Advances in Bioscience and Bioengineering

ISSN 2201-8336

Volume 2, Number 1, 2014, 66-103

© Copyright 2014 the authors. 66

Impact of New Microbial PR/PGP Inducers on Increase of Resistance

to Parasitic Nematode of Wild and RNAi Transgenic Rape Plants

Tsygankova V.A.1, Biliavska L.O.2, Andrusevich Ya.V.1, Bondarenko O.N.1,

Galkin A.P.3, Babich O.A.4, Kozyritska V.E.2, Iutynska G.O.2, Blume Ya.B.3

1Department for Chemistry of Bioactive Nitrogen-Containing Heterocyclic Compounds, Institute of

Bioorganic Chemistry and Petrochemistry, National Academy of Sciences of Ukraine, 02660, 1,

Murmanskaya str., Kyiv, Ukraine

2Department of General and Soil Microbiology, Zabolotny Institute of Microbiology and Virology,

National Academy of Sciences of Ukraine, 154, Zabolotnogo str., D 03680, DSP, Kyiv, Ukraine

3Department Genomics and Molecular Biotechnology, Institute of Food Biotechnology and Genomics,

National Academy of Sciences of Ukraine, 04123, 2a, Osypovskogo str., Kyiv, Ukraine

4National University of Life and Environmental Sciences of Ukraine, 15, Heroyiv Oborony str.,

03041, Kyiv, Ukraine

Corresponding author: Victoria Anatolyivna Tsygankova, PhD, ScD, Department for Chemistry of

Bioactive Nitrogen-Containing Heterocyclic Compounds, Institute of Bioorganic Chemistry and

Petrochemistry, National Academy of Sciences of Ukraine, 1, Murmanskaya Str., Kyiv, 02660,

Ukraine

Abstract. The bioprotective effect against parasitic nematode Heterodera schachii Schmidt of new

microbial PR/PGP inducers on the wild spring rape plants of Kalinivsky cultivar and RNAi

transgenic rape plants was studied. In the field conditions on the artificially created invasive

background the considerable diminishing of amount of nematodes on the roots of wild rape plants up

to 89.7 % - under action of Avercom and up to 38.5 % - under action of Avercom-nova 2 was found.

The increasing of productivity of grain yields of infected by nematode wild rape plants up to 2775.6

pounds/ha - under action of Avercom and up to 2156.1 pounds/ha - under action of Avercom-nova 2

compared to control was observed. Using Dot-blot hybridization method the increase of difference in

the degree of homology between the cytoplasmic mRNA from control rape plants and small

regulatory si/miRNA from infected by nematode and treated with PR/PGP inducers Avercom,

Avercom nova-1 and Avercom nova-2 rape plants: up to 30-39 % - for wild and up to 38-47 % - for

RNAi transgenic plants was found. In the wheat embryo cell-free system of protein synthesis the

considerable increase of silencing activity of si/miRNA isolated from infected by nematode and

treated with these PR/PGP inducers rape plants: up to 26-43 % - for wild and up to 58-86 % - for

RNAi transgenic rape plants on the templates both of nematode mRNA and rape plant mRNA was

revealed. Results of the study indicate that PR/PGP inducers increase synthesis of si/miRNA with

specific anti-nematodic activity in the rape plants. Owing to this process the increase of resistance of

wild and RNAi-transgenic rape plants to parasitic nematode occurs.

Keywords: plant parasitic nematode, wild and RNAi transgenic rape plants, PR/PGP inducers,

plant immunity, wheat embryo cell-free system, anti-nematodic activity of si/miRNA.

Advances in Bioscience and Bioengineering 67

Introduction

The actual problem of successful economic development of countries, which

have effective agriculture, is an increase of productivity of agricultural crops under

the maintenance of the ecological state and fertility of cultivated soils. The

ecologically safe biotechnological means play an important role in realization of this

strategy aimed to obtaining high-quality products of plant-growing and

improvement of the ecology of environment due to diminishing of pesticide toxicity,

which is used for chemical protection of plants from diseases and pests (Compant et

al., 2005; Doran et al., 1996). For today application of pesticides reached dangerous

amounts. Getting to soils these compounds violate natural physical, chemical and

biological processes (Downing et al., 2004; Gerhardson 2002). Pesticides have a row

of adverse effects such as pest dependability to them, nonselective action and

oppression of useful biological objects, and pollution of environment as well.

Ecologically safe biological means are more promising, in particular Microbial Plant

Resistance (PR) and plant growth promotion (PGP) inducers (PR/PGP inducers), to

which there is no habit-forming of pests. In addition these formulations promote to

increase of plant systemic resistance to biotic and abiotic stresses (Gupta and

Dikshit, 2010; Kerry, 1987).

Plant parasitic nematodes belong to the most widespread and dangerous

factors for grain, vegetable and industrial crops. These pests cause diseases of

mixed nematode-microbial etiology that result in diminishing of crop productivity.

In different countries, crop losses reach 25 - 70% and at extreme conditions they can

reach 90 - 100 % (Rivoal and Cool, 1993; Stevens and May, 2010). Therefore,

creation of modern meanses for biocontrol of crop pests spread is very actual

direction of development of plant-growing (Bleve-Zacheo et al., 2007). Sugar beet

cyst-forming nematode Heterodera schachtii Schmidt is dangerous pests that cause

heteroderosis of sugar beet in almost 40 countries worldwide (Gerhardson, 2002;

Stevens and May, 2010). The largest yield losses because of this nematode are

observed during sugar beet growing in a monoculture and in high concentrations

(up to 60 - 80 %) in the crop rotation of plant- host cultures. Risk of increasing of

68 Advances in Bioscience and Bioengineering

harmfulness of sugar beet nematode for rape plants arises in recent years as a

result of increasing areas of growing of rape plants at the crop rotations of sugar

beet. Therefore the question of limiting the spread of these dangerous pests becomes

more actual (Curto, 2008).

The currently existing means for control of nematodes’ distribution and

protection of crops from pests’ invasion are soil fumigants, nematicides and

insecticides of synthetic origin (Fuller et al., 2008; Oka, 2010). However, in most

countries, a tendency limiting their practical applications is observed because of

their high toxicity to humans and contamination of the environment. Excessive use

of chemical facilities of plant protection and mineral fertilizers causes a number of

negative consequences such as forming of resistant races of causative disease agents;

reduction of quantitative and quality composition of natural microbial cenosis,

mainly, due to diminishing to the quantity of useful microbial populations; an

accumulation of toxic compounds in the environment (Downing et al., 2004).

Among the biological antiparasitic facilities the bio-formulations on the basis

of antiparasitic macrolide antibiotics avermectins – metabolite products of soil

streptomycete Streptomyces avermitilis are the most promising. Currently

avermectins are considered as the most effective biological substances against

parasitic nematodes, claws and harmful insects (Burg et al., 1979; Fisher, 1990).

The new effective nematicidic PR/PGP inducers Avercom and its derivates

Avercom nova-1 and Avercom nova-2 were created on the basis of metabolite

products of soil streptomycete Streptomyces avermitilis UCM Ac-2179 in Zabolotny

Institute of Microbiology and Virology, National Academy of Sciences of Ukraine.

These nematicidic PR/PGP inducers contain antiparasitic antibiotic avermectin and

the complex of biologically active compounds, namely, lipids (including free fatty

acids), aminoacids, vitamins of the B group and phytohormones: indole-3-acetic acid,

isopentenyl adenine, zeatin, zeatin riboside, brassinosteroids (Iutynska, 2012). High

nematicide and growth stimulating activity of the PR/PGP inducers is confirmed on

vegetable crops, however their biological efficiency was not investigated on rape

plants.

Advances in Bioscience and Bioengineering 69

New promising strategy for prevention of plant defeat by nematodes and

increase of their resistance to these pests is application of RNA interference

technology (RNAi) for control of expression of genes that are accountable for

reproduction cycle of pathogenic organisms and pests by the way of Post-

Transcriptional Gene Silencing (PTGS) (Felippes et al., 2012; Jagtap et al., 2011;

Llave et al., 2002; Yu and Kumar, 2003).

Gene silencing was first demonstrated for the free-living nematode,

Caenorhabditis elegans, and the underlying mechanism of RNAi has subsequently

been studied in depth for this nematode (Fire et al., 1998). The impact of this work

was recognized in 2006 by the award of the Nobel Prize in Physiology and Medicine

to Andrew Fire and Craig Mello. Gene silencing mediated by either degradation or

translation arrest of target RNA has roles in adaptive protection against pathogenic

organisms, genome defense against mobile DNA elements and developmental

regulation of gene expression (Bakhetia et al., 2005; Fabian et al., 2010; Filipowicz

et al., 2005; Vaucheret, 2006). These silencing systems involve processing of two

type small regulatory RNAs: short interfering RNA (siRNA) and microRNA (miRNA)

(Hamilton and Baulcombe, 1999; Hamilton et al., 2002; Katiyar-Agarwal and Jin,

2010; Zhang et al., 2007).

Now a great success in cloning of parasitic organisms’ genes is reached.

During the last years the vectors containing dsRNA (double stranded RNA) which is

specific to pest genes have been constructed with the application of RNAi technology

and transgenic plants (transformed by these vectors) with increasing resistance to

pathogenic and parasitic organisms have been obtained (Agrawal et al., 2003; Dinh

et al., 2014; Hirai and Kodama, 2008; Jiarui et al., 2011; Klink and Matthews, 2009;

Liu and Zhu, 2014; Rosso et al., 2005; Senthil-Kumar and Mysore, 2010; Sindhu et

al., 2009; Thakur et al., 2014; Tsygankova, Yemets et al., 2013).

In our earlier researches (Tsygankova, Yemets, et al. 2013) we have

constructed vector containing dsRNA that is antisence to the conservative region of

8Н07 gene of nematode H. schachtii. Using A. tumefaciens-mediated genetic

transformation we have obtained RNAi transgenic rape plants with increased

70 Advances in Bioscience and Bioengineering

resistance to this nematode. Considering the fact that obtained rape plants contain

vector with dsRNA that is specific for only one major gene for the life cycle of pest,

therefore a very important question is to verify the possibility of additional increase

of resistance of RNAi transgenic rape plants against nematode H. schachtii with use

of new microbial nematicidic PR/PGP inducers. The aim of this work was study of

impact of new nematicidic PR/PGP inducers Avercom, Avercom nova-1 and Avercom

nova-2 on morpho-physiological and genetic indexes of resistance to parasitic

nematode H. schachtii of wild and RNAi transgenic rape plants containing dsRNA

that is specific to the conservative region of 8Н07 gene of this nematode.

Materials and Methods

Plant Growing and Treatment

Nematicidic PR/PGP inducer Avercom was obtained by ethanol extraction

from of 7-days biomass of Streptomyces avermitilis UCM Аc-2179, the concentration

of avermectin is 100 μg/ml. Its modifications are: Avercom nova-1 that contains 50

ml of Avercom with antibiotic avermectin at concentration 100 μg/ml with adding 50

ml of supernatant of cultural liquid of Streptomyces avermitilis UCM Ас-2179 and

0.05 mM of salicylic acid (the total content of avermectin is 50 μg/ml) and Avercom

nova-2 that contains 50 ml of Avercom with antibiotic avermectin in concentration

100 μg/ml and 50 ml of supernatant of cultural liquid of Streptomyces avermitilis

UCM Ас-2179 and 0.01 mM of water-soluble chitosan of “Sigma” Company (the total

concentration of avermectin is 50 μg/ml) (Iutynska, 2012). Systemic insecticide

Confidor Maxi (ingredient is Imidacloprid - 700 g/kg) was used also. Field

experiments were conducted on spring wild rape plants of Kalinivsky cultivar, the

molecular-biological experiments were conducted in the laboratory conditions on

RNAi transgenic rape plants Brassica rapa ssp. оleifera obtained according to

technique described in detail in the work (Tsygankova, Yemets et al., 2013). Wild

spring rape plants of Kalinivsky cultivar were grown in field conditions on black

earth with insignificant content of soil organic matter (soil layer is a 0-20 cm)

having the following agrochemical characteristics: content of soil organic matter –

Advances in Bioscience and Bioengineering 71

4.53%, total nitrogen – 0.27 – 0.31 %, total phosphorus – 0.15 – 0.25 %, movable

(changing) phosphorus – 3.3 – 3.4 mg, movable (changing) potassium – 9.8 – 10.3

mg in 100 gm of soil, рН of salt extraction – 6.9. Experiments were carried out on

plot of land with size 13.5 m2 and randomized disposition. An artificial invasive

background was created by bringing in soil before sowing of highly infectious

material taken from natural soil samples contaminated with sugar beet nematode H.

schachtii.

In the field experiments rape seeds were treated with nematicidic PR/PGP

inducers Avercom and Avercom nova-2 before sowing and during a vegetation

period in phase of plant bushing out as well. Efficiency of nematicidic PR/PGP

inducers was compared to the action of chemical insecticide Confidor Maxi. In the

control seeds were processed with the equivalent volume of water. All the

experiments were performed in three replicates as follows (Table 1).

Soil samples for nematological analysis were taken using manual drill

according to standard techniques (Southey, 1986), nematodes were isolated from the

soil by watering-can method (Southey, 1986), and determination of nematode

species was carried out according to the methods described in the methodical

guidelines (Chen et al., 2004). The quantity of nematodes (plant parasitic,

mycohelmints and saprophytic nematodes) was determined in the rhizosphere soil

of plants in middle (July and August) and at the end of vegetation period

(September) according to a method described in the methodical guidelines (Chen et

al., 2004). Diminishing of nematode population density in the soil (in %) was

calculated according to difference of the nematode amounts (pieces/100g of soil)

determined in rhizosphere soil of experimental wild rape plants compared to control

plants.

Determination of amount of viable bacteria in the rhizosphere soil of rape

plants was executed during flower and maturation phases by Plate Count

Techniques (on pour or spread plate): a soil sample was decimally diluted and

subsamples from the dilution tubes were plated on an appropriate agar culture

medium. Colonies were counted after a suitable incubation period and the number

72 Advances in Bioscience and Bioengineering

of colony forming units (CFU) per gram of dry soil was determined (Zuberer, 1994).

The soil respiration intensity was estimated as the CO2 production (mg CO2 per 1 g

of soil during 1h) by chemical titration (Alef et al., 1995).

All the experiments were performed in three replicates. The statistical

analysis of the data was carried out by the methods described in methodical

guidelines (Statistical Methods in Biology, 1995) and computer programs Statistica

6.0 and Microsoft Excel ’00.

In the molecular-biological experiments we verified impact of nematicidic

PR/PGP inducers on morpho-physiological signs and genetic indexes of resistance to

nematode H. schachtii of wild rape plants of Kalinivsky cultivar and RNAi

transgenic rape plants Brassica rapa ssp. оleifera. RNAi transgenic rape plants

were obtained using A. tumefaciens-mediated genetic transformation by our

constructed vector containing dsRNA with antisense sequences to the conservative

region of 8Н07 gene of nematode H. schachtii (Tsygankova, Yemets, et al. 2013).

Seeds of wild and RNAi transgenic rape plants were sprouted in Petri dishes

(9.5 cm in diameter) in nematode-free dechlorinated aqueous medium (control

variant). In the experimental variants the impact of nematicidic PR/PGP inducers:

Avercom, Avercom nova-1 and Avercom nova-2 on additional increase of resistance

of wild and RNAi transgenic rape plants to nematode invasion was verified. For this

aim seeds of these plants were sprouted with a suspension of nematodes H.

schachtii eggs (at the concentration 20-50 nematode eggs/per 20 seeds, from which

the nematode larvae was appeared during incubation process in 5-7 days later) and

on aqueous medium with a solution of each nematicidic PR/PGP inducers (at the

concentration 20 μl of each nematicidic PR/PGP inducers/ml of distilled water with

final content of avermectin – 0.02 μg/ml).

Isolation of Small Regulatory si/miRNA Populations

To determine the impact of PR/PGP inducers on molecular-genetic indexes of

plant resistance to parasitic nematodes (i.e. on characteristics of mRNA and

si/miRNA populations as well as on silencing activity of si/miRNA populations) we

Advances in Bioscience and Bioengineering 73

isolated total RNA from the cells of control and experimental wild and RNAi

transgenic rape plants and separated si/miRNA populations according to our

elaborated and earlier published method (Tsygankova, Andrusevich, Ponomarenko

et al., 2012). The size (of 21-25 nt) of isolated si/miRNA populations labeled before

isolation in vivo with 33P using Na2HP33O4 (Tsygankova, Stefanovska, Galkin et al.,

2012) was verified by electrophoresis in a 15% polyacrylamide gel (PAGE) stained

with ethidium bromide solution prior to photographing RNA fractions under UV

light (Sambrook et al., 1989). Obtained gel was dried out in the thermal vacuum

dryer (LKB, Sweden). The fluorography of gel was carried out according to the

method described in guideline (Bonner and Laskey, 1974), in the gel we added

fluorescent reagent 2,5-diphenyl-1,3-oxazole (Osterman, 1981) and exposed gel with

X-ray film during two months at - 70°C.

Identification of Degree of Homology Between si/miRNA and mRNA

Populations

To determine the degree of homology between cytoplasmic mRNA and small

regulatory si/miRNA populations, isolated from control and experimental wild and

RNAi transgenic rape plants, we performed hybridization of si/miRNA with fraction

of cytoplasmic RNA populations using Dot-blot method described in methodical

guidelines (Promega Protocols and Applications Guide, 1991; Sambrook et al., 1989).

Hybridization was conducted on modified and activated cellulose filters (Whatman

50, 2-aminophenylthioether paper of Company Amersham-Pharmacia Biotech, UK).

These filters form covalent linkages with deposited DNA or RNA unlike the

cellulose and nitrocellulose filters that form hydrogen bonds with DNA or RNA

enabling to avoid losses of the nucleic acids in the course of the filter washing out

(Sambrook et al., 1989). Radioactivity of hybrid molecules was detected (imp./count

per min/20 μg ± SE of mRNA) on glass Millipore AP-15 filter in toluene scintillator

using Beckman LS 100C scintillation counter. Degree of homology (in %) was

determined according to the difference of hybridization between mRNA and

si/miRNA populations isolated from experimental and control wild and RNAi

74 Advances in Bioscience and Bioengineering

transgenic rape plants (Tsygankova, Stefanovska, Galkin et al., 2012; Tsygankova

et al., 2014).

Determination of Silencing Activity of si/miRNA Populations in the

Cell-Free System of Protein Synthesis

Investigation of silencing activity (i.e. inhibition of translation of own plant

mRNA or nematode mRNA) of si/miRNA populations, isolated from wild and RNAi

transgenic rape plants (that were not treated or treated by nematicidic PR/PGP

inducers) was conducted in the wheat embryo cell-free system of protein synthesis,

which is widely used along with the rabbit reticulocyte lysate cell-free system and

cell-free system from syncytial blastoderm Drosophila embryos (Marcus et al., 1974;

Sambrook et al., 1989; Tang et al., 2003; Tuschl et al., 1999). Preparation of cell

free-system is described in details elsewhere (Marcus et al. 1974; Sambrook et al.

1989). Reagents of different companies, namely Amersham-Pharmacia Biotech, UK;

New England Biolab, USA; Promega Corporation Inc, USA and Boehringer, Dupont,

NEN, USA and Mannheim GmbH, Germany were used in our researches. In the

cell-free system the determination of inhibition of protein synthesis by si/miRNA

populations on the template of own plant mRNA or nematode mRNA was carried

out according to index of decreasing of incorporation [35S] methionine into proteins

and was accounted (in imp./count per min/1mg of protein) on glass filter Millipore

AP-15 in toluene scintillator in the scintillation counter LS 100C (Bonner and

Laskey, 1974; Osterman, 1981). Unlabelled si/miRNA populations were used for

testing of their inhibitory activity in the cell-free system of protein synthesis. The

silencing activity of si/miRNA populations (%) isolated from control and

experimental wild and RNAi transgenic rape plants was determined as a difference

of radioactivity of polypeptides (in imp./count per min/1mg of proteins), which were

synthesized on template of nematode mRNA or own plant mRNA, isolated from

control and experimental variants (Tsygankova et al., 2014).

The statistical analysis of the data was carried out by dispersive (Student)

method (Statistical Methods in Molecular Biology, 2010).

Advances in Bioscience and Bioengineering 75

Results

Impact of Nematicidic PR/PGP Inducers on Morpho-Physiological

Signs of Wild and RNAi Transgenic Rape Plant Resistance to Parasitic

Nematodes and on Soil Microflora

Biometric researches that were conducted in the field conditions showed that

wild rape plants of Kalinivsky cultivar, which were grown on artificial invasive

background (created by nematode Heterodera schachtii Schmidt) and treated (pre-

sowing treatment of seeds and crop spraying) with nematicidic PR/PGP inducers

Avercom and Avercom nova-2, are more viable according to morpho-physiological

signs and resistant to nematodes compared to control wild rape plants that were not

treated with PR/PGP inducers (Table 2). Avercom exerted the most positive effect

on biometric indexes of rape plants. Therein height of treated with PR/PGP inducer

plants exceeded height of control plants up to 14.7 %. The same results were

obtained on plants treated with Avercom nova-2: their height exceeded similar

index of control plants up to 8.4 % likewise. Comparative study of impact of

insecticide Confidor Maxi on plant height showed increase of this index up to 12.6 %

at plants treated with insecticide compared to control plants. It should be noted that

crops of wild rape plants, which were grown on control plots and were not treated

with nematicidic PR/PGP inducers, were rarefied with the signs of defeat by

nematodes and had low height.

The analysis of density of nematode population complex showed the

considerable diminishing of total amount of parasitic nematodes on roots of wild

rape plants that were treated either with Avercom (up to 87.9 %) and Confidor Maxi

(up to 74.4 %). Avercom nova-2 decreased of nematode population on fewer amounts

(up to 38.5 %) compared to control plants (Table 3). All investigated PR/PGP

inducers diminished of total amount of mycohelmints in rhizosphere of wild rape

plants (up to 57.9 − 88.0 %) compared to control plants. Amount of saprophytic

nematodes (which are harmless for plants) decreased under action of Avercom and

Confidor Maxi.

76 Advances in Bioscience and Bioengineering

The soil state is important for agro-ecosystem stable functioning, in

particular in cause of use of bio-formulations in plant growing. Healthy soils were

maintained by the microbial communities that help to form associations of plant

growth promoting rhizobacteria with plant roots, to recycle plant nutrients, to form

soil organic matter (Arias et al., 2005). It is known some indicators for evaluating

its current status, in particular the soil respiration activity recognized as general

index of soil biological activity. Soil respiration activity (CO2 emission) is biological

oxidation of organic matter to CO2 by aerobic organisms, notably microorganisms. It

is positively correlated with soil organic matter content, and often with microbial

biomass and microbial activity.

Obtained data suggest that at the rhizosphere of wild rape plants treated with

PR/PGP inducers the biological activity of soil was changed (Fig. 1).

At an average during vegetation period the statistically reliable increase in

activity of CO2 emission up to 54 % compared to control was marked in the variant

with application of Avercom nova-2. Meanwhile, application of chemical insecticide

Confidor Maxi repressed activity of CO2 emission up to 42 % compared to control

variant.

Study of rhizosphere microbial communities of wild spring rape showed

activation of development of quantity of microorganisms of some ecologically-trophic

groups at use of PR/PGP inducers containing avermectin (Fig. 2).

It was found out the greater quantity of microorganisms, which participate in

transformation of organic compounds and fixing of atmospheric nitrogen, in the

rhizosphere of wild spring rape plants treated with PR/PGP inducers compared to

control. The quantity of nitrogen-fixing microorganisms that improve nitrogen

nutrition of plants due to fixing of atmospheric nitrogen increased in rhizosphere of

wild rape plants treated both with Avercom – up to 1.2 times and Avercom nova-2 -

up to 2.2 times compared to rhizosphere of control plants. In these variants the

quantity of phosphate mobilizing bacteria (that release for plants of soluble

phosphorus from inconsiderably available compounds) increased up to 1.6 - 3.1

times. At conditions of application of PR/PGP inducers the quantity of

Advances in Bioscience and Bioengineering 77

ammonificating and amylolytic microorganisms that are able to hydrolyze of

complex organic compounds (proteins and starch) in rhizospheric soil increased up

to 1.7 - 2 times compared to rhizosphere of control plants.

It should be noted that use of chemical insecticide Confidor Maxi impinged on

development of phosphate mobilizing, ammonificating and amylolytic

microorganisms, which quantity was below of control variant in 1.2 - 1.7 times.

Such difference in action of PR/PGP inducers and insecticide could be explained

those that PR/PGP inducers contain the complex of physiological active substances

that influence positively on soil microorganisms and stimulate development of wild

rape plants and increase of root quantity.

In the field experiments we obtained results that testify to high bio-protective

and stimulating action of PR/PGP inducers Avercom and Avercom nova-2 on

increase of yield indexes of wild spring rape plants of Kalinivsky cultivar (Table 4).

At use of PR/PGP inducers Avercom and Avercom nova-2 the yield of wild rape

plants exceeded up to 2775.6 and 2156.1 pounds/ha (up to 199.5 and up to 155 %

accordingly) of control variant. Chemical insecticide Confidor Maxi increased a yield

less - up to 1895.9 pounds/ha (up to 136.3 %). Obtained results could be explained

those that unlike chemical insecticide the PR/PGP inducers contained not only

antibiotic avermectin (which possesses specific anti-nematodic activity), but also

physiologically active substances (i.e. phytohormones, lipids, fatty acids, vitamins

etc.) stimulating height and development of plants, and promoting plant resistance

against pathogens as well. As a result of these processes the positive action on plant

development and bio-protective efficiency PR/PGP inducers was higher compared to

chemical insecticide.

In the laboratory conditions the impact of the nematicidic PR/PGP on the

morpho-physiological signs and genetic indexes of resistance of wild and RNAi

transgenic rape plants to parasitic nematode Heterodera schachtii was studied. The

wild and RNAi transgenic rape plants that were not treated and treated with

PR/PGP inducers Avercom, Avercom nova-1 and Avercom nova-2 and grown on an

infectious background (created by the larvae of parasitic nematode H. schachtii) are

78 Advances in Bioscience and Bioengineering

shown in Fig. 3. The considerable increase of viability and resistance to nematode

invasion of wild and RNAi transgenic rape plants that were treated with PR/PGP

inducers compared to control plants was found.

Impact of Nematicidic PR/PGP Inducers on Changes in the Degree of

Homology between si/miRNA and mRNA Populations

Using Dot-blot hybridization method (Promega Protocols and Applications

Guide, 1991; Sambrook et al., 1989) the difference in the degree of homology (in %)

between the populations of cytoplasmic mRNA isolated from control uninfected by

the larvae of nematode H. schachtii and untreated with PR/PGP inducers wild and

RNAi transgenic rape plants and si/miRNA isolated from experimental infected by

the larvae of nematode H. schachtii and treated with PR/PGP inducers wild and

RNAi transgenic rape plants was obtained (Fig. 4). The most difference in the

percent of homology was observed between the populations of cytoplasmic mRNA

isolated from control uninfected plants and si/miRNA isolated from experimental

infected wild and RNAi transgenic rape plants which were treated with PR/PGP

inducers as follows: Avercom (up to 39 % - for wild and up to 47 % - for RNAi

transgenic plants), Avercom nova-2 (up to 34 % - for wild and up to 43 % - for RNAi

transgenic plants) and Avercom nova-1 (up to 30 % - for wild and up to 38 % - for

RNAi transgenic plants). Less difference in the percent of homology was obtained

between the populations of cytoplasmic mRNA from control uninfected plants and

si/miRNA from experimental infected and untreated with PR/PGP inducers wild

and RNAi transgenic rape plants: up to 29 % - for RNAi transgenic and up to 12 % -

for wild rape plants compared to control plants: up to 99 % - for wild and up to 97 %

- RNAi transgenic rape plants. Obtained results testify that these PR/PGP inducers

activate synthesis of populations of immune-protective si/miRNA in the cells of wild

and RNAi transgenic rape plants.

Advances in Bioscience and Bioengineering 79

Impact of Nematicidic PR/PGP Inducers on Silencing Activity of

si/miRNA Populations

Comparative analysis of silencing activity (i.e. inhibiting of translation of

mRNA isolated from cells of control and infected plants) of si/miRNA populations

isolated from control and experimental wild and RNAi transgenic rape plants was

carried out in the wheat embryo cell-free system of protein synthesis (Marcus et al.,

1974; Sambrook et al., 1989) (Fig. 5). It was found that according to index of

inhibition of translation of mRNA isolated from cells of infected plants the most

higher silencing activity showed si/miRNA populations isolated from experimental

wild and RNAi transgenic rape plants, which were grown on infectious background

(created by nematode H. schachtii larvae) and treated with PR/PGP inducers:

Avercom nova-1 (up to 43 % - for wild and up to 73 % - for RNAi transgenic plants);

Avercom-nova-2 (up to 37 % - for wild and up to 69 % - for RNAi transgenic plants)

and Avercom (up to 32 % - for wild and up to 58 % - for RNAi transgenic plants). At

the same time si/miRNA populations, isolated from experimental RNAi transgenic

rape plants infected and untreated with PR/PGP inducers, showed silencing activity

which reached up to 52 %, lowest activity which reached up to 18 % showed

si/miRNA populations isolated from wild rape plants infected and untreated with

PR/PGP inducers compared to activity of si/miRNA isolated from control uninfected

and untreated with PR/PGP inducers wild and RNAi transgenic rape plants: up to

99 % - for wild and up to 100 % - for RNAi transgenic rape plants.

In the wheat embryo cell free system of protein synthesis it was carried out

also a comparative analysis of silencing activity (i.e. inhibiting of translation of

mRNA isolated from larvae of nematode H. schachtii) of si/miRNA populations,

isolated from the same control and experimental wild and RNAi transgenic rape

plants (Fig. 6). We have found the increase of differences in the silencing activity

between si/miRNA populations isolated from cells of wild and RNAi transgenic rape

plants that were not treated or treated with PR/PGP inducers. It was found that

insignificant silencing activity showed si/miRNA populations isolated from the cells

of wild rape plants grown in absence and in presence of infectious background and

80 Advances in Bioscience and Bioengineering

untreated with PR/PGP inducers: up to 11 % - for control and up to 19 % - for

experimental rape plants. This fact testifies insignificant homology between

nematode mRNA and si/miRNA populations isolated from wild rape plants.

Considerably higher activity showed si/miRNA populations isolated from the cells of

RNAi transgenic rape plants grown in absence and in presence of infectious

background and untreated with PR/PGP inducers: up to 43 % - for control and up to

68 % - for experimental rape plants. These results testify higher homology between

nematode mRNA and si/miRNA populations isolated from RNAi transgenic rape

plants obtained using A. tumefaciens-mediated genetic transformation by vector

containing dsRNA with antisense sequences to the conservative region of 8Н07 gene

of nematode H. schachtii (Tsygankova, Yemets et al., 2013). In the wheat embryo

cell-free system of protein synthesis on the template of nematode mRNA the

considerable increasing of silencing activity of si/miRNA populations isolated from

cells of wild and RNAi transgenic rape plants that were treated with nematicidic

PR/PGP inducers was found. The highest activity showed si/miRNA populations

isolated from cells of experimental wild and RNAi transgenic rape plants grown on

infectious background (created by larvae of nematode H. schachtii) and treated with

PR/PGP inducers: Avercom nova-1 (up to 34 % - for wild and up to 86 % - for RNAi

transgenic plants); Avercom nova-2 (up to 30 % - for wild and up to 82 % - for RNAi

transgenic plants) and Avercom (up to 26 % - for wild and up to 75 % - for RNAi

transgenic plants).

Obtained results confirm that PR/PGP inducers cause reprogramming of

plant genome, i.e. induce synthesis of si/miRNA populations with specific silencing

activity both to own plant mRNA (which increasing level of expression promotes of

nematode infection) and to homologous mRNA of nematodes.

Discussion

Modern methodical approaches for the decision of problem of increase of plant

resistance to pathogenic organisms are based on revealing of molecular-genetic

mechanisms of interaction between plant-host and pests with the aim of

Advances in Bioscience and Bioengineering 81

identification of gene families that control plant resistance and immune-protective

properties against pathogenic and parasitic organisms, or genes which increasing

level of expression, on contrary, promotes defeat of plants by pests (Ellis and Jones,

2003; Heikal et al., 2008; Klink and Matthews, 2009; Mang et al., 2009; Molecular

plant-microbe interactions, 2009; Shamray, 2003; Sindhu et al., 2009; Thakur et al.,

2014; Quentin et al., 2013; Williamson, 1999). Plant hormones and bioregulators of

natural origin as well as genetic engineering methods are used for upregulation or

downregulation of expression of these genes (Atkinson and Urwin, 2012; Block et al.,

2005; Denancé et al., 2013; Erb et al., 2012; Fuller et al., 2008; Gachomo et al., 2003;

Giron et al., 2013; Groot and Dicke, 2002; Heil and Bostock, 2002; Jia et al., 2013;

Jouanin et al., 1998; Kästner et al., 2014; Keresa et al., 2008; Khan et al., 2009;

Liao et al., 2006; Naseem and Dandekar, 2012; Nelson et al., 2014; Olaiya et al.,

2013; Pieterse et al., 2009; Studham and MacIntosh, 2012; Vavrina et al., 2004).

Today it is known that in the immune protection of plants from pathogenic

and parasitic organisms a leading role is carried out by a small regulatory si/miRNA

that take part in realization of signals of plant hormones, which regulate expression

of genes that are accountable for plant immune defense both on the transcription

level by the way of TGS (i.e. by the way of methylation of DNA and modifications of

histones) and on the post-transcription level by the way of PTGS (i.e. by the way of

silencing of translation of mRNA) (Zhang et al., 2007; Zhang et al., 2011; Katiyar-

Agarwal et al., 2006; Katiyar-Agarwal et al., 2007; Li et al., 2010; Li et al., 2012). In

the nature conditions in the plants defeated by pathogens or parasitic organisms

the synthesis of small regulatory si/miRNA, which play the main role in plant

resistance to pathogenic organisms, considerably increases (Hewezi et al., 2008;

Katiyar-Agarwal and Jin, 2010; Li et al., 2012; Lуpez et al., 2012; Padmanabhan et

al., 2009; Shen et al., 2014; Vaucheret, 2006; Yang and Huang, 2014; Zhang et al.,

2010). Unfortunately a level of its synthesis is not always sufficient for prevention

of plant invasion by pests.

Promising strategy is creation of plants with enhanced resistance to pests by

induction of RNA-interference process in the plant cells, i.e. increase of synthesis of

82 Advances in Bioscience and Bioengineering

endogenous small regulatory si/miRNA populations (which contains antisense

(complementary) sequences both to the mRNA of plants and pests) using of plant

hormones, bioregulators of synthetic and natural origin, or genetic engineering

methods (Agrawal et al., 2003; Bakhetia et al., 2005; Dinh et al., 2014; Fuller et al.,

2008; Gheysen and Vanholme, 2006; Hewezi et al., 2008; Hirai and Kodama, 2008;

Jagtap et al., 2011; Jiarui et al., 2011; Júnior et al., 2013; Karakas, 2008; Khan et

al., 2009; Klink and Matthews, 2009; Nelson et al., 2014; Olaiya et al., 2013; Rosso

et al., 2005; Senthil-Kumar and Mysore, 2010; Pieterse et al., 2012; Sindhu et al.,

2009; Spoel and Dong, 2012; Tamilarasan and Rajam, 2013; Tsygankova,

Andrusevich, Ponomarenko et al., 2012; Tsygankova, Роnоmаrenko, Hrytsaenko,

2012; Tsygankova, Stefanovska, Galkin et al., 2012; Tsygankova, Andrusevich et al.,

2013; Tsygankova, Yemets et al., 2013; Tsygankova et al., 2014; Vavrina et al., 2004;

Wani et al., 2010).

It is found that the most important for nematode life cycle genes (that have

high homology to many eukaryotic genes) can be considered as target genes for

silencing (Baum et al., 2007; Bleve-Zacheo et al., 2007; Tamilarasan and Rajam,

2013; Tsygankova, Yemets et al., 2013). These genes control various cellular

processes: DNA replication, transcription, RNA processing, synthesis of tRNA,

translation, control of ribosome functioning and tRNA, control of modification

processes, secretion and protein transfer, control of their stability and degradation,

and control of the functioning of mitochondrion and the metabolism of protein

mediators; genes for the control of the cell cycle (proteins of cytoskeleton, i.e.,

tubulins) and the cell structure, transfer of endo- and intercellular signals,

processes of endocytosis, and ionic regulation; genes of nematodes enzymes,

destroying cell walls in plant roots; genes of the secretory proteins of esophageal

glands (the expression of which is necessary for the penetration of nematode stylet

into special plant root cells, i.e., feeding sites); genes of the reproductive cycle (major

sperm proteins) and the genes of chitin synthetase (which controls formation of the

solid chitin cover on the nematode eggs) (Ajjappala et al., 2012; Bradley et al., 2009;

Bleve-Zacheo et al., 2007; Dinh et al., 2014; Fanellia et al., 2005; Klink and

Advances in Bioscience and Bioengineering 83

Matthews, 2009; Patel et al., 2010; Quentin et al., 2013; Rosso et al., 2005; Sindhu

et al., 2009; Tamilarasan and Rajam, 2013; Tsygankova, Andrusevich,

Ponomarenko et al., 2012; Tsygankova, Yemets et al., 2013; Tsygankova et al.,

2014).

The plant host genes, which express at highly level during process of plant

invasion and thereof promote the penetration of parasitic nematodes in the roots,

were also identified as target genes for silencing. The most important ones include

the following gene families: pathogenesis-related proteins (PRPs), protein

transporter of auxin PIN2 (EIR), proteins of ethylene-responsive factors (ERFs) and

of some transcription factors (AP2, MADS-box, bZIP, bHLH, and NAC genes),

gibberellin regulatory proteins, enriched with the proline the extension-similar

proteins, proteins-transporters of nitrates NTP2, as well as the genes of

pectinesterase, ferritin, cytochrome P450, chalcon synthaze, cell wall metabolism

proteins, etc. (Ascenzi, 2010; Favery et al., 2008; Ithal et al., 2007; Jammes et al.,

2005; Tamilarasan and Rajam, 2013; Wiig, US Patent No. 2010115660, 2010; Wiig,

US Patent No. 2010107276, 2010).

In our early conducted field and greenhouse experiments it was found that

PR/PGP inducers Avercom and its modifications considerably promote (up to 85-

100 %) resistance of many agricultural plants such as wheat, tomatoes, potato and

cucumbers to the defeat by parasitic nematodes as follows Tylenchorbynchus dubius,

Pratylenchus pratensis, Meloidogyne incognita та Diethylenchus destructor

(Iutynska, 2012; Iutynska et al., 2013; Tsygankova, Andrusevich, Beljavskaja et al.,

2012; Tsygankova et al., 2014). In these investigations using Dot-blot method of

hybridization of cytoplasmic mRNA and small regulatory si/miRNA from control

and experimental wheat, tomatoes, potato and cucumbers plants infected by

nematodes and also testing silencing activity of small regulatory si/miRNA isolated

from control and experimental plants in the wheat embryo cell-free system, it was

shown that the PR/PGP inducer Avercom and its modifications promote RNA-

interference process in the cells of the plants defeated by nematodes, i.e. PR/PGP

inducers increase synthesis of small regulatory si/miRNA with specific anti-

84 Advances in Bioscience and Bioengineering

nematodic properties (Iutynska et al., 2013; Tsygankova, Andrusevich, Beljavskaja

et al., 2012; Tsygankova et al., 2014). Obviously, this immune-protective effect of

PR/PGP inducers is connected with presence in their composition, foremost, of

phytohormones (i.e. auxins, cytokinins, gibberellins and brassinosteroids) that

stimulate synthesis of specific to the genes of plants and pests si/miRNA in the

plant cells.

The results that were obtained in this work are correlated with above

mentioned data which indicate the impact of nematicidic PR/PGP inducer Avercom

and its modifications on reprogramming of genome both of wild rape plants of

Kalinivsky cultivar and RNAi transgenic rape plants Brassica rapa ssp. оleifera. As

a result, there is intensification of synthesis in the plants of small regulatory

si/miRNA with specific (antisense) sequences to major for the life cycle of parasitic

nematode H. schachtii genes as well as genes of infected plants (which increasing

level of expression promotes defeat of plants by nematodes). Owing to this process

the plant resistance to these pests considerably increases.

Conclusion

In the field and laboratory conditions according to morpho-physiological signs

of plants treated with nematicidic PR/PGP inducer of microbial origin Avercom and

its modifications it was shown that them use promote decrease of defeat by the

parasitic nematode H. schachtii both of wild rape plants of Kalinivsky cultivar and

RNAi transgenic rape plants Brassica rapa ssp. оleifera. The positive changes in

development of rhizosphere microflora and productivity of wild rape plants treated

with PR/PGP inducers were found. In the molecular-biological experiments the

increase of difference in the percent of homology between the populations of

cytoplasmic mRNA isolated from control rape plants and si/miRNA isolated from

control and experimental wild and RNAi transgenic rape plants: up to 30-39 % for

wild and up to 38 - 47 % for RNAi transgenic plants was shown. Obviously, obtained

difference at the percent of homology is explained by the fact that these PR/PGP

inducers activate synthesis of small regulatory si/miRNA with high anti-nematodic

Advances in Bioscience and Bioengineering 85

activity in the wild and RNAi transgenic rape plants. The confirmation of this fact

is our results witnessing about the considerable increase in the wheat embryo cell-

free system of silencing activity (i.e. inhibiting of translation of mRNA of infected

rape plants and mRNA of nematode H. schachtii) of small regulatory si/miRNA

populations: up to 26-43 % - for wild and up to 58-86 % - for RNAi transgenic rape

plants that were treated with nematicidic PR/PGP inducers.

Acknowledgements

The research has been carried out with support of the project “Molecular

bases of creation of biologically active and ecologically safe preparations with

bioprotective and immune-modulating properties” of the special purpose complex

interdisciplinary program of the scientific researches of the National Academy of

Sciences of Ukraine “Fundamentals of molecular and cellular biotechnology”

(confirmed by the decision of Presidium of the National Academy of Sciences of

Ukraine from 07.07.10, No. 222).

86 Advances in Bioscience and Bioengineering

References

[1] Agrawal, N., Dasaradhi, P.V.N., Mohmmed, A., Malhotra, P., Bhatnagar, R.K. and Mukherjee,

S.K. (2003). RNA Interference: Biology, Mechanism, and Applications. Microbiol. Mol. Biol. Rev.,

67(4): 657-685. DOI: 10.1128/MMBR.67.4.657-685.2003

[2] Ajjappala, H., Sim, J.-S., and Hahn, B.-S. (2012). RNA Interference Silencing in Root-knot

Nematodes. Korean J. Intl. Agri., 24(4), 485-493. http://www.intagrijournal.org/pdf/24(4)-15.pdf

[3] Arias, M.E., Gonzalez-Perez, J.A., Gonzalez-Vila, F.J., Ball, A.S. (2005). Soil health - a new

challenge for microbiologists and chemists. Int Microbiol., 8(1): 13-21.

[4] Ascenzi, R. (2010). Compositions and Methods Using RNA Interference of Cdpk-Like Genes for

Control of Nematodes, US Patent No. 2010017910. http://www.google.es/patents/US20100017910

[5] Atkinson, N.J. and Urwin, P.E. (2012). The interaction of plant biotic and abiotic stresses: from

genes to the field. Journal of Experimental Botany, 63 (10): 1–21.

[6] Bakhetia, M., Charlton, W.L., & Urwin, P.E. (2005). RNA interference and plant parasitic

nematodes. Trends Plant Sci., 10(8): 362-367. http://dx.doi.org/10.1016/j.tplants.2005.06.007.

[7] Baum, T.J., Hussey, R.S., and Davis, E.L. (2007). Root-Knot and Cyst Nematode Parasitism

Genes: The Molecular Basis of Plant Parasitism. Genet. Engin., 28: 17–43.

[8] Bleve-Zacheo, T., Melillo, M.T., & Castagnone-Sereno, P. (2007). The Contribution of

Biotechnology to Root-Knot Nematode Control in Tomato Plants. Pest Technology, Global Science

Books, 1(1): 1-16. http://globalsciencebooks.info/JournalsSup/images/SF/PT_1(1)1-16.pdf.

[9] Block, A., Schmelz, E., O’Donnell, P.J., Jones, J.B., and Klee, H.J. (2005). Systemic Acquired

Tolerance to Virulent Bacterial Pathogens in Tomato. Plant Physiology, 138: 1481–1490.

[10] Bonner, W. M., Laskey, R. A. (1974). A film detection method for tritium-labelled proteins and

nucleic acids in polyacrylamide gels. Eur. J. Biochem., 46(1): 83–88.

[11] Bradley, J.D., Chiapelli, B., Gao, B., McCarter, J.P., Verbsky, M.L., and Williams, D.J. (2009).

Methods and Compositions for Root Knot Nematode Control, WO Patent No. 2009126896.

http://www.lens.org/lens/patent/WO_2009_126896_A2.

[12] Burg, R.W., Miller, B.M., Baker, E.E., Birnbaum, J., Currie, S.A., Hartman, R., Kong,

Y.L., Monaghan, R.L., Olson, G., Putter, I., Tunac, J.B., Wallick, H., Stapley, E.O., Oiwa, R., Omura,

S. (1979). Avermectins, new family of potent antіhelmintic agents: producing organism and

fermentation. Antimicrob. Agents Chemother., 15 (3): 361-367.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC352666/pdf/aac00273-0045.pdf

[13] Curto, G. (2008). Sustainable methods for management of cyst nematodes. In: Ciancio A. &

Mukerji K.G. (Eds.), Integrated Management and Biocontrol of Vegetable and Grain Crops

Nematodes. Springer. pp. 221–237.

Advances in Bioscience and Bioengineering 87

[14] Chen, Z.X., Chen, S.Y. and Dickson, D.W. (2004). Nematology: Advances and Perspectives. Vol. 1:

Nematode Morphology, Physiology and Ecology. CABI Publishing. 656 p.

http://onlinelibrary.wiley.com/doi/10.1111/j.1365-3059.2005.01180.x/pdf

[15] Compant, S., Duffy, B., Nowak, J., Clément, C., Barka, E. A. (2005). Use of plant growth-

promoting bacteria for biocontrol of plant diseases: principles, mechanisms of action, and future

prospects. Appl. Environ. Microbiol., 71, (9): 4951-4959.

[16] Denancé, N., Sánchez-Vallet, A., Goffner, D., and Molina, A. (2013). Disease resistance or

growth: the role of plant hormones in balancing immune responses and fitness costs. Frontiers in

Plant Science. Plant Cell Biology, 4 (Article 155): 1–12.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3662895/

[17] Dinh, P.T.Y., Zhang, L., Brown, C.R., and Elling, A.A. (2014). Plant-mediated RNA interference

of effector gene Mc16D10L confers resistance against Meloidogyne chitwoodi in diverse genetic

backgrounds of potato and reduces pathogenicity of nematode offspring. Nematology, 1-14. DOI:

10.1163/15685411-00002796. (In Press.)

[18] Doran, J.W., Sarrantonio, M., Liebig, M. (1996). Soil health and sustainability. In: Sparks D.L.

(Eds.), Advances in Agronomy. Academic Press. San Diego. 56, pp. 1–54.

[19] Downing, H.F., DeLorenzo, M.E., Fulton, M.H., Scott, G.I., Madden, C.J., Kucklick, J.R. (2004).

Effects of the agricultural pesticides atrazine, chlorothalonil, and endosulfan on South Florida

microbial assemblages. Ecotoxicology, 13 (3): 245-260.

[20] Ellis, J.G. and Jones, D.A. (2003). Plant Disease Resistance Genes. In: R.A.B. Ezekowitz and J.A.

Hoffmann (Eds.). From: Infectious Disease: Innate Immunity. Humana Press Inc. Totowa, N.J. XI.

pp. 27–45.

[21] Erb, M., Meldau, S. and Howe, G.A. (2012). Special Issue: Specificity of plant–enemy

interactions. Role of phytohormones in insect-specific plant reactions. Trends in Plant Science, 17(5):

250–259.

[22] Fabian, M.R., Sonenberg, N., & Filipowicz, W. (2010). Regulation of mRNA Translation and

Stability by microRNAs. Annu. Rev. Biochem., 79: 351-79. http://dx.doi.org/10.1146/annurev-

biochem-060308-103103

[23] Fanellia E., Vitob M.D. , Jonesc J.T., Giorgia C.D. (2005). Analysis of chitin synthase function in

a plant parasitic nematode, Meloidogyne artiellia, using RNAi. Gene, 349: 87–95.

[24] Favery, B., Abad P., and Caillaud M.C. (2008). Method for Increasing the Resistance of a Plant

to Endoparasitic Nematodes, WO Patent No. 2008139334.

[25] Felippes, F. F., Wang, J., Weige, D. (2012). MIGS: miRNA-induced gene silencing. Plant J., 70:

541–547.

[26] Filipowicz, W., Jaskiewicz, L., Kolb, F.A., & Pillai, R. S. (2005). Post-transcriptional gene

silencing by siRNAs and miRNAs. Curr. Opin. Struct. Biol., 15: 331-341.

88 Advances in Bioscience and Bioengineering

http://dx.doi.org/10.1016/j.sbi.2005.05.006

[27] Fire, A., Xu, S., Momtogomery, M.K, Kostas, S.A, Driver, S.E, Mello, C.C. (1998). Potent and

specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, 391: 806–

811.

[28] Fisher, M.H. (1990). Recent advances in avermectin sesearch. Pure & Apple. Chem., 62 (7):

1231-1240.

[29] Fuller, V.L., Lilley, C.J., and Urwin, P.E. (2008). Nematode resistance. New Phytol., 180: 27-44.

http://dx.doi.org/10.1111/j.1469-8137.2008.02508.x.

[30] Gachomo, E.W., Shonukan, O.O.,

and Kotchoni, S.O. (2003). The molecular initiation and

subsequent acquisition of disease resistance in plants. African Journal of Biotechnology, 2(2): 26–32.

[31] Gerhardson, B. (2002). Biological substitutes for pesticides. Trends in Biotechnol., 20: 338-343.

[32] Gheysen, G., & Vanholme, B. (2006). RNAi from plants to nematodes. Trends in Biotechnol.,

25(3): 89-92. http://dx.doi.org/10.1016/j.tibtech.2007.01.007.

[33] Giron, D., Frago, E., Glevarec, G., Pieterse, C.M.J., and Dicke, M. (2013). PLANT-MICROBE-

INSECT INTERACTIONS. Cytokinins as key regulators in plant–microbe–insect interactions:

connecting plant growth and defence. Functional Ecology: 1–11.

[34] Groot, A.T., and Dicke, M. (2002). Insect-resistant transgenic plants in a multi-trophic context.

The Plant Journal, 31(4): 387–406.

[35] Gupta, S., Dikshit, A.K. (2010). Biopesticides: An eco-friendly approach for pest control. Journal

of Biopesticides 3, (1 Special Issue): 186–188.

http://www.jbiopest.com/users/lw8/efiles/suman_gupta_v31.pdf

[36] Hamilton, A.J., Baulcombe, D.C. (1999). A species of small antisense RNA in posttranscriptional

gene silencing in plants. Science, 286: 950–952.

[37] Hammilton, A., Voinnet, O., Chapell, L., Baulcombe, D. (2002). Two classes of short interfering

RNA in RNA silencing. EMBO J., 21: 4671–4679.

[38] Heikal, A.M., Solliman, M.E.-D., Aboul-Enein, A.A., Ahmed, F.A., Abbas, A., Taha, H.S., and

Handa, A.K. (2008). Tfg-Mi, a root-knot nematode resistance gene from fenugreek (Trigonella

foenum-graecum) confers nematode resistance in tomato. Arab J. Biotech., 11(2): 139-158.

http://www.acgssr.org/BioTechnology/V112July2008/Full_Paper/013.pdf

[39] Heil, M., and Bostock, R.M. (2002). Induced Systemic Resistance (ISR) Against Pathogens in the

Context of Induced Plant Defences. Annals of Botany, 89: 503–512.

[40] Hewezi, T., Howe, P., Mairer, T.R., & Baum, T.J. (2008). Arabidopsis small RNAs and their

targets during cyst nematode parasitism. Mol. Plant-Microbe Interact., 21: 1622-1634.

http://dx.doi.org/10.1094/MPMI-21-12-1622.

[41] Hirai, S., Kodama, H. (2008). RNAi Vectors for Manipulation of Gene Expression in Higher

Plants. The Open Plant Science Journal, 2: 21–30.

Advances in Bioscience and Bioengineering 89

[42] Ithal, N., Recknor, J., Nettleton, D., Hearne, L., Maier, T., Baum, T. J., & Mitchum, M. G. (2007).

Parallel genome wide expression profiling of host and pathogen during soybean cyst nematode

infection of soybean. Mol. Plant–Microbe Interact., 20(3): 293-305. http://dx.doi.org/10.1094/MPMI-

20-3-0293.

[43] Iutynska, G.O. (2012). Elaboration of natural polyfunctional preparations with antiparasitic

and biostimulating properties for plant growing. Mikrobiol. J., 74 (4): 3–12 (In Ukr.).

[44] Iutynska, G.A., Tsygankova, V.A., Biliavska, L.A., Kozyritskaja, V.Е. (2013). Impact of new

biopreparations on the basis of Avercom on plant development and productivity and expression of

genes of si/miRNA synthesis. J. Microbiology and Biotechnology, 1: 48–58 (In Ukr.).

[45] Jagtap, U.B., Gurav, R.G., Bapat, V.A. (2011). Role of RNA interference in plant improvement,

Naturwissenschaften, 98: 473–92.

[46] Jammes, F., Lecomte, F., Almeida-Engler, J., Bitton, F., Martin-Magniette, M.-L., Renou, J.P.,

Abad, P., and Favery, B. (2005). Genome-wide expression profiling of the host response to root-knot

nematode infection in Arabidopsis. Plant J., 44: 447–458. doi: 10.1111/j.1365-313X.2005.02532.x.

http://onlinelibrary.wiley.com/doi/10.1111/j.1365-313X.2005.02532.x/full

[47] Jia, C., Zhang, L., Liu, L., Wang, J., Li, C., Wang, Q. (2013). Multiple phytohormone signalling

pathways modulate susceptibility of tomato plants to Alternaria alternata f. sp. Lycopersici. Journal

of Experimental Botany, 64 (2): 637–650.

[48] Jiarui, Li J., Todd, T.C., Lee, J., and Trick, H.N. (2011). Biotechnological application of

functional genomics towards plant-parasitic nematode control. Plant Biotechnology Journal, 9: 936–

944. doi: 10.1111/j.1467-7652.2011.00601.x

[49] Jouanin, L., Bonadeґ-Bottino, M., Girard, C., Morrot, G., Giband, M. (1998). Transgenic plants

for insect resistance. Plant Science, 131: 1–11.

[50] Júnior, J.D.A.S., Coelho, R.R., Lourenço, I.T., Fragoso, R.R., Viana, A.A.B., Macedo, L.L.P.,

Silva, M.C.M., Carneiro, R.M.G., Engler, G., Almeida-Engler, J., Grossi-de-Sa, M.F. (2013).

Knocking-Down Meloidogyne incognita Proteases by Plant-Delivered dsRNA Has Negative

Pleiotropic Effect on Nematode Vigor. PLOS ONE, 8 (Iss.12): e85364

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0085364

[51] Karakas, M. RNA interference in plant parasitic nematodes. (2008). African Journal of

Biotechnology, 7 (15): 2530-2534. http://www.ajol.info/index.php/ajb/article/viewFile/59073/47382

[52] Kästner, J., Knorre, D., Himanshu, H., Erb, M., Baldwin, I.T., Meldau, S. (2014). Salicylic Acid,

a Plant Defense Hormone, Is Specifically Secreted by a Molluscan Herbivore. PLOS ONE, 9 (Iss.1),

e86500.

http://pubman.mpdl.mpg.de/pubman/item/escidoc:1878033/component/escidoc:1909822/ITB451.pdf

[53] Katiyar-Agarwal, S., Gao, S., Vivian-Smith, A., and Jin, H. (2007). A novel class of bacteria-

induced small RNAs in Arabidopsis. GENES & DEVELOPMENT, 21: 3123–3134.

90 Advances in Bioscience and Bioengineering

[54] Katiyar-Agarwal, S. and Jin, H. (2010). Role of Small RNAs in Host-Microbe Interactions. Annu

Rev Phytopathol, 48: 225–246. doi:10.1146/annurev-phyto-073009-114457.

[55] Katiyar-Agarwal, S., Morgan, R., Dahlbeck, D., Borsani, O., Villegas, A., Zhu, J.K., Jin, H.L.

(2006). A pathogen-inducible endogenous siRNA in plant immunity. Proc Natl Acad Sci USA, 103(47):

18002-18007. http://dx.doi.org/10.1073/pnas.0608258103.

[56] Keresa, S., Grdisa, M., Baric, M., Barcic, J.I., Marchetti, S. (2008). TRANSGENIC PLANTS

EXPRESSING INSECT RESISTANCE GENES. Sjemenarstvo, 25(2): 139–153.

http://www.mendeley.com/research/transgenic-plants-insect-resistance/

[57] Kerry, B. R. (1987). Biological control. In: Brown R. H. & Kerry B. R. (Eds). Biological control of

nematodes: prospects and opportunities. Principles and practice of nematode control in crops. Sydney,

Australia: Acad. Press. pp. 233-263.

[58] Khan, W., Rayirath, U.P., Subramanian, S., Jithesh, M.N., Rayorath, P., Hodges, D.M, Critchley,

A.T., Craigie, J.S., Norrie, J., Prithviraj, B. (2009). Seaweed Extracts as Biostimulants of Plant

Growth and Development. J. Plant Growth Regul., 28: 386-399.

http://faculty.ksu.edu.sa/76158/documents/jpgr%20review.pdf.

[59] Klink, V.P., and Matthews, B.F. (2009). Emerging Approaches to Broaden Resistance of Soybean

to Soybean Cyst Nematode as Supported by Gene Expression Studies. Plant Physiology, 151: 1017–

1022. http://www.plantphysiol.org/content/151/3/1017.full.pdf+html

[60] Li, X., Wang, X., Zhang, S., Liu, D., Duan, Y., & Dong, W. (2012). Identification of Soybean

MicroRNAs Involved in Soybean Cyst Nematode Infection by Deep Sequencing. PloS ONE, 7(6):

e39650. http://dx.doi.org/10.1371/journal.pone.0039650.

[61] Li, Y., Zhang, Q.Q., Zhang, J., Wu, L., Qi, Y., and Zhou, J.-M. (2010). Identification of

MicroRNAs Involved in Pathogen-Associated Molecular Pattern-Triggered Plant Innate Immunity.

Plant Physiology, 152: 2222–2231.

[62] Liao, Y.C., Li, H.P., Zhao, C.S., Yao, M.J., Zhang, J.B., and Liu, J.L. (2006). Plantibodies: a novel

strategy to create pathogen-resistant Plants. Biotechnology and Genetic Engineering Reviews, 23:

253-271. http://dx.doi.org/10.1080/02648725.2006.10648087.

[63] Liu, R., and Zhu, J.-K. (2014). Non-coding RNAs as potent tools for crop improvement. National

Science Review: 1–4. doi: 10.1093/nsr/nwu006.

http://nsr.oxfordjournals. org/content/early/ 2014/05/14/nsr.nwu006.full.

[64] Llave, C., Kasschau, K.D., Rector, M.A., Carrington, J.C. (2002). Endogenous and silencing-

associated small RNAs in plants. Plant Cell, 14: 1605–1619.

[65] Lуpez, C., Perez-Quintero, B. L., and Szurek, B. (2012). Small Non-Coding RNAs in Plant

Immunity. In: N. K. Dha and S.C. Sahu (Eds.). Agricultural and Biological Sciences "Plant Science".

pp. 169-190.

http://www.intechopen.com/books/plant-science/small-non-coding-rnas-in-plant-immunity.

Advances in Bioscience and Bioengineering 91

[66] Mang, H.G., Laluk, K.A., Parsons, E.P., Kosma, D.K., Cooper, B.R., Park, H.C., AbuQamar, S.,

Boccongelli, C., Miyazaki, S., Consiglio, F., Chilosi, G., Bohnert, H.J., Bressan, R.A., Mengiste, T.,

and Jenks, M.A. (2009). The Arabidopsis RESURRECTION 1 Gene Regulates a Novel Antagonistic

Interaction in Plant Defense to Biotrophs and Necrotrophs. Plant Physiology, 151: 290–305.

[67] Marcus, A., Efron, D., Week, D.P. (1974). The wheat embryo cell free system. Meth. Enzymol.,

30: 749–754.

[68] Molecular plant-microbe interactions. (2009). In: K. Bouarab, N. Brisson and F. Daayf (Eds.).

CAB International. 340 p.

[69] Naseem, M., Dandekar, T. (2012). The Role of Auxin-Cytokinin Antagonism in Plant- Pathogen

Interactions. PLOS Pathogens, 8 (Iss. 11): e1003026.

http://www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.1003026

[70] Nelson, D., Beattie, K., McCollum, G., Martin, T., Sharma, S., Rao, J.R. (2014). Performance of

Natural Antagonists and Commercial Microbiocides towards in Vitro Suppression of Flower Bed

Soil-Borne Fusarium oxysporum. Advances in Microbiology, 4: 151-159.

http://dx.doi.org/10.4236/aim.2014.43020.

[71] Oka, Y. (2010). Mechanisms of nematode suppression by organic soil amendments. Appl. Soil

Ecol., 44: 101–115.

[72] Olaiya, C.O., Gbadegesin, M. A., & Nwauzoma, A. B. (2013). Bioregulators as tools for plant

growth, development, defence and improvement. African Journal of Biotechnology, 12(32): 4987-4999.

[73] Osterman, L.A. (1981). Methods of research of proteins and nucleic acids. Мoscow: Nauka, 250 p.

(In Russ.)

[74] Padmanabhan, Ch., Zhang, X., & Jin, H. (2009). Host small RNAs are big contributors to plant

innate immunity. Curr. Opin. in Plant Biol., 12: 465-472. http://dx.doi.org/ 10.1016/j.pbi.2009.06.005.

[75] Patel, N., Hamamaouch, N., Li, C., Hewezi, T., Hussey, R.S. (2010). A nematode effector protein

similar to annexins in host plants. J. Exp. Bot., 61: 235-248.

[76] Pell, M., Stenström, J., Granhall, U. (2006) Soil respiration. In: Bloem J., Hopkins D.W.,

Benedetti A. (Eds), Microbiological Methods for Assessing Soil Qality. CAB International. pp. 117-

126.

[77] Pieterse, C.M.J., Leon-Reyes, A., Van der Ent, S., and Van Wees, S.C.M. (2009). Networking by

small-molecule hormones in plant immunity. Nature chemical biology, 5(5): 308–316.

[78] Pieterse, C. M.J., Van der Does, D., Zamioudis, C., Leon-Reyes, A., and Van Wees, S. C.M.

(2012). Hormonal Modulation of Plant Immunity. Annual Review of Cell and Developmental Biology,

28: 489-521. DOI: 10.1146/annurev-cellbio-092910-154055.

http://www.annualreviews. org/doi/pdf/10.1146/annurev-cellbio-092910-154055

[79] Promega Protocols and Applications Guide. (1991). 2nd Edn.,. USA: Promega Corporation. 422 p.

92 Advances in Bioscience and Bioengineering

[80] Quentin, M., Abad, P., and Favery, B. (2013). Plant parasitic nematode effectors target host

defense and nuclear functions to establish feeding cells. Front. Plant Sci., 4 (Article 53): 1–7. doi:

10.3389/fpls.2013.00053.

[81] Rivoal, R., Cool, R. (1993). Nematode pests of cereals. In: Svans K., Trudgill D.I. and Webster

J.M. (Eds). Plant parasitic nematodes in temperate agriculture. Oxford: CAB International. pp. 259-

303.

[82] Rosso, M.N., Dubrana, M.P., Cimbolini, N., Jaubert, S., and Abad, P. (2005). Application of RNA

interference to root-knot nematode genes encoding esophageal gland proteins. Mol. Plant Microbe

Interact., 18: 615–620.

[83] Sambrook, J., Fritsch, E.F., Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual. 2nd

Ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press., 480 р.

[84] Senthil-Kumar, M., and Mysore, K.S. (2010). RNAi in Plants: Recent Developments and

Applications in Agriculture. In: Catalano A.J. (Ed.), Gene Silencing: Theory, Techniques and

Applications. Nova Science Publishers, Inc. pp.183–199.

[85] Shamray, S. N. (2003). Plant Resistance Genes: Molecular and Genetic Organisation, Function

and Evolution. Journal of General Biology, 64(3): 195-214 (In Rus.).

http://dspace.univer.kharkov.ua/bitstream/123456789/5006/2/shamray.pdf

[86] Shen, D., Suhrkamp, I., Wang, Y., Liu, S., Menkhaus, J., Verreet, J.-A., Fan, L. and Cai, D.

(2014). Identification and characterization of microRNAs in oilseed rape (Brassica napus) responsive

to infection with the pathogenic fungus Verticillium longisporum using Brassica AA (Brassica rapa)

and CC (Brassica oleracea) as reference genomes. New Phytologist. doi: 10.1111/nph.12934.

http://onlinelibrary.wiley.com/doi/10.1111/nph.12934/abstract

[87] Sindhu, A.S., Maier, T.R., Mitchum, M.G., Hussey, R.S., Davis, E.L., and Baum, T.J. (2009).

Effective and specific in planta RNAi in cyst nematodes: expression interference of four parasitism

genes reduces parasitic success. Journal of Experimental Botany, 60(1): 315–324.

doi:10.1093/jxb/ern289

http://jxb.oxfordjournals.org/content/60/1/315.full

[88] Southey, J.F. (1986). Laboratory methods for work with plant and soil nematodes. Her Majesty`s

Stationery Office, 202 р.

[89] Spoel, S.H., & Dong, X. (2012). How do plants achieve immunity? Defence without specialized

immune cells. Nature Reviews, Immunology, 12: 89-100. http://dx.doi.org/10.1038/nri3141

[90] Statistical Methods in Biology. (1995). Bailey N.T.J. (Ed.). 3rd Edn., Cambridge University Press.

255 p.

[91] Statistical Methods in Molecular Biology. (2010). In H. Bang, X. K. Zhou, H. L. Epps, M.

Mazumdar (Eds.). Series: Methods in Molecular Biology. Humana Press, XIII (620). 636 p.

Advances in Bioscience and Bioengineering 93

[92] Stefanovska, T., Pidlisnyuk, V. (2009).Challenges to grow oilseed rape Brassica napus in sugar

beet rotations. Commun. Agricult. Appl. Biol. Sci., 74 (2): 573−579.

[93] Stevens, M., May, M. J. (2010). Pests, diseases and weeds review 2009. British Sugar Beet

Review, 78(1): 7–10.

[94] Studham, M.E., and MacIntosh, G.C. (2012). Phytohormone signaling pathway analysis method

for comparing hormone responses in plant-pest interactions. BMC Research Notes, 5 (392): 1-7.

[95] Tamilarasan, S., and Rajam, M.V. (2013). Engineering Crop Plants for Nematode Resistance

through Host-Derived RNA Interference. Cell Dev Biol., 2: 114. doi:10.4172/2168-9296.1000114

http://omicsgroup.org/journals/2168-9296/2168-9296-2-114.php?aid=16585

[96] Tang, G., Reinhart, B. J., Bartel, D. P., & Zamore, P. D. (2003). A biochemical framework for

RNA silencing in plants. Genes & Development, 17: 49-63. http://dx.doi.org/10.1101/ gad.1048103.

[97] Thakur, N., Upadhyay, S.K., Verma, P.C., Chandrashekar, K., Tuli, R., Singh, P.K. (2014).

Enhanced Whitefly Resistance in Transgenic Tobacco Plants Expressing Double Stranded RNA of

v-ATPase A Gene. PLOS ONE 9 (Iss. 3): e87235. DOI: 10.1371/journal.pone.0087235.

http://www.plosone.org/article/info%253Adoi%252F10.1371%252Fjournal.pone.0087235

[98] Tsygankova, V.A., Andrusevich, Ya. V., Babayants, O. V., Ponomarenko, S.P., Medkov, A. I., &

Galkin, A. P. (2013). Stimulation of plant immune protection against pathogenic fungi, pests and

nematodes with growth regulators. Physiol. and biochem. cultivated plants, 45(2): 138-147. (In Ukr.).

https://www.academia.edu/4562192/Stimulation_of_plant_immune_protection_against_phytopathoge

ns

[99] Tsygankova, V.A. Andrusevich, Ya.V., Beljavskaja, L.A., Kozyritskaja, V.Е., Iutinskaja, H.A.,

Galkin, A.P., Galagan, T.O., Boltovskaya, E.V. (2012). Growth stimulating, fungicidal and

nematicidal properties of new microbial substances and their impact on si/miRNA synthesis in plant

cells. Mikrobiol. J., 74(6): 36-45 (In Ukr.).

http://www.imv.kiev.ua/images/doc/MBJ/ 2012/MB_6_2012.pdf

[100] Tsygankova, V.A., Andrusevich, Ya.V., Ponomarenko, S.P., Galkin, A.P., & Blume, Ya.B. (2012).

Isolation and Amplification of cDNA from the Conserved Region of the Nematode Heterodera

schachtii 8H07 Gene with a Close Similarity to Its Homolog in Rape Plants. Cytology and Genetics,

46(6): 335-341.

http://dx.doi.org/10.3103/S0095452712060114

https://www.academia.edu/4874047/Cloning_of_antinematodic_Si-miRNA

[101] Tsygankova, V.A., Iutynska, G.A., Galkin, A.P., Blume, Ya.B. (2014). Impact of New Natural

Biostimulants on Increasing Synthesis in Plant Cells of Small Regulatory si/miRNA with High Anti-

Nematodic Activity. Internat. J. Biol., 6(1): 48-64. doi:10.5539/ijb.v6n1p48.

http://dx.doi.org/10.5539/ijb.v6n1p48

94 Advances in Bioscience and Bioengineering

[102] Tsygankova, V.A., Роnоmаrenko, S.P., Hrytsaenko, Z.M. (2012). Increase of plant resistance to

diseases, pests and stresses with new biostimulants. Acta Horticulturae (Strasburg, France), 1009:

225–233. https://www.academia.edu/5192515/New_natural_biostimulants_as_effective

_bioprotectors_from_phytopathogens

[103] Tsygankova, V. A., Stefanovska, T.R., Galkin, A.P., Ponomarenko, S.P., & Blume, Ya. B. (2012).

Inducing effect of PGRs on small regulatory si/miRNA in resistance to sugar beet cyst nematode.

Comm. Appl. Biol. Sci. Ghent University (Belgium), 77(4): 779-788.

http://www.researchgate.net/profile/V_Tsygankova/publication/252323155Inducing_effect_of_PGRs_o

...

[104] Tsygankova, V.A., Yemets, A.I., Iutynska, H.O., Beljavska, L.O., Galkin, A.P., Blume, Ya.B.

(2013). Increasing the resistance of rape plants to the parasitic nematode Heterodera schachtii using

RNAi technology. Cytology and Genetics, 47(4): 222-230.

http://link.springer.com/article/10.3103%2FS0095452713040105

https://www.academia.edu/4344993/Increasing_of_plant_resistance_to_nematodes_using_RNAi-

technology

[105] Tuschl, T., Zamore, P. D., Lehmann, R., Bartel, D. P., & Sharp, P. A. (1999). Targeted mRNA

degradation by double-stranded RNA in vitro. Genes and development, 13: 3191-3197.

http://dx.doi.org/10.1101/gad.13.24.3191.

[106] Vavrina, C.S., Roberts, P.D., Kokalis-Burelle, N., & Ontermaa, E.O. (2004). Greenhouse

Screening of Commercial Products Marketed as Systemic Resistance and Plant Growth Promotion

Inducers. HortScience, 39(2): 433-437. http://hortsci.ashspublications.org/content/39/2/433.abstract

[107] Vaucheret, H. (2006). Post-transcriptional small RNA pathways in plants: mechanisms and

regulations, Genes Dev, 20: 759-771. doi:10.1101/gad.1410506

[108] Wani, S.H., Sanghera, G.S., Singh, N.B. (2010). Biotechnology and Plant Disease Control-Role

of RNA Interference. American Journal of Plant Sciences, 1: 55-68. doi:10.4236/ajps.2010.12008

(http://www.SciRP.org/journal/ajps).

[109] Wiig, A. (2010). Compositions and Methods using RNA Interference of Opr3-Like Gene for

Control of Nematodes, US Patent No. 2010115660.

[110] Wiig, A. (2010). Compositions and Methods using RNA Interference Targeting Mthfr-Like

Genes for Control of Nematodes, US Patent No. 2010107276.

[111] Williamson, V.M. (1999). Plant nematode resistance genes. Curr. Opin. in Plant Biol., 2: 327–

331.

[112] Yang, L., Huang, H. (2014). Roles of small RNAs in plant disease resistance. J. Integr Plant

Biol., XX, XX–XX.

http://www.jipb.net/pubsoft/content/2/10.1111/jipb.12200.pdf. doi: [10.1111/jipb.12200].

Advances in Bioscience and Bioengineering 95

[113] Yu, H., Kumar, P.P. (2003). Post-transcriptional gene silencing in plants by RNA. Plant Cell

Rep., 22: 167–174.

[114] Zhang, B., Wang, Q., & Pan, X. (2007). MicroRNAs and their regulatory roles in animals and

plant. J. Cell. Physiol., 210: 279-289. http://dx.doi.org/10.1002/jcp.20869.

[115] Zhang, W., Gao, Sh., Zhou, X., Xia, J., Chellappan, P., Zhou, X., Zhang, X., Jin, H. (2010).

Multiple distinct small RNAs originate from the same microRNA precursors. Genome Biology, 11:

R81. http://genomebiology.com/2010/11/8/R81.

[116] Zhang, W., Gao, Sh., Zhou, X., Chellappan, P., Chen, Z., Zhou, X., & Zhang, X. (2011). Bacteria-

responsive microRNAs regulate plant innate immunity by modulating plant hormone networks.

Plant Mol. Biol., 75: 93-105. http://dx.doi.org/10.1007/s11103-010-9710-8

[117] Zuberer, D.A. (1994). Recovery and enumeration of viable bacteria. In: Weaver R.W. et al. (Eds).

Methods of Soil Analysis. Part 2, Microbiological and Biochemical Properties. Number 5 in Soil

Science Society of America Book Series. Soil Science Society of America. Inc. Madison. Wisconsin.

USA. pp. 119-144.

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Table 1. Conditions of treatment of seeds of wild spring rape plant of

Kalinivsky cultivar by nematicidic PR/PGP inducers

Nematicidic PR/PGP

inducers

Presowing treatment,

of 1 t of seeds

Treatment during of vegetation period

at the phase of bushing out on 1

hectare of plant sowing

Control 10 L of water 200 - 250 L of water

Avercom 1 ml/10 L of water 2-2,5 ml/200 - 250 L of water

Avercom nova-2 0,5 ml/10 L of water 1-1,5 ml/ 200 - 250 L of water

Confidor Maxi 4 kg/10 L of water 0,06 kg/200 - 250 L of water

Table 2. Biometric indexes of control and experimental wild spring rape

plants of Kalinivsky cultivar

Experience variant Average height of plants at the flowering phase

Cm ± % in relation to control

Control (water) 95±2,8* −

Confidor Maxi

(0,06 kg/per 1 ha on 200-250 L of water) 107±3,4** +12,6**

Avercom

(2-2,5 ml/per 1 ha on 200-250 L of water) 109±2,8** +14,7**

Avercom nova-2

(1-1,5 ml/per 1 ha on 200-250 L of water) 103±3,0** +8,4**

Note. **Significant differences from control values*, p < 0.05, n =3, (-) decreasing; (+)

- increasing

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Table 3. Amount of nematodes on roots of wild spring rape plants of

Kalinivsky cultivar

Experience

variant

Average amount of nematodes at the vegetation period

(in 100 g of plant roots)

Plant parasitic nematodes Mycohelmints Saprophytic nematodes

Control (water) 390±13.1* 190±19.2* 580±16.0*

Avercom 40±4.2** 23±3.2** 30±3.7**

Avercom nova-2 240±12.3** 80±6.0** 600±16.3**

Confidor Maxi 100±6.7** 70±5.6** 150±8.2**

Note. **Significant differences from control values*, p < 0.05, n =3, (-) decreasing; (+)

- increasing

Table 4. Productivity of wild spring rape plants of Kalinivsky cultivar

Experience variant

Productivity of grain yield, pounds/ha Increase of

yield, % to

control Frequency

Average

value

Control (water) 1415.3 1494.7 1402.1 1256.6 1391.1 100

Avercom 3822.8 4096.1 4512.8 4239.4 4166.7 199.5

Avercom nova-2 3238.5 3467.8 3891.1 3584.6 3547.2 154.9

Confidor Maxi 2784.4 3789.7 3573.6 3000.4 3287.0 136.3

Note: LSD05 (the Least Substantial Difference) =679.0

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