THE ARTICLE WAS PUBLISHED IN JOURNAL: J. REP. ACAD. SCI. UKR. ( JOURNAL Dopovidi Akade ii !a"k Uk#ai!$%& ''.

(()'(&. P. '*+ , '-) (IN UKR.&.

.ENETIC /ECHANIS/S O0 WHEAT AND CHICKPEA (CICER ARIETINU/ L.& INHERITANCE O0 RESISTANCE TO PATHO.ENIC /ICRO/1CETE O0 0USARIU/ L. .ENUS
V. . Tsygankova Institute of Bioorganic Chemistry and Petrochemistry, NAS of Ukraine, Kyiv E-mail: vTsygankova@ukr.net In vivo experiments the inheritance of resistance to pathogenic micromycetes (Fusarium graminearum and Fusarium oxysporum f. ciceris of !heat and "icer arietinum #. plants of the next generation$ o%taining from the seeds of plants the first generation$ that at ontogenesis process !ere processed %y gro!th regulators &iolan$ 'egoplant$ (timpo on an infectious %ackground$ !as investigated. )ccording to morpho-physiological indexes the heterosis-like effect of resistance to pathogens of experimental "icer arietinum #. and !heat plants is set. The molecular-genetic analysis !ith using *+T-%lot hy%ridi,ation method sho!ed the considera%le difference in the percent of homology %et!een populations of si-mi'.) !ith immune-protective properties of experimental plants and m'.) of control plants. Introduction. Plant-gro ing of almost all orld countries suffers from harmful organisms! insects and mites , "athogenic and "arasitic ell as eeds &'() *iseases hich are caused $y "athogenic micromycetes of Fusarium heat, $arley, oat, corn and other

microorganisms #$acteria, fungi, viruses, nematodes% as

#. genus, including Fusarium graminearum #. #se+ual stage, /i%%erella ,eae%, damage grain "roduction of

cereals, es"ecially in recent , decades) According to -ood Agricultural organi.ation at UN #-A/% data, the raise of damage of cereal cro"s is o$served in different countries) -or e+am"le, in the USA the yield shortage caused $y this disease resulted in an economic detriment accounting '

$illion 0 in '112 and 344 million 0 in '115) Not less annual economic losses are o$served in China, Canada, countries of South America, and also in Ukraine, here damage of agricultural cro"s $y this "athogens amounts u" 64-74 8 &',,, 2, 5() 9armfulness of cereals affection $y Fusarium graminearum #. consists in considera$le yield loss and decline of so ing "ro"erties and nourishing grain :uality) ;he dis"lays of sick "lant "athogenesis are "hysiological signs that sho u" in thinning and in $ro ning "lant stems #defeat at early gro th stages% or in "lant ithering or fading #the day $efore or in the "eriod of flo ering%) As a result of "lant vessels corking $y fungal mycelium and e+cretions $y its to+ic su$stances and ethylene the vascular system #fusarial fading% and "lant tissues #rot of roots, fruit and seed% are struck) <+ce"t it the "lant "athologies are dangerous for the health of man and agricultural animals due to mycoto+in accumulation in cereals and "lant stems, hich can not $e used on a feed to the cattle for the receiving of high-:uality meat, milk and "roducts after its "rocessing &3() *iseases orsen the $read $aker=s "ro"erties of flour, and the $read $aked from fusarial grain has su$.ero :uality, to+ic and do"ey properties. F. graminearum #. "roduces several dangerous mycoto+ins, including deo+ynivalenol #*/N% and derivatives, .earalenone, fusarin C, and aurofusarin &2, 3-'4() */N is a "otent "rotein $iosynthesis inhi$itor affecting the digestive system and ma>or organ function in humans and animals hen ingested in sufficient :uantities &?() */N levels are regulated in food su""lies of many countries@ for e+am"le, the <uro"ean Community limits */N levels to 4)3 AgBg for cereals and the United States limits */N levels to ' AgBg for finished "roducts for human consum"tion &2, ?() ;he "resence of F. graminearum in mai.e has resulted in high levels of .earalenone and other mycoto+ins in the Cdistillers= dried grains ith solids,C remnants from mai.e - $ased ethanol "roduction fed to cattle and "igs &7() Dearalenone increses the estrogen level in animals, including humans, and levels of this mycoto+in in food "roducts are regulated in some countries of <uro"ean Community &2, '4() Although .earalenone is of concern to the U)S) -ood and *rug Administration, there are currently no regulatory standards limiting its levels in grain &2, ?() Another '', ',() In the Ukraine chick"ea is idely cultivated in south areas! in Kherson, /dessa, Fykolaiv and on the south coast of Crimea) ;here are hich causes this "athogen at "lants such as! turning yello of l eaves #chlorosis%, drying out and necrosis of all, that results in corking and $lockade of "lant different "athological signs of disease, ides"read "athogenic micromycete of Fusarium #. genusis Fusarium oxysporum f. ciceris that causes fusariosis of leguminous hich is im"ortant for agriculture of Eestern and Fiddle Asia, North Africa, and North America countries &', culture chick"ea #"icer arietinum #.%,

leaves due to a defeat $y fungal mycelium of vessel system and en.yme cleavage of cellular

trans"ort system &'', ',() According to -A/ data, the damage of agricultural so ing of chick"ea in different countries arrives at 34 8)

Garge la$iality of micromycetes of Fusarium #. genus to the environmental factors and out-of-control a""lication of highly to+ic fungicides of chemical origin have resulted in the increase of different varieties of Fusarium due to deterioration of the com"etitive activity $et een the different varieties of "athogens) A "ro$lem of agricultural "lant "rotection against fusariosis is very actual for Ukraine) At the Institute of Biorganic Chemistry and Petrochemistry NAS of Ukraine in coo"eration F<S of Ukraine the ne ith the National <nter"rise Interde"artmental Science H ;echnology Center CAgro$iotechC NAS and ith a $io"rotective effect Biolan, Iego"lant and Stim"o ere ell as of ecologically safe com"osition "olycom"onent "re"arations

created) ;hey contain meta$olism "roducts in culture in vitro ofroot fungus-endo"hyte from ginseng Pana+ Jinsed F roots as soil stre"tomycete (treptomyces avermitilis meta$olites ith "hytostimulating, anti"arasitic and anti"athogenic action&'()

/ur molecular-genetic e+"eriments &'2, '5( have sho ed that these "re"arations considera$ly "romoted "lant resistance to the different "athogens due to stimulation in the "lant cells synthesis of small regulatory siBmiINA, hich in the "rocess of "osttranscri"tional gene silencing ith #P;JS% either $lock the translation of im"erfect on the structure o n mINA and com"lementary mINA of "athogens or destroy them >ointly the en.ymes of IISC com"le+ #INA-induced silencing com"le+% $y the ay of en.ymatic degradation &'3, '?() ;he "ur"ose of this resistance of Stim"o) Materials and methods. Ee determined the "ositive or negative conse:uences of treatment Stim"o of first generation treatment ith gro th regulators Biolan, Iego"lant and hich ere gro n ithout ere heat and chick"ea "lants gro n on an infection $ackground #at "resence of "athogenic micromycetes Fusarium ork as the investigation of molecular-genetic mechanisms of increasing of immune-"rotective "ro"erties and heat and chick"ea to "athogenic micromycetes of Fusarium #. genus $y means of "lant gro th regulators Biolan, Iego"lant and

graminearum and Fusarium oxysporum "iceriae% on gro th and molecular-genetic inde+es of ne+t generation "lants, investigated%) Ee used in e+"eriments , different varieties of chick"ea and heat seeds that

ith gro th regulators on infection $ackground #thus the conse:uences of "ost-action of gro th regulators as immune-modulators

ere kindly "rovided $y /) K) Ba$ayants, *octor of

Biological Sciences, Selection H Jenetic Institute L the National Centre of Seed-gro ing and Kariety Study of NAAS of Ukraine, /dessa) Plant seeds ere s"routed in Petri-dishes on a filter "a"er moistened $y the distilled ater on infection $ackground) Ee determined! '% the integral inde+es of gro th and develo"ment of seedlings@

,% the molecular-genetic inde+es L "ercent of homology of $asic constituents of "lant immune system - siBmiINA #isolated from seedlings of the ,nd generation of heat and chick"ea "lants gro n on infection $ackground o$tained from seeds of the ' st generation of "lants treated ith gro th regulators and gro n on an infection $ackground, i)e) in the "resence of Fusarium graminearum and Fusarium oxysporum f. ciceris% to mINA of control seedlings of the ,nd generation of "lants that "lants gro n on infection $ackground ere gro n on infection $ackground o$tained from seeds of the 'st generation of ere isolated from "lants $y as determined according to difference in the degree ithout treatment $y gro th regulators) Small regulatory siBmiINAs

our earlier ela$orated and "u$lished method &'5() S"ecificity of gro th regulatorsM "ost-action

of homology $et een siBmiINA and mINA of the control and e+"erimental heat and chick"ea "lants of different varieties) Results and discussion) As one can see from -ig) ' and -ig) , the seedlings o$tained from seeds of $oth varieties of chick"ea "lants, infected $y "athogenic micromycete F. oxysporum f. ciceris, and seedlings o$tained from seeds of $oth varieties of "athogenic micromycete F. graminearum, hich ere treated heat "lants, infected $y ith gro th regulators Biolan, Iego"lant and Stim"o, according to integral inde+es of

seed germination s"eed and gro th, "assed ahead shar"ly the gro th and develo"ment of the control seedlings, o$tained from the seeds of "lants that ere gro n ithout regulators on an infectious $ackground) Already, starting from the 3th day to 6th day from the $eginning of germination the gro th inde+es of the e+"erimental seedlings e+ceeded almost three times inde+es of control seedlings) It testifies a$out increasing via$ility of ne regulators and a$out high $iological "otential of seeds o$tained from chick"ea and and treated regulators) -or greater demonstrativeness the ty"ical si.es of control and e+"erimental chick"ea seedlings taken from Petri-dishes A and C #-ig) ,% are sho ed on -ig) 2) ;he e+"lanation of this difference on gro th inde+es $et een control and e+"erimental "lants can $e results of molecular-$iological e+"eriments that are "resented in a ;a$le) ;he differences of mor"hological inde+es sho ed in a ;a$le testify in favor of the circumstance that seedlings o$tained from the seed of first generation "lants hich during the vegetation stage ere treated ith the gro th regulators Biolan, Iego"lant and Stim"o on infection $ackground #in the "resence of "athogenic micromycetes F. graminearum and F. oxysporum f. ciceris% according to integral inde+es of germination, gro th and generation of "lants due to their treatment hich ithout treatment ith gro th ith gro th heat "lants, ere gro n on an infectious $ackground

ith gro th regulators, as com"ared to "lants of first generation gro n on an infectious $ackground

develo"ment have higher via$ility in com"arison treated ith gro th regulators%) A considera$le difference

ith control seedlings #o$tained from seeds of infected "lants of first generation

hich

ere not

as found also on molecular-genetic inde+es, namely in all the e+"erimental variants the "ercent of homology as isolated from seedlings o$tained from seeds of infected and treated ith gro th regulators "lants% heat and chick"ea

$et een immune-"rotective siBmiINA #that

and mINA #isolated from control seedlings% differs considera$ly) It is "ossi$le to see also varietals differences in the effects of "ost-action of different gro th regulators #on differences in the homology level $et een siBmiINA and mINA of the control and e+"erimental "eriod of forming of "lant em$ryos the genome re"rogramming occurs $y a the same functional "ur"ose, $ut s"ecific to concrete "hysiology conditions o$tained in our earlier e+"eriments &'6, '7( as ell as data "lants of different varieties%) /$tained difference on the a$ove-mentioned inde+es confirms circumstance that in the em$ryogenesis during the ay of s itching of one ada"tation genes on others in gene families of hich are formed under action of gro th stimulants) Iesults that ere

hich suggests an e+istence of the fle+i$le system of "lant cell genome re"rogramming

under action of gro th regulators $oth on the different stages of ontogenesis &'1( and during the ada"tation mechanismsM formation of "lant resistance to the different "athogenic organisms &,4( confirm this su""osition) Conclusions. The results of conducted experiments testify in favor of the mechanism of epigenetic inheritance of resistance to pathogenic micromycetes of Fusarium L. genus by the investigated wheat and chickpea plants of next generation obtained from seeds of first generation plants which were treated on infection background by growth regulators with bioprotective properties Biolan, Regoplant and Stimpo. We

concluded that at the embryogenesis during forming of plant seeds there is observed the process of reprogramming genome of plant seed embryos through the way of switching on! genes of the antipathogenic si"miR#$ which synthesis is induced under %&Rs' action.

References

')0onomarenko (. 0.$ Terek +. I.$ 1rytsayenko 2. 3. etal.Bioregulation of "lant gro th and develo"ment) In Cha"ter 5 of the Fonogra"h NBioregulation of micro$ial-"lant systemsO B <d) Ponomarenko S) P), Iutynska 9) /) L Kiyv! Nichlava, ,4'4) L P) ,3' L ,1') ,) Fuller 4.#.$ #illey ".5.$ 6r!in 0.E. Nematode resistance BB Ne Phytologist) L ,447) L K) '74) L P) ,6 L 55) 2) Trail F. -or Blighted Eaves of Jrain! -usarium graminearum in the Postgenomics <ra BB Plant Physiology) L ,441) - K) '51) L P) '42 L ''4) 5. &a%ayants #. T.$ &a%ayants +. 4.$ 3iros (. #.$ Totsky 4. .. Jenetic determination and inheritance of a sign of graminearum -- Cytology and genetics) L ,44') L Kol) 23, N 2) L P) ,,-,1) 3) /eraldo 3.'.$ Tessmann *.5.$ 7emmelmerier ". Production of mycoto+ins $y -usarium graminearum isolated from small cereals # heat, triticale and $arley% affected ith sca$ disease in Southern Bra.il BB Bra.ilian Q) Ficro$iol) L ,44?) L K) 26) L P) 37 L ?2) ?) Fycoto+ins! Iisks in Plant, Animal and 9uman Systems, ;ask -orce Ie"ort B Council for Agricultural Science and ;echnology, Ames, IA L ,442) 6) (tarkey *.E.$ 8ard T.5.$ )oki T.$ /ale #.'. et al. Jlo$al molecular surveillance reveals novel -usarium head $light s"ecies and trichothecene to+in diversity BB -ungal Jenet) Biol)L ,446)L K) 55) L P) ''1'L',45) 7) 8u F.$ 3unkvold /.0. Fycoto+ins in ethanol co-"roducts! modeling economic im"acts on the livestock industry and management strategies BB Q) Agr) -ood Chem) L ,447) L K) 3?) L P) 2144 L 21'') 1) 4oigt ".).$ (chafer 8.$ (alomon (. A secreted li"ase of -usarium graminearum is a virulence factor re:uired for infection of cereals BB Plant Q) L ,443) L K) 5,) L P) 2?5 L 263) '4) 6termark 5.$ 7arlovsky 0. Iole of .earalenone lactonase in "rotection of Jliocladium roseum from fungito+ic effects of the mycoto+in .earalenone BB A""l) <nviron) Ficro$iol) L ,446) L K) 62) L P) ?26 L ?5,) '') "outeaudier 9.$ )la%ouvette ". Survival and inoculumsM "otential of conidia and chlamydos"ores of -usarium o+ys"orum f) s") lini in soilBB Can) Q) Ficro$iol) L '114) L K) 2?) L P) 33' L 33?) ',. #eslie 5.F.$ (ummerell &.). ;he -usarium Ga$oratory manual B Black ell Pu$lishing! Io a, USA) L ,44?) L 26, ") '2) Tsygankova 4. ).$ /alkin ). 0.$ /alkina #. +. et al. Jene e+"ression under regulatorsM stimulation of "lant gro th and develo"ment) In Cha"ter 2 of the Fonogra"h NNe "lant gro th regulators! $asic research and technologies of a""licationO B <d) Ponomarenko S) P), Iutynska 9) /) L Kyiv! Nichlava, ,4'') L P) 15 L '3,) heat resistance to Fusarium

'5) Tsygankova 4.).$ )ndrusevich 9a.4.$ &lume 9a.&. Isolation from "lant cells the small regulatory siBmiINA ith antinematode activity BB *o"ovidi Academii Nauk Ukrainy) -,4'') L R 1) L P) '31 - '?5) '3. Filipo!ic, 8.$ 5askie!ic,. #.$ 7ol% F.). and 0illai '.(. Post-transcri"tional gene silencing $y siINAs and miINAs BB Curr) /"in) Struct) Biol) L ,443) L K) '3) L P) 22'L25') '?) 4oinnet +. Post-transcri"tional INA silencing in "lant-micro$e interactions! a touch of ro$ustness and versatility BB Curr) /"in) PlantBiol) - ,447) L K) '', R 5) L P) 5?5 - 564) '6) Tsygankova 4. ).$ 3usatenko #. I.$ 0onomarenko (. 0. et al.Change of functionally active cyto"lasmic mINA "o"ulations in "lant cells under gro th regulators action and $iological "ers"ectives of cell-free systems of "rotein synthesis BB Biotechnology #ukr)%) L ,4'4)L S) 2, R ,) L T) '1L2,) '7) Tsygankova4.).$0onomarenko (.0.$ /alkin ).0.$ Titova #.4.$BeljavskajaL.A., Leonova N.O.,IutinskajaH.A. Gene e+"ression changes in "lant cells under influence of e+ogenous gro th regulators BB Plant "hysiology, "ro$lems and "ros"ect of develo"ment B Ukr) Society of "lant "hysiol) - In , vol) L Kyiv! Gogos, ,441) L K) ,) L P) 36? - 375) '1) .am I. 9an-/. /"timi.ation of "lant gro th and develo"ment regulators in $iotechnology in vitro B *isser) on com"etition of graduate degree of *octor $iol) sciences) L Fosco , ,445) L 2', ") ,4) )ndreeva E.).$ 7hoyd,ayova #.T. Investigation of cytokinin role in develo"ment of sign resistance to diseases in the model system "otato "hytoftora BB ,nd Congress of Jenetic and Selectioneers) L Sanct- Peters$urg, ,444) L K) ') L P) ,63)

Fig. 1. The 7 day old seedlings of !heat "lants #variety $%astivka&' ! (, ) L seedlings, o$tained from seeds of "lants, untreated gro th regulators #control%@ *, + - seedlings, o$tained from seeds of "lants, treated ith Stim"o@ ,, F seedlings, o$tained from seeds of "lants, treated ith Iego"lant

Fig. -. The 7 day old seedlings of Cicer arietinum L. #variety $Trium"h&'. ( L seedlings, o$tained from seeds of "lants, untreated gro th regulators #control%@ ) - seedlings, o$tained from seeds of "lants, treated ith Stim"o@ * seedlings, o$tained from seeds of "lants, treated ith Iego"lant@ + seedlings, o$tained from seeds of "lants, treated ith Biolan

) ere treated #A%

Fig. /. Com"arative si.es of 6 day old "icer arietinum #. seedlings, o$tained from seeds of "lants that during the vegetation stage and ere not treated #B% $y gro th regulator Iego"lant on infectious $ackground #Fusarium oxysporum f. ciceris%

Table
P2a!3 va#ie3ie4 a!d 5#o637 #e5"2a3o#4 W7ea3 p2a!3 va#ie3ie4 1 8La43ivka9 va#ie3$ (va#ia!34 o: e;pe#i e!34& Control (si/mi RNA and mRNA from control plants) "# $ 1.% #& $ 1.'( ()1'*) #' $ 1.&( ()1&*) "# $ 1.' #% $ 1.%( ()1%*) ## $ +."'( ()1+*) C7i<kpea p2a!3 va#ie3ie4 & 8Ro4a!!a9 va#ie3$ (va#ia!34 o: e;pe#i e!34& Control (si/mi RNA and mRNA from control plants) "# $ 1.& #' $ 1.'( ()1&*) .# $ 1.%( ()&+*) ## $ +."#( ()1+*) Treat,ment Treat,ment wi Treatment with with -iolan th Re oplant !timpo Control (si/miRNA and mRNA from control plants) "# $ 1.% #& $ 1.#( ()1'*) #+ $ 1.'( ()1#*) .' $ &.%&( ()&&*) 8T#i" p79 va#ie3$ (va#ia!34 o: e;pe#i e!34& Treat,ment w Treat,ment with ith -iolan Re o,plant Treatment with !timpo Treatment with Re oplant Treatment with !timpo 8P#i!<e44 O25a9 va#ie3$ (va#ia!34 o: e;pe#i e!34& Control (si/mi RNA and mRNA from control plants) Treatment with Re oplant Treatment with !timpo