growth regulators - inductors si-mirna

8/13/2019 Growth Regulators - Inductors Si-miRNA

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economica!!y im&ortant 91:.(he tota! !osses of agricu!tura! harvest due to diseases

caused y different &athogens in Ukraine is u& 64 7.

Nematodes are one of ides&read and harmfu! &!ant &athogen. ;ie!d !osses

due to &arasitic nematodes in different countries are from 26 7 to 45144 7 91:. (he tota! annua! g!oa! cro& !osses caused

y nematodes in money e-uiva!ent are 144 i!!ion US+ 92:. #ife cyc!e of a!! the

soi! nematodes /&hyto&arasitic and free !iving0 are c!ose!y connected, on the one

side, ith &!ants, and on the other side, nematodes can e vectors for transmitting

of &hyto&athogenic and sa&ro&hyte acteria, fungi, viruses and other organisms.

(oday the re&resentatives of over 24 genera of &!ant nematodes are knon

as o!igate &arasites of higher &!ants. *ost of them e!ong to order Tylenchida,

fami!yHeteroderidae. In the Centra! and $astern countries, inc!uding ?ussia and

Ukraine the cyst nematodes Globodera rostochiensis, Globodera pallida and

Globodera tabaccum are most dangerous for cro&s. irst to s&ecies cause

@!oodera desiase on &otato. (he cro& !oss cou!d reach in some year u& to 47.

Both s&ecies are inc!uded to the !ist of -uarantine !ist of the $uro&ean P!ant

Protection 3rganiation /$PP30. Besides the aove mentioned nematodes there are

severa! nematodes of economica! im&ortance the tuer nematode /Ditylenchus

destructor0, nematodes vectors for transmitting virus, such asParatrichodorus

teres, iphinema di!ersicaudatum, "on#isporus elon#ates and root nematodes

/Paratylenchus spp., Helycotylenchus spp.0. Association ith viruses= disease

aove !isted &arasitic nematodes contriutes to increasing of &otato yie!d of &otato

in Centra! $uro&e. In some years it can cause DD 7 yie!d reduction 9":. In

greenhouses three ty&es of root5knot nematodes $eloido#yne inco#nita

$eloido#yne %a!anica and $eloido#yne arenaria are most destructive &!ant

&arasite. (hey cause a dangerous *e!oidogyne desease. (hese &arasitic organisms

affect the severa! vegeta!es /tomato, egg5&!ants, &otato, sugar and red eet, carrot,

ater5me!ons etc0 in o&en fie!d a!so 9%:.

Sugar Beet cyst nematode Heterodera schachtii Schmidt is eEtreme!y

harmfu!. It is dangerous &est hich caused Feterodera disease of sugar eet in

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a!most %4 countries 96:. (his nematode contriutes >6 7 of cro& !osses caused y

a!! harmfu! organisms. Sugar eet nematode in Ukraine is ides&read in Ginnitsa,

Sumy, Chernihiv, Cherkassy, Kharkov, Kyiv, Hhitomir regions here it might

cause "4 7 cro& !osses 9:. An im&ortant feature of this &arasite is ai!ity to

re&roduce on &!ants of 26 fami!ies, in &articu!ar, onBrassicaceaecu!tivated cro&s

/oi!seed ra&e, caage, Chinese radish, mustard0 and eeds. A!so nematode can

deve!o& at cu!tivated cro&s and eeds of Chenopodiaceaefami!y 9:. (he cro&

rotation is most re!ia!e &reventive too! to contro! sugar eet cyst nematode. (he

most significant !osses of nematode are oserved during groing of sugar eet as a

monocu!ture and in case !arge concentration /to 45D4 70 of its host cro&s in cro&

rotation. (he most effective method of sugar eet cyst nematode contro! is the

rotation of sugar eet ith non host toH. scachtiicro&s cerea!s, grain !egumes,

corn, &otato, &erennia! grasses. 3ver the !ast years the risk of increase of H.

shcachtiidamage in sugar eet cro& rotations has een increased significant!y. It

occurs as a resu!t of increase soing of oi!seed ra&e and eEtension of groing area

of this cu!ture /hich is suita!e host for H. schachtii0, hich use for otaining of

io5fue!. Conse-uent!y the contro! of this dangerous &est -uantity is high &riority

9:, anana

914:, cotton &!ant 911:, toacco 912: and gar!ic 91":. (he use of avermectines

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against the root5knot nematodes&. inco#nita, &. arenaria, &. %a!anicais the most

effective. It is knon a!so aout efficiency of &re&arations action against the stem

nematode Ditilenchus dipcasi, root nematode 'otylenchulus reni(ormis and

&hyto&arasite Tylenchulus semipenetrans. (he cotton seed treatment ith

aamectine against root5knot nematodes, sugar eet against eet nematode and

corn againstP. pratensis911, 1%: is effective a!so.

In Ukraine over the !ast years the domestic anti&arasitic &re&arations are

used more ide!y. Avercom is most effective among them. It as created on the

asis of avermectines com&!eE anti&arasitic antiiotics /&roducer ground

stre&tomycete Streptomyces a!ermitilis0 at the Institute of *icroio!ogy and

Giro!ogy NAS of Ukraine 916, 1:. It as found that this &re&aration &romotes

immuno5&rotecting &ro&erties of &!ants, forards their groth and !imits

harmfu!ness of &arasitic organisms nematodes 916:. (he conducted !aoratory

researches shoed that in the high concentrations the Averkom caused D4 7 death

of$eloido#yne inco#nita!arvae. Soi! treatment ith this &re&aration in !aoratory

conditions decreased nematode &o&u!ation y four times. 91:.

(he com&osition &o!yfunctiona! &re&arations Biogen, Stim&o and ?ego&!ant

are to ne effective domestic &re&arations ith nematocida! and anti&athogenic

action. (hey ere created at the Institute of Bioorganic Chemistry and

Petrochemistry NAS of Ukraine together ith the State $nter&rise

Interde&artmenta! Science ' (echno!ogy Center )Agroiotech) NAS and *$S of

Ukraine. Bio&rotective &ro&erties a&&eared ecause of the synergistic effect of root

fungus5endo&hyte &roducts in the cu!ture in !itroof ginseng PanaE @insed *. /miE

of amino acids, carohydrates, fatty acids, &o!ysaccharides, &hytohormones and

microe!ements0 and aversectines com&!eE anti&arasitic macro!ide antiiotics of

soi! stre&tomycete Streptomyces a!ermitilis 91:. Stre&tomycetes are knon as

&roducers of not on!y antiiotics ut a!so such ioactive com&ounds hich have

&hyto&rotecting and groth stimu!ating action on &!ants. It is different hormones,

vitamins, amino acids, carotenoids, enymes, toEins and other matters hich

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inf!uence on the groth &rocesses of &!ants, name!y, stimu!ate the seed

germination and &romote the &roductivity 916, 1:.

?esu!ts of mo!ecu!ar5genetic research 9152%:. @ene si!encing mediated y either degradation

or trans!ation arrest of target ?NA has ro!es in ada&tive &rotection against viruses,

genome defense against moi!e +NA e!ements and deve!o&menta! regu!ation of

gene eE&ression. Sma!! regu!atory siJmi?NA of 2252%5nt 91>52%: carries out a

!eading ro!e in si!encing. (hey are generated from the &redecessors &re5mi?NA

of 5"2: or on ce!!u!ar m?NA /ith aerrant and

defective structure or hich eE&ressed at high !eve! during infection0 9"": as e!!

as enymatic c!eave Loint!y ith the enymes of ?ISC com&!eE /?NA5induced

si!encing com&!eE0 these target m?NA mo!ecu!es that resu!ts in their degradation.

(he &ur&ose of our ork as determination of &ossii!ity of enhancement

y &o!ycom&onent &re&arations Biogen, Stim&o and ?ego&!ant of sugar eet and

s&ring heat immune5&rotective &ro&erties through increasing synthesis of sma!!

regu!atory siJmi?NA and, as a resu!t, to achieve increased &!ant resistance to the

&!ant5&arasitic nematode, Heterodera schachtii and to &athogenic micromycete

Fusarium #raminearum.

In eE&eriments e used the &!ants of sugar eet Beta !ul#aris ". infected in

the !aoratory or in greenhouse conditions y sugar eet nematode H. schachtii

and root5knot nematode &. inco#nita. e used a!so &!ants of s&ring heat of

@rio variety infected y &athogenic micromycete Fusarium #raminearum. In

eE&eriments for determination of variety sensitiveness to groth regu!ators e

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used the varieties of inter heat ;atran 4, Go!odarka, Smuh!ianka and

Podo!ianka hich ere kind!y given y an academician NAS of Ukraine G.G.

*orgun. (he eE&erimenta! &!ants ere &rocessed y com&osition &o!ycom&onent

&re&arations ?ego&!ant, Stim&o, Biogen.

$E&eriments on the study of efficiency to decrease nematode &o&u!ation

nematodes using groth regu!ators e conducted at first in the conditions of

greenhouse. (he &!ant seeds ere treated ith &re&arations ?ego&!ant, Stim&o,

Biogen here the concentration of Aversectine as 4.468 4.18 4.68 1 and 14 MgJm!.

(reated seeds ere &!anted in diameter 14 cm &ots ith and the tem&erature of

266 O. or the infection of &!ants e used the infectious !arvae of cyst

nematodes hich ere eEtracted from cysts. Cysts of nematodes ere iso!ated

from soi! using decantation on sieves. Cysts ere carried in the 3ostenrinkQs cu&s

and fi!!ed y the saturated so!ution of inc ch!oride 9"%:. (he cyst incuation as

carried out during < days at a room tem&erature. (he !arvae of nematodes ere

removed from a sus&ension ith the use of a sieve ith the diameter of screen

mesh 26 Mm.

Nematodes in an amount 1 444 !arvae on every &ot ere inc!uded in to

eeks after soing of the seed treated ith regu!ators, and in to eeks the &!ants

ere eEcavated and the amount of !arvae in roots as determined. As contro! e

used &!ants otained from the seeds hich ere not treated ith groth regu!ators.

(he roots of sugar eets ere carefu!!y ashed in f!oing ater. (hen roots ere

di&&ed in so!ution of !actic acid, g!ycerin, ani!ine and disti!!ed ater on a fe

minutes, ere armed u& 2 minutes in microave and ere dried out on air.

Pieces of roots 1,6 cm ere &!aced in homogenier. (he homogenied roots ere

transfer in cy!inders ith vo!ume 164 m! and 144 m! of ater, ere carefu!!y

shake. (hen e counted the amount of nematodes hich &enetrated in a root.

e studied the im&act of seed treatment ith groth regu!ators !ooking at

&ercent of nematode !arvae &enetration in sugar eet roots.

+etermination of !etha! concentration #+64 and #+>4 e conducted in

!aoratory conditions. e registered the death rate of eet and root5knot


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nematodes on 2nd and 2%thhour after treatment ith &re&arations ?ego&!ant and

Stim&o in the concentrations of Aversectine 4.468 4.18 4.68 1 and 14 MgJm!. (he

test as carried out on atch5g!ass in four reiterations. e rought 644 M! of

testa!e concentration of groth regu!ators and %4 !arvae of cyst or root5knot

nematodes on every g!ass. (he !arvae of second age of cyst nematode ere

otained y aove5descried method. or the se!ection of !arvae of second age of

root5knot nematode e used a fi!ter screen ith the &ores 24 Mm 9"6:.

By means of method of mo!ecu!ar hyridiation of m?NA from siJmi?NA

e !ooked u& &ossii!ity of induction y groth regu!ators of synthesis of

siJmi?NA ith nematocida! activity. ith this &ur&ose the seeds of sugar eet

ith high germinating ca&acity ere s&routed in the Petri dishes on the nematode5

free a-uatic medium /contro!0 and ith the sus&ension of cysts herefrom the

!arvae of nematodes /a&&roEimate!y on 65: /ge!s ere stained y so!ution of ethidium romide efore

&hotogra&hing of ?NA fractions in u!travio!et0.

20 +ivision of &o!y/A0R

m?NA /m?NA0 and &o!y/A0

m?NA on o!igo/d(05

ce!!u!ose co!umn ith the &ur&ose of the further use of &o!y/A0 Rm?NA for testing


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of functiona! activity of siJmi?NA in heat germ ce!!5free systems of &rotein

synthesis 9", "D:8

"0 Preci&itation of high mo!ecu!ar &o!y/A0m?NA from an e!uate as

eEecuted y 14 7 so!ution of &o!yethy!ene g!yco! /mo!. mass D4440 from 4.6 * of

NaC!, and siJmi?NA y > 7 ethano! at 522 OC during the day8 from a co!umn

the &o!y/A0Rm?NA as &reci&itated y 25" vo!umes of uffer of such

com&osition 14 * tris5T! /&F


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of homo!ogy &ercent m?NA &o&u!ations from eE&erimenta! and contro! &!ants 9%4,

%1:.

or the synthesis of sing!e5stranded c+NA as a&&!ied the uffer so!ution

containing 144 m* tris5FC! /&F D."0 at %2 O, 14 m* *gC! 2, 1%4 m* KC!, 144

MgJm! o!igo/dt01251D &rimer, 2 m* methy!mercuric hydroEide, 24 m*

V5merca&toethano!, 1 m* vanady!5rionuc!eoside com&!eEes, or 4.6 unitsJmk! of

?Nase, 1 m* so!ution of the a!! four dN(P, 144 MgJm! &o!y/A0 R?NA, %445D44

unitsJm! of reverse transcri&tase and 9W5"2P:5dC(P /D44 CiJm*0 9", %4, %1:.

or transferring "2Xc+NA on the fi!ters it as disso!ved in a concentration

24 MgJm! in a uffer of 4." * NaC! 4.4" * sodium citrate, YT


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of m?NA hyridiation according!y ith homo!ogous and hetero!ogous c+NA.

Fyridiation as com&ared in different time interva!s during tenty5four hours.

After hyridiation com&!etion the sma!! ott!es ith fi!ters ere coo!ed,

fi!ters ere taken out and ashed in a crater from every side y 64 m! 2ZSS.

After ashing the fi!ters ere &!aced in so!ution 2ZSSC that contained ?Nase in a

concentration 24 MgJ!. After incuation ith ?Nase at a room tem&erature during

1 hour the fi!ters ere again ashed y so!ution 2ZSSC, then y ethy! a!coho!.

?adio5activity of sam&!es as determined on fi!ter g!asses *i!!i&ore AP516 in

to!uene scinti!!ator in the scinti!!ation counter #S 144C of the firm Beckman. (he

statistica! &rocessing of the otained data as conducted y dis&ersive /Student0

and corre!ation5regressive methods.

It as found in the conducted !aoratory, greenhouse and fie!d eE&eriments

that the com&!eE &o!ycom&onent &re&arations ?ego&!ant and Stim&o considera!y

&romote &!ant stai!ity to the dangerous &arasites nematodes /cyst and root5knot0

and &athogenic fungus /Fusarium my)omycete* 9%", %%:.

ig. 1 shos images ith infected y cyst nematode +. schachtiisugar eet

&!ants Beta !ul#aris ". and a!so adu!t fema!es and cysts of this nematode in the

fie!d condition.

@reenhouse eE&eriment resu!ts shoed /(a!e 10 that seed treatment ith

groth regu!ators ?ego&!ant and Stim&o inhiite &enetration of Luveni!es of eet

nematode in the root of sugar eet in the first month of cro& vegetation. (he

amount of nematode !arvae hich otained in a root under action of Stim&o

diminished y


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Table

9fficiency of sugar beet seed treatent &ith gro&th regulators against the

beet neatode . schachtii

Preparation 5oncentration,

>g$l

3ecrease of penetration of larvae in a

root coparatively to control, ?

Stim&o

4.46 %>.


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concentrations #+64and #+>4of these &re&arations are shon in the (a!e 1 and

a!so on ig. " and %. (here is a difference eteen o&erating of regu!ators Stim&o

and ?ego&!ant on eet and root5knot nematodes. ?ego&!ant &redetermines 64 7

death rate of !arvae of root5knot nematode in 2 hours in a concentration y "4 7

!ess than Stim&o, and in 2% hours tice !ess. *ore significant difference in

resu!ts as otained for a eet nematode. In 2 hours 64 7 of !arvae &erished in the

concentrations of groth regu!ator ?ego&!ant y D4 7 !ess than regu!ator Stim&o,

and in 2% hours siE fo!d !ess.

>4 7 death rate of oth ty&es of nematodes as oserved for groth

regu!ators Stim&o and ?ego&!ant on!y in 2% hours /(a!e 20. A !etha! concentration

#+>4for a eet nematode as y D4 7 !ess than for root5knot. ?ego&!ant caused >4

7 death rate of root5knot nematode !arvae in tice !ess concentration than Stim&o.

3n a eet nematode the >4 7 death rate is registered in a&&!ication a&&roEimate!y

of identica! concentration of &re&arations.

(hus, the resu!ts of eE&eriments conducted in greenhouse testify that under

the action of groth regu!ators ?ego&!ant and Stim&o the risk of &enetration and

accumu!ation of nematode !arvae in the root of sugar eet at the eginning of cro&

vegetation is decreased significant!y.

Table *

5oncentration 83-+and 83@+of gro&th regulators for sugar beet and rootknot neatodes

Preparation

Root cyst neatode. Schachtii Rootknot neatode

. inco!nita

#+64/ MgJm!0 #+>4/ MgJm!0 #+64/ MgJm!0 #+>4/MgJm!0

2

hours

2% hours 2

hours

2% hours 2 hours 2% hours 2 hours 2% hours

Stim&o ".> 4. % 1.> 1 "4.D%]]/670 D>4.70

6.4 >44.2]]/D70 >44.D]]/D70

Regoplant

'Radosti D

Aversectines(

4.2 >%4."D]]/%70 D4.%D]]/1270

2.6 D>4.


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] Presence of significant differences in contro!, X [ 4.46, n^".

(he !eve! /70 of differences as studied y means of method of +ot5!ot

hyridiation. In eE&eriments e used 25month &!ants of s&ring heat of @rio

variety gron from seeds that as not treated and treated ith groth regu!ators

on an infectious ackground ith micromyceteFusarium #raminearum.

Sustantia! changes in &o&u!ation descri&tions of siJmi?NA of s&ring heat

s&routs of @rio variety are educed in the determination of &ercent of siJmi?NA

homo!ogy to m?NA in adu!t &!ants gron from seeds treated ith groth

regu!ators, not infected and infected y micromycete Fusarium #raminearum. In

this case e oserved changes de&ended on the concentration of groth regu!ators.

5onclusions. (hus, e found &o&u!ation differences de&endent on groth

regu!ator doe, varieta! differences of cyto&!asmic m?NA of heat &!ant y the

method of +ot 5 !ot hyridiation and ith the use of the heat emryo ce!! 5 free

system of &rotein synthesis, hich can e eE&!ained y genetic nature of different

stai!ity of &!ant varieties to iotic and aiotic eEterna! factors.

References

. Ponomaren/o S. P., Tere/ 0. 1., Hrytsayen/o 2. $. et al. Bioregu!ation of &!ant

groth and deve!o&ment. In Cha&ter % of the *onogra&h `Bioregu!ation of

microia!5&!ant systems J $d. Ponomarenko S. P., Iutynska F. 3. Kiyv

Nich!ava, 2414. P. 261 2>1.

2. Fuller V. "., "illey C. 3., 4r5in P. 6.Nematode resistance JJ Ne Phyto!.

244D. G. 1D4. P. 2< %%.

". 2ino!ye!a S. V. *o!ecu!ar mechanisms of &!ant and &arasitic nematodes

interaction theoretica! and a&&!ied as&ects J Parasitic nematodes of &!ants and

insects /(o the 64 anniversary of the &hyto5&arasito!ogic research at the Institute of

Parasito!ogy of the ?ussian Academy of Sciences0. *osco Nauka, 244%. P.

64 5 D6.

%. Chi7ho! V..+iagnostic of ga!!ic nematodes of$eloido#ynegenus /Nematoda

(y!enchida0 in the &rotected soi! J Iid. 5 *osco Nauka, 244%. 5 P. 26" 5 2


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6. Ste!ens $., $ay $. 3.Pests, diseases and eeds revie 244> JJ British Sugar

Beet ?evie. 2414. G. . Garabedian S., Van Gundy S. D. Use of avermectins for the contro! of

$eloido#yne inco#nitaon tomatoes JJ Iid. 1>D". G. 16. P. 64"61>.

14. 3ansson '. 9., 'abatin S. Curative and residua! efficacy of inLection

a&&!ications of avermectins for contro! of &!ant5&arasitic nematodes on anana JJ

Iid. 1>>. P. >6>6. G. 2.

16.Bilya!s/a ". 0., 9o7yryts/a V. ;e., Valahuro!a V. 0., 1utyns/a H. 0.Avercom

ne domestic &re&aration ith nematocida! and &hytostimu!ative action JJ

2%
http://www.ncbi.nlm.nih.gov/pubmed/20222620http://www.ncbi.nlm.nih.gov/pubmed/20222620


25/27

Agricu!tura! microio!ogy /Interde&artmenta! thematic research co!!ection0.

Chernihiv CN($I, 244D. Issue .

1.1utyns/a H. 0., Valahuro!a 6. V, 9o7yryts/a V. 6. and oth. Avercom ne

anti&arasitic &re&aration.

Cha&ter 1 of the monogra&h `Bioregu!ation of themicroia! vegeta!e systems J $d. Iutynska F. 3. and Ponomarenko S. P. 5 Kyiv

Nich!ava, 2414. 5 %% &.

1 5 1%.

1>.6lbashir S. $., "endec/el :., Tuschl T.?NA interference is mediated y 215

and 225nuc!eotide ?NAs JJ @enes +ev. 2441. G. 16. P. 1DD 244.

24.Hamilton A., Voinnet 0., Chappell ". et al. (o c!asses of short interfering

?NA in ?NA si!encing JJ $*B3 . 2442. G. 21, b 1.

22. $ourelatos 2., Dostie 3., Paush/in S. et al. mi?NPs a nove! c!ass of

rionuc!eo&roteins containing numerous micro?NAs JJ @enes +ev. 2442. G.

1. P.


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26.Ba/hetia $., Charlton :. "., 4r5in P. 6. et al. ?NA interference and &!ant

&arasitic nematodes JJ (rends P!ant Sci. 2446. G. 14, b D. P. "2". G. 12. P.

%6 % 261.

2


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". Tsy#an/o!a V. A., Blume ;a. B. et al.An unusua! minor &rotein a&&earing in

emryonic aEis ce!!s of haricot ean seeds fo!!oing germination &rocess

stimu!ated y 5methy!thiouraci! JJ Bio&o!ymers and ce!!. 1>>D. G. 1%, b 6.

P. %"D%%D.

""2.

">."oc/er 3.Ana!ytica! and &re&arative e!ectro&horesis of ?NA in agarose5urea JJ

Ana!. Biochem. 1>. G. >D, N 2. P. "6D"D2. %D4 &.

%1. Promega &rotoco!s and a&&!ications guide. Second edition. USA Promega

Cor&oration, 1>>1. %22 &.

%2. *ethods of deve!o&ment io!ogy J $d +et!af (. A., Brodsky G. ;a, @aue @.

@. *osco Nauka. 1>

%". Tsy#an/o!a V. A., Ste(ano!s/a T.'., Ponomaren/o S. P., Blume ;a. B.

*o!ecu!ar mechanisms of sugar eet resistance to &hyto&arasitic nematodes JJ

Naukovy do&ovidi NUBiP. 2412, " /"20 htt&JJ.nuv.gov.uaJe5

Lourna!sJNdJ2412"J12cva.&df

%%. Tsy#an/o!a V.A., Ste(ano!s/a T.'., Andruse!ich ;a.V., Ponomaren/o S.P.,

Gal/in A.P., Blume ;a.B. Induction of sma!! regu!atory siJmi?NA iosynthesis in

&!ant ce!!s y groth regu!ators ith anti&athogenic and anti&arasitic &ro&erties JJ

Biotechno!ogy /ukr.0. 2412. G. 6, b ". P. 2

growth regulators - inductors si-mirna

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