Patent Application
20230104477
The present invention relates to the use of dehydroascorbic acid (DHA) for protecting plants against pathogens, in particular against infection with nematodes. The invention also provides compositions comprising the same, methods of making the same, and methods of controlling plant disease.
The agricultural industry is under constant pressure to efficiently enhance food production in order to cope with ever increasing food demands. The only durable way to meet up with that demand is through increasing agricultural productivity from the same—or ideally even reduced—acreages of arable land. It has been estimated that 80% of all crop problems start with root problems and healthy development of plant roots is the key to improve crop yield (Root Health—the key to improving yield; White paper, 2011). Stronger, healthier roots are better suited for uptake of water and nutrients, two of the most critical resources. More robust root systems help to produce stronger stems and foliage that can better withstand environmental stress, especially in challenging climates, and ultimately lead to improved yield consistency (EIP-Agri focus group report, 2015). Plants are subject to multiple belowground disease-causing agents, including abiotic stress such as drought and biotic stress such as e.g. plant-pathogenic nematodes, which are responsible for significant economic losses in agriculture.
Sugar beet (Beta vulgaris L.) is the primary source of sugar in the temperate zone and it accounts for 20% of the world's total sugar production. According to the European Commission the EU is the world's leading beet sugar producer with 16.84 million tonnes of sugar production in year 2016/2017, however it exports around 8% of its sugar production. Below ground plant parasitic nematodes cause significant damage to sugar beet production. Generally, crop losses caused by plant-parasitic nematodes are worth of at least 80 billion dollars per year globally. Heterodera schachtii (beet cyst nematode (BCN)) is responsible for 90% of all nematode-related sugar beet infestations worldwide. BCN are sedentary endoparasites feeding within the roots of their host (Moens et al., 2018). Cyst nematodes have the ability to survive in the soil for many years in the absence of a suitable host plant (Perry et al., 2018). The presence of root exudates provides a cue for eggs inside the cysts, causing second-stage juveniles (J2) to hatch and invade the roots of the host plant (Moens et al., 2018). The infective J2 stage invades plants primarily in the elongation zone above the root tips (Wyss and Grundler, 1992). Upon invasion, the J2 migrates further into the cortex where it initiates a feeding structure (syncytium) in the root vascular tissue (Sobczak and Golinowski, 2009).
Rice has been cultivated as a staple crop for many years, and sustains approximately half of the world's population. Yield losses in rice because of nematodes have been estimated to range between 10 and 25% worldwide (Kyndt et al., 2014). Meloidogyne graminicola or the rice root-knot nematode (RKN) is one of the most prevalent nematodes in various rice growing agrosystems. The infective second stage juveniles (J2s) of M. graminicola enter the root in the elongation zone and move intercellular until they reach the vascular cylinder where they induce the formation of feeding sites containing 5-8 giant cells, leading to root galls, preferentially at the root tips (Kyndt et al., 2014). Once the giant cells are established, the nematodes become sedentary, start feeding, and molt three times to reach the adult stage.
Upon pathogen attack, the plant innate immune system responds with a series of local and systemic defence mechanisms (Dhankher et al., 2018; Jones and Dangl, 2006). The plant immune system can be activated externally, for example by pathogens, or applying hormone analogues (systemic acquired resistance, dependent on salicylate (SA)) or by chemicals or beneficial micro-organisms (induced systemic resistance, ISR).
Previously, it has been shown that root application of β-aminobutyric acid (BABA) (Jisha and Puthur, 2016), thiamine (Huang et al., 2016) or silicon (Zhan et al., 2018) enhances the rice plant defence system, leading to improved tolerance or resistance against root parasitic nematodes. Systemic defence activation has also been observed: foliar application of methyl jasmonate (MeJA), ethephon, SA-analogue BTH, or COS-OGA (Fytosave®, a mixture of chitosan oligomers (COS) and pectin-derived oligogalacturonides (OGA)) can activate root defence pathways with varying efficacy (Nahar et al., 2011; Singh et al., 2019).
Several studies show a negative correlation between the amount of ascorbic acid (AA) and resistance to aboveground pathogens. Pavet et al., (2005) showed that Arabidopsis vtc (vitamin C)-mutants exhibit enhanced basal resistance against Pseudomonas syringae, accompanied by increased cell death activation. However, there are also reports on the role of ascorbic acid in defence against pathogens. Brassica rapa cultivars with naturally high amounts of AA showed enhanced resistance to Turnip Mosaic Virus, while vtc1 plants are more susceptible than wild-type lines (Fujiwara et al., 2016). Furthermore, Fujiwara et al., 2013, reports an antiviral effect of DHA applied to Brassica rapa plants. Also, effects of L-ascorbic acid on plant root elongation in non-infected conditions have been described in literature (e.g. Li et al., 2018; Kka et al., 2018).
In the present invention it was demonstrated that the application of dehydroascorbic acid (DHA) improves general plant tolerance to belowground infection, in particular by parasitic nematodes. Moreover it was shown that application of DHA has a positive effect on plant root elongation in infected plants.
The present invention provides the use of dehydroascorbic acid (DHA), or a stereoisomer, tautomer, hydrate, salt, ester, solvate, complex and/or functional analog thereof, for controlling nematode infections in plants and/or for promoting root growth in (nematode-)infected plants. The use of DHA was demonstrated to be effective against root parasitic nematodes, in particular root-knot nematodes and/or cyst-nematodes. Application of exogenous DHA or compound provided herein can be to the leaves of the plant, as a seed coating or as a soil drench. In one embodiment the present invention encompasses a spraying formulation comprising DHA or a compound of the invention.
A particular advantage is that a compound of Formula I or II, more specific DHA, can be applied to the plant as a pre-emptive measure or as a priming agent, i.e. before infection or visible damage/signs of infection. In a particular embodiment, the invention provides the use of DHA, or a stereoisomer, tautomer, hydrate, salt, ester, solvate, complex and/or functional analog thereof, for stimulating the immune defence of a plant against nematode infection, in particular against root-parasitic nematodes.
Susceptibility assay on rice plants soil drenched with AA (20 mM), AO (20 U/mL), DHA (20 mM) or water as a negative control. Fifteen-day-old plants were inoculated with 250-300 nematodes. 24 hours before the inoculation, plants were soil drenched with respective chemicals. The number of galls per plant (A) and number of nematodes per plant (B) was counted at 12 days after inoculation. Bars represent means and standard deviation of 8 plants. Different letters indicate statistically significant differences (Duncan; α=0.05). Data represent one of three independent experiments with similar results. AO, ascorbate oxidase; DHA, dehydroascorbate, AA ascorbic acid.
Wild type Nipponbare (Nip), and JA signaling mutant jar1 were sprayed with water, AO (20 U/mL), DHA (20 mM), and DIECA (JA biosynthesis inhibitor, 100 μM) or AoA (ET biosynthesis inhibitor, 12.5 mM) either alone or in combination with AO (20 U/mL) or DHA (20 mM). Plant roots were inoculated with 300 M. graminicola juveniles at 24 hours after spraying with respective chemicals. Bars represent means±standard error of the number of galls of 8 plants at 12 days after inoculation. Different letters indicate statistically significant differences (Duncan's multiple range test with α=0.05). MeJA, methyl jasmonate; AO, ascorbate oxidase; DHA, dehydroascorbate; AoA, aminooxyacetic acid; DIECA, diethyldithiocarbamic acid.
As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise. The terms “comprising”, “comprises” and “comprised of” as used herein are synonymous with “including”, “includes” or “containing”, “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The term “about” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of +/−20% or less, preferably +1-10% or less, more preferably +/−5% or less, of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” refers is itself also specifically, and preferably, disclosed. Whereas the terms “one or more” or “at least one”, such as one or more or at least one member(s) of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any >3, >4, >5, >6 or >7 etc. of said members, and up to all said members. All references, and teachings specifically referred to, cited in the present specification are hereby incorporated by reference in their entirety. Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the present invention. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous. Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention.
The present invention relates to a method to control nematode infection in plants. More specific, the present invention relates to a method for reducing or preventing root damage by or due to nematode infections in plants, said method comprising applying dehydroascorbic acid (DHA), or a stereoisomer, tautomer, hydrate, salt, ester, solvate, complex and/or functional analog thereof, to said plant. Moreover, it was demonstrated that the application of DHA to healthy as well as (nematode-) infected plants results in enhanced root growth (e.g. longer and more roots when compared to control plants). It was furthermore shown that increase or accumulation of DHA inside roots of plants results in tolerance against nematode infection and promotes root growth. Hence the invention provides a method to control nematode infections in a plant and/or to stimulate root development in a plant by applying a compound of the invention, in particular dehydroascorbic acid (DHA), to the plant, thereby inducing accumulation of DHA in the roots of said plant.
Dehydroascorbic acid (DHA) (C6H6O6; IUPAC (5R)-5-[(1S)-1,2-dihydroxyethyl]furan-2,3,4(5H)-trione) is an oxidized form of ascorbic acid (vitamin C), having the following structural formula:
The present invention provides methods and uses of DHA as presented in Formula I, or a stereoisomer, tautomer, hydrate, ester, solvate, and/or complex thereof. Functional analogs of DHA are also part of the present invention and can be used in the methods and uses provided herein.
Accordingly and in one embodiment, the present invention provides methods and uses of a compound of the Formula II, or a stereoisomer, tautomer, hydrate, salt, ester, solvate, and/or complex thereof, wherein the compound of Formula II is:
wherein R1 and R2 each independently represents a hydrogen atom, —SO3H, —PO3, H2, a glycosyl group, or —COX wherein X represents an unsubstituted or substituted C1 to C30 alkyl group or an unsubstituted or substituted C2 to C30 alkenyl group. In one embodiment R2 represents xylose. More specific, at least one of R1 and R2 represents —COX, wherein X represents an unsubstituted or substituted C1 to C30 alkyl group or an unsubstituted or substituted C2 to C30 alkenyl group. The glycosyl group is a sugar residue such as a monosaccharide e.g. glucose, galactose, fructose, rhamnose and the like; or a low molecular weight oligosaccharide, e.g. rutinose, vicianose, lactose, maltose, sucrose and the like.
The number of substituents in the alkyl or alkenyl groups may be 1, or may be 2 or more. Two or more substituents may be the same, or may be different. Examples of groups which can be a “substituent” in the C1 to C30 alkyl group or the C2 to C30 alkenyl group include a hydroxyl group; a mercapto group; an amino group; a nitro group; a halogen atom; an alkoxy group; an aryloxy group; a haloalkoxy group; an alkylthio group; an alkylamino group; an arylamino group; a cyano group and the like.
A “C1 to C30 alkyl group” in X is a saturated hydrocarbon group comprising 1 to 30 carbon atoms. A C1 to C30 alkyl group may be a linear chain, or may be a branched chain. Examples include methyl, ethyl, n-propyl, etc. A “C2 to C30 alkenyl group” in X is an unsaturated hydrocarbon group comprising 2 to 30 carbon atoms having at least one carbon-carbon double bond. A C2 to C30 alkenyl group may be a linear chain, or may be a branched chain. Examples include propenyl, allyl, butenyl, pentenyl, etc.
Suitable agrochemical acceptable salts may include alkali metal salts, e.g. sodium or potassium salts; alkaline earth metal salts, e.g. calcium or magnesium salts; and salts formed with suitable organic ligands, e.g. quaternary ammonium salts. A salt can be any ionic compound that can be formed by the neutralization reaction of an acidic compound and a base. Examples include, but or not limited to, an ammonium, a potassium or isopropylamine salt. In another embodiment, base salts are formed from bases which form non-toxic salts, including aluminum, arginine, benzathine, choline, diethylamine, diolamine, glycine, lysine, meglumine, olamine, tromethamine and zinc salts. In one embodiment, hemisalts of acids and bases may also be formed, for example, hemisulphate and hemicalcium salts. In a particular embodiment, the invention also encompasses esters derived from the compounds of the invention. One example of an ester is a compound wherein at least one —OH (hydroxyl) group is replaced by an —O-alkyl (alkoxy) or other organic group. Examples include, but or not limited to, methyl, ethylhexyl or ethyl esters. In one embodiment, the ester is palmitoyl ascorbate or ascorbyl stearate. Said esters can be of particular interest for a foliar application in view of enhanced penetration properties.
In one embodiment, the invention provides the use of L-dehydroascorbic acid (CAS Number 490-83-5).
In a further embodiment, the compound of Formula I or II, in particular DHA, is used to control nematode infections in plants. More specific, the compound of the invention is used to improve tolerance of plants or crops to nematodes, in particular root nematodes. The present invention demonstrates that exogeneous application of the compound, in particular DHA, to a plant protects plants against infection with nematodes, and more specific inhibits the penetration of plant parasitic nematodes (resulting in reduced numbers of nematodes on the plants), improves root growth and development under infected conditions and/or reduces number of feeding sites such as e.g. the production of galls or syncytia by such nematodes. Hence, the compounds of the invention can be used as a “plant stimulator”, i.e. a compound increasing the immune defence of a plant and stimulating root growth.
The terms “controlling nematodes” or “protecting against nematode infection” refers to reducing the overall negative effect of plant pathogenic nematodes on plants such that the plants experience a decreased amount of negative effects by said nematodes as compared to plants not treated with the compound, in particular DHA. The overall negative effect by plant pathogenic nematodes may be reduced, such as by reducing the overall number or density of plant parasitic nematodes (i.e., decrease in nematode population) on the plant or by reducing the severity or extent of negative effects of the nematodes (e.g. number of lesions, spots, wilting, gall formation, rot, seedling death, growth reduction, yield losses, etc.). Of particular advantage is that the compound, in particular DHA, stimulates the systemic immune defence of the plant and can be used pre-emptively or preventive as a priming agent (e.g. to seedlings or non-infected plants), as well as curative and require only a simple formulation. The use as a priming agent will delay, or even prevent, the damage to the plant when becoming infected.
As used herein, the term “nematode” refers to multicellular animals in the phylum Nematoda. “Plant parasitic nematode” refers to nematode parasites of plants which can be found in/on plant roots, seeds, flowers, leaves, stems, or the soil in which the plant is growing. Plant parasitic nematodes feed on all parts of the plant, including roots, stems, leaves, flowers and seeds and include microorganisms from the genera Meloidogyne, Heterodera, Globodera, Pratylenchus, Aphelenchoides, Xiphinema, Radopholus, Bursaphelenchus, Rotylenchulus, Nacobbus, Longidorus, Ditylenchus and Trichodorus. In one embodiment, the parasitic nematodes are from the genera Meloidogyne, Heterodera and Pratylenchus. “Cyst nematodes” (genera Heterodera and Globodera) and “root-knot nematodes” (genus Meloidogyne), in particular, cause significant economic loss in plants, especially crop plants. Examples of cyst nematodes include, H. schachtii (sugar beet cyst nematode), H. avenae (cereal cyst nematodes), H. glycines (soybean cyst nematode), H. sacchari (sugarcane cyst nematode), H. carotae (carrot cyst nematode), G. pallida (white potato cyst nematode) and G. rostochiensis (yellow potato cyst nematode). Root-knot nematodes include, for example, M. graminicola, M. javanica, M. incognita, M. arenaria, M. chitwoodi, M. artiellia, M. fallax, M. hapla, M. microtyla, M. partityla, M. panyuensis, M. naasi, M. exigua, M. enterolobii and M. paranaensis. These pathogens establish “feeding sites” in the plant, by causing the morphological transformation of plant cells into giant cells or syncytia, which block the flow of nutrients and photosynthesis products. Hence, nematode “infection” refers to invasion of and feeding upon the tissues of the host plant. Other nematodes that cause significant damage include the “root-lesion” nematodes such as Pratylenchus, particularly P. penetrans, which infects maize, rice and vegetables, P. brachyurus which infects pineapple, P. zeae, which infects cereals, sugarcande and coffee, P. coffeae, which infects coffee and banana, and P. thornei, which infects wheat.
In a further embodiment, the invention provides the use of a compound of Formula I or II, more specific DHA, or a stereoisomer, tautomer, hydrate, salt, ester, solvate, and/or complex thereof, for controlling infection of a plant with a root-knot nematode selected from the group consisting of: M. graminicola, M. javanica, M. incognita, M. arenaria, M. chitwoodi, M. artiellia, M. fallax, M. hapla, M. microtyla, M. partityla, M. panyuensis, M. naasi, M. exigua, M. enterolobii and M. paranaensis, in particular M. graminicola. In another embodiment, the invention provides the use of a compound of Formula I or II, more specific DHA, or a stereoisomer, tautomer, hydrate, salt, ester, solvate, and/or complex thereof, for controlling infection of a plant with a cyst nematode selected from the group consisting of H. schachtii, H. avenae, H. glycines, H. sacchari, H. carotae, G. pallida and G. rostochiensis, in particular H. schachtii.
In a further embodiment, the present invention discloses a composition or formulation comprising the compound as provided herein and a diluent, an additive, a plant (micro) nutrient, an emulsion stabilizer, a surfactant, a buffer, a crop oil, a drift inhibitor and/or an (inert) substratum, in particular water and/or a surfactant. In particular, the composition is a foliar spray, root drench or a seed coating. Furthermore, the compound of the invention, or a composition comprising it can further include or be used in combination with one or more fertilizers, biostimulants, herbicides, fungicides, bactericides, acaricides, nematicides and/or insecticides. In particular the composition is an agrochemical composition comprising DHA or functional analog as provided herein, and at least one excipient, in particular an agriculturally and/or horticulturally acceptable excipient. The term “agriculturally and/or horticulturally acceptable” indicates that the formulation is non-toxic and otherwise acceptable for application to a plant, whether applied indoors (e.g. in a contained environment) or outdoors (e.g. in a non-contained environment that is exposed to other plant, animal and human life).
In a further embodiment, the compounds of Formula I or II, or as provided herein, is isolated (e.g. in substantially purified form) or synthetically made using standard techniques, or commercially available. In a particular embodiment, the compound is not part of a natural extract made by extracting a part of a raw material, often by using a solvent such as ethanol or water (e.g. aqueous or ethanolic extracts of seeds or plants; oils). More in particular, the compound, in particular DHA (e.g. as part of a composition) is employed in a purity of from 90% to 100%, preferably from 95% to 100% (according to their NMR spectrum).
Examples of suitable excipients are Tween® 20 (comprising polyoxyethylene (20) and sorbitan monolaurate (polysorbate 20)), castor oil ethoxylate, rapeseed methyl ester, alkyl phosphates, tributyl phosphate, tripropyl phosphate, naphthalene sulphonic acid salts, organic sulfonate/2-methylpentane-2,4-diol, alkylpolyglucoside, siloxanes derivates, alkylsulfonates, polycarboxylates, lignosulfonates, alkoxylated triglycerides, fatty amines polymers, and dioctylsulfosuccinates.
The composition or formulation will typically contain effective amounts of a compound as described herein. An “effective amount” means that it is used in a quantity which allows to obtain the desired effect but which does not give rise to any significant phytotoxic symptom on the treated plant. In one embodiment, the concentration of the compound (e.g. DHA) administered on or to the plant ranges from 0.5 to 1000 mM, from 1 to 500 mM, in particular from 1 to 200 mM, more in particular from about 5 to about 150 mM, even more in particular from about 10 to about 100 mM.
Any plant/crop can be treated. The term “plant (or plants)” is a synonym of the term “crop” which is to be understood as a plant of economic importance and/or a men-grown plant. The methods, compounds and compositions of the present invention may be applied to any monocot or dicot plant or a tree. In one embodiment, the treated plant belongs to the amaranth family (Amaranthaceae), e.g. sugar beet (Beta vulgaris) or spinach (Spinacia oleracea). In another embodiment, the treated plant is a monocot plant, in particular a grass, more in particular a cereal grass such as rice. Rice is a monocot and includes the genera Oryza, Zizania and Porteresia.
In a further embodiment of the present invention, a compound of the invention, or a composition comprising it is applied to a plant, directly or indirectly. Any appropriate plant part can be treated or used including plant organs (e.g., leaves, stems, roots, etc.), seeds, and plant cells and progeny of the same. In the alternative, the compound or a composition can be applied to the soil or substrate surrounding the plant, however with direct contact with the roots. The applying of the compound is prior to planting, at planting, or after planting. In one embodiment, contacting includes direct application to a plant. All or part of a plant including, without limitation, leaves, stems, roots, propagules (e.g., cuttings), fruit, seeds etc., may be contacted with the compound as provided herein. Contacting may also be carried out indirectly, via application, e.g., to soil or other plant substrates but making uptake by the plant possible.
Preferred application methods are foliar treatment (e.g. by spraying), seed treatment (e.g. by seed coating) or soil treatment (e.g. by soil drenching), and in particular foliar treatment. Plant parts to be treated can be a seed, fruit, fruit body, leaf, stem, shoot, stalk, flower, root, tuber or rhizome, in particular leaves.
Suitable application methods include high or low-pressure spraying, immersion, atomizing, foaming, fogging, coating, and encrusting. Other suitable application procedures can be envisioned by those skilled in the art. In a particular embodiment, the compound of the invention is applied to the parts of the plant above ground or to the foliage of the plant by spraying e.g. by the use of mechanical sprayers. Sprayers convert a formulation of the invention which is mixed with a liquid carrier, such as water or fertilizer, into droplets. The droplets can be any size. Boom sprayers and air blast sprayers can also be used to apply formulations of the invention to pre-emerging or post-emerging crops. Air blast sprayers inject formulations of the invention mixed with a liquid carrier into a fast-moving air stream. Where the formulations of the invention are in a solid, powder or granule form, they can be applied with granule or dust application equipment.
In another embodiment, seeds of a plant are coated with a compound as provided herein (“coated seeds”). Any appropriate seed coating method known the skilled person can be used.
In another aspect, the compound of the invention, or a composition comprising it can be applied to the substrate of the plant (e.g. in hydroponics) or to the soil directly, e.g. by drip irrigation or drench application (soil drench). A soil drench applies the compound, such as DHA, optionally mixed with water, to the soil around the base of a plant so that its roots can absorb the extract.
According to the method or use of the present invention, the compound provided herein, or the composition comprising it according to the invention can be applied once to a plant (part)/crop, or it can be applied two or more times after each other with an interval between every two applications as can be determined by the person skilled in the art.
The following examples are set forth below to illustrate the methods, compositions, and results according to the disclosed subject matter. These examples illustrate representative methods, compositions, and results. These examples are not intended to exclude equivalents and variations of the present invention, which are apparent to one skilled in the art.
Rice—Seeds of wild-type rice Oryza sativa cv. Nipponbare were obtained from USDA (GSOR-100). Seeds were germinated on wet filter paper for 4-5 days at 30° C. and seedlings were then transferred to SAP-substrate (Sand mixed with Absorbent Polymer AquaPerla®, DCM, Belgium) and further grown at 28° C., under a light regime of (day/night) 12/12. Hoagland solution was given as a source of nutrients 3 times a week.
Sugar beet—seeds of cv. Amarok were germinated on moist tissue for 4-5 days at 25° C. For longer experiments seedlings were transplanted to polyvinyl-chloride (PVC) tubes containing fine soil particles. All experiments were conducted in a plant growth room at 25° C., with a 16/8 h day/night regime. The plants were fertilized by supplying Hoagland solution as a source of nutrients 3 times a week.
Root Knot Nematode Meloidogyne graminicola
M. graminicola originally isolated in the Philippines (Batangas) was cultured on susceptible rice plants. Second stage juveniles (J2s) were extracted using a sieve/tray extraction method and inoculation was done as described in Nahar et al., (2011). For susceptibility assays, at least two independent experiments were carried out, each time including eight plants per treatment. Twelve days after inoculation (dai), roots were stained using acid fuchsine (Nahar et al., 2011) to count galls and number of nematodes in the roots.
Beet Cyst Nematode (BCN), Heterodera schachtii
BNC cysts were obtained from Iran and were maintained on sugar beet (cv. Amarok) plants. Cysts were obtained from stock cultures, by washing the infected soil and letting the cysts to float in water. Full cysts with eggs were hand-picked and allowed to dry before setting up for hatching. For second-stage juveniles (J2) to hatch, cysts were placed in 3 mM of ZnCl3 and transferred to an incubator/shaker at a temperature of 25.5° C. at 70 rpm for 4 days (Kammerhofer et al., 2015). J2 were re-suspended in water and counted under a stereomicroscope. Around 250-300 J2 nematodes were inoculated per plant. One day before and after inoculation, plants were not supplied with any nutrients or water. To investigate the plant tolerance to BCN infection root length, shoot height, fresh root weight as well as the number of cysts were measured at different time points after spraying and/or inoculation. For all experiments, 8 individual plants were used per treatment.
Reduced Ascorbic acid (AA, 20 mM), dehydroascorbic acid (DHA, 20 mM), ascorbate oxidase (AO, 20 U/ml), methyl jasmonate (MeJA, 100 μM), aminooxyacetic acid (AoA, 12.5 mM), diethyldithiocarbamic acid (DIECA, 100 μM) and ethephon (100 μM) were purchased from Sigma and dissolved in water (except MeJA that was first dissolved in a drop of ETOH) to attain the indicated concentration.
3-days old rice seedlings were drenched with 1 mL of AA (20 mM), AO (20 U/mL), DHA (20 mM) or water (H2O) as a negative control. Rice root length and dry root weight as well as the number of embryonic crown roots (ECR) were measured 5 days after drenching.
For foliar application, all chemicals were dissolved in water and 0.02% (v/v) of Tween20 as a surfactant was added prior to spraying on aerial portions of the rice plants. Control plants were sprayed with distilled water containing 0.02% Tween20. Eight plants per treatment were sprayed until run-off with 6.5 mL of solution (at 24 h prior to nematode inoculation, in cases where infected plants were used). The substrate was covered to avoid direct contact with the roots and/or nematodes.
For soil application, 6.5 mL of each solution was drenched on the substrate, 24 h prior to nematode inoculation.
Ascorbate measurements—Plant roots were collected from a pool of 5-6 plants per treatment and crushed in liquid nitrogen. In each assay, 100 mg of crushed material was used per sample, and at least 4 biological replicates were analyzed per treatment. Reduced and oxidized AA were measured according to the method described in Ueda et al., 2013. Absorbance was monitored using a microplate reader (TECAN—Infinite200pro).
H2O2 measurements—14 days old rice plants were foliar sprayed with 20 mM DHA and 1 day later, plants were inoculated with Meloidogyne graminicola nematodes (Mg+) or mock inoculated (Mg−). One day after inoculation, samples were collected. Root samples were flash-frozen and ground under liquid nitrogen. Per sample, 100 mg of material was used to execute the xylenol orange method for H2O2 quantification described by Cheeseman (2006). After 5% trichloroacetic acid extraction, and 30 minutes reaction with the ferrous ammonium sulphate/xylenol orange (FOX) reagent, the absorbance was measured at 560 nm. The concentration was calculated by comparison with a standard curve of known H2O2 concentrations. The assay was done with 3 biological replicates, which each consisted of pooled material of 4 plants.
PAL activity measurements—14 days old rice plants were foliar sprayed with 20 mM DHA and 1 day later, plants were inoculated with Meloidogyne graminicola nematodes (Mg+) or mock inoculated (Mg−). Three or seven days after inoculation, samples were collected. Both galls and systemic tissue (shoots) were collected. Samples were flash-frozen and ground under liquid nitrogen. Per sample, 100 mg of material was used to execute PAL-activity measurement according to Camacho-Cristobal et al. (2002). PAL was assayed by measuring the formation of trans-cinnamic acid at 290 nm. Samples were dissolved in 800 μl of 50 mM sodium phosphate as assay buffer containing 2% (w/v) poly-vinylpolypyrrolidone (PVPP), 2 mM EDTA, 18 mM-mercaptoetanol and 0.1% (v/v) Triton X-100. The homogenate was centrifuged at 8000 rpm, 4° C. for 10 min. In different 2 ml tubes, 135 μl of reaction buffer, 50 μl of 5 mM of L-phenlyalanine, and 20 μl of supernatant were mixed and absorbance at 290 nm was measured before incubation at 40° C. for 30 minutes. Then, 10 μl of hydrochloric acid was added and measurement was again done at the same wavelength. The assay was done with 4 biological replicates, which each consisted of pooled material of 4 plants.
Fourteen-day-old rice plants were foliar sprayed with 20 mM DHA as described above. Root material from 3 biological replicates of control plants and 3 biological replicates of treated plants were sampled at 1 day after treatment. Each biological replicate consists of at least 4 individual plants that were pooled to minimize inherent biological variability. RNA was extracted using the RNeasy Mini Kit by Qiagen. Quality and concentration of the samples was assessed using NanoDrop 2000. Approximately one microgram of total RNA was used for 3′ mRNA-Seq library construction using the QuantSeq 3′ mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen). To minimize lane effects the samples were multiplexed, using the multiplexing sequencing adapters provided in the Multiplexing Sample Preparation Oligo Kit (Illumina). Size selection of the library was performed on a 2% agarose gel (Low Range Ultra Agarose, Biorad 161-3107). The denatured library was diluted to a final concentration of 6 pM and loaded on a flow cell (Illumina). After cluster generation, the multiplexed library was sequenced on an Illumina NextSeq500 (75 cycles, single end, high output). Data-analysis was performed using Microsoft Open R (version 3.5.2). The raw data underwent a quality control via the fastqc function. Following the quality control, samples were trimmed (using Trimmomatic (Trimmomatic Manual: V0.32) and again their quality was checked. Trimmed data was mapped onto the annotated genome by making use of the STAR-mapper function (Dobin, 2019). The counttable of the data was generated using the summarize Overlaps function in R. Differentially expressed genes were detected using the DESEQ2 function to analyse the countable and applying a false discovery rate of 0.01 (adjusted p-values <0.01). The gene lists were analysed using the Wilcoxon Signed Rank test in MapMan to identify significantly induced pathways.
Three days after germination, rice seedlings were drenched with 1 mL of AA (reduced ascorbic acid, 20 mM), DHA (20 mM), or ascorbate oxidase (AO, 20 U/ml) or water as control. 5 days after this application, the root length (
14-days old rice plants were foliarly sprayed with AA (20 mM), AO (20 U/mL), DHA (20 mM) or water (H2O) as a negative control (Ctrl). One day later, half of the plants were inoculated with 250 nematodes per plant, while the other half stayed uninfected. The root length of all plants was monitored at three different time points: 4 days, 8 days and 12 days after spraying (DAS). Under uninfected conditions (
Rice plants were foliar sprayed with AA (20 mM), DHA (20 mM), or ascorbate oxidase (AO, 20 U/ml), 24 h prior to nematode inoculation. We investigated the effect of these treatments on number of galls and number of nematodes at 12 dai. There were no significant differences in number of galls and total number of nematodes between the control and AA-sprayed plants (
We also evaluated the effect of foliar application with these chemicals (AA, AO & DHA) on endogenous reduced AA and oxidized AA (=DHA) levels, 24 h after their application. The amount of reduced AA was significantly increased in the shoots of AA sprayed plants (
In this experiment the same treatments as in example 3 were applied as soil drenching. Twelve days after root-knot nematode inoculation (M. graminicola), we observed no significant difference in number of galls and total number of nematodes between control and AO soil drenched rice plants (
To investigate whether the induction of a systemic induced defence in rice against root-knot nematodes works through activation of ethylene (ET) and jasmonic acid (JA), an infection experiment was carried out using foliar application of DHA. This treatment was done on the jasmonate signalling Osjarl mutant, and on wild-type Nipponbare (Nip) plants. Also, DHA was sprayed on wild-type Nipponbare in combination with JA biosynthesis inhibitor ammonium diethyldithiocarbamic acid (DIECA) or ET biosynthesis inhibitor aminooxy acetic acid (AoA) (
Two-week old sugar beet plants were foliarly sprayed with AA (20 mM), AO (20 U/mL), DHA (20 mM) or water as a negative control (Ctrl). Seven days later, the length of root and shoot and the fresh root weight was measured. Results, shown in
Three-weeks old sugar beet plants were foliarly sprayed with AA (20 mM), AO (20 U/mL), DHA (20 mM) or water as a negative control (Ctrl). One day later, the plants were inoculated with 250 Heterodera schachtii nematodes per plant. 3 days later, which corresponds to 4 days after spraying (DAS), the root and shoot parameters were evaluated. Results, shown in
The same plants were also evaluated at a later time point, namely 43 DAS, corresponding to 42 days after nematode inoculation. At this time point, the DHA treated plants still had significantly more fresh root weight (
Biochemical measurements of defence responses, more specifically levels of H2O2 and PAL-activity were investigated in 20 mM DHA-treated plants, which were either uninfected or infected with root-knot nematode M. graminicola. PAL-activity, which is an indication for activation of the phenylpropanoid pathway is strongly enhanced upon DHA treatment in both locally infected tissue—i.e. galls in comparison with uninfected root tips—as in systemic shoot tissues of infected versus uninfected plants, at 3 and 7 dpi (
To confirm defence induction by DHA, gene expression analysis (‘transcriptome analysis’;
| Number | Date | Country | Kind |
|---|---|---|---|
| 19218568.4 | Dec 2019 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/086690 | 12/17/2020 | WO |
