Background
An emerging strategy of pest control is to use (natural) compounds that elicit plant defenses, rather than to use biocides. In the case of biotic stress, plants implement a plurality of endogenous defense mechanisms that can develop in space and time from the point of infection or infestation, and contribute to reduce the spread of the disease: hypersensitivity reaction, local acquired resistance, systemic acquired resistance. Plant defense mechanisms that are inducible by plant pathogens involve molecules such as ones with direct antimicrobial or insecticide activity, or molecules involved in the reinforcement of cell walls, thus limiting plant colonization.
The detailed knowledge of these defense mechanisms led to the development and use of plant care-products that no more act directly on the cause of the stress, but have the property to act indirectly by stimulating the natural defense mechanisms. These pesticides called plant elicitors are classified in two main categories: the direct elicitors (which lead, when applied to the plant, to a direct activation of the defense responses whether or not attack of pathogens), and the potentiators (which will activate the defenses in response to a subsequent stress).
Very few approvals to market have been issued to date for such products. In contrast, a wide variety of products available (officially or not) claim more or less explicitly to stimulate the plant defenses without evidences, and are sold mixed with fertilizers. Finally, some products registered as phytopharmaceuticals with effects other than plant-protection would be likely to have also an effect on the stimulation of the natural plant defenses.
To accurately characterize existing and future products for their ability to elicit plant defenses and therefore to be considered as real plant elicitors, there is a need to develop new devices that are complementary or alternative to the phenotypic assays of plant protection, which give an indication of efficiency without prejudging their mode of action. In other words, the different phytopharmaceutical stakeholders are seeking a device or a versatile tool allowing to quickly and easily identify the state of stimulation of the natural defenses of plants, to screen compounds for their properties of plant defense stimulation, or to select plants that are sensitive to elicitors.
Description of the innovation and industrial applications
The research team of Marie-Noëlle Brisset within the Plant Pathology Research Unit of INRA Angers has developed an innovative system allowing the simultaneous analysis of the different metabolic pathways of plant defense against biotic and / or abiotic stress.
The molecular diagnostic tool called "qPFD" (quantitative low-density microarray: microplate quantitative RT-PCR / low-density DNA microarray) is able to evaluate a set of nine groups representing 28 target genes whose expression alone or in combination provides information on the stimulation state of the natural defenses of plants. Results of the qPCR are operated by the ΔΔCT method which provides relative expressions of defense genes in a given sample towards a control sample. Expressions are normalized by the geometric mean of 3 genes of reference (TUA, Actin, GAPDH) in this sample (Figure 1: gene expression profile viewed as a heat map). Researchers have specifically chosen the 28 defense genes by analogy to model plants in order to cover the different signaling pathways (e.g. salicylic acid, jasmonic acid, ethylene) or downstream defense (e.g. PR proteins or enzymes of the secondary metabolism) of apple tree (Malus domestica), the species used for the validation of the qPFD concept in trials performed under controlled conditions with 10 products (elicitors, tree thinners, growth regulators) known for their protective effectiveness (pathosystem: apple - Erwinia amylovora, the causal agent of fire blight).
The tests performed on populations of apple seedlings have shown the correlation between the lack of stimulation of the selected defense genes and the lack of protective effect. Moreover, researchers have shown that overexpression of a combination of target genes is most often associated with protection against the pathogen.
Industrial applications of qPFD:
- Screening and selection of new elicitor molecules, or the screening of existing products
- Optimization of their use alone or as compositions (dose, delay of response, duration of effect, etc.)
- Selection of plants susceptible to elicitors and having an improved stress resistance (screening of genotypes for their reactivity to elicitors)
This invention can be implemented by various stakeholders of the phytopharmaceutical sector:
- Organizations of fruit tree growers to determine whether the announced properties of so-called elicitors are real
- Professional or official committees, and control laboratories to evaluate, select or approve products useful for fruit tree cultivation
- Plant protection firms to analyze their own molecules and optimize their use
- Plant breeders
Advantages and benefits provided by qPFD include:
- Substantial gain of time and economy of field experiments, unnecessary and expensive for products that do not have any effect on plant defenses
- qPFD avoids redundant processing, including implementation of compounds eliciting the same defense mechanisms, and limits negative interactions (some elicitors have antagonistic effects regarding the molecular pathways they regulate)
- Distinction between direct elicitors and potentiators
- qPFD overcomes all the stages of development of the qPCR
This versatile device targeting a defined set of genes is capable to determine the stimulation state of the natural defenses of plants exposed to one or more elicitors, and / or exposed to a wide variety of biotic or abiotic stress, and / or exposed to a combination of these two exposures
- Easy, fast and efficiently interpretable results on the stimulation state of plant natural defenses
- Ready to use, the qPFD can be routinely implemented for analyzing induction levels of the different defense pathways of Rosaceae (e.g. apple tree), or other plant species
Furthermore, it should be noted that the team of Dr. Brisset has initiated further work on vine (Vitis vinifera), which is also of great economic interest and subject to many treatments, in order to consolidate the molecular approach on other plant species.
Intellectual property and technology transfer
These results have been protected by the French patent application No. 1055042 filed by INRA on June 24, 2010 and the PCT/FR2011/051470 filed on June 24, 2011 and entitled "Device for determining or analysing the state of stimulation of natural defenses of plants or part of plant".
INRA Transfert is in charge of the technology transfer of this innovation towards stakeholders of the phytopharmaceutical sector via the concession of licenses for commercial applications.
Moreover, collaborative research projects on the numerous potential applications of qPFD (molecular screening, other pathosystems, other plant species, etc.) will be studied with interest by INRA.
Scientific leader
Marie-Noëlle BRISSET
Research unit
UMR0077 Pathologie Végétale: Biodiversité, Ecologie, Interactions Bioagresseurs / Plantes
INRA
42 rue Georges Morel
49071 Beaucouzé Cedex
France
Technology Transfer Officer
Claire NODET
INRA Transfert
28, rue du Docteur Finlay
75015 Paris
France
Phone: +33 (0) 1 42 75 93 44
Fax: +33 (0) 1 45 77 63 90
Email: claire.nodet@paris.inra.fr
Figure1: Results of qPFD analysis of the stimulation state of the natural defenses of apple seedlings treated with different products (elicitors, tree thinners, growth regulators). The columns correspond to the 9 groups of 28 genes of plant natural defense, the lanes correspond to trials of treatment with 10 products tested as potential elicitors. The heat map shows the levels of overexpression of genes (red) or inhibition of genes (blue) in leaves of apple seedlings. Note that the products C, G, H show a very similar activation of the same combination of genes, correlated with protection against Erwinia amylovora. No other product is effective in terms of protection against this pathogen, except streptomycin, used here as a biocide control.
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