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(ll)l'atentNumber: KE 378

(45) Date org,.nt 23/03/2010

(51) lntCI.8:  A OIN 47/40,51/00

(21) Application Number: KE/P/2008/ 000719

(22) Filing Date: 19/09/2006

(30) Priority data: 102005045174.8  21/09/2005  DE

(86) Pcrdata PCT/EP06/009072 19/09/2006  wo 2007/033810 A2    29/03/2007

(54) Title: PLANT PATHOGENIC DEFENCE INCREASE
 
(73)0wner: BAYER CROPSCIENCE AG  of  Alfred-Nobel-Str. 50, 40789 Monheirn , Gennany

(72) Inventor: ANDERSCH, Wolfram; THIELERT, Wolfgang; ECKES, Peter and BENTING, JUrgen

(74) Agent/address for correspondence: Kaplan & Stratton Advocates, P.O. Box 40111-00100, Nairobi
 
(57) Abstract: The invention relates to a method for increasing a plant pathogenic defence by cbloronicotinyl treatment. The chloronicotinyls

make it possible to obtain a good protection of plants against damages caused by fungal, bacterial or viral pathogens independently of insect control. The anti-pathogenic defences are obtainable by inducing PR proteins following the treatment by at least one type of cbloronicotinyl

Increasing pathogen defence in plants

The present invention relates to a method of increasing the defence of plants against pathogens.

It is known that plants react to natural stress conditions, such as for example cold. heat, dryness, wounding and pathogenic infections (caused by viruses, bacteria, fungi or insects) etc. as well as to herbicides by specific or non•specific defence mechanisms [cf. Pflanzenbiochemie (Plant Biochemistry), pp. 393-462, Spektrum Akademischer Verlag, Heidelberg, Berlin, Oxford, Hans W
Heldt, 1996~ Biochemistry and Molecular Biology of Plants, pp. 1102-1203, American Society of

10    Plant Physiologists, Rockville, Maryland. eds. Buchanan, Gruissem, Jones, 2000]. In this process cell wall components formed for example by wounding or specific pathogen-derived signal substances act as inducers of plant signal transduction chains which in the end lead to the fonnation of defence molecules directed against the stress factor. These substances can for

example be  (a)  low  molecular weight substances,  such  as for example  phytoalexins,  (b) non-IS    enzymatic  proteins,  such  as  for  example  "pathogenesis-related  proteins"  (PR  proteins),  (c) enzymatic  proteins,  such  as  for example chitinases or glucanases or (d) specific  inhibitors  of essential proteins, such as for example protease inhibitors or xylnnase inhibitors, which attack the pathogen directly or hinder its proliferation (cf. Dang! and Jones, 2001, Nature 411:  826-833;

Kessler and Baldwin, 2003, Annual Review of Plant Biology, 53: 299-328).

20

An additional defence mechanism is the so-called hypersensitive reaction (HR.) which is induced by oxidative stress and leads to the dying.-off of plant tissue in the region of the site of an infection, thus preventing the spread of plant pathogens, which need living cells to survive [cf. Pennazio,
1995, New Microbial. 18, pp. 229-240].

25

In the further course of an infection, the plant's inherent messenger substances send signals to non-affected tissue, in which they also cause defensive responses to be triggered and hinder the formation of secondary infections (System Acquired Resistance, SAR) [Ryals et al., 1996, The
Plant CellS: 1809-1819].

30

A number of plant-endogenous signal substances are already known which are involved in stress tolerance or pathogen defence. These include for example salicylic acid, benzoic acid, jasmonic acid or ethylene (Biochemistry and Molecular Biology of Plants, pp. 850-929, American Society of Plant Physiologists, Rockville, Maryland, eds. Buchanan, Gruissem, Jones, 2000]. Some of

35    these substances or their stable synthetic derivatives and derived structures are also effective when applied externally to plants or seed dressings and they activate defence reactions which produce
increased stress or pathogen tolerance by plants [Sembdner and Parthier,  1993, Ann. Rev. Plant

Physiol. Plant Mol. Bioi. 44:  569~589). This salicylate-induced defence is directed specifically

against phytopathogenic fungi, bacteria and viruses [Ryals et al., 1996, The Plant Cell 8: 1809-1819].

One well-known synthetic product which has a similar function to salicylic acid and can induce a protective effect against phytopathogenic fungi, bacteria and viruses, is benzothiadiazole (trade name Bion"l [Achuo et al., 2004, Plant Pathology 53 (1): 65-72].

10    Other compounds belonging to the oxylipin group, such as for example jasmonic acid, and the protective mechanisms triggered thereby are particularly effective against insect pests [Walling, 2000, J Plant Growth Regul. 19, 195-216].

It is therefore known that plants have several endogenous reaction mechanisms which can produce

15    effective defence against the most diverse types of hannful organisms (biotic stress) and/or natural abiotic stress.

It is already known that chloronicotinyl insecticides can be used for combating animal pests, and in

particular  insects.   It  is  also  known  that  the  treatment  of plants  with  insecticides  from  the

20    chloronicotinyl series produces increased resistance of plants to abiotic stress. This applies in particular to imidacloprid (cf. Brown et al., 2004, Beltwide Cotton Conference Proceedings: 2231-2237). This protection ftmctions by influencing the physiological and biochemical properties of the plant cells, such as for example by improving membrane stability, increasing the carbohydrate

concentration and increasing the polyol concentration and antioxidant activity (Gonias et at., 2004,

25    Beltwide Cotton Conference Proceedings: 2225-2229).

Only occasional references can be found in the literature to the activity of chloronicotinyls against biotic stress factors (Crop Protection (2000), 19(5), 349-354; Journal of Entomological Science (2002), 37(1), 101-112; Annals of Biology (Hisar, India) (2003),19(2), 179-181).

30

Chloronicotinyls can be defined by the following general fonnula (D,

in which

Het    represents a heterocycle which is in each case optionally mono- or polysubstituted by fluorine, chlorine, methyl or ethyl and is selected from the following group of

heterocycles:

pyrid-3-yl,  pyrid-5-yl,  3-pyridinio,  l-oxido-5-pyridinio,  1-oxido-5-pyridinio,  tetrahydro-

furan-3-yl and thiazol-5-yl,

A    represents C1-C,-alkyl, -N(R 1)(R2)  or S(R2),

wherein

represents  hydrogen,  C1-4-alkyl,  phenyi..C1-C4-alkyl,  C3-4-cycloalkyl,  C2-C6-

10    alkenyl or CrC6-alkinyl and

R'

R    represents hydrogen, C1-C6-alkyl, CrC6-alkenyl, C2-C6-alkinyl, -C(=O)-CH3 or benzyl or, together with R2, represents one of the following groups:

-CH2-CHr.  -CH2-CHrCHr,  -CHrO-CHr,  -CH2-S-CHr.  -CHz-NH-CHr  and  -CHz-

15    N(CH3)-CH2- and

X    represents N-N02, N-CN or CH-N02•

Het    particularly  preferably  represents  a  heterocycle  selected  from  the  following  group  of

heterocycles:

2-chloropyrid-5-yl, 2-methylpyrid-5-yl, 1-oxido-3-pyridinio, 2-chloro-1-oxtdo-5-pyridinio,

20    2,3-dichloro-1-oxido-5-pyridinio, tetrahydrofuran-3-yl, 5-methyl-tetrahydrofuran-3-yl and 2-chlorothiazol-5-yl.

particularly preferably represents -N(R 1)(R2).

particularly preferably represents hydrogen, methyl or ethyl.

particularly preferably represents methyl, ethyl, n-or i-propyl, n-, i-, s-ort-butyl, ethenyl, !-propenyl, 2-propenyl, ethinyl, 1-propinyl, 2-propinyl, -C(=O)-CH3 or benzyl.
 

R    particularly preferably represents hydrogen, methyl, ethyl or -C(=O)-CH3 or particularly preferably, together with R2, represents one of the following groups:

This class of compounds includes for example the following list of compounds, which must not

however be understood to be final:

imidacloprid of the formula (I)

Cl--0-cH;-N?-H

N..._N02
(1), cf. EP 0 192 060,

clothianidine of the formula (II)
!j    H    ~
cl-'(").-cH;-~N,
S    n  CH3

N..._N02
(II), cf. EP 0 376 279,

dinotefuran of the formula (lll)
1
0--CH-N  H
I
'  y"-cH,

N......_N02
(ill), cf. EP 0 649 845,

10    thiamethoxam of the formula (IV)



(IV), cf. EP 0 580 553,

thiacloprid of the formula (V)
Cl-o-~ CH;-N'l.
N-    y

N.......,_CN
(V), cf. EP 0 235 725,


15    acetamiprid of the formula (VI)
-o- 'i"•
Cl    CHr-N    CHs
y

N........ CN
(VI), cf. WO 91/04965 and

nitenpyram of the formula (VII)
\'•"• ~
Cr-0--CH;-N'(N-cH
N    '
'l_
NO,    (VII), cf. EP 0 302 389.

Surprisingly it has now been found that chloronicotinyls, and in particular imidacloprid, result in the increased expression of genes from the group comprising "pathogenesis-related proteins" (PR proteins). PR proteins assist plants primarily in their defence against biotic stressors, such as for example phytopathogenic fungi, bacteria and viruses. This means that, after applying imidacloprid. plants are more effectively protected from infections by phytopathogenic fungi, bacteria and viruses. Where it is necessary to use fungicides and bactericides as mixtures with imidacloprid, or

where the  latter is used sequentially to fungicides and  bactericides, their activity  is supported

lO    thereby.

Definitions of terms used hereinbelow

The term "eDNA" (£omplementary DNA), as used herein, describes a single DNA strand which is synthesized with a sequence complementary to an RNA and in vitro by enzymatic
reverse  transcriptioiL   The  eDNA can either correspond to the  whole RNA  length  or

15    represent only a partial sequence of the RNA serving as the matrix.

The terms "DNA chip" and "DNA rnicroarray.., which are used synonymously herein, refer to a matrix, the basic material of which consists, for example, of glass or nylon, onto which DNA fragments are immobilized, it being possible for the application of the DNA to be carried out for example by (a) a photolithographic process (DNA is synthesized directly on

20    the array matrix), (b) a microspotting process (externally synthesized oligonucleotides or PCR products are applied to the matrix and covalently bonded thereto), or (c) by a microspraying process (externally synthesized oligonucleotides or PCR products are

sprayed onto the matrix contactlessly by an ink-jet printer) (cf. R. Rauhut, Bioinformatik

(Bioinfonnatics), pp 197-199, ed: Wiley-VCH Verlag GmbH, Weinheim, 2001). A DNA

25    chip, which represents genomic sequences of an organism. is referred to as a "genomic DNA chip". The analysis of the measured values obtained with the aid of these DNA chips is referred to as "DNA chip analysis".

The term "DNA chip hybridization", as used herein, refers to the pairing of two single•strand complementary nucleic acid molecules, one of the base•pairing molecular partners being locali1.ed

as DNA (deoxyribonucleic acid) on the DNA chip, preferably in a covalently bonded form, whereas the other is present in solution in the form of the RNA (ribonucleic acid) or the eDNA corresponding thereto (complementary DNA). The bound and non-bound nucleic acids are hybridized on the DNA chip in an aqueous buffer solution, optionally under additionally denaturizing conditions, such as for example in the presence of dimethyl sulphoxide, at temperatures of 30-60°C, preferably 40-SOOC, and particularly preferably 45°C for 10-20 hours, preferably 14-18 hours, and particularly preferably 16 hours with constant movement. The

hybridization  conditions  can  be  kept  constant,  for  example,  in  a  hybridization  oven.  Under

standard conditions movements of 60 rpm (rounds per minute) are obtained in such a hybridization lO oven.

The terms "expression patterns", "induction patterns" and "expression profile" used synonymously herein describe the time-differentiated and/or tissue-specific expression of the plant mRNA, tbis pattern being obtained directly by the intensity of the hybridization signal produced by the RNA obtained from the plant or its corresponding eDNA with the aid of DNA chip technology. The

15    "expression values" measured are obtained by directly offsetting the resulting signals against corresponding signals obtained using a synonymous chip by hybridization with a non-treated control plant.

The term "expression state'\ which is obtained by the "gene expression profiling" process, as used

herein, describes the whole transcriptional activity recorded for cellular genes and measured with

20    the aid of a DNA chip.

The term "whole RNA", as used herein, describes the possible representation, due to the digestion

process used. of various plant-endogenous RNA groups which can be present in a plant cell, such as for example cytoplasmic rRNA (ribosomal RNA), cytoplasmic tRNA (transfer RNA), cytoplasmic mRNA (messenger RNA), and their respective nuclear precursors ctRNA

25    (chloroplastic RNA) and mtRNA (mitochondrial RNA), although it also includes R..~A molecules which can be derived from exogenous organisms, such as for example viruses or parasitic bacteria and fungi.

The term "useful plants", as used herein, refers to crop plants which are used as plants for obtaining food- or feedstuffs or for technological purposes.

30    The active compounds can be converted into the customary formulations such as solutions, emulsions, spray powders, water- and oil-based suspensions, powders, dusting agents, pastes, soluble powders, soluble granules, scatter granules, suspension/emulsion concentrates, natural


substances impregnated with active compounds, synthetic substances impregnated with active compounds, fertilizel"S and microencapsulations in polymeric materials.

These formulations are produced in a known manner, for example by mixing the active compounds

with extenders, i.e. liquid solvents and!or solid carriers, optionally using surface~active agents, i.e.

emulsifying  agents  and/or  dispersants  andlor  foam-producing  agents.   The  formulations  are

produced either in suitable machines or before or during use.

Auxiliaries which may be used are substances which are suitable for providing the agent itself

and/or preparations derived therefrom (such as spray  mixtures or seed dressings) with  special

properties, such as specific technical properties., and/or also special biological properties. Typical

10    auxiliaries which can be used are: extenders, solvents and carriers.

Suitable extenders are for example water, polar and non-polar organic chemical liquids, for example from the classes comprising aromatic and non-aromatic hydrocarbons (such as paraffins, alkylbenzenes, alkylnaphthalenes and chlorobem:enes), alcohols and polyols (which can optionally
also be substituted, etherified andlor esterified), ketones (such as acetone and cyclohexanone),

15    esters (including fats and oils) and (poly-)ethers, simple and substituted amines, amides, lactams (such as N-alky\pyrrolidones) and lactones, sulphones and sulphoxides (such as dimethyl sulphoxide ).

Where water is used as an extender organic solvents can for ex.ampte also be used as CQso\vents.

Liquid solvents mainly suitable are the following: aromatic compounds such as xylene, toluene or

20    alkylnaphthalenes, chlorinated aromatic compounds and chlorinated aliphatic hydrocarbons such as chlorobenzenes, chloroethy\enes or methylene chloride, aliphatic hydrocarbons such as cyclohexane or paraffins, such as for example petroleum fractions, mineral and vegetable oils, alcohols, such as butanol or glycol and ethers and esters thereof, ketones such as acetone, methyl

ethyl  ketone,  methyl  isobutyl  ketone  or  cyclohexanone  and  strongly  polar  solvents  such  as

25    dimethyl sulphoxide and water.

Suitable solid carriers are the following:

e.g. ammonium salts and natural rock powders, such as kaolins, clays, talcum, chalk. quartz, attapulgite, montmorillonite or diatomaceous earth and synthetic rock powders such as highly

disperse silica, aluminium oxide and smcates; suitable solid carriers for granules are: for example

30    crushed and fractionated natural rocks such as calcite, marble, pumice, sepiolite, dolomite and synthetic granules of inorganic and organic powders and granules of organic materials such as paper, sawdust, coconut shells, com stalks and tobacco stalks; suitable emulsifiers andlor foam-fanning agents are: for example non-ionic and anionic emulsifters, such a.s polyox)'ethylene fatty
 
acid esters, polyoxyethylene fatty alcohol ethers, for example alkylaryl  polyglycol ethers, alkyl

sulphonates, alkyl sulphates, aryl sulphonates and protein hydrolyzates; suitable dispersants are

non-ionic and/or ionic substances, for example from the classes comprising alcohol POE and/or

POP ethers, acid and/or POP or POE esters, alkyl-aryl and/or POP or POE ethers, fatty and/or

POP-POE  adducts,  POE  and/or  POP  polyol  derivatives,  POE  and/or  POP/sorbitan  or  sugar

adducts,   alkyl  or aryl  sulphates,  sulphonates  and  phosphates  or  the corresponding PO  ether adducts.  Furthennore,  suitable oligomers or polymers, for example based on vinyl  monomers, acrylic acid, EO and/or PO alone or in combination with for example (poly-)alcohols or (poly-amines.  Use  can  also  be  made  of lignin  and  sulphonic  acid  derivatives  thereof,  simple  and lO     modified  celluloses,  aromatic  and/or  aliphatic  sulphonic  acids  and  adducts  thereof  with

formaldehyde.

It is possible to use in the fonnulations adhesives such as carboxymethylcellulose, natural and synthetic powdered, granular or latex-like polymers such as gum arabic, polyvinyl alcohol, polyvinyl acetate and natural phospholipids, such as cephalins and lecithins and synthetic
15    phospholipids.

It is possible to use colouring agents such as inorganic pigments, for example iron oxide, titanium oxide, Prussian blue and organic colouring agents such as alizarin, azo and metal phthalocyanine dyes and trace nutrients such as salts of iron, manganese, boron, copper, cobalt, molybdenum and zinc.

20    Further additives can be odorants, mineral or vegetable, optionally modified, oils, waxes and nutrients (including trace nutrients), such as salts of iron, manganese, boron, copper, cobalt, molybdenum and zinc.

The fonnulations can also contain stabilizers such as low-temperature stabilizers, preservatives, antioxidants, light-protecting agents or other agents improving chemical and/or physical stability.

25    The fonnulations generally contain between 0.01 und 98 wt.-% of active compound, preferably between 0.5 and 90 %.

The active compound according to the invention can be used in its commercially available fonnulations and in the use fonns prepared from these fonnulations as mixtures with other active compounds such as insecticides, attractants, sterilants, bactericides, acaricides, nematicides,

30    fungicides, growth-regulating substances, herbicides, safeners, fertilizers or semiochemicals.

The present invention relates to the use of chloronicotinyls to protect plants from fungi, bacteria

and  viruses.  Chloronicotinyls  produce,  independently  of  their  control  of  insects,  effective

protection of plants against damage by fungal, bacterial or viral pathogens.

Advantages over other possible methods are the low application rates for obtaining such protection, the high pbytocompatibility and the already existing approvals for the use of

chloronicotinyls in agriculture.  In addition, a single active compound can be used for protecting

plants from a large number of pathogens.

In order to obtain  protection  from  pathogens, the plants can be  treated with individual  active

compounds or with combinations of chloronicotinyls.

I 0 In addition, the abovementioned positive effects of chloronicotinyls on the inherent defence mechanisms of plants can be supported by additional treatment with fungicidal or bactericidal active compounds.

In a preferred embodiment this protection is obtained by the induction of PR proteins as a result of treatment with chloronicotinyls.

15    Preferred chloronicotinyls are imidacloprid, clothianidine, dinotefuran, thiamethoxam, thiacloprid, acetamiprid and nitenpyram. Particularly preferred chloronicotinyls are imidacloprid, thiacloprid, clothianidine and thiamethoxam. lmidacloprid is very particularly preferred.

According to the invention, plants particularly preferably treated are those of the plant varieties in

each case commercially available or in use. Plant varieties are understood to be plants with new

20    properties ("traits"), which have been bred both by conventional methods, by mutagenesis or with the aid of recombinant DNA techniques. Crop plants can therefore be plants which can be obtained by conventional breeding and optimization methods or by biotechnological and genetechnological methods or combinations of these methods, including transgenic plants and including plant varieties which are or are not protectable under plant variety laws.

25    Preferred plants are barley, tobacco, tomatoes, wheat, com, rice, soya, cotton, rape, potatoes, brassicas, paprika, aubergines, cucumbers, lettuce, melons. turf, citrus, vines, coffee, tea, hops, pomaceous fruit, stone fruit and soft fruit.

Barley is particularly preferred.

The methods according to the invention are particularly also suitable for use on transgenic plants

30    and transgenic seeds. Preferred chloronicotinyls for this use are imidacloprid, clothianidine and thiamethox:am. Imidacloprid is very particularly preferred for this use.

Preferred pathogens are Phytophthora nicotianae, Peronospora tabacinae, Phytophthora infestans,

Sphaerotheca fuliginea, Phakopsora pachyrhizi, Ramularia gossypii, Rhizoctonia solani, Curvularia spec., Pyrenophora spec., Sclerotinia homoeocarpa, Erysiphe graminis and

Colletotrichum graminicola.

Preferred points in time for the application of chloronicotinyls for defending plants against pathogens are seed, soil, nutrient solution, stem and/or leaf treatments with the approved
application rates.

The quantities of a chloronicotinyl for obtaining the properties according to the invention can be

varied over a relatively large range. Concentrations of 0.00001% to 0.05%, particularly preferably

10    0.000025% to 0.025% and very particularly preferably 0.000025% to 0.005%, are preferred for obtaining the inventive effect. If mixtures are used, the concentration of the active compound combinations is preferably between 0.000025% and 0.005%, and particularly preferably between 0.00005% and 0.001%. The indicated values above and below are, unless otherwise specified, percentages by weight.

15    The following example describes the invention in detail.

The induction of PR proteins in barley after treatment with imidacloprid

Barley seeds (of the Baroness variety) were sown in pots containing soil about 2 em deep  (1250 g

of sandy loam/per pot; the soil moisture content was adjusted to 70% of the maximum water-holding capacity) and cultivated in a c!imatized chamber under specified light, moisture and temperature conditions (15 h white light, 70% atmospheric humidity, 23-I~C day/night).

14 days after the emergence of the barley plants 10 mg of imidacloprid per plant, dissolved in 100 ml water, were applied by means of a pipette to the soil around the base of the shoot. The same
volume of water without any active compound was applied to the control  pots.  After the soil

10    treatment the plants were no longer watered. At various times after application (0.25; 1; 6; 8; II; 13; 15; 16 and 17 days) the leaves were harvested, quick-frozen in liquid nitrogen and stored at -80°C until further treatment.

The  labelled  RNA  probes  for  the  DNA  chip  hybridization  were  produced  according  to  the

protocols (Expression Analysis, Technical Manual) of the Affymetrix company (Affymetrix Inc.,

15    3380 Central Expressway, Santa Clara, CA, USA). Whole RNA was first of all isolated from in each case 500 mg of the harvested leaves. 10 J.Lg portions of whole RNA were used for the first and second strand eDNA synthesis. The eDNA was amplified with 17 polymerase and simultaneously labelled with biotin-UTP. 20 J.Lg portions of this biotinylated eDNA were used for
the  hybridization  of  the  barley  genome  array  (Barleyl  Gene  Chip,  Item  no:  511012)  from

20    Affymetrix. This DNA microarray contains DNA sequences of 22840 genes composed of a total of 400000 EST (Expressed Sequence Tag) sequences. Then the DNA microarrays were washed in the Mfymetrix Fluidics Station, stained with streptavidin/phycoerythrin (Molecular Probes, PIN S-866) and scanned with the Affymetrix Gene Chip Scanner 3000. The fluorescence data obtained

were analyzed with the Microarray Suite 5 software from Affymetrix and the Expressionist Pro

25    software from the GeneData company. After a quality check. all of the DNA chip analyses were stored in a database. Since the Affymetrix Gene Chip System is based on measuring the absolute expression values of the genes contained in the chip, the expression values of the biological

replicates    of  treated  and  non-treated  plants  were  averaged  after  nonnalization  (median

calculation).  With the aid of the statistical ANOVA method. those genes were identified whose

30    expression was increased in the plants treated with imidacloprid but remained relatively constant in the untreated controls. The assembly of gene groups from specific metabolic pathways, signal transduction chains or functions was carried out by a keyword search through the gene annotations supplied by Affymetrix and by linking the genes to their corresponding Gene Ontology Annotations (Gene Ontology Consortium).

On  searching  through  gene  groups  from  signal  transduction  chains  and metabolic  pathways

associated  with  stress tolerance and pathogen defence, a powerful  induction of genes for PR

proteins was found in treated plants compared with non~treated plants (tables l-3).


"Pathogenesis-related protein" genes induced by imidacloprid

Affymetrix no.    Description of the genes   
       
       
Contig2990_at    CHITINASE (EC 3.2.l.t4)   
       
Contig2788_x_at    TRAUMATIN-LIKE PROTEIN 1LP7   
       
Contig2214_s_at    PATHOGENESIS RELATED PROTEIN (HV-lA)   
       
Contig2209_at    PATHOGENESIS RELATED PROTEIN (HV-lA)   
       
Contig639_at    PATHOGENESIS-RELATED PROTEIN PRECURSOR   
       
Contig2210_at    PATHOGENESIS-RELATED PROTEIN 1 PRECURSOR   
       
Contigl7082_at    TRYPSIN INHIBITOR   
       
Contig2212_s_at    PATHOGENESIS-RELATED PROTEIN PRB 1-3   
       
Contigll773_at    REGULATOR OF PATHOGENICITY FACTORS   
       
Contig\2046_at    PATHOGENESIS RELATED PROTEIN-\   
       
Contig4405_x_at    PATHOGENESIS-RELATED PROTEIN PR-lOA   
       
       
HV_CEb0020COlr2_at    INTEGUMENTARY MUCIN B.\   
       
Contig2550_x_at    PATHOGENESIS-RELATED PROTEIN 4   
       
       
Contig221l_at    PATHOGENESIS-RELATED PROTEIN PRECURSOR   

Toble2a

Median of the raw expression data (in each case 3 biological replicates)

            0.25days        I day    6days    8days    11 days       
Affymetrix no.        treated    treated        treated    treated    treated    treated    treated    treated    untreated    treated       
Contig2990  at        35.51        31.12        31.38    29.13        66.82    561.20    43.07        830.74        49.01    664.56       
Contig2788    x    at    90.09    71.22        47.02    70.44        445.30    5432.88    989.41        7952.61        1940.45    12190.31       
Contig2214    s    at    96.03        86.29        39.68    44.68        338.95    3322.93    433.24        4807.28        479.17    4463.75       
                                                                           
Contig2209_at        239.67        187.60        106.74    153.35        489.30    4773.49    711.28        8173.99        71l.ll    8587.82       
                                                                               
Contig639  at        218.37        157.66        41.56    38.68        289.31    2482.57    269.13        3944.01        479.62    4273.57       
                                                                               
Contig2210  at        241.38        143.08        61.60    78.12        866.31    6881.20    1017.88        896l.l7    1460.36    7946.25       
                                                                               
Contig 17082    at    173.51        161.42        182.26    162.50        197.68    745.06    230.64        853.21        25o.42    911.63       
                                                                       
Contig2212_s    at    165.29        153.25        137.70    135.85        363.79    3508.08    382.89        3936.08        430.16    3866.40       
Contig 11773    at    187.82        175.49        71.90    70.99        194.41    1513.72    191.42        2102.86        212.15    1580.30       
Contigl 2046  at    85.31        76.89        57.89    46.22        121.37    786.00    184.88        1336.13        101.84    792.96       
Contig4405    x    at    57.15        60.34        55.27    55.96        68.93    146.82    64.94        181.30        56.60    125.79       
HV  CEb0020CO I r2  at    68.25        96.81        86.20    87.44        96.17    418.12    150.33        428.35        180.21    389.74       
Contig2550    x    at    86.98        68.63        47.12    54.66        122.48    716.14    152.46        ll21.6l    126.26    771.21       
Contig22ll_at        217.60        223.31        221.83    206.49        263.78    824.50    252.D7    928.65        290.17    868.02       


Median of the raw expressions data (in each case 3 biological replicates), continued from table 2a

                13    days        15 days    16    days    17    days
    Affymetrix no.    treated    treated    treated    treated    treated    treated    treated    treated
    Contig2990 at    77.68    235.46    216.16    227.32    31.04    76.80    52.55    32.84
    Contig2788    x  at    2083.35    4800.62    3248.12    4880.14    721.89    1836.52    998.31    1166.53
    Contig2214    s    at    125.46    636.20    273.34    808.24    516.41    1241.52    756.38    472.04
    Contig2209_at    287.59    1723.94    633.29    1689.29    823.13    2069.04    1406.75    888.58
    Contig639    at        132.32    1496.21    322.95    920.86    298.41    1300.36    416.44    1196.37
    Contig2210 at    426.37    1553.93    706.30    1970.04    845.77    2013.49    1627.64    1121.55
    Contigl7082    at    126.15    281.55    137.96    209.34    174.85    276.41    208.27    225.25
    Contig2212    s    at    126.72    470.29    167.28    378.35    316.94    756.91    531.25    376.36
Contigll773_at    59.01    208.99    96.55    182.27    185.22    694.71    148.57    340.39
Contig 12046  at    51.84    140.26    60.92    150.31    211.37    622.77    254.76    220.80
Contig4405    x at    41.99    55.42    50.58    57.66    62.61    88.69    56.98    87.06
HV  CEb0020CO I r2  at    107.82    180.00    175.74    170.35    132.88    161.77    191.56    106.72
Contig2550    x  at    48.92    150.42    112.63    162.60    145.82    389.62    190.59    174.42
Contig22ll_at    166.66    296.99    221.25    252.25    231.97    340.64    267.64    219.24

Induction factor= Expression (treated) I Expression (untreated) as a function of the times in days

(d).

        0.25d    ld    6d    8d    lld    l3d    lSd    I6d    l7d   
Affymetrix no.                                           
                                           
Contig2990_at        0.9    0.9    8.4    19.3    13.6    3.0    1.1    2.5    0.6   
Contig2788_x    at    0.8    0.7    12.2    8.0    6.3    2.3    1.5    2.5    1.2   
Contig2214_s    at    0.9    0.5    9.8    11.1    9.3    5.1    3.0    2.4    0.6   
Contig2209_at        0.8    0.6    9.8    11.5    12.1    6.0    2.7    2.5    0.6   
Contig639_at        0.7    0.3    8.6    14.7    8.9    11.3    2.9    4.4    2.9   
Contig2210  at        0.6    0.4    7.9    8.8    5.4    3.6    2.8    2.4    0.7   
Contigl7082_at    0.9    1.1    3.8    3.7    3.6    2.2    1.5    1.6    1.1   
Contig2212  s at    0.9    0.9    9.6    103    9.0    3.7    2.3    2.4    0.7   
Contigl1773_at    0.9    0.4    7.8    11.0    7.4    3.5    1.9    3.8    2.3   
Contigl2046_at    0.9    0.8    6.5    7.2    7.8    2.7    2.5    2.9    0.9   
Contig4405_x    at    1.1    0.9    2.1    2.8    2.2    1.3    1.1    1.4    1.5   
HV_CEb0020C01r2  at    1.4    0.9    4.3    2.8    2.2    1.7    1.0    1.2    0.6   
Contig2550  x at    0.8    0.7    5.8    7.4    6.1    3.1    1.4    2.7    0.9   
Contig22ll_at        1.0    1.0    3.1    3.7    3.0    1.8    1.1    1.5    0.8   
                                           

1.    Use of at least one compound selected from the class of chloronicotinyls of the fonnula (0,

wherein

Het    represents a heterocycle which is in each case optionally mono- or polysubstituted

by fluorine, chlorine, methyl or ethyl and is selected from the following group of

heterocycles:

pyrid-3-yl,   pyrid-5~yl,  3-pyridinio,   1-oxido-5-pyridinio,   l-oxido-5-pyridinio,

tetrahydrofumn-3-yl and thiazol-5-yl,

A    1    2    2   
    represents C1-{\-alkyl, -N(R )(R )  or S(R ),   

10    wherein

R1 represents hydrogen, c,-C,-aikyl, pheny!.C,-C4-alkyl, C3-C,-cycloalkyl, C,-C6-alkenyl or C,-C,-alkinyL and

R2 represents C1-C6-alkyl, C2-C6-alkenyl, Cr4-alkinyl, -C(::::O)-CH3 or benzyl,

15    R represents hydrogen, C1.C,-alkyl, C2-C6-alkenyl, C2.C,-alkinyl, .C(=O)-CH3 or benzyl or, together with R2, represents one of the following groups:

-CHrCH2-, -CH2-CHrCH2-. -CH2-0-CHr. -CHrS-CHr, -CHrNH-CHz- and

CH,-N(CH3)-CH2-, and

X    represents N-N~. N-CN or CH-N02,  optionally in combination with one or more

20    additional plant protection active compounds, for increasing the inherent defence mechanisms of plants.

2.    Use of at least one compound selected from the class of chloronicotinyls of the fonnula (I) according to claim I, characterized in that the chloronicotinyls are imidacloprid, clothianidine, dinotefuran, thiamethox.am, thiacloprid, aetamiprid or nitenpyram.


3.    Use of at least one compound selected from the class of chlomicotinyls of the formula (I) according to claim 2, characterized in that the chloronicotinyl is imidacloprid.

4.    Use of at least one compound selected from the class of chloronicotinyls of the formula (I) according to one of claims t-3, characterized in that the plants are transgenic.

S.    Use of at least one compound selected from the class of chloronicotinyls of the formula (n

according to claim 1 for  protecting plants against biotic stress facto~.

6.    Method of inducing PR proteins in plants by treating the plants with chlomicotinyls, characterized in that plants are treated with at least one compound selected from the class of chlomicotinyls of the formula (1),

(I)

in which

Het    represents a heterocycle which is in each case optionally mono- or polysubstituted

10    by fluorine, chlorine, methyl or ethyl and is selected from the following group of heterocycles:

pyrid~3-yl,  pyrid~5~yl,  3~pyridinio,  l-oxido~5~pyridinio,  1-oxido~5-pyridinio,

tetrahydrofuran-3-yl and tlliazol-5-yl,

A    represents C1-C,-alkyl, -N(R1)(R2)  or S(R2),

15    wherein

R1 represents  hydrogen,  C,-C,-alkyl,  phenyl-C,-C,-alkyl,  c,-C,-cycloalkyl,
CrC6~alkenyl or C1:~C;-alklnyl and

R1    represents  C1-C,-alkyl,  C2-C.-alkenyl,  C1-C,-alkinyl,  -C(,.Q)-CH3   or

benzyl,

20    R represents hydrogen, C1-C6-alkyl, C2-C6-alkeny1, CrCt;~alk:inyl, -C(==O)-CH3 or benzyl or, together with R2, represents one of the following groups:

-CH,-CH,-,  -CH,-CH,-CH,-,  -CH1-0-CH1-,  -CH,-S-CH2-,  -CH2-NH-CH2-,  -CH1-

N(CH3)-CH2- and
 

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