 μM, about 60 μM, about 70 μM, about 80 μM, about 90 μM or about 100 μM.

[0085] In certain embodiments, the concentration of one or more C1 oxidized cellodextrins or salts thereof (which may include cellobionic acid or a salt thereof) (e.g., in the (phytodrug) compositions taught herein) may be 0.10 μM to 1000 μM, such as 0.10 μM to 900 μM, 0.10 μM to 800 μM, 0.10 μM to 700 μM, or 0.10 μM to 600 μM, preferably 0.10 μM to 500 μM, e.g. 0.10 μM to 400 μM, or 0.10 μM to 300 μM, more preferably 0.10 μM to 200 μM, even more preferably 0.10 μM to 100 μM, for example about 0.10 μM, about 0.50 μM, about 1.0 μM, about 5.0 μM, about 10 μM, about 20 μM, about 30 μM, about 40 μM, about 50 μM, about 60 μM, about 70 μM, about 80 μM, about 90 μM or about 100 μM.

[0086] In certain embodiments, the concentration of one or more C4 oxidized cellodextrins (e.g., in the (phytodrug) compositions taught herein) may be 0.10 μM to 1000 μM, such as 0.10 μM to 900 μM, 0.10 μM to 800 μM, 0.10 μM to 700 μM, or 0.10 μM to 600 μM, preferably 0.10 μM to 500 μM, such as 0.10 μM to 4 00 μM, or 0.10 μM to 300 μM, more preferably 0.10 μM to 200 μM, even more preferably 0.10 μM to 100 μM, for example about 0.10 μM, about 0.50 μM, about 1.0 μM, about 5.0 μM, about 10 μM, about 20 μM, about 30 μM, about 40 μM, about 50 μM, about 60 μM, about 70 μM, about 80 μM, about 90 μM or about 100 μM.

[0087] In certain embodiments, the concentration of one or more C1 and C4 oxidized cellodextrins (e.g., in the (phytodrug) compositions taught herein) may be 0.10 μM to 1000 μM, such as 0.10 μM to 900 μM, 0.10 μM to 800 μM, 0.10 μM to 700 μM, or 0.10 μM to 600 μM, preferably 0.10 μM to 500 μM, such as 0.10 μM to 1000 μM. 400 μM, or 0.10 μM to 300 μM, more preferably 0.10 μM to 200 μM, even more preferably 0.10 μM to 100 μM, for example about 0.10 μM, about 0.50 μM, about 1.0 μM, about 5.0 μM, about 10 μM, about 20 μM, about 30 μM, about 40 μM, about 50 μM, about 60 μM, about 70 μM, about 80 μM, about 90 μM or about 100 μM.

[0088] In certain embodiments, the total concentration of cellodextrins, C1 oxidized cellodextrins including cellobionic acid, C4 oxidized cellodextrins, and C1 and C4 oxidized cellodextrins (e.g., in the (phytodrug) compositions taught herein) can be from 0.10 μM to 1000 μM, e.g., from 0.10 μM to 900 μM, from 0.10 μM to 800 μM, from 0.10 μM to 700 μM, or from 0.10 μM to 600 μM, preferably from 0.10 μM to 500 μM. 00 μM, for example, 0.10 μM to 400 μM or 0.10 μM to 300 μM, more preferably 0.10 μM to 200 μM, even more preferably 0.10 μM to 100 μM, for example about 0.10 μM, about 0.50 μM, about 1.0 μM, about 5.0 μM, about 10 μM, about 20 μM, about 30 μM, about 40 μM, about 50 μM, about 60 μM, about 70 μM, about 80 μM, about 90 μM or about 100 μM.

[0089] The present inventors have discovered that natural cello dextrins, C1 oxidized cello dextrins, and C4 oxidized cello dextrins, and optionally combinations or mixtures of C1 and C4 oxidized cello dextrins, exhibit advantageous plant elicitor activity when included in a wide variety of relative molar ratios.

[0090] In certain particularly preferred embodiments, a strong activator effect is observed with a combination comprising a molar ratio of natural cellodextrin: C1 oxidized cellodextrin: C4 oxidized cellodextrin of about 4:5:1. In such a combination, the absence of C1 and C4 oxidized (i.e., doubly oxidized) cellodextrins is preferred.

[0091] Many other molar amounts are possible in the context of the present invention, such as, but not limited to, about 6.5:3:0.5, about 3:6.5:0.5, about 4:3:3, about 4:1:5, about 0.5:9:0.5 (molar ratio of natural fiber dextrin: C1 oxidized fiber dextrin: C4 oxidized fiber dextrin) or about 0.5:4:0.5:5, about 2.5:2.5:2.5 (molar ratio of natural fiber dextrin: C1 oxidized fiber dextrin: C4 oxidized fiber dextrin: C1 and C4 oxidized fiber dextrins).

[0092] By way of further non-limiting example, in a composition comprising natural cello dextrin, C1 oxidized cello dextrin, and C4 oxidized cello dextrin:

[0093] 0.25 to 9.50 mole parts may be natural fiber dextrin, 0.25 to 9.50 mole parts may be C1 oxidized fiber dextrin, and 0.25 to 9.50 mole parts may be C4 oxidized fiber dextrin, for a total of 10 parts;

[0094] 2.50 to 6.00 mole parts may be natural fiber dextrin, 3.50 to 7.00 mole parts may be C1 oxidized fiber dextrin, and 0.50 to 3.00 mole parts may be C4 oxidized fiber dextrin, for a total of 10.00 parts; or

[0095] 3.33 mole parts can be natural cellodextrin, 3.33 mole parts can be C1 oxidized cellodextrin, and 3.33 mole parts can be C4 oxidized cellodextrin.

[0096] By way of further non-limiting example, in a composition comprising natural cello dextrin, C1 oxidized cello dextrin, C4 oxidized cello dextrin, and C1 and C4 oxidized cello dextrins:

[0097] 0.25 to 9.25 mole parts may be natural fiber dextrin, 0.25 to 9.25 mole parts may be C1 oxidized fiber dextrin, 0.25 to 9.25 mole parts may be C4 oxidized fiber dextrin, and 0.25 to 9.25 mole parts may be C1 and C4 oxidized fiber dextrins, for a total of 10 parts;

[0098] 2.50 mole parts can be natural fiber dextrin, 2.50 mole parts can be C1 oxidized fiber dextrin, 2.50 mole parts can be C4 oxidized fiber dextrin, and 2.50 mole parts can be C1 and C4 oxidized fiber dextrins; or

[0099] 0.25 to 4.75 mole parts can be natural fiber dextrin, 2.00 to 5.00 mole parts can be C1 oxidized fiber dextrin, 0.25 to 4.75 mole parts can be C4 oxidized fiber dextrin, and 3.00 to 6.00 mole parts can be C1 and C4 oxidized fiber dextrins, for a total of 10 parts.

[0100] In certain particularly preferred embodiments, cellobionic acid, cellodextrins, C1-oxidized cellodextrins, C4-oxidized cellodextrins, and / or C1 and C4-oxidized cellodextrins can be produced by decomposing cellulose with one or more lytic polysaccharide monooxygenases (LPMOs). In certain embodiments, compositions, such as the botanical pharmaceutical compositions taught herein, can include decomposition products of cellulose by one or more LPMOs, such decomposition products typically comprising native, C1-oxidized, and C4-oxidized cellodextrins, and optionally a mixture of C1 and C4-oxidized cellodextrins.

[0101] Lytic polysaccharide monooxygenases (LPMOs) broadly encompass a class of copper-containing enzymes that catalyze the cleavage of cellulose in an oxidative manner. These include C1 hydroxylating LPMOs (EC 1.14.99.54), which form cellulose fragments containing a D-glucono-1,5-lactone group at the reducing end that are rapidly and spontaneously hydrolyzed to aldonic acid. These also include C4 dehydrogenating LPMOs (EC 1.14.99.56), which form cellulose fragments containing a 4-dehydro-D-glucose group at the non-reducing end. These also include C1 hydroxylating / C4 dehydrogenating LPMOs (EC 1.14.99.54 and EC 1.14.99.56), which form cellulose fragments containing a 4-dehydro-D-glucose group at the non-reducing end and a gluconic acid group at the reducing end.

[0102] LPMOs are widely distributed in the bacterial and fungal kingdoms and are currently classified into seven “accessory-active” (AA) families (AA9—formerly glycoside hydrolase (GH) family 61, AA10—formerly carbohydrate-binding module (CBM) 33, AA11, AA13, AA14, AA15, and AA16) according to the Carbohydrate-Active Enzymes (www.cazy.org) database. The CAZY database entry for the AA9 family currently lists nearly 600 GenBank (http: / / www.ncbi.nlm.nih.gov / ) accession numbers for LPMOs from microorganisms, including, among others, Arthrobotrys oligospora, Aspergillus fumigatus, Aspergillus oryzae, Botrytis cinerea, Fusarium fujikuroi, Fusarium graminearum, Magnaporthe grisea, Neurospora crassa, and many others. While any suitable LPMO can be used to prepare the plant elicitor compounds and compound combinations taught herein, in certain embodiments, at least one of the one or more LPMOs can belong to adjuvant active (AA) Family 9. In certain preferred embodiments, at least one of the one or more LPMOs may be derived from Botrytis cinerea, Thielavia terrestris, or Myceliophthora thermophila. Exemplary LPMO enzymes of this type, including GenBank accession numbers for their protein sequences, their isolation, and how they are used in enzymatic reactions, are discussed in the experimental section and will be understood by those skilled in the art.

[0103] In certain embodiments, LPMO activity can be enhanced by including one or more cellobiose dehydrogenases (CDHs) in the reaction. CDHs are auxiliary activity 3 enzymes (EC 1.1.99.18) that catalyze the oxidation to cellobiose-1,5-lactone and also act more slowly on cellodextrins in addition to cellobiose.

[0104] The CAZY database entry for the AA3 family currently lists over 1300 GenBank accession numbers for CDHs from many different sources. In certain preferred embodiments, the CDH may be from Botrytis cinerea or Myceliophthora thermophila. Exemplary protein sequences for such enzymes are included those annotated under GenBank accession number AF074951.1 (Myceliophthora thermophila ATCC 42464 cellobiose dehydrogenase (cdh) mRNA, complete cds). Such proteins may be isolated, for example, from a microbial source that endogenously expresses the protein, or may be produced by recombinant expression as is known in the art. LPMO enzymes and enzyme mixtures are also commercially available (NZYTech, Lisabon, Portugal, LPMO-AA9 product code CZ0959, "Cilytic Cellulose Monooxygenase 9B" from Geotrichum candidum; Novozymes, Denmark, Products CTec 2 and CTec 3).

[0105] In certain embodiments, the cellulose to be acted upon by the LPMO enzyme and the optional CDH enzyme can be from lignocellulosic waste or pulping paper. Lignocellulose or lignocellulosic biomass refers to a plant biomass primarily composed of carbohydrate polymers (primarily comprising cellulose and hemicellulose), and the aromatic polymer lignin. Lignocellulosic biomass is an abundant raw material and is used, for example, in biorefining applications to produce biofuels, such as primarily bioethanol. Another common use of lignocellulosic biomass is the production of wood pulp and paper. Therefore, lignocellulosic waste or pulping paper advantageously represent an abundant source of cellulose.

[0106] In certain embodiments, the lignocellulosic waste can be a lignocellulosic byproduct from a biorefinery application. Typically, the production of biofuels involves enzymatic hydrolysis of lignocellulosic biomass using a mixture containing LPMO to decompose cellulose for downstream fermentation. Thus, the biowaste product from such a biorefinery already contains a certain amount of cellobionic acid and / or cellodextrins and oxidized cellodextrins (e.g., about 1 to 3 w / v% aldonic acids, such as cellobionic acid), which can be used as taught herein, optionally (but not necessarily) after some process of separation or purification from the biowaste.

[0107] Thus, in certain embodiments, the (phytopharmaceutical) compositions taught herein may be prepared from, comprise, or consist essentially of, LPMO-treated biorefinery lignocellulosic by-products.

[0108] In another embodiment, one or more additional plant defense elicitors can be combined with the novel plant defense elicitors taught herein. Thus, combination treatments are also contemplated. The administration of different elicitors to plants can be simultaneous or sequential in any order, and they can be contained in the same composition (e.g., mixed) or can be provided in physically separate forms, e.g., in different vials or packaging, optionally forming kits-of-parts.

[0109] In certain embodiments, such one or more additional plant defense elicitors may be selected from compounds of plant or fungal cell wall origin. In certain preferred embodiments, such one or more additional plant defense elicitors may be selected from pectin fragments, oligogalacturonosides, cellobiose, xyloglucans, unbranched β-1,3-glucans, chitin fragments, arabinose, arabinan, rhamnose, homogalacturonans, rhamnogalacturonans I and II, xylogalacturonans, starch, and combinations thereof.

[0110] According to the present disclosure, the compounds, combinations of compounds or compositions comprising the compounds taught herein are used as plant pathogen defense exciters. This term broadly encompasses any compound or composition that, when applied to plants, plant seeds or plant organs, can trigger natural plant defenses, activate plant defenses and resistance responses against plant pathogens, stimulate the production of plant defense molecules against plant pathogens and / or prevent, control or treat plant resistance to plant pathogen infection. Therefore, when perceived by plants, exciters can trigger molecular, biochemical and / or physiological defensive plant cell reactions, such as the synthesis or increase in the synthesis of plant defense molecules (such as ethylene and / or salicylic acid and jasmonic acid), the generation of reactive oxygen species (ROS), and / or the expression of specific defense-related genes and proteins (such as polygalacturonase inhibitor protein (PGIP)). The activation of signal transduction pathways can subsequently lead to the expression of lasting defense genes and the generation of secondary metabolites. Therefore, plant pathogen defense exciters can enhance the ability of plants to resist or antagonize given pathogens. Plant defense elicitors are generally classified into the group of agents commonly known as biopesticides, which primarily include bioinsecticides, biofungicides, bionematicides, and the like.

[0111] Thus, in certain embodiments, plant infections caused by plant pathogens can be prevented, controlled, or treated by the compounds or compositions of the present invention. Although these terms are well known per se, by way of further guidance, "prevention" can particularly mean avoiding the occurrence of at least one adverse effect or symptom, preferably all adverse effects or symptoms induced by a plant pathogen infection; "control" can particularly mean stopping the progression of a plant pathogen infection, more precisely reducing or eliminating the spread of a plant pathogen in healthy parts of a plant or plant organ, or from an infected plant to another plant, typically to a neighboring plant; and "treatment" can particularly mean ameliorating the symptoms of an infection, or completely curing an infection, typically by reducing or completely eliminating the plant pathogen (typically a fungus or bacteria), i.e., by eliminating any viable plant pathogen in a plant or an organ, or in several or each organ of a plant.

[0112] Plants can be contacted with an effective amount of a compound or composition of the present invention, for example, through an organ of the plant (preferably selected from leaves, roots, and / or fruits) or through the seeds of the plant. The contacting step can be performed once or several times (e.g., regularly or periodically, for example, at an appropriate season or at an appropriate stage of plant development). The term "effective amount" refers to an amount of one or more (active) compounds taught herein that induces or triggers the plant's natural defenses, activates the plant's defense and resistance responses against plant pathogens, and / or stimulates the production of plant defense molecules against plant pathogens, thereby obtaining a plant resistant to pathogens. The effective amount is considered variable because it can be affected by many factors, including but not limited to the type of plant treated, the treatment dose and application rate, the contact method, the weather and seasonal conditions experienced during the plant's growth cycle, pathogen sensitivity, etc. Such variations are generally encountered and understood by skilled artisans, who can adjust the preventive or treatment regimen, such as the application rate, application time and / or frequency, and mode of application. Specific suitable amounts or concentration ranges are discussed and exemplified elsewhere in this specification. The terms "organ," "plant organ," or "organ of a plant" refer interchangeably to a part of a plant or plant propagation material. The example of plant organ includes but not limited to leaf, stem, fruit, seed, cutting (cutting), tuber, root, bulb, rhizome etc.The contact step with plant or organ can be carried out in many ways, for example, by spraying, soaking, immersing, dipping, injecting, by soil feeding and any combination thereof.Or, compound or composition can be by providing volatility or compound or composition based on the form of steam near plant tissue, and allow to be diffused to plant or organ by atmosphere, thereby be applied on plant or organ.Technician knows how to make application mode adapt to specific purposes.

[0113] In the context of the present invention, the term "plant" generally refers to a plant that is infected by a plant pathogen or shows susceptibility to infection by a plant pathogen. For example, the plant may belong to the Angiosperm branch.

[0114] In certain embodiments, the plant may belong to the dicotyledonous branch. Examples of plants from the dicotyledonous branch include, but are not limited to, the Solanaceae family, including Solanum lycopersicum (tomato), Solanum tuberosum (potato), Solanum melongena (eggplant), Capsicum genus (pepper), and Nicotiana tabacum (tobacco); the Vitaceae family, including the Vitis genus (grapevine); the Brassicaceae family, including Brassica oleracea (cabbage), Brassica rapa (turnip and Chinese cabbage), mustard seeds, and Arabidopsis thaliana; the Rosaceae family, including Malus pumila (apple), Pyrus species (pear), and Fragaria tiliacea. ananassa) (strawberry); Fabaceae, including legumes such as peas, beans, and soybeans; Asteraceae, including sunflowers; Amaranthaceae, including sugar beets.

[0115] In certain embodiments, the plant may belong to the monocotyledonous branch. Examples of plants from the monocotyledonous branch include, but are not limited to, Gramineae or Poaceae, such as corn, rice, barley, or wheat.

[0116] Thus, in certain embodiments, the plant may be a dicot, preferably selected from the family Cruciferae, Solanaceae or Rosaceae, such as Arabidopsis, cabbage, tomato, grapevine, soybean, apple, pear or strawberry, or wherein the plant is a monocot, preferably selected from the family Poaceae, such as corn, rice, barley or wheat.

[0117] In some preferred embodiments, the plant pathogen may be a fungus or a bacterium. Thus, in such cases, the compositions taught herein may also conveniently be referred to as antifungal and / or antibacterial adjuvants, i.e., products that aid in the prevention or treatment of plant diseases typically caused by fungi or bacteria.

[0118] In certain embodiments, the pathogen can be a necrotrophic fungus or bacteria, a hemitrophic fungus or bacteria, or a biotrophic fungus or bacteria.

[0119] During colonization of a plant host, most fungal pathogens exhibit one of two modes of nutrition: biotrophy, in which nutrients are obtained from living host cells, and necrotrophy, in which nutrients are obtained from host cells previously killed by the fungus. A third mode of nutrition is hemibiotrophy, in which the pathogen has an initial biotrophic phase followed by a necrotrophic phase. Thus, plant pathogenic pathogens, particularly fungi, can be distinguished based on their mode of nutrition: necrotrophic (e.g., Botrytis cinerea), biotrophic (e.g., Ustilago maydis), or hemibiotrophic (e.g., Colletotrichum higginsianum).

[0120] In some particularly preferred embodiments, the plant pathogen may be a fungus, typically a plant pathogenic fungus. The expression "plant pathogenic fungus" refers to a fungal pathogen that infects plant organs. Examples of plant pathogenic fungi include, but are not limited to, fungi belonging to the classes Ascomycetes and Basidiornycetes, such as, for example, fungi of the order Helotiales, such as, for example, Sclerotiniaceae, Botrytis / Botryotinia, such as gray mold species; fungi of the order Hypocreales, such as, for example, Nectriaceae, Fusarium Fungi of the genus Fusarium, Uredinales, for example, Pucciniaceae, Puccinia; fungi of the order Ustilaginales, for example, Ustilaginaceae, Ustilago); fungi of the order Sordariomycetes, for example, Glomerellaceae, Colletotrichum.

[0121] In another embodiment, the plant pathogen may be a bacterium, typically a phytopathogenic bacterium.The expression "phytopathogenic bacterium" refers to a bacterial pathogen that infects plant organs. Examples of phytopathogenic bacteria include, but are not limited to, bacteria of the order Pseudomonadales, such as the family Pseudomonadaceae, the genus Pseudomonas, for example, species Pseudomonas syringae; bacteria of the order Burkholderiales, such as the family Burkholderiaceae, the genus Ralstonia, for example, species Ralstonia solanacearum; bacteria of the order Enterobacterales, such as the family Erwiniaceae, the genus Erwinia, for example, species Erwinia amylovora, or of the family Pectobacteriaceae, the genus Pectobacterium, for example, species Pectobacterium carrotis. carotovorum species (formerly known as Erwinia carotovora), bacteria of the order Xanthomonadale, such as the family Xanthomonadaceae, the genus Xylella, such as the species Xylella fastidiosa, or the genus Xanthomonas, such as the species Xanthomonas campestris. Similar to fungi, and based on the type of plant colonization, bacteria can also be classified into necrotrophic, biotrophic, and hemibiotrophic subclasses.

[0122] Particularly preferred may be necrotrophic fungi, preferably such as Botrytis cinerea. Thus, in certain embodiments, the plant pathogen is a fungus or bacterium, such as a necrotrophic fungus or bacterium, such as Botrytis cinerea, a hemitrophic fungus or bacterium, or a biotrophic fungus or bacterium, such as Pseudomonas syringae.

[0123] In the context of the present invention, a plant infection caused by a plant pathogen generally refers to a plant infection caused by at least one plant pathogen. The infection can occur on any organ of the plant. For example, but not limited to, the plant infection can be, for example, a Botrytis cinerea infection, such as a Botrytis cinerea infection in Arabidopsis thaliana, tomato, strawberry, sunflower, grapevine or apple; a Higgins's anthracnose infection, such as a Higgins's anthracnose infection in turnip, Chinese cabbage, mustard, Arabidopsis thaliana or apple; a Ustilago mays infection, such as a Ustilago mays infection in corn; a Ralstonia solanacearum infection, such as a Ralstonia solanacearum infection in tomato, potato, eggplant, pepper or tobacco; a Pseudomonas syringae infection, such as a Pseudomonas syringae infection in apple or pear; or any combination thereof, such as Botrytis cinerea and / or Higgins's anthracnose in Arabidopsis thaliana, an infection of apple with Botrytis cinerea and / or Higgins's anthracnose, or an infection of tomato with Botrytis cinerea and / or Ralstonia solanacearum.

[0124] In view of the foregoing discussion, the present application therefore also provides, inter alia, aspects and embodiments as set forth in Statements 1 to 17 below:

[0125] Statement 1. Use of cellobionic acid or a phytologically acceptable salt thereof as a plant pathogen defense elicitor.

[0126] Statement 2. A method for activating a plant's defense against plant pathogens, comprising contacting the plant with an effective amount of cellobionic acid or a phyto-pharmaceutically acceptable salt thereof.

[0127] Statement 3. The use according to Statement 1 or the method according to Statement 2, wherein the cellobionic acid or its salt is contained in a composition that also contains one or more cellodextrins; one or more C1 oxidized cellodextrins or their plant-acceptable salts; one or more C4 oxidized cellodextrins; and / or one or more C1 and C4 oxidized cellodextrins or their plant-acceptable salts.

[0128] Statement 4. The use according to statement 1 or 3 or the method according to statement 2 or 3, wherein the cellobionic acid or its salt is contained in a composition that also contains one or more additional plant defense elicitors, such as plant elicitors selected from compounds derived from plant or fungal cell walls, such as in particular plant elicitors selected from the following: pectin fragments, oligogalacturonosides, cellobiose, xyloglucan, unbranched β-1,3-glucan, chitin fragments, arabinose, arabinan, rhamnose, homogalacturonan, rhamnogalacturonan I and II, xylogalacturonan, starch, and combinations thereof.

[0129] Statement 5. The use or method of Statement 3 or 4, wherein the degree of polymerization (DP) of the cellodextrin, C1 oxidized cellodextrin, C4 oxidized cellodextrin, and C1 and C4 oxidized cellodextrins are each independently 2 to 10, preferably 2 to 5.

[0130] Statement 6. The use according to any one of Statements 1 or 3 to 5 or the method according to any one of Statements 2 to 5, wherein the cellobionic acid or the cellodextrins, C1 oxidized cellodextrins, C4 oxidized cellodextrins, and C1 and C4 oxidized cellodextrins are produced by breaking down cellulose with one or more lytic polysaccharide monooxygenases (LPMOs).

[0131] Statement 7. The use or method of Statement 6, wherein at least one of said one or more LPMOs belongs to Adjuvant Active (AA) Family 9, such as LPMO-AA9 from Botrytis cinerea, Thielavia terrestris, or Myceliophthora thermophila.

[0132] Statement 8. The use or method of Statement 6 or 7, wherein one or more cellobiose dehydrogenases (CDHs), such as CDHs from Botrytis cinerea or Myceliophthora thermophila, are used to enhance the activity of the LPMO.

[0133] Statement 9. The use or method of any one of Statements 6 to 8, wherein the cellulose is derived from lignocellulosic waste or pulp paper, e.g., wherein the composition comprises or consists essentially of a LPMO-treated biorefinery lignocellulosic by-product.

[0134] Statement 10. The use according to any one of Statements 1 or 3 to 9 or the method according to any one of Statements 2 to 9, wherein the concentration of cellobionic acid or its salt is 0.10 μM to 1000 μM, such as 0.10 μM to 500 μM, preferably 0.10 μM to 100 μM, such as 0.10 μM to 10 μM.

[0135] Statement 11. The use or method of any one of Statements 3 to 10, wherein the concentration of the cellodextrin, the C1 oxidized cellodextrin including the cellobionic acid, the C4 oxidized cellodextrin, and the total concentration of the C1 and C4 oxidized cellodextrins is from 0.10 μM to 1000 μM, for example, from 0.10 μM to 500 μM, preferably from 0.10 μM to 100 μM, for example, from 0.10 μM to 10 μM.

[0136] Statement 12. The use or method of any one of Statements 3 to 11, wherein the composition comprises cellodextrin, C1 oxidized cellodextrin, and C4 oxidized cellodextrin in a molar ratio of about 4:5:1.

[0137] Statement 13. The use according to any one of Statements 1 or 3 to 12 or the method according to any one of Statements 2 to 12, wherein:

[0138] Prevent, control or treat plant infections caused by plant pathogens;

[0139] The plant pathogen is a fungus or bacterium, such as a necrotrophic fungus or bacterium, such as Botrytis cinerea, a hemibiotrophic fungus or bacterium, or a biotrophic fungus or bacterium, such as Pseudomonas syringae;

[0140] The plant is a dicotyledonous plant, preferably selected from the family Cruciferae, Solanaceae or Rosaceae, such as Arabidopsis thaliana, cabbage, tomato, grapevine, soybean, apple, pear or strawberry, or wherein the plant is a monocotyledonous plant, preferably selected from the family Poaceae, such as corn, rice, barley or wheat; and / or

[0141] The plant is brought into contact via an organ of the plant, preferably an organ selected from the group consisting of leaves, roots and / or fruits, or via a seed of the plant.

[0142] Statement 14. A botanical pharmaceutical composition comprising cellobionic acid or a botanically pharmaceutically acceptable salt thereof and a botanically pharmaceutically acceptable carrier.

[0143] Statement 15. A phytodrug composition according to Statement 14, wherein said composition comprises 0.10 μM to 1000 μM, such as 0.10 μM to 500 μM, preferably 0.10 μM to 100 μM, such as 0.10 μM to 10 μM of cellobionic acid or a salt thereof.

[0144] Statement 16. The botanical composition of Statement 14 or 15, wherein:

[0145] The composition further comprises one or more cellodextrins; one or more C1 oxidized cellodextrins or plant pharmaceutically acceptable salts thereof; one or more C4 oxidized cellodextrins; and / or one or more C1 and C4 oxidized cellodextrins or plant pharmaceutically acceptable salts thereof;

[0146] The composition further comprises one or more additional plant defense elicitors, for example plant elicitors selected from compounds of plant or fungal cell wall origin, for example in particular plant elicitors selected from the group consisting of pectin fragments, oligogalacturonosides, cellobiose, xyloglucans, unbranched β-1,3-glucans, chitin fragments, arabinose, arabinan, rhamnose, homogalacturonans, rhamnogalacturonans I and II, xylogalacturonans, starch, and combinations thereof;

[0147] The degree of polymerization (DP) of the cellodextrin, C1 oxidized cellodextrin, C4 oxidized cellodextrin, and C1 and C4 oxidized cellodextrins are each independently 2 to 10, preferably 2 to 5;

[0148] The cellobionic acid or the cellodextrins, C1 oxidized cellodextrins, C4 oxidized cellodextrins, and C1 and C4 oxidized cellodextrins are produced by decomposing cellulose with one or more lytic polysaccharide monooxygenases (LPMOs), optionally wherein:

[0149] At least one of the one or more LPMOs belongs to adjuvant active (AA) family 9, such as LPMO-AA9 from Botrytis cinerea, Thielavia terrestris, or Myceliophthora thermophila;

[0150] One or more cellobiose dehydrogenases (CDHs), such as CDHs from Botrytis cinerea or Myceliophthora thermophila, are used to enhance the activity of the LPMO; and / or

[0151] The cellulose is derived from lignocellulosic waste or pulp paper, for example, wherein the composition comprises or consists essentially of a LPMO-treated biorefinery lignocellulosic by-product;

[0152] The total concentration of the cellodextrin, C1 oxidized cellodextrin including cellobionic acid, C4 oxidized cellodextrin, and C1 and C4 oxidized cellodextrin is 0.10 μM to 1000 μM, such as 0.10 μM to 500 μM, preferably 0.10 μM to 100 μM, such as 0.10 μM to 10 μM; and / or

[0153] The composition comprises cellodextrin, C1 oxidized cellodextrin, and C4 oxidized cellodextrin in a molar ratio of about 4:5:1.

[0154] Statement 17. A botanical pharmaceutical composition according to any one of Statements 14 to 16, wherein the composition comprises one or more anionic, nonionic, amphoteric or cationic surfactants or a combination thereof, such as Triton X-100, Tween-20, Tween-40 and / or Silwet L-77.

[0155] The present application also provides aspects and embodiments as set forth in statements 1' to 15' below:

[0156] Statement 1'. Use of a composition comprising:

[0157] (i) one or more cellodextrins (i.e., natural or non-oxidized cellodextrins) and

[0158] (ii) one or more C1 oxidized cellodextrins or their plant pharmaceutically acceptable salts, one or more C4 oxidized cellodextrins, and / or one or more C1 and C4 oxidized cellodextrins or their plant pharmaceutically acceptable salts.

[0159] Statement 2'. A method for activating a plant's defense against a plant pathogen, comprising contacting the plant with an effective amount of a composition comprising:

[0160] (i) one or more cellodextrins, and

[0161] (ii) one or more C1 oxidized cellodextrins or their plant pharmaceutically acceptable salts, one or more C4 oxidized cellodextrins, and / or one or more C1 and C4 oxidized cellodextrins or their plant pharmaceutically acceptable salts.

[0162] Statement 3'. Use according to statement 1' or method according to statement 2', wherein the composition further comprises one or more additional plant defense elicitors, for example plant elicitors selected from compounds of plant or fungal cell wall origin, for example in particular plant elicitors selected from the following: pectin fragments, oligogalacturonosides, cellobiose, xyloglucans, unbranched β-1,3-glucans, chitin fragments, arabinose, arabinan, rhamnose, homogalacturonan, rhamnogalacturonan I and II, xylogalacturonan, starch, and combinations thereof.

[0163] Statement 4'. The use according to Statement 1' or 3' or the method according to Statement 2' or 3', wherein the degree of polymerization (DP) of the cellodextrin, C1 oxidized cellodextrin, C4 oxidized cellodextrin, and C1 and C4 oxidized cellodextrins are each independently 2 to 10, preferably 2 to 5.

[0164] Statement 5'. The use according to any one of Statements 1', 3', or 4' or the method according to any one of Statements 2' to 4', wherein the cellodextrins, C1 oxidized cellodextrins, C4 oxidized cellodextrins, and C1 and C4 oxidized cellodextrins are produced by breaking down cellulose with one or more lytic polysaccharide monooxygenases (LPMOs).

[0165] Statement 6'. The use or method according to Statement 5', wherein at least one of said one or more LPMOs belongs to Adjuvant Active (AA) Family 9, such as LPMO-AA9 from Botrytis cinerea, Thielavia terrestris, or Myceliophthora thermophila.

[0166] Statement 7'. The use or method according to Statement 5' or 6', wherein one or more cellobiose dehydrogenases (CDHs), such as CDHs from Botrytis cinerea or Myceliophthora thermophila, are used to enhance the activity of the LPMO.

[0167] Statement 8'. The use or method of any one of Statements 5' to 7', wherein the cellulose is derived from lignocellulosic waste or pulp paper, eg, wherein the composition comprises or consists essentially of a LPMO-treated biorefinery lignocellulosic by-product.

[0168] Statement 9'. The use according to any one of Statements 1' or 3' to 8' or the method according to any one of Statements 2' to 8', wherein the total concentration of the cellodextrin, C1 oxidized cellodextrin, C4 oxidized cellodextrin and C1 and C4 oxidized cellodextrin is 0.10 μM to 1000 μM, for example 0.10 μM to 500 μM, preferably 0.10 μM to 100 μM, for example 0.10 μM to 10 μM.

[0169] Statement 10'. The use according to any one of Statements 1' or 3' to 9' or the method according to any one of Statements 2' to 9', wherein the composition comprises cellodextrin, C1 oxidized cellodextrin, and C4 oxidized cellodextrin in a molar ratio of about 4:5:1.

[0170] Statement 11'. The use according to any one of Statements 1' or 3' to 10' or the method according to any one of Statements 2' to 10', wherein:

[0171] Prevent, control or treat plant infections caused by plant pathogens;

[0172] The plant pathogen is a fungus or bacterium, such as a necrotrophic fungus or bacterium, such as Botrytis cinerea, a hemibiotrophic fungus or bacterium, or a biotrophic fungus or bacterium, such as Pseudomonas syringae;

[0173] The plant is a dicotyledonous plant, preferably selected from the family Cruciferae, Solanaceae or Rosaceae, such as Arabidopsis thaliana, cabbage, tomato, grapevine, soybean, apple, pear or strawberry, or wherein the plant is a monocotyledonous plant, preferably selected from the family Poaceae, such as corn, rice, barley or wheat; and / or

[0174] The plant is brought into contact via an organ of the plant, preferably an organ selected from the group consisting of leaves, roots and / or fruits, or via a seed of the plant.

[0175] Statement 12'. A botanical composition comprising:

[0176] (i) one or more cellodextrins (i.e., natural or non-oxidized cellodextrins), and

[0177] (ii) one or more C1 oxidized cellodextrins or their plant pharmaceutically acceptable salts, one or more C4 oxidized cellodextrins, and / or one or more C1 and C4 oxidized cellodextrins or their plant pharmaceutically acceptable salts, and

[0178] Plant-based pharmaceutically acceptable carrier.

[0179] Statement 13'. A phyto-pharmaceutical composition according to Statement 12', wherein the concentration of the cellodextrin, C1 oxidized cellodextrin, C4 oxidized cellodextrin and the total concentration of C1 and C4 oxidized cellodextrin is 0.10 μM to 1000 μM, such as 0.10 μM to 500 μM, preferably 0.10 μM to 100 μM, such as 0.10 μM to 10 μM.

[0180] Statement 14'. The phytopharmaceutical composition according to Statement 12' or 13', wherein:

[0181] The composition further comprises one or more additional plant defense elicitors, for example plant elicitors selected from compounds of plant or fungal cell wall origin, for example in particular plant elicitors selected from the group consisting of pectin fragments, oligogalacturonosides, cellobiose, xyloglucans, unbranched β-1,3-glucans, chitin fragments, arabinose, arabinan, rhamnose, homogalacturonans, rhamnogalacturonans I and II, xylogalacturonans, starch, and combinations thereof;

[0182] The degree of polymerization (DP) of the cellodextrin, C1 oxidized cellodextrin, C4 oxidized cellodextrin, and C1 and C4 oxidized cellodextrins are each independently 2 to 10, preferably 2 to 5;

[0183] The cellodextrins, C1 oxidized cellodextrins, C4 oxidized cellodextrins, and C1 and C4 oxidized cellodextrins are produced by decomposing cellulose with one or more lytic polysaccharide monooxygenases (LPMOs), optionally wherein:

[0184] At least one of the one or more LPMOs belongs to adjuvant active (AA) family 9, such as LPMO-AA9 from Botrytis cinerea, Thielavia terrestris, or Myceliophthora thermophila;

[0185] One or more cellobiose dehydrogenases (CDHs), such as CDHs from Botrytis cinerea or Myceliophthora thermophila, are used to enhance the activity of the LPMO; and / or

[0186] The cellulose is derived from lignocellulosic waste or pulp paper, for example, wherein the composition comprises or consists essentially of a LPMO-treated biorefinery lignocellulosic by-product;

[0187] The composition comprises cellodextrin, C1 oxidized cellodextrin, and C4 oxidized cellodextrin in a molar ratio of about 4:5:1.

[0188] Statement 15'. A botanical pharmaceutical composition according to any one of Statements 12' to 14', wherein the composition comprises one or more anionic, nonionic, amphoteric or cationic surfactants or a combination thereof, such as Triton X-100, Tween-20, Tween-40 and / or Silwet L-77.

[0189] The present application also provides aspects and embodiments as set forth in statements 1* to 17* below:

[0190] Statement 1*. Use of one or more C4 oxidized cellodextrins as plant pathogen defense elicitors.

[0191] Statement 2*. A method for activating a plant's defense against a plant pathogen, comprising contacting the plant with an effective amount of one or more C4 oxidized cellodextrins.

[0192] Statement 3*. The use according to Statement 1* or the method according to Statement 2*, wherein the one or more C4 oxidized cellodextrins are contained in a composition that also contains one or more cellodextrins; one or more C1 oxidized cellodextrins or their plant-acceptable salts; and / or one or more C1 and C4 oxidized cellodextrins or their plant-acceptable salts.

[0193] Statement 4*. The use according to statement 1* or 3* or the method according to statement 2* or 3*, wherein the one or more C4 oxidized cellodextrins are contained in a composition that also contains one or more additional plant defense elicitors, such as plant elicitors selected from compounds derived from plant or fungal cell walls, such as in particular plant elicitors selected from the following: pectin fragments, oligogalacturonans, cellobiose, xyloglucans, unbranched β-1,3-glucans, chitin fragments, arabinose, arabinan, rhamnose, homogalacturonans, rhamnogalacturonans I and II, xylogalacturonans, starch, and combinations thereof.

[0194] Statement 5*. The use according to any one of Statements 1*, 3* or 4* or the method according to any one of Statements 2* to 4*, wherein the one or more C4 oxidized fiber dextrins, or the fiber dextrin, C1 oxidized fiber dextrin, C4 oxidized fiber dextrin, and C1 and C4 oxidized fiber dextrins have a degree of polymerization (DP) of each independently from 2 to 10, preferably from 2 to 5.

[0195] Statement 6*. The use according to any one of Statements 1* or 3* to 5* or the method according to any one of Statements 2* to 5*, wherein the one or more C4 oxidized cellodextrins, or the cellodextrins, C1 oxidized cellodextrins, C4 oxidized cellodextrins, and C1 and C4 oxidized cellodextrins are produced by breaking down cellulose with one or more lytic polysaccharide monooxygenases (LPMOs).

[0196] Statement 7*. The use or method according to Statement 6*, wherein at least one of the one or more LPMOs belongs to Adjuvant Active (AA) Family 9, such as LPMO-AA9 from Botrytis cinerea, Thielavia terrestris, or Myceliophthora thermophila.

[0197] Statement 8*. The use or method of Statement 6* or 7*, wherein one or more cellobiose dehydrogenases (CDHs), such as CDHs from Botrytis cinerea or Myceliophthora thermophila, are used to enhance the activity of the LPMO.

[0198] Statement 9*. The use or method of any one of Statements 6* to 8*, wherein the cellulose is derived from lignocellulosic waste or pulp paper, e.g., wherein the composition comprises or consists essentially of a LPMO-treated biorefinery lignocellulosic by-product.

[0199] Statement 10*. The use according to any one of Statements 1* or 3* to 9* or the method according to any one of Statements 2* to 9*, wherein the concentration of the one or more C4 oxidized cellodextrins is from 0.10 μM to 1000 μM, for example from 0.10 μM to 500 μM, preferably from 0.10 μM to 100 μM, for example from 0.10 μM to 10 μM.

[0200] Statement 11*. The use or method of any one of Statements 3* to 10*, wherein the total concentration of the cellodextrin, C1 oxidized cellodextrin, C4 oxidized cellodextrin, and C1 and C4 oxidized cellodextrin is 0.10 μM to 1000 μM, for example, 0.10 μM to 500 μM, preferably 0.10 μM to 100 μM, for example, 0.10 μM to 10 μM.

[0201] Statement 12*. The use or method of any one of Statements 3* to 11*, wherein the composition comprises cellodextrin, C1 oxidized cellodextrin, and C4 oxidized cellodextrin in a molar ratio of about 4:5:1.

[0202] Statement 13*. The use according to any one of Statements 1* or 3* to 12* or the method according to any one of Statements 2* to 12*, wherein:

[0203] Prevent, control or treat plant infections caused by plant pathogens;

[0204] The plant pathogen is a fungus or bacterium, such as a necrotrophic fungus or bacterium, such as Botrytis cinerea, a hemibiotrophic fungus or bacterium, or a biotrophic fungus or bacterium, such as Pseudomonas syringae;

[0205] The plant is a dicotyledonous plant, preferably selected from the family Cruciferae, Solanaceae or Rosaceae, such as Arabidopsis thaliana, cabbage, tomato, grapevine, soybean, apple, pear or strawberry, or wherein the plant is a monocotyledonous plant, preferably selected from the family Poaceae, such as corn, rice, barley or wheat; and / or

[0206] The plant is brought into contact via an organ of the plant, preferably an organ selected from the group consisting of leaves, roots and / or fruits, or via a seed of the plant.

[0207] Statement 14*. A botanical pharmaceutical composition comprising one or more C4 oxidized cellodextrins and a botanical pharmaceutically acceptable carrier.

[0208] Statement 15*. A phytodrug composition according to Statement 14*, wherein said composition comprises 0.10 μM to 1000 μM, such as 0.10 μM to 500 μM, preferably 0.10 μM to 100 μM, such as 0.10 μM to 10 μM of said one or more C4 oxidized cellodextrins.

[0209] Statement 16*. A phytopharmaceutical composition according to Statement 14* or 15*, wherein:

[0210] The composition further comprises one or more cellodextrins; one or more C1 oxidized cellodextrins or plant pharmaceutically acceptable salts thereof; and / or one or more C1 and C4 oxidized cellodextrins or plant pharmaceutically acceptable salts thereof;

[0211] The composition further comprises one or more additional plant defense elicitors, for example plant elicitors selected from compounds of plant or fungal cell wall origin, for example in particular plant elicitors selected from the group consisting of pectin fragments, oligogalacturonosides, cellobiose, xyloglucans, unbranched β-1,3-glucans, chitin fragments, arabinose, arabinan, rhamnose, homogalacturonans, rhamnogalacturonans I and II, xylogalacturonans, starch, and combinations thereof;

[0212] The one or more C4 oxidized cellodextrins, or the cellodextrins, C1 oxidized cellodextrins, C4 oxidized cellodextrins, and C1 and C4 oxidized cellodextrins each independently have a degree of polymerization (DP) of 2 to 10, preferably 2 to 5;

[0213] The one or more C4 oxidized cellodextrins, or the cellodextrins, C1 oxidized cellodextrins, C4 oxidized cellodextrins, and C1 and C4 oxidized cellodextrins are produced by decomposing cellulose with one or more lytic polysaccharide monooxygenases (LPMOs), optionally wherein:

[0214] At least one of the one or more LPMOs belongs to adjuvant active (AA) family 9, such as LPMO-AA9 from Botrytis cinerea, Thielavia terrestris, or Myceliophthora thermophila;

[0215] One or more cellobiose dehydrogenases (CDHs), such as CDHs from Botrytis cinerea or Myceliophthora thermophila, are used to enhance the activity of the LPMO; and / or

[0216] The cellulose is derived from lignocellulosic waste or pulp paper, for example, wherein the composition comprises or consists essentially of a LPMO-treated biorefinery lignocellulosic by-product;

[0217] The total concentration of the cellodextrin, C1 oxidized cellodextrin, C4 oxidized cellodextrin, and C1 and C4 oxidized cellodextrin is 0.10 μM to 1000 μM, such as 0.10 μM to 500 μM, preferably 0.10 μM to 100 μM, such as 0.10 μM to 10 μM; and / or

[0218] The composition comprises cellodextrin, C1 oxidized cellodextrin, and C4 oxidized cellodextrin in a molar ratio of about 4:5:1.

[0219] Statement 17*. A botanical pharmaceutical composition according to any one of Statements 14* to 16*, wherein the composition comprises one or more anionic, nonionic, amphoteric or cationic surfactants or a combination thereof, such as Triton X-100, Tween-20, Tween-40 and / or Silwet L-77.

[0220] The present application also provides aspects and embodiments as set forth in statements 1# to 16# below:

[0221] Statement #1. Use of one or more C1 and C4 oxidized cellodextrins or their phytologically acceptable salts as plant pathogen defense elicitors.

[0222] Statement #2. A method for activating a plant's defense against a plant pathogen comprising contacting the plant with an effective amount of one or more C1 and C4 oxidized cellodextrins or a phytologically acceptable salt thereof.

[0223] Statement 3#. The use according to Statement 1# or the method according to Statement 2#, wherein the one or more C1 and C4 oxidized cellodextrins or their salts are contained in a composition that also contains one or more cellodextrins; one or more C1 oxidized cellodextrins or their plant-acceptable salts; and / or one or more C4 oxidized cellodextrins.

[0224] Statement 4#. The use according to statement 1# or 3# or the method according to statement 2# or 3#, wherein the one or more C1 and C4 oxidized cellodextrins or their salts are contained in a composition that also contains one or more additional plant defense stimulants, such as plant stimulants selected from compounds derived from plant or fungal cell walls, such as, in particular, plant stimulants selected from the following: pectin fragments, oligogalacturonans, cellobiose, xyloglucans, unbranched β-1,3-glucans, chitin fragments, arabinose, arabinan, rhamnose, homogalacturonans, rhamnogalacturonans I and II, xylogalacturonans, starch, and combinations thereof.

[0225] Statement 5#. The use according to any one of Statements 1#, 3# or 4# or the method according to any one of Statements 2# to 4#, wherein the one or more C1 and C4 oxidized cellulosic dextrins, or the cellulosic dextrins, C1 oxidized cellulosic dextrins, C4 oxidized cellulosic dextrins and C1 and C4 oxidized cellulosic dextrins have a degree of polymerization (DP) of each independently from 2 to 10, preferably from 2 to 5.

[0226] Statement 6#. The use according to any one of Statements 1# or 3# to 5# or the method according to any one of Statements 2# to 5#, wherein the one or more C1 and C4 oxidized cellodextrins, or the cellodextrins, C1 oxidized cellodextrins, C4 oxidized cellodextrins and C1 and C4 oxidized cellodextrins are produced by breaking down cellulose with one or more lytic polysaccharide monooxygenases (LPMOs).

[0227] Statement 7#. The use or method of Statement 6#, wherein at least one of the one or more LPMOs belongs to Adjuvant Active (AA) Family 9, such as LPMO-AA9 from Botrytis cinerea, Thielavia terrestris, or Myceliophthora thermophila.

[0228] Statement 8#. The use or method of Statement 6# or 7#, wherein one or more cellobiose dehydrogenases (CDHs), such as CDHs from Botrytis cinerea or Myceliophthora thermophila, are used to enhance the activity of the LPMO.

[0229] Statement 9#. The use or method of any one of Statements 6# to 8#, wherein the cellulose is derived from lignocellulosic waste or pulp paper, e.g., wherein the composition comprises or consists essentially of a LPMO-treated biorefinery lignocellulosic byproduct.

[0230] Statement 10#. The use according to any one of Statements 1# or 3# to 9# or the method according to any one of Statements 2# to 9#, wherein the concentration of the one or more C1 and C4 oxidized cellodextrins or their salts is 0.10 μM to 1000 μM, for example 0.10 μM to 500 μM, preferably 0.10 μM to 100 μM, for example 0.10 μM to 10 μM.

[0231] Statement 11#. The use or method of any one of Statements 3# to 10#, wherein the total concentration of the cellodextrin, C1 oxidized cellodextrin, C4 oxidized cellodextrin, and C1 and C4 oxidized cellodextrin is 0.10 μM to 1000 μM, for example, 0.10 μM to 500 μM, preferably 0.10 μM to 100 μM, for example, 0.10 μM to 10 μM.

[0232] Statement 12#. The use according to any one of Statements 1# or 3# to 11# or the method according to any one of Statements 2# to 11#, wherein:

[0233] Prevent, control or treat plant infections caused by plant pathogens;

[0234] The plant pathogen is a fungus or bacterium, such as a necrotrophic fungus or bacterium, such as Botrytis cinerea, a hemibiotrophic fungus or bacterium, or a biotrophic fungus or bacterium, such as Pseudomonas syringae;

[0235] The plant is a dicotyledonous plant, preferably selected from the family Cruciferae, Solanaceae or Rosaceae, such as Arabidopsis thaliana, cabbage, tomato, grapevine, soybean, apple, pear or strawberry, or wherein the plant is a monocotyledonous plant, preferably selected from the family Poaceae, such as corn, rice, barley or wheat; and / or

[0236] The plant is brought into contact via an organ of the plant, preferably an organ selected from the group consisting of leaves, roots and / or fruits, or via a seed of the plant.

[0237] Statement 13#. A botanical pharmaceutical composition comprising one or more C1 and C4 oxidized cellodextrins or a botanically pharmaceutically acceptable salt thereof and a botanically pharmaceutically acceptable carrier.

[0238] Statement 14#. A botanical pharmaceutical composition according to Statement 13#, wherein said composition comprises 0.10 μM to 1000 μM, e.g., 0.10 μM to 500 μM, preferably 0.10 μM to 100 μM, e.g., 0.10 μM to 10 μM of said one or more C1 and C4 oxidized cellodextrins or salts thereof.

[0239] Statement 15#. The botanical composition of Statement 13# or 14#, wherein:

[0240] The composition further comprises one or more cellodextrins; one or more C1 oxidized cellodextrins or plant pharmaceutically acceptable salts thereof; and / or one or more C4 oxidized cellodextrins;

[0241] The composition further comprises one or more additional plant defense elicitors, for example plant elicitors selected from compounds of plant or fungal cell wall origin, for example in particular plant elicitors selected from the group consisting of pectin fragments, oligogalacturonosides, cellobiose, xyloglucans, unbranched β-1,3-glucans, chitin fragments, arabinose, arabinan, rhamnose, homogalacturonans, rhamnogalacturonans I and II, xylogalacturonans, starch, and combinations thereof;

[0242] The one or more C1 and C4 oxidized cellodextrins, or the cellodextrin, C1 oxidized cellodextrin, C4 oxidized cellodextrin, and C1 and C4 oxidized cellodextrins each independently have a degree of polymerization (DP) of 2 to 10, preferably 2 to 5;

[0243] The one or more C1 and C4 oxidized cellodextrins, or the cellodextrins, C1 oxidized cellodextrins, C4 oxidized cellodextrins, and C1 and C4 oxidized cellodextrins are produced by decomposing cellulose with one or more lytic polysaccharide monooxygenases (LPMOs), optionally wherein:

[0244] At least one of the one or more LPMOs belongs to adjuvant active (AA) family 9, such as LPMO-AA9 from Botrytis cinerea, Thielavia terrestris, or Myceliophthora thermophila;

[0245] One or more cellobiose dehydrogenases (CDHs), such as CDHs from Botrytis cinerea or Myceliophthora thermophila, are used to enhance the activity of the LPMO; and / or

[0246] The cellulose is derived from lignocellulosic waste or pulp paper, e.g., wherein the composition comprises or consists essentially of a LPMO-treated biorefinery lignocellulosic by-product; and / or

[0247] The total concentration of the cellodextrin, C1 oxidized cellodextrin, C4 oxidized cellodextrin, and C1 and C4 oxidized cellodextrin is 0.10 μM to 1000 μM, such as 0.10 μM to 500 μM, preferably 0.10 μM to 100 μM, such as 0.10 μM to 10 μM.

[0248] Statement 16#. A botanical pharmaceutical composition according to any one of Statements 13# to 15#, wherein the composition comprises one or more anionic, nonionic, amphoteric or cationic surfactants or a combination thereof, such as Triton X-100, Tween-20, Tween-40 and / or Silwet L-77.

[0249] Although the present invention has been described in conjunction with specific embodiments thereof, it is apparent that, based on the foregoing, many substitutions, modifications and variations are apparent to those skilled in the art. Therefore, it is intended to encompass all such substitutions, modifications and variations that fall within the spirit and broad scope of the appended claims.

[0250]

[00146] The aspects and embodiments of the invention disclosed herein are further supported by the following non-limiting examples.

[0251] Example

[0252] Materials and methods used in Examples 1-2

[0253] Plant material and fungal inoculation

[0254] Arabidopsis thaliana Columbia (Col-0) is the deposited material used for all experiments. Seeds were surface sterilized by subsequently treating with 50% v / v Et-OH and 3% bleach solution with stirring for 5 minutes and 10 minutes, respectively. The seeds were then washed three times with distilled water and placed in the dark at 4°C for 3 days. For qPCR analysis and microarray experiments, seeds were sown on square petri dishes (13×13 mm) containing half-strength Murashige and Skoog (MS) salts, 2.5 mM MES, 1% w / v sucrose and 0.8% w / v plant agar. The cells were grown at 22±0.5°C under short-day conditions (8 / 16 hours light / dark; 100 μmol photons m -2 s -1 The experiments were performed on 14-day-old seedlings grown vertically in a controlled growth chamber under 4% CO. Treatments were performed by applying 40 to 50 μl of freshly prepared and filtered solution to each seedling.

[0255] For the "in planta" growth test and histochemical assay of Botrytis cinerea, five-week-old Col-0 plants were germinated in soil and grown in a controlled room (8 / 16 h light / dark, 22 / 20° C. day / night) at a relative humidity of 70%. Treatment was performed by applying 40 to 50 μl of freshly prepared and filtered solution to each leaf.

[0256] Botrytis cinerea (strain B05.10, from INRA / IJPB Institute, Versailles, France) was grown on potato dextrose agar at 21°C with a photoperiod of 12 h light / 12 h dark. Two drops of 5 μL of Botrytis cinerea spores (5 × 10 5 spores ml -1 At the indicated time points, genomic DNA from whole leaves was extracted according to a modification of the protocol of Edwards et al. (A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucl. Acid. Res. 1991, vol. 19, 1349-1991), and fungal biomass was quantified as described by Gachon and Saindrenan (Real-time PCR monitoring of fungal development in Arabidopsis thaliana infected by Alternaria brassicicola and Botrytis cinerea. Plant Physiol Biochem. 2004, vol. 42, 367-371).

[0257] RNA extraction, cDNA preparation, and qPCR analysis

[0258] For RNA extraction, 14-day-old leaves from 10 independent plants were collected at the indicated time points after treatment, pooled, and frozen in liquid nitrogen. Total RNA was extracted from 100 mg of ground-frozen samples using the LEV Plant RNA kit (Promega, Madison, WI, USA), and RNA integrity was determined using a NanoDrop 2000 UV-Vis spectrophotometer (Thermo Scientific, Loughborough, UK). RT cDNA synthesis MIX (Solis Biodyne, Tartu, Estonia) was used to synthesize the first-strand cDNA. Selected MasterMix 2x (Applied Biosystems, Thermo Fisher Scientific, Waltham, USA) is carried out qPCR according to the manufacturer's protocol. Cycling conditions consist of 40 two-step cycles of 7 minutes at an initial 95°C, followed by 5 seconds at 95°C and 30 seconds at 60°C. Melting curve analysis is performed after the cycle is complete to verify amplicon identity. Relative expression levels are calculated by using actin (ACTIN) as a reference according to a standard curve-based method (Larionov et al. A standard curve based method for relative real time PCR data processing, BMC Bioinformatics. 2005, vol. 6, 1). The gene-specific primers used in this study are shown below.

[0259] Microarray hybridization and data analysis

[0260] RNA sample preparation for microarray hybridization such as Applied Biosystems TM GeneChip TM The whole transcript (WT) PLUS Reagent Kit User Guide (Thermo Fisher Scientific, Waltham, MA, USA) was performed as described. Briefly, 200 ng of total RNA was used to generate double-stranded cDNA. 12 μg of subsequently synthesized cRNA was purified and reverse transcribed into single-stranded (ss) cDNA, into which non-natural dUTP residues were incorporated. The purified and labeled sscDNA was hybridized to the Arabidopsis thaliana gene 1.0 ST array (Affymetrix, Santa Clara, CA, USA) and detected using an Applied Biosystems TM Fluorescence signals were measured using a GeneChip Scanner 3000 7G system. Sample processing was performed at the Genomic Core Facility "KFB - Center of Excellence for Fluorescent Bioanalysis" (Regensburg, Germany).

[0261] Normalized probeset signals were calculated on a log2 scale using the RMA algorithm. Average linkage hierarchical clustering of differentially expressed genes was generated using Cluster 3.0 and visualized using a Java TreeView.

[0262] Gene ontology and pathway enrichment analysis

[0263] According to Reimand et al. (Pathway enrichment analysis and visualization ofomics data using g:Profiler, GSEA, Cytoscape and EnrichmentMap. Nat Protoc. 14: 482-517.2019), gene ontology analysis of up-regulated and down-regulated genes related to the biological processes analyzed by cytoscape was performed by gProfiler software using Benjamini and Hochberg FDR (<0.05) as a filtering step and only using annotated genes (log2 fold change >1.5 or <-1.5, t-test p value <0.05).

[0264] Callose staining

[0265] Callose deposition quantification was performed essentially as described by Zarattini et al. (The bile acid deoxycholate elicits defences in Arabidopsis and reduces bacterial infection. Mol Plant Pathol. 2017, vol. 18(4), 540-554). Briefly, 5-week-old Arabidopsis leaves were stained with 0.01% w / v aniline blue in 150 mM K2HPO4 (pH 9.5) buffer for 30 minutes and subsequently decolorized overnight in lactophenol clearing solution. Leaves were examined by stereofluorescence microscopy using an Azio Zoom V.16 (Carl Zeiss Inc., Oberkochen, Germany), and callose spots were quantified using ImageJ software.

[0266] Hydrogen peroxide (H2O2) detection and determination

[0267] Hydrogen peroxide was determined in both in vitro and soil-grown Arabidopsis plants. In situ detection of H2O2 was performed according to the protocol described by Daudi and O'Brien (Detection of Hydrogen Peroxide by DAB Staining in Arabidopsis Leaves. Bio Protoc. 2012, vol. 2 (18), e263). Briefly, leaves of 5-week-old Col-O plants were drop-treated with the indicated compounds (40 to 50 μl drops per leaf). 24 hours after treatment, the leaves were treated with 1 mg ml dissolved in 10 mM sodium phosphate buffer and 0.05% v / v Tween 80. -1 Four rosette leaves from five independent plants were gently vacuum-infiltrated with 3,3'-diaminobenzidine (DAB) for 5 minutes. The staining reaction was terminated 5 hours after DAB infiltration and the leaves were mounted in ethanol: glycerol: acetic acid 3: 1: 1. Chlorophyll was removed by several washing steps with 70% v / v ethanol and photographs were taken using a stereomicroscope (Discovery V8, Zeiss).

[0268] Elicitor-induced H2O2 was also detected by a luminol-peroxidase-based assay as described by Albert et al. (Chemiluminescence Detection of the Oxidative Burst in Plant Leaf Pieces. Bio-protocol 2015, vol. 5(6), e1423). Briefly, 20 leaves were excised from 10 independent 4- to 5-week-old Col-0 plants and incubated overnight in ddH2O. Time-course luminescence was evaluated using a SpectraMax iD3 multi-mode microplate reader (Molecular devices, San Jose, CA, USA) with an integration time / well of 1000 milliseconds.

[0269] Superoxide (O2 - ) Detection and determination

[0270] Superoxide anions were determined by performing the nitro blue tetrazolium (NBT) assay. Briefly, 5-week-old Col-O plants were drop-treated with the indicated compounds. 24 h after treatment, 4 leaves were cut from 5 plants and placed in a 5-well plate containing NBT solution (3.5 mg ml -1The leaves were placed in a tube containing NBT (50 mM sodium phosphate buffer, pH 7.5) and vacuum-infiltrated for 5 minutes. The leaves were then incubated overnight in the dark. Chlorophyll was removed by several washing steps with 70% v / v ethanol, and photographs were taken using a stereomicroscope (Discovery V8, Zeiss).

[0271] AA9 cloning, expression, and oligosaccharide characterization

[0272] Several enzymes of the lytic polysaccharide monooxygenase (LPMO) auxiliary activity (AA) family-9 (AA9) were cloned to generate a variety of AA9-produced cello-oligosaccharides (AA9_COS) for use as elicitors. Particularly active examples include: TtAA9E from Thielavia terrestris (ACE10234.1), which was synthesized from Eurofins Genomic (Ebersberg, Germany), the gene sequence

[0273]

[0274] The synthetic gene was cloned into pPICZa-A containing the AOX1 methanol-inducible promoter, the α-factor secretion signal, the C-terminal c-myc epitope, and the polyhistidine tag (6His-Tag). The resulting plasmid (approximately 10 μg pPICZT::lpmo) was inserted into Pichia pastoris X-33 by transformation. Competent cells were prepared as follows: yeast cells from 100 mL YPD (1% (w / v) yeast extract, 2% (w / v) peptone, and 2% (w / v) dextrose) medium were cultured to an OD of 600 The cells were pelleted to a density of 1 to 2 and precipitated by centrifugation at 1,500 × g for 5 minutes. The cells were then washed twice with water and suspended in 8 mL of 0.1 M LiCl, 10 mM DTT, 0.6 M sorbitol, and 10 mM Tris-HCl pH 7.5 for 30 minutes at room temperature. The cells were then pelleted and resuspended in 1 M sorbitol to a density of approximately 10 10The final concentration of cells / mL was 400 cells / mL. The transformation method was performed as described by Invitrogen. Transformed Pichia pastoris colonies were cultured in YPDS (1% (w / v) yeast extract, 2% (w / v) peptone, 2% (w / v) dextrose, 1M sorbitol and 2% (w / v) agar) medium containing 0.2 mg / mL zeocin at 30°C for 3 days and selected (S. Wu, GJ Letchworth, High efficiency transformation by electroporation of Pichia pastoris pretreated with lithium acetate and dithiothreitol, Biotechniques. 36: 2004, 152-154, doi.org / 10.2144 / 04361dd02). Protein expression was performed as follows: isolated zeocin-resistant transformants were selected in YPD containing 0.2 mg / mL zeocin and incubated at 30°C and 200 rpm for 16 hours. 50 μL of YPD preculture was inoculated into 5 ml of BMGY (1% yeast extract, 2% (w / v) peptone, 1.34% (w / v) YNB, 4×10 -5 The cells were precipitated and suspended in 1 ml of BMMY (1% (w / v) yeast extract, 2% (w / v) peptone, 1.34% (w / v) YNB, 4×10 -5 The cells were then incubated in a 5% (v / v) culture medium containing 0.1% biotin, 0.1 M potassium phosphate (pH 6.0), and 1% (v / v) methanol. The culture medium was then supplemented with 2% (v / v) methanol daily for 3 days. Enzyme expression was determined by SDS-PAGE analysis of the supernatant contents (M. Haon, S. Grisel, D. Navarro, A. Gruet, J. G. Bergin, C. Bignon, Recombinant protein production facility for fungal biomass-degrading enzymes using the yeast Pichia pastoris, Front Microbiol. 6: 2015, 1002, doi.org / 10.3389 / fmicb.2015.01002).

[0275] In addition, a second example of an active LPMO used was the MtAA9A gene (MYCTH_112089) of Thermothelomyces thermophilus M77 (formerly known as Myceliophthora thermophila), which was obtained from the Fungal Genetic Stock Center (University of Missouri, Kansas City, Missouri) and amplified by PCR from genomic DNA using the original signal peptide. The PCR product was amplified using an oligonucleotide forward primer (5'-AGCATCATTACACCTCAGCAATGAAGTCCTTCACCCTCAC-3'; SEQ ID NO: 2) and a reverse primer (5'-TAAATCACTAGATATCTCTATTAGACGCACTGCGAGTAGT-3'; SEQ ID NO: 3) and cloned into the pEXPYR vector (Kadowaki et al. Functional characterization of a lytic polysaccharide monooxygenase from the thermophilic fungus Myceliophthora thermophila. Plos One 2018, vol. 13, e0202148) using a Ligation-Independent cloning protocol (LIC). The clone was transformed into A. nidulans A773 as described (Segato et al. High-yield secretion of multiple client proteins in Aspergillus. Enzyme Microb. Technol. 2012, vol. 51, 100-106). 7 spores ml -1The culture medium was inoculated into a liquid minimal medium containing 50 ml / l Clutterbuck salts (120 g / l NaNO3, 10.4 g / l KCl, 10.4 g / l MgSO4.7H2O and 30.4 g / l KH2PO4), 1 ml / l trace elements (22 g / l ZnSO4.7H2O, 11 g / l H3BO3, 5 g / l MnCl2.4H2O, 5 g / l FeSO4.7H2O, 1.6 g / l CoCl2.5H2O, 1.6 g / l CuSO4.5H2O, 1.1 g / l Na2MoO4.4H2O and 50 g / l Na2EDTA) at pH 6.5, supplemented with 1 mg / L pyridoxine and 2% (w / v) maltose, and maintained as a static culture at 37°C for 48 hours. The culture medium was filtered using a Miracloth membrane (Calbiochem, San Diego, CA, USA).

[0276] For all secreted proteins, identical following steps are used to carry out its separation and purification. By VivaFlow 200 tangential cross-flow concentrators (molecular weight cut-off, 10kDa; Sartorius Stedim Biotech GmbH, Germany), protein is concentrated 10 times, and it is immediately applied to the 10ml DEAE-agarose column (GE Healthcare) with 20mM Tris / HCl buffer pH 8.0 pre-equilibration. Enzyme is collected in the flow-through fraction, and by ultrafiltration (10kDa cut-off Centricon-Millipore, Billerica, MA, USA), concentrate. By at room temperature by protein solution in 50mMTris-HCl pH 8.0 with 3 times of molar excess Cu (II) SO 4 together, hatched 10 minutes and made MtAA9A copper saturated, and used size exclusion chromatography on HiLoad 16 / 60 Sephadex75 posts (GE Healthcare) to be further purified with the running buffer consisting of 150mM NaCl and 20mMTris-HCl pH 8.0. Total protein was quantified using the Bradford method (Bradford. A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Anal. Biochem. 1976, vol. 72, 248-254.). Protein purity was analyzed by SDS-PAGE, stained with Coomassie Blue G-250. A cellulose matrix, PASC-phosphoric acid-swollen cellulose, was prepared as follows: 4 grams of Avicel cellulose (Sigma Aldrich Saint-Luis) was suspended in 100 mL (86% w / v) phosphoric acid at room temperature and stirred for 1 hour. The mixture was then washed several times with 20 L of ice-cold water to remove H3PO4, and a 1% NaHCO3 solution was added to neutralize any residual H3PO4. The final dry matter was analyzed in a moisture analyzer MA45 (Sartorius, Germany) and the preparation was carried out according to the standard procedure described by Wood (Preparation of Crystalline, Amorphous, and Dyed Cellulase Substrates. Methods Enzymol. 1988, vol. 160, 19-25).PASC (0.5%, w / v) was incubated with 1 μM MtAA9A in 20 mM sodium acetate buffer (pH 5.0) using 1 mM ascorbic acid as a reducing agent in a final volume of 10 ml. The reaction was incubated at 50°C and shaken at 800 rpm for 16 hours. The reaction was then centrifuged at 13,000 × g for 3 minutes, and the supernatant was transferred to a new tube. 60 μL of sample was added to an HPLC conical flask, and the LPMO reaction products were analyzed by high performance anion exchange chromatography (HPAEC) on a Dionex ICS6000 instrument equipped with pulsed amperometry detection (PAD) and a CarboPac PA1 column (2 × 250 mm) and a CarboPac PA1 guard column (2 × 50 mm). Elution was performed as described by Westereng et al. (Efficient separation of oxidized cello-oligosaccharides generated by cellulose degrading lytic polysaccharide monooxygenases. J Chromatogr A. 2013, vol. 1271, 144-152). The cellulose oligomer peaks in the chromatogram were assigned according to Westereng et al. 2013 (supra).

[0277] β-Glucosidase enzymatic digestion assay

[0278] The natural cellodextrins cellobiose, cellotriose and cellotetraose, each at 100 μM, the C1-oxidized cellobionic acid, cellotriosenic acid and cellotetrasomic acid, each at 100 μM, and a mixture of AA9-COS (100 μm) were incubated at 50 degrees Celsius for 1 hour using 50 mM sodium acetate buffer at pH 5.5 and 1 μM β-glucosidase from Aspergillus niger (Megazymes, Ireland, product code: E-BGLUC). The reaction volume was set to 500 μl and placed in a 2 ml airtight tube, with constant shaking at 800 rpm, and 100 μl of the hydrolyzate was sampled after 5 minutes, 30 minutes and 60 minutes. The hydrolyzate was analyzed by HPAEC analysis according to Westereng et al. 2013 (see above).

[0279] Example 2 - Oxidized Cellodextrins and Mixtures of Oxidized and Natural Cellodextrins as Potent Plant Defense Elicitors

[0280] In this example, members of the AA9LPMO family, namely TtAA9E and MtAA9A, were cloned from the fungi Thielavia terrestris and Myceliophthora thermophila, respectively, and their active products on cellulose substrates were shown for the first time and characterized as plant defense stimulants. In order to understand the impact of LPMO-derived cellooligosaccharides (hereinafter referred to as AA9_COS) in plants, whole genome analysis was performed in Arabidopsis seedlings. Analysis of early transcriptional responses showed a powerful regulation of the plant immune system. In addition, single components, particularly cellobionic acid, cellotriosaccharic acid and cellobiose, were isolated to characterize the specific effects of oxidation and natural cellodextrins from the AA9_COS mixture. For several readouts related to plant defense, synergistic effects were observed, resulting from the combined effects of cellobionic acid and other oxidized cellodextrins, or the combined effects of oxidized cellodextrins (such as cellobionic acid) and natural cellodextrins (as found in the AA9_COS mixture). To the best of our knowledge, the smallest oxidized cellodextrin, cellobionic acid, has never been shown to activate plant defenses and has never even been shown to achieve an effect different from, superior to, or stronger than that of cellobiose. Interestingly, no oxidative burst was detected after AA9-COS treatment, but a 60% reduction in gray mold was observed in plant growth. In summary, the data confirm that mixtures of oxidized and natural cellodextrins, as well as certain individual components, such as cellobionic acid in particular, can be used as powerful plant defense elicitors, for example in agricultural and horticultural applications.

[0281] Expression of Botrytis cinerea lytic polysaccharide monooxygenase (LPMO) auxiliary activity (AA) family 9 (BcAA9) enzymes during pathogen attack

[0282] To understand the involvement of AA9 during pathogenicity, the expression of the Botrytis cinerea auxiliary activity 9 (BcAA9) gene family was measured 48 hours after inoculation of Arabidopsis leaves. Among the 10 genes in this family, BCIN_o5g08230, BCIN_06g00480, BCIN_06g07050, and BCIN_09g06730 were expressed at least threefold relative to the BcTUB genes ( Figure 1 a), while BCIN_12g03920 showed even higher expression. To predict the regioselectivity of AA9 deployed by B. cinerea during pathogenicity, a phylogenetic tree comparing the BcAA9 catalytic module sequence with the structurally resolved AA9 enzyme was constructed ( Figure 1b). This analysis showed that three of the five BcAA9 genes (BCIN_06g07050, BCIN_09g06730, and BCIN_12g03920) expressed BcAA9 in plants, clustering with AA9 enzymes from other species characterized by C1 / C4 regioselectivity. In addition, prediction of the structural catalytic domain of the BCIN_12g03920 enzyme (the most highly expressed BcAA9) showed a high degree of similarity to the structure of TtAA9E from the fungus Thermothielavioides terrestris ( Figure 1 c). Therefore, to generate a library of cellodextrins that mimic the in vivo activity of BcAA9, the enzymes TtAA9E and MtAA9A were selected from the collection. The cellodextrins obtained after incubation of TtAA9 with a cellulosic substrate (phosphoric acid swollen cellulose, PASC) were purified using a molecular sieve (3 kDa) and characterized using high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Figure 1 d). HPAEC characterization of individual components of AA9_COS revealed a 4:5:1 ratio between native, C1-oxidized, and C4-oxidized cellodextrins, respectively. The products were also tested for their resistance to the cellodextrin-degrading enzyme β-glucosidase. Degradation assays revealed that cellobionic acid and 4-keto-cellobiose (C1 and C4 oxidation products of cellobiose, respectively) were the only products resistant to β-glucosidase digestion after transient (less than 5 minutes) incubation.

[0283] Treatment with a mixture of oxidized and natural cellodextrins induces transcriptome changes in Arabidopsis seedlings

[0284] In Arabidopsis, DAMP-dependent PTI stimulation is known to be accompanied by profound transcriptional reprogramming of immune genes. Here, cDNA microarray analysis was performed in 14-day-old Arabidopsis plants 1 hour after treatment with AA9-COS or cellobiose. For both substances, a concentration of 100 μM was used because, at this concentration, cellobiose was reported to activate defense in Arabidopsis roots (Souza et al. 2017, supra), and also because dose-response analysis of selected defense marker genes such as FRK1 and WRKY18 ( Figure 2 ) showed satisfactory activation at this concentration. Transcriptome analysis was performed using Arabidopsis thaliana 1.0ST microarray chips (Affymetrix, Santa Clara, CA, USA), and differentially expressed genes (DEGs) were selected using the following two criteria: log2 expression ratio ≥1.5-fold or ≥-1.5-fold, and Student t-test p value ≤0.05. Based on these criteria, it was observed that exogenous application of AA9_COS had a strong effect on the Arabidopsis transcriptome.

[0285] Figure 3 Shown is a general overview of the statistical analysis of the microarray data obtained after treatment. This analysis highlighted changes in expression of 935 genes in the AA9-COS treated plants, of which 664 genes were upregulated and 271 genes were downregulated ( Figure 3 a). On the other hand, cellobiose treatment triggered transcriptional changes in 440 genes, of which 346 and 94 were up-regulated or down-regulated, respectively ( Figure 3 a). Interestingly, by plotting the log-based 10 The gene expression ratios of the transformed p-values ​​were observed to show higher expression and significance levels of the up-regulated genes triggered by both treatments compared to the down-regulated genes ( Figure 3 c, d). In addition, principal component analysis also explained 73% of the variance in the combined and individual samples between treatments ( Figure 3 b). The control plants clustered separately from the two treatments. Considering that the first principal component (PC1) was associated with the largest possible variance (39%), it clearly explained the effect of each treatment. However, the second principal component (PC2), associated with 19% of the variance, could be attributed to the different responses induced between cellobiose and AA9_COS application ( Figure 3 b) This data is consistent with other analyses where transcriptome changes were clearly distinct between treatments.

[0286] Transcriptome changes induced by treatment with a mixture of oxidized and natural cellodextrins were primarily associated with defense responses

[0287] In order to identify the biological processes of differentially expressed genes (DEGs) triggered by AA9_COS, gene ontology (GO) and pathway enrichment analysis were performed as described in Reimand et al. (Pathway enrichment analysis and visualization of omics data using g:Profiler, GSEA, Cytoscape and EnrichmentMap.Nat Protoc.2019, vol.14, 482-517). Typically, enrichment maps incorporate clustering of similar GO terms to create networks of nodes and edges representing gene sets and overlapping genes between gene sets, respectively. GO analysis was performed using g:Profiler software (Raudvere et al. g:Profiler: A web server for functional enrichment analysis and conversions of gene lists.Nucl.Acids Res.2019, vol.47, 191-198). Most GO terms are related to plant defense, including responses to injury, incompatible interactions, and bacterial and pathogen attacks. AA9_COS also activated genes involved in JA and SA signaling pathways, callose deposition, and response to the fungal elicitor chitin. For example, GO terms with particularly significant p-values / FDR values ​​included:

[0288] GO:0002376 Immune system processes, GO:0042742 Defense response to bacteria, GO:0009408 Response to heat, GO:0006955 Immune response, GO:0045087 Innate immune response, GO:0046677 Response to antibiotics, GO:0010200 Response to chitin, GO:0050832 Defense response to fungi, GO:009620 Response to fungi, GO:0042542 Response to hydrogen peroxide, GO:0034605 Cellular response to heat, GO:00467 77 protein autophosphorylation, GO:0009817 defense response to incompatible interactions with fungi, GO:0052542 defense response to callose deposition, GO:0009644 response to high light intensity, GO:0031347 regulation of defense response, GO:0010243 response to organic nitrogen compounds, GO:0009814 defense response to incompatible interactions, GO:0000302 response to reactive oxygen species, GO:0009636 response to toxic substances, and GO:0006979 response to oxidative stress.

[0289] To better visualize the GO analysis, GO terms were clustered using the “Autonotate” cytoscape tool, resulting in 10 clusters related to defense responses ( Figure 4 Among them, the five most enriched groups were defined as innate immune regulation (22 nodes), cell wall regulation (12 nodes), incompatible interactions (9 nodes), glucosinolate metabolism (8 nodes), and response to oxidative stress (5 nodes) ( Figure 4 In summary, pathway enrichment analysis revealed that exposure of Arabidopsis plants to AA9_COS increased the expression of genes associated with several plant defense levels.

[0290] Genome-wide comparison shows that oxidized cellodextrins are more potent than natural cellodextrins in triggering defense-related genes

[0291] To elucidate the differences and overlaps in transcriptome responses between AA9_COS and cellobiose application, pairwise transcriptome comparisons were performed ( Figure 5 Hierarchical clustering revealed a gene set that was more strongly activated by AA9_COS compared to cellobiose treatment (clusters I and II), whereas comparable gene expression was detected in genes belonging to cluster III ( Figure 5 a, Table II).

[0292] Table II. Hierarchical clustering of differentially expressed genes in Col-0 plants after 100 μM AA9_COS or 100 μM cellobiose (Cluster I). Values ​​represent fold change in gene expression compared to control.

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

[0300] These clusters contain most core defense marker genes, such as FRK1, WRKY33, and NAC055, as well as several LRR receptors and wall-associated receptor kinases ( Figure 5b and 5c, Table 1). However, cluster V, which showed opposite expression profiles between cellobiose (downregulated) and AA9_COS (upregulated), contained WRKY40, which is known to be important in signaling immune responses against biotrophic pathogens. This analysis suggests that AA9_COS plays a more powerful role in triggering plant defense responses than cellobiose.

[0301] Cluster IV ( Figure 5 a) is characterized by genes associated with cellulose and carbohydrate metabolism and cell growth GO terms, indicating that cellobiose can be more readily metabolized by plants than AA9_COS.

[0302] Cellobiocid alone showed similar or even higher elicitor capacity than the mixture of oxidized and natural cellodextrins (AA9_COS) and cellotriacylglycerol in eliciting the specific plant defense gene markers CYB81F2 and RBOHD ( Figure 6 ), whereas the universal WRKY30 marker, known to be activated by the natural cellulosic dextrin, was only activated by AA9_COS.

[0303] Regarding the total number of co-induced genes, the number of up-regulated genes (22.1%, 184 genes) was almost four times greater than that of down-regulated genes (15.1%, 48 genes). Figure 5 d). Interestingly, even among a large number of shared upregulated genes related to defense responses (including SA (salicylic acid) and JA (jasmonic acid) response genes ( Figure 5 e)), genes upregulated by AA9_COS were more enriched in defense responses, as indicated by much higher -log2(FDR) values ​​( Figure 5 e).

[0304] Similarly, SA- and JA-dependent genes were found to be considerably enriched among genes upregulated by AA9_COS treatment, whereas negligible enrichment was detected among ET (ethylene) responsive genes ( Figure 5 e). In summary, these results indicate that AA9_COS triggers a significantly higher magnitude of defense gene expression than cellobiose, suggesting that oxidized cellodextrins, such as aldonic acids, such as cellobionic acid in particular, contribute significantly to defense gene induction. For several readouts related to plant defense, the observation that this data even allows the hypothesis of a synergistic effect resulting from the combined action of cellobionic acid and other oxidized cellodextrins, or from the combined action of oxidized cellodextrins (e.g., cellobionic acid) and natural cellodextrins (as found in the AA9_COS mixture).

[0305] These predictions were further confirmed by liquid chromatography tandem mass spectrometry (LC-MS) quantification of SA and JA and gas laser detection of ethylene associated with AA9_COS-treated plants ( Figure 16 ). Compared to the simulation, SA and JA levels were detected 24 hours after AA9_COS treatment and increased by about 4-fold and 8-fold, respectively. In contrast, ethylene was monitored in the time course analysis from immediately after AA9_COS application until the next 24 hours. Compared to the simulation, an early two-fold increase in ET was observed 2 hours after AA9_COS treatment, and significantly higher ET levels persisted until 4 hours after treatment, which is typical for this volatile hormone ( Figure 16 ).

[0306] Signal transduction components modulated by a mixture of oxidized and natural cellodextrins

[0307] Transcription factors (TFs) are regulatory elements that act upstream of signal transduction pathways. Transcriptome analysis showed that AA9_COS activated specific defense-related TFs belonging to different families, such as MYB, NAC, and WRKY.

[0308] WRKY TF genes are used as important regulators of pathogen-triggered responses in many plant species. Ten members of the WRKY TF family were significantly upregulated by AA9_COS, with WRKY18 expressing the most (Table I). Previously, it has been shown that WRKY18 functions in combination with WRKY40 and WRKY60, and WRKY18, 33, and 40 are core players in early FLG22-dependent defense response signaling (Birkenbihl et al. Induced Genome-Wide Binding of ThreeArabidopsis WRKY Transcription Factors during Early MAMP-TriggeredImmunity. Plant Cell. 2017, vol. 29 (1), 20-38). Although AA9_COS did not significantly (p > 0.05) induce WRKY60 expression, the expression of WRKY40 and WRKY33 was about 2 times higher than that of mock-treated plants. In contrast, it was found that only WRKY18, 22, and 11 were significantly upregulated by cellobiose (Table I).

[0309] Another class of TFs that play an important role in regulating both biotic and abiotic stress responses is represented by the NAC TF family (Nuruzzaman et al. Roles of NAC transcription factors in the regulation of biotic and abiotic stress responses in plants. Front Microbiol. 2013, vol. 4, 248). Five NAC genes were significantly upregulated by AA9_COS, three of which were also significantly induced by cellobiose (Table 1). Importantly, it has been previously shown that the NAC gene most induced by AA9_COS (i.e., NAC055) serves as a key regulator of JA response genes (Hickman et al. Architecture and dynamics of the jasmonic acid gene regulatory network. Plant Cell 2017, vol. 29, 2086-2105). This may also be the basis for the higher enrichment of JA-dependent responses observed in AA9_COS-treated plants compared to cellobiose-treated plants ( Figure 5 e).

[0310] Similarly, members of the MYB TF family are induced by AA9_COS. Although many MYB genes are also significantly regulated by cellobiose, the expression of the MYB51 gene was observed to be significantly increased after AA9_COS treatment. Because MYB51 is the main regulator of MAMP-dependent indole glucosinolate accumulation in Arabidopsis thaliana (Frerigmann et al. Regulation of Pathogen-Triggered Tryptophan Metabolism in Arabidopsis thaliana by MYBTranscription Factors and Indole Glucosinolate Conversion Products. Mol Plant. 2016, vol. 9 (5), 682-695), this suggests that AA9_COS can also protect plants through this secondary metabolic pathway.

[0311]

[0312] In addition, to determine the reproducibility of the microarray gene expression data of the present invention, aliquots of RNA hybridized to the same array were used to further analyze 10 genes (listed in Table S1) by qPCR. The values ​​determined by qPCR correlated well with the ratios generated by the microarray results (R2 =0.91, FDR=3.9e -8 ; Figure 7 ), even though expression values ​​obtained from microarray data generally underestimate the fold enrichment determined by qPCR.

[0313]

[0314] A mixture of oxidized and natural cellodextrins does not induce oxidative burst in Arabidopsis leaves

[0315] The transient and rapid production of reactive oxygen species (ROS) in the apoplast (called oxidative burst) is a hallmark of early response to treatment with several MAMPs and chemical elicitors (Torres et al. Reactive oxygen species signaling in response to pathogens. Plant Physiol. 2006, vol. 141 (2), 373-8; Zarattini et al. 2017, supra). To evaluate the in situ production of hydrogen peroxide (H2O2) and superoxide anions (O2-), two classic histochemical DAB and NBT assays were performed in 5-week-old plants 24 hours after treatment. Figure 8 As shown in Figures 8a and 8b, no measurable production of H2O2 and O2- was detected after treatment with AA9-COS or cellobiose. In addition, to evaluate short-term extracellular H2O2 production, a luminol-based assay was performed in 14-day-old plants ( Figure 8 c). Similarly, no H2O2 production was detected in this assay following exposure to AA9_COS or cellobiose, suggesting that AA9_COS or cellobiose treatment triggers the plant immune system independently of the oxidative burst.

[0316] Unlike natural cellodextrins, oxidized cellodextrins trigger the plant's basal defenses and improve protection against gray mold

[0317] To demonstrate that the treatments of the invention induce protection against necrotrophic fungi, the gene expression data ( Figure 4 ), 5-week-old plants were treated with 100 μM AA9_COS or 100 μM cellobiose droplets and 24 h after treatment with 10 μl of Botrytis cinerea spore suspension (5×10 5 spores ml -1 Three days after fungal inoculation, the area of ​​developing necrotic lesions was analyzed by ImageJ software, and the growth of Botrytis cinerea “in planta” was evaluated by qPCR. Figure 9a shows representative photographs of necrotic lesions formed on treated and untreated plants. The average size of necrotic lesions developed on leaves pretreated with AA9-COS was significantly reduced by 30% when compared to untreated leaves, whereas little protection against Botrytis cinerea was observed in plants treated with cellobiose ( Figure 9 b). The morphological data obtained correlated well with the "in planta" growth performed 3 days after infection. In fact, a reduction of fungal biomass growth of about 50% was observed in plants pretreated with AA9_COS, while a slight reduction in the growth of Botrytis cinerea was detected in plants pretreated with cellobiose ( Figure 9 c). Therefore, in this experiment, both treatments had a positive effect on protecting Arabidopsis from Botrytis cinerea infection, with the effect of AA9_COS being much stronger than that of cellobiose ( Figure 9 c).

[0318] In fact, GO term enrichment analysis showed that the cluster of upregulated genes in AA9_COS was related to “cell wall modification”. Among them, several genes were related to callose deposition in the cell wall ( Figure 4 To experimentally illustrate this, callose was stained in 5-week-old Arabidopsis plants 6 and 24 hours after treatment with 100 μM AA9-COS, 100 μM cellobiose, or 1 μM FLG22 ( Figure 9 d). At both time points, a significant increase in callose accumulation was detected after AA9-COS treatment. In contrast, no visible callose spots were detected after cellobiose treatment. Interestingly, different kinetics and amplitudes of callose deposition were observed between AA9-COS and FLG22 treatments, with the former having a stronger effect 24 hours after treatment and the latter having a stronger effect at 6 hours ( Figure 9 d).

[0319] Taken together, these results establish that AA9_COS induces resistance to the necrotrophic fungus Botrytis cinerea that is more significant or stronger than the resistance induced by cellobiose.

[0320] A mixture of oxidized and natural cellodextrins triggers phytoalexin biosynthesis and signaling

[0321] Previous studies have shown that the accumulation of tryptophan-derived phytoalexins is a key response to enhance resistance to gray mold. The regulation of phytoalexin biosynthesis that occurs during pathogen attack has been well described (He et al. The Arabidopsis Pleiotropic Drug Resistance Transporters PEN3 and PDR12 MediateCamalexin Secretion for Resistance to Botrytis cinerea. Plant Cell. 2019, vol. 31 (9), 2206-2222). This pathway involves the activation of the MPK3-MPK6-WRKY33 signaling module, which in turn induces the expression of PAD3 as well as PDR12 and PEN3 (two pleiotropic drug resistance transporter genes).

[0322] Real-time qPCR was performed and the expression profiles of these genes were analyzed 1 hour and 24 hours after treatment with 100 μM AA9-COS or 100 μM cellobiose in 14-day-old Arabidopsis plants. In addition to PDR12 (data not shown), significant mRNA accumulation of MPK3 / 6, WRKY33, PAD3, and PEN3 was observed after AA9-COS application compared to mock- and cellobiose-treated samples ( Figure 10 With the exception of PAD3, comparable gene expression profiles were observed after cellobiose treatment, even though expression levels were lower compared to AA9-COS-treated plants ( Figure 10 ).

[0323] Figure 10 c shows the quantification of phytoalexins in 14-day-old Col-0, sif2 and sif4KO mutants 24 hours after 100 μM AA9_COS, 100 μM cellobiose or mock treatment. For each treatment, phytoalexins were extracted from a pool of 14-day-old Arabidopsis. Briefly, 100 mg of ground-frozen samples were soaked in 80% (v:v) methanol at a ratio of 1:1 (m / v). After homogenization, the supernatant was recovered by centrifugation at 17,000 × g for 5 minutes and evaporated at 50°C under a nitrogen atmosphere. The residue was finally resuspended in high-performance LC-grade methanol and filtered with a 0.22 μm filter. Then, a reverse phase Each pool was analyzed in triplicate by LC-MS using an Omega 5μm Polar C18 column (250×4.6 mm). The mobile phase consisted of 0.1% water (Solvent A) and acetonitrile (Solvent B). The applied gradient was as follows: 0 to 2 minutes, 10% B; 2 to 13 minutes, 10 to 98% B; 13 to 23 minutes, 98% B; 23 to 25 minutes, 98 to 10% B; 25 to 30 minutes, 98% B; 1 minute after the run.

[0324] In Col-0 plants, cellobiose and mock produced comparable levels of phytoalexins (mock = 2.4 ± 1.4 ng g-1, cellobiose = 3.7 ± 1.8 ng g-1), while AA9-COS produced 35.0 ± 5.4 ng g-1. In contrast, no increase in phytoalexin production or growth of Botrytis cinerea was observed in either sif2 or sif4 plants treated with AA9-COS.

[0325] Figure 10 d shows early immunohistochemical quantification or Western blot analysis of MPK3 / 6 phosphorylation after the specified treatment. 14-day-old Arabidopsis wild type, sif2 and sif4 mutant plants were treated with 100 μM AA9_COS, 100 μM cellobiose or mock. Leaves were collected 5 minutes after treatment, immediately frozen in liquid nitrogen, and stored at -80°C for analysis. After protein extraction, 20 μg of protein per sample was denatured and separated on a 12% acrylamide / bisacrylamide SDS-PAGE gel. The separated proteins were transferred to a 0.45 μM Immobilon-PPVDF membrane (Millipore). Blocking was performed in TBST containing 5% skim milk powder, and the blots were incubated with specific antibodies (anti-AtMPK6 and anti-phospho-p44 / 42 MPK (Thr202 / Tyr204)) at 4°C overnight, washed in TBST, and incubated with secondary antibody anti-rabbit IgG conjugated to horseradish peroxidase (HRP) enzyme at a dilution of 1 / 10,000. TM Western EC Substrate and ChemiDoc TM The MP Imaging System was used for chemiluminescent detection of HRP.

[0326] In Col-0 plants, early MPK3 / 6 phosphorylation was detected after treatment with 100 μM AA9_COS, whereas no phosphorylation was observed after treatment with 100 μM cellobiose ( Figure 10d and data not shown). Therefore, the MPK3 / 6 protein and its phosphorylated form (pMPK3 / 6) were hybridized in Col-0, sif2, and sif4 mutant lines. After AA9-COS treatment, the present inventors observed pMPK3 / 6 signals in both Col-0 and sif2 and sif4 KO lines, although a slight decrease in the pMPK3 / 6 form was observed in the sif4 KO line ( Figure 10 d and data not shown).

[0327] SIF2 and SIF4 (two LRR-RLKs) were shown to be involved in defense activation driven by a mixture of oxidized and natural cellodextrins

[0328] Transcriptome analysis highlighted several PRR genes that were regulated by AA9_COS treatment. Among them, two leucine-rich repeat receptor-like kinases (LRR-RLKs), stress-inducible factors 2 and 4 (SIF2 and SIF4), were strongly upregulated and appeared in cluster 1 ( Figure 5 d). To evaluate whether SIF2 and SIF4 play a role in the signaling of AA9_COS-dependent defense gene activation, sif2-1 and sif4-1 loss-of-function mutants were treated with 100 μM AA9_COS drops, and the expression of four marker genes, FRK1, WRKY18, WRKY22, and WRKY33, was evaluated 1 hour after treatment ( Figure 11 ). In sif2-1( Figure 11 ) and sif4-1 (not shown) mutants, gene expression of all tested marker genes was not upregulated by AA9_COS, whereas the increased expression of these genes was significant in wild-type plants, suggesting that these LRR-RLKs may be involved in key regulators of signaling transduction for AA9_COS-dependent activation of PTI gene expression.

[0329] Protection in tomatoes

[0330] The protection of tomato plants against the pathogen Botrytis cinerea was demonstrated using the assay for Bc symptoms (described above) using AA9-COS cellodextrins (natural cellodextrin, C1-oxidized and C4-oxidized cellodextrins in a 4:5:1 ratio) at different concentrations (negative control (CTR), 0.1 μM, 1 μM, 10 μM, 100 μM). The values ​​represent the size of the lesion area produced during infection with the pathogen, i.e. values ​​below the control indicate that the defense mechanism has been triggered. Beneficial effects were already observed at the lowest concentration of AA9-COS (i.e. 0.1 μM) and were already significant at 10 μM ( Figure 12 ).

[0331] Comparison of Cellobiose in Tomatoes vs. a Mixture of Oxidized and Natural Cellodextrins

[0332] The protection of tomato plants against the pathogen Botrytis cinerea was compared using 100 μM cellobiose or 100 μM AA9-COS ("CTR" is a negative control). The values ​​represent the size of the lesion area produced during infection with the pathogen, i.e. values ​​below the control indicate that the defense mechanism has been triggered. Cellobiose alone showed less protection than the AA9-COS mixture ( Figure 13 All experiments were performed on 10 6 spores / ml of Botrytis cinerea on 4-week-old tomato plants.

[0333] Comparison of plant elicitor activities of mixtures of oxidized cellodextrins and natural cellodextrins with different molar ratios of the components

[0334] Several combinations of natural cellodextrins, and C1-oxidized, C4-oxidized, and optionally C1- and C4-oxidized (double-oxidized) cellodextrins were tested for protection of tomato plants against the pathogen Botrytis cinerea. Figure 14 ). In particular, Figure 14 The numbers in represent a mixture of the cellodextrin components having the following molar ratios:

[0335] Natural: C1 oxidation: C4 oxidation

[0336] (1) 4:5:1; (2) 6.5:3:0.5; (3) 3:6.5:0.5; (4) 4:3:3; (5) 4:1:5; (6) 0.5:9:0.5;

[0337] Natural: C1 oxidation: C4 oxidation: double oxidation

[0338] (7)0.5∶4∶0.5∶5;(8)2.5∶2.5∶2.5∶2.5.

[0339] The molar amounts of the components were adjusted using a combination of known techniques to separate and combine the components."CTR" stands for negative control, water.

[0340] The values ​​represent the size of the lesion area produced during pathogen infection, i.e. values ​​below the control indicate that the defense mechanism has been triggered. All experiments were performed after infection with 10 6 All mixtures were applied at the same molar mass concentration (100 μM).

[0341] In general, all mixtures used successfully triggered plant defenses. In this experimental model, the most active mixture was 4:5:1 and the least active was 4:3:3, with the other mixtures providing intermediate protection.

[0342] Example 3 - Obtaining AA9_COS from Biorefinery Waste or Specially Engineered Waste Bioprocessing Using Cellulosic Substrates and AA9 Enzyme

[0343] Oxidized cellodextrins or mixtures of native and oxidized cellodextrins, or components thereof such as cellobiose, illustrating embodiments of the present invention, can be obtained from several bioresources, typically waste products of lignocellulose-based biorefining industries, such as those described below, which generate large amounts of waste products comprising, in whole or in part, or consisting of AA9-COS. Lignocellulosic bioethanol production:

[0344] The production of lignocellulosic bioethanol uses the non-edible parts of wood or crops as a source of cellulose to be converted into fermentable sugars through a chemical-enzymatic reaction step. Commercially available and widely used enzymes contain considerable amounts of AA9 or similar cellulose-active family members of the AA family that are considered LPMOs. The remaining lignocellulose digested after fermentation and ad distillation produces a mixture of phenols and undigested sugar-derived molecules, of which cellobionic acid is typically present at a percentage of 0.1% to 2% of the total wet volume. Since these enzyme mixtures are designed to maximize monosaccharide separation, they contain large amounts of β-glucosidase, which converts most of the oxidized cellodextrins into non-fermentable C1 or C4 oxidized disaccharide forms (cellobionic acid and 4-ketobic acid), as well as fermentable glucose molecules. Some examples of such processes are the Poet-DSM plant in the United States, SEKAB technology in Sweden, GranBio in Brazil, ST1 in Finland, all run by the Novozymes Cellic Ctec family, Metgen and Graanul biotech joint venture at Sweetwoods technology (Estonia), using their proprietary enzymes including the AA9 enzyme.

[0345] Production of Nanocellulose Fibers:

[0346] In the emerging nanocellulose fiber industry, great interest has been given to the use of cellulases (especially comprising AA9, such as the enzyme required to initiate fiber opening). Similar to lignocellulose biorefining, these processes are generating residual sugar streams. Among them, cellobiocides are present in an amount estimated to be 0.1% to 2% of the total wet volume of the sugar waste stream. The adjustable composition of the enzyme to be used in this process makes it possible to configure in the absence of β-glucosidase, because the fiber must remain intact, so that longer oxidized cellodextrins or longer mixtures of natural and oxidized cellodextrins can be obtained, rather than as the main or only disaccharide form in the case of lignocellulose biorefining.

[0347] Specific biological processes for waste paper valorization:

[0348] A new specific bioprocess is envisioned for the direct or maximized production of oxidized cellodextrins or a mixture of native and oxidized cellodextrins by using low-cost cellulosic substrates such as waste paper, which can be enzymatically pretreated with AA9 enzyme to obtain several oxidized cellodextrins or a mixture of native and oxidized cellodextrins before their normal recycling chain (pulping).

[0349] The material thus obtained comprising oxidized cellodextrins or a mixture of native and oxidized cellodextrins can be used in agricultural technology directly or after optional enrichment or purification or formulation with a suitable carrier.

[0350] Example 4 - Ethylene Emission ( Figure 15 ).

[0351] Three Arabidopsis seedlings were cultured in vitro for 3 weeks in glass bottles containing sterile Murashige Skoog medium and treated by spraying with 100 μM AA9-COS, 100 μM cellobiose, 100 μM cellobionic acid, or mock (H O). In three independent experiments, ethylene production was analyzed using an ETD 300 detector (Sensor-Sense, Nijmegen, the Netherlands) under a stop-and-flow program, in which gas from one bottle was accumulated over 10-minute intervals and then flushed to the detector. Figure 15 The bars represent the mean and standard deviation. Ethylene production was higher in Arabidopsis seedlings treated with either AA9-COS or cellobionic acid than in mock-treated seedlings. This suggests that the oxidized oligosaccharides contained in the AA9-COS mixture, particularly cellobionic acid, are responsible for inducing the key defense-related plant hormone, ethylene.

Claims

1. Use of cellobionic acid or a plant pharmaceutically acceptable salt thereof as a plant pathogen defense elicitor.

2. A method for activating a plant's defense against plant pathogens, comprising contacting the plant with an effective amount of cellobionic acid or a phyto-pharmaceutically acceptable salt thereof.

3. The use according to claim 1 or the method according to claim 2, wherein the cellobionic acid or its salt is contained in a composition that further comprises one or more cellodextrins; one or more C1 oxidized cellodextrins or their plant pharmaceutically acceptable salts; one or more C4 oxidized cellodextrins; and / or one or more C1 and C4 oxidized cellodextrins or their plant pharmaceutically acceptable salts.

4. The use according to claim 1 or the method according to claim 2, wherein the cellobionic acid or its salt is contained in a composition further comprising one or more additional plant defense stimulants selected from the group consisting of: pectin fragments, oligogalacturonosides, cellobiose, xyloglucan, unbranched β-1,3-glucan, chitin fragments, arabinose, arabinan, rhamnose, homogalacturonan, rhamnogalacturonan I and II, xylogalacturonan, starch, and combinations thereof.

5. The use or method of claim 3, wherein the cellodextrin, C1 oxidized cellodextrin, C4 oxidized cellodextrin, and C1 and C4 oxidized cellodextrin each independently has a degree of polymerization (DP) of 2 to 10.

6. The use or method of claim 5, wherein the cellodextrin, C1 oxidized cellodextrin, C4 oxidized cellodextrin, and C1 and C4 oxidized cellodextrin each independently has a degree of polymerization (DP) of 2 to 5.

7. The use according to claim 1 or the method according to claim 2, wherein the cellobionic acid is produced by breaking down cellulose with one or more lytic polysaccharide monooxygenases (LPMOs).

8. The use or method of claim 3, wherein the cellodextrins, C1 oxidized cellodextrins, C4 oxidized cellodextrins, and C1 and C4 oxidized cellodextrins are produced by breaking down cellulose with one or more lytic polysaccharide monooxygenases (LPMOs).

9. The use or method of claim 7, wherein at least one of the one or more LPMOs belongs to adjuvant activity (AA) family 9.

10. The use or method of claim 8, wherein at least one of the one or more LPMOs belongs to adjuvant activity (AA) family 9.

11. The use or method of claim 9, wherein at least one of the one or more LPMOs is LPMO-AA9 from Botrytis cinerea, Thielavia terrestris, or Myceliophthora thermophila.

12. The use or method of claim 7, wherein one or more cellobiose dehydrogenases (CDHs) are used to enhance the activity of the LPMO.

13. The use or method of claim 8, wherein one or more cellobiose dehydrogenases (CDHs) are used to enhance the activity of the LPMO.

14. The use or method according to claim 12, wherein CDH from Botrytis cinerea or Myceliophthora thermophila is used to enhance the activity of the LPMO.

15. The use or method of claim 7, wherein the cellulose is derived from lignocellulosic waste or pulp paper.

16. The use or method of claim 8, wherein the cellulose is derived from lignocellulosic waste or pulp paper.

17. The use or method of claim 15, wherein the cellulose is derived from a biorefinery lignocellulosic by-product treated with LPMO.

18. The use according to claim 1 or the method according to claim 2, wherein the concentration of cellobionic acid or a salt thereof is 0.10 μM to 1000 μM.

19. The use or method of claim 18, wherein the concentration of cellobionic acid or a salt thereof is 0.10 μM to 500 μM.

20. The use or method of claim 18, wherein the concentration of cellobionic acid or a salt thereof is 0.10 μM to 100 μM.

21. The use or method of claim 18, wherein the concentration of cellobionic acid or a salt thereof is 0.10 μM to 10 μM.

22. The use or method of claim 3, wherein the concentration of the cellodextrins, C1 oxidized cellodextrins including the cellobionic acid, C4 oxidized cellodextrins, and the total concentration of C1 and C4 oxidized cellodextrins is 0.10 μM to 1000 μM.

23. The use or method of claim 22, wherein the concentration of the cellodextrins, C1 oxidized cellodextrins including the cellobionic acid, C4 oxidized cellodextrins, and the total concentration of C1 and C4 oxidized cellodextrins is 0.10 μM to 500 μM.

24. The use or method of claim 22, wherein the concentration of the cellodextrins, C1 oxidized cellodextrins including the cellobionic acid, C4 oxidized cellodextrins, and the total concentration of C1 and C4 oxidized cellodextrins is 0.10 μM to 100 μM.

25. The use or method of claim 22, wherein the concentration of the cellodextrins, C1 oxidized cellodextrins including the cellobionic acid, C4 oxidized cellodextrins, and the total concentration of C1 and C4 oxidized cellodextrins is 0.10 μM to 10 μM.

26. The use or method of claim 3, wherein the composition comprises cellodextrin, C1 oxidized cellodextrin, and C4 oxidized cellodextrin in a molar ratio of 4:5:

1.

27. The use according to claim 1 or the method according to claim 2, wherein: Prevent, control or treat plant infections caused by plant pathogens; The plant pathogen is a necrotrophic fungus or bacterium, a hemitrophic fungus or bacterium, or a biotrophic fungus or bacterium; The plant is a dicotyledonous plant selected from the family Brassicaceae, Solanaceae or Rosacea, or wherein the plant is a monocotyledonous plant selected from the family Gramineae; and / or The plant is contacted via an organ of the plant selected from the group consisting of leaves, roots and / or fruits, or via a seed of the plant.

28. A botanical pharmaceutical composition comprising 0.10 μM to 1000 μM of cellobionic acid or a botanical pharmaceutically acceptable salt thereof and a botanical pharmaceutically acceptable carrier comprising one or more anionic surfactants, nonionic surfactants, amphoteric surfactants or cationic surfactants, or a combination thereof.

29. The botanical pharmaceutical composition of claim 28, wherein the composition comprises 0.10 μM to 500 μM cellobionic acid or a salt thereof.

30. The botanical pharmaceutical composition of claim 28, wherein the composition comprises 0.10 μM to 100 μM cellobionic acid or a salt thereof.

31. The botanical pharmaceutical composition of claim 28, wherein the composition comprises 0.10 μM to 10 μM cellobionic acid or a salt thereof.

32. The botanical pharmaceutical composition of claim 28, wherein: The composition further comprises one or more cellodextrins; one or more C1 oxidized cellodextrins or plant pharmaceutically acceptable salts thereof; one or more C4 oxidized cellodextrins; and / or one or more C1 and C4 oxidized cellodextrins or plant pharmaceutically acceptable salts thereof; The composition further comprises one or more additional plant defense elicitors selected from the group consisting of pectin fragments, oligogalacturonosides, cellobiose, xyloglucans, unbranched β-1,3-glucans, chitin fragments, arabinose, arabinans, rhamnose, homogalacturonans, rhamnogalacturonans I and II, xylogalacturonans, starch, and combinations thereof; The degree of polymerization (DP) of the cellodextrin, C1 oxidized cellodextrin, C4 oxidized cellodextrin, and C1 and C4 oxidized cellodextrins are each independently 2 to 10; The cellobionic acid or the cellodextrins, C1 oxidized cellodextrins, C4 oxidized cellodextrins, and C1 and C4 oxidized cellodextrins are produced by decomposing cellulose with one or more lytic polysaccharide monooxygenases (LPMOs), optionally wherein: at least one of the one or more LPMOs belongs to adjuvant activity (AA) family 9; One or more cellobiose dehydrogenases (CDHs) are used to enhance the activity of the LPMO; and / or The cellulose is derived from lignocellulosic waste or pulp paper; The cellodextrin, C1 oxidized cellodextrin including cellobionic acid, C4 oxidized cellodextrin, and the total concentration of C1 and C4 oxidized cellodextrins is 0.10 μM to 1000 μM; and / or The composition includes cellodextrin, C1 oxidized cellodextrin, and C4 oxidized cellodextrin in a molar ratio of 4:5:

1.

33. The botanical pharmaceutical composition of claim 32, wherein the cellodextrins, C1 oxidized cellodextrins, C4 oxidized cellodextrins, and C1 and C4 oxidized cellodextrins each independently have a degree of polymerization (DP) of 2 to 5.

34. The botanical pharmaceutical composition of claim 32, wherein at least one of the one or more LPMOs is LPMO-AA9 from Botrytis cinerea, Thielavia terrestris, or Myceliophthora thermophila.

35. The phyto-pharmaceutical composition according to claim 32, wherein CDH from Botrytis cinerea or Myceliophthora thermophila is used to enhance the activity of the LPMO.

36. The botanical pharmaceutical composition of claim 32, wherein the composition comprises or consists essentially of a LPMO-treated biorefinery lignocellulosic by-product.

37. The botanical pharmaceutical composition of claim 32, wherein the concentration of the cellodextrins, C1 oxidized cellodextrins including the cellobionic acid, C4 oxidized cellodextrins, and the total concentration of C1 and C4 oxidized cellodextrins is 0.10 μM to 500 μM.

38. The botanical pharmaceutical composition of claim 32, wherein the concentration of the cellodextrins, C1 oxidized cellodextrins including the cellobionic acid, C4 oxidized cellodextrins, and the total concentration of C1 and C4 oxidized cellodextrins is 0.10 μΜ to 100 μΜ.

39. The botanical pharmaceutical composition of claim 32, wherein the concentration of the cellodextrins, C1 oxidized cellodextrins including the cellobionic acid, C4 oxidized cellodextrins, and the total concentration of C1 and C4 oxidized cellodextrins is 0.10 μM to 10 μM.

40. The botanical pharmaceutical composition of claim 28, wherein the surfactant is Triton X-100, Tween-20, Tween-40 and / or Silwet L-77.

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