Impact of New Microbial PR/PGP Inducers on Increase of Resistance to Parasitic Nematode of Wild and RNAi Transgenic Rape Plants

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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
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   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 1 Department 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 2 Department 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 3 Department Genomics and Molecular Biotechnology, Institute of Food Biotechnology and Genomics, National Academy of Sciences of Ukraine, 04123, 2a, Osypovskogo str., Kyiv, Ukraine 4 National 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 srcin (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. оleife ra 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  –   
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