climate change

Adaptation to Climate Change

in the Baltic Sea Region:

Contributions from Plant

and Microbial Biotechnology

MIKKELI, FINLAND

July 12-17, 2010

2

Citation:

Adaptation to Climate Change in the Baltic Sea Region: Contributions from Plant and

Microbial Biotechnology

Valkonen, J.P.T., Weinheimer, I., Nupponen, S., Santala, J., Lojkowska, E. & Tikhonovich, I. (eds.)

Published by Department of Agricultural Sciences, University of Helsinki, Helsinki, Finland

ISBN 978-952-10-6368-8

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The joint 5th Postgraduate Course and Minisymposium of AB-RMS, 16th Biotechnology Summer School of University of Gdansk, and

2nd Workshop of PAS and RAAS on Plant Molecular Biotechnology

PhD Course and Minisymposium

Adaptation to Climate Change in the Baltic Sea Region: Contributions from Plant and

Microbial Biotechnology

July 12-17, 2010

Mikkeli University Consortium Finland

Local organizer:

University of Helsinki

Department of Agricultural Sciences

Helsinki, Finland

Co-organizers:

University of Gdansk, Poland

The Polish Academy of Science (PAS)

The Russian Academy of Agricultural Sciences (RAAS)

Ruralia Institute, Mikkeli Unit, University of Helsinki

MTT AgriFood Research Finland, Karila research station, Mikkeli

The town of Mikkeli

The Regional Council of Etelä-Savo

Organizing committee:

Chairperson

Academy Prof. Jari Valkonen

Department of Agricultural Sciences

PO Box 27 (Latokartanonkaari 7)

FI-00014 University of Helsinki, FINLAND

Tel: +358-40-7432479

Fax: +358-9-1915 8727

e-mail: jari.valkonen~at~helsinki.fi

Prof. Ewa Lojkowska


e-mail: lojkowsk~at~biotech.ug.gda.pl

Prof. Andrzej Legocki

The Polish Academy of Science (PAS), Warsaw, Poland

e-mail: Andrzej.Legocki~at~pan.pl

Prof. Igor Tikhonovich

All-Russia Institute of Agricultural Microbiology (ARRIAM),

St. Petersburg, Russia

e-mail: contact~at~arriam.spb.ru

4

Accomodation & Directions

walking route to the conference site

driving route to the Suomen nuoriso-opisto

The conference site is in the auditorium at Mikkeli University Campus (Lönnrotinkatu 5).

Accommodation for most of the participants is arranged in Suomen nuoriso-opisto (25

min walking distance to the conference site). Some participants are accommodated in

Cumulus Hotel Mikkeli (5 min walking distance to the conference site).

Supermarket

Suomen

nuoriso-opisto Cumulus Hotel

Ruralia Institute –

Conference Site

Railway Station

Harbour

Bus station

5

PhD Course and Minisymposium

The joint 5th Postgraduate Course and Minisymposium of AB-RMS,

16th Biotechnology Summer School of University of Gdansk, and

2nd Workshop of PAS and RAAS on Plant Molecular Biotechnology

Programme in brief

Morning Afternoon Evening

Monday, July 12 Arrival, registration,

scientific program

Settling in,

get-together

Tuesday, July 13 Scientific program Scientific program Reception

Wednesday, July 14 Field excursion Scientific program Evening free

Thursday, July 15 Scientific program Scientific program Cultural excursion

Friday, July 16 Scientific program Scientific program Social program

Saturday, July 17 Scientific program Departure

Adaptation to Climate Change in the Baltic Sea Region:

Contributions from Plant and Microbial Biotechnology

July 12-17, 2010

Mikkeli, Finland

Secretariat:

Sirpa Nupponen (Course secretary)

Ruralia Institute, Mikkeli

e-mail: sirpa.nupponen~at~helsinki.fi

Johanna Santala (Course assistant)

Dept. Agricultural Sciences, Univ. Helsinki

Tel: +358-40-5361473

e-mail: johanna.aura~at~helsinki.fi

Isabel Weinheimer (Course assistant)

Dept. Agricultural Sciences, Univ. Helsinki

Tel: +358-46-6268449

e-mail: isabel.weinheimer~at~helsinki.fi

Country coordinators:

Anna Ihnatowicz


e-mail:

ihnatowicz~at~biotech.ug.gda.pl

Igor Tikhonovich

ARRIAM, Russia

e-mail: Contact~at~arriam.spb.ru

6

Programme

Monday 12.07. 2010

Time

Activity

Place

13:30 Departure to Mikkeli by bus Helsinki airport, arrivals hall no. 2

16:15 Arrival in Mikkeli, accommodation Suomen Nuoriso-Opisto

(and Mikkeli Hotel Cumulus)

18:00 Opening session Suomen Nuoriso-Opisto

Chair:

18:00

18:30

19:15

Jari Valkonen, Ewa Lojkowska, Igor Tikhonovich Welcome Jari Valkonen, University of Helsinki, Finland Ewa Lojkowska, University of Gdansk, Poland Igor Tikhonovich, All-Russia Research Institute for Agricultural Microbiology, Russia O1: Contributions from plant and microbial biotechnology to mitigation and utilization of the climate change Jari Valkonen, University of Helsinki, Finland O2: Effect of climate change and management on plant-associated microbial communities Gabrielle Berg, Graz University of Technology, Austria

20:00 Get-together Suomen Nuoriso-Opisto

Tuesday 13.07. 2010

08:45 Bus to the University Campus Suomen Nuoriso-Opisto

09:15 Presentation of the Mikkeli University Consortium and Ruralia Institute

Pirjo Siiskonen

Mikkeli University Campus

09:35 Session 1 Mikkeli University Campus

Chair:

09:35

10:20

10:45

Fred Stoddard O3: Rhizobial Molecules that Control Nodulation of Legumes William Broughton, LBMPS, Université Genève, Switzerland Coffee O4: Genetic integration of plants and microbes into the beneficial symbiotic systems Igor Tikhonovich, All-Russia Research Institute for Agricultural Microbiology, Russia

7

11:30

O5: Multipartite symbiotic systems in legumes: genetics, natural history and the use in sustainable agriculture Oksana Shtark, All-Russia Research Institute for Agricultural Microbiology, Russia

12:15 Lunch Mikkeli University Campus

13:15 Setting up poster display Mikkeli University Campus


Chair:

13:30

14:15

14:40

Günter Adam O6: A new group of bacteria from Dickeya genus - an emerging risk factor for potato crops in Poland Ewa Lojkowska, University of Gdansk, Poland O7: An extracellular and a membrane glycopolymer of the bacterium Herbaspirillum seropedicae Z78 Natalya Shishonkova, Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences, Russia Coffee

15:00 Poster session 1 Rooms 1, 2, 3


16:30 Free time Mikkeli

18:30 Departure for informal reception Suomen Nuoriso-Opisto

(and Mikkeli Hotel Cumulus)

19:00 Informal reception by invitation of Mr. Matti Viialainen, Excecutive Director of the Regional Council of Etelä-Savo

Kekkolan Kartano (Kekkola Manor)

22:15 Return to Mikkeli

Wednesday 14.07. 2010



Chair:

09:20

10:05

William Broughton O8: Legumes in the agricultural and food context: Nitrogen fixation, climate change, and local food and feed supplies Frederick Stoddard, Dept. of Agricultural Sciences, University of Helsinki, Finland O9: Sequencing the pea (Pisum sativum L.) regulatory symbiotic genes controlling nodulation Vladimir Zhukov, All-Russia Research Institute for Agricultural Microbiology, Russia

8

10:50

Coffee

11:10 Departure to Karila research station

11:25 Investigations of the field demonstration plots

MTT Karila research station, Mikkeli

13:15 Departure to Mikkeli University Campus

13.30 Lunch Mikkeli University Campus

14:30 Poster session 2 Rooms 1, 2 and 3


16:00 Coffee Mikkeli University Campus


Chair:

16:20

16:50

17:35

18:20

Gabrielle Berg O10: Use of rhizobial Nod factors as biofertilizers for legumes Dominika Kidaj, M. Curie-Sklodowska University, Poland O11: Climate change, salinization and soil microbial community adaptive evolution Evgeny Andronov, All-Russia Research Institute for Agricultural Microbiology, Russia O12: Insight into microbial world – molecular biology research in environmental microbiology Aleksandra Ziembińska, The Silesian University of Technology, Poland Closure of the session

18:20 - Evening free

Thursday 15.7. 2010



Chair:

09:20

10:05

10:30

11:10

Igor Tikhonovich O13: LSD1 is the Darwinian Fitness Regulator in Arabidopsis Stanislaw Karpiński, Warsaw University of Life Sciences, Poland O14: Nitric oxide as a key regulator of induced resistance in potato leaves to Phytophthora infestans Dariusz Abramowski, Poznań University of Life Sciences, Poland O15: A high-efficiency medium-free technology for monocot transformation by genes increasing water deficit, salinization and resistance to plant diseases Mikhail Chumakov, Saratov State University, Russia; RAS, Russia Coffee

9

11:25 Poster session 3 Rooms 1, 2 and 3


12:55 Removal of posters (no. 1-27)



Chair:

14:00

14:30

15:00

Ewa Lojkowska O16: The environmental role of associations of cyanobacterium and actinomycetes – the components of plant symbioses Ekaterina Ivanova, Lomonosov Moscow State University, Russia O17: New approaches for the control of common scab Lea Hiltunen, MTT Agrifood Research Finland, Finland Coffee

16:45 Departure to Lake Saimaa (evening meal on the boat)

Mikkeli Harbour, m/s Jaarli

23:15 Arrival in Mikkeli Suomen Nuoriso-Opisto (Mikkeli Hotel Cumulus)

Friday 16.7. 2010


09:20 Setting up new posters (no. 28-54) Mikkeli University Campus

09:30 Poster session 4 Mikkeli University Campus

11:00 Coffee


Chair:

11:20

12:00

Stanislaw Karpínski O18: Expression analysis of genes involved in scopoletin biosynthesis in Arabidopsis thaliana wild accessions Joanna Siwińska, Intercollegiate Faculty of Biotechnology UG-MUG, Poland O19: Elaboration of a vaccine protecting wild animals from borreliosis Anna Urbanowicz, Polish Academy of Sciences, Poland



Chair:

13:40

14:10

Sigute Kuusiene O20: Distribution, detection and genetic variability of Potato mop-top virus Johanna Santala, University of Helsinki, Finland O21: PCR technologies as tools for diagnostic and identification of plant pathogens Sergey Zavriev, Russian Academy of Sciences, Russia

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14:50

15:20

O22: Application of FTA®Cards to Sample Microbial Plant Pathogens for PCR and RT-PCR Guenter Adam, University of Hamburg, Germany Coffee


17:10 Free time Mikkeli

18:30 Social programme Mikkelipuisto (offered by the town of Mikkeli)

Saturday 17.07. 2010




Chair:

10:45

11:25

Jari Valkonen O23: siRNA sequencing: reconstructing viral genomes from defence molecules Jan Kreuze, International Potato Center (CIP), Peru Closure of the symposium

11:30 Lunch

12:00 Departure

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Abstracts of oral presentations

O1

Contributions from plant and microbial biotechnology to mitigation and utilization of the

climate change

Jari Valkonen

Department of Agricultural Sciences, University of Helsinki, Finland

Predictions of climate change suggest that, in the coming decades, agriculture in the Baltic Sea region

may benefit from milder winters and longer growing seasons. The climate may also allow growing

crops that are not yet playing any important role in the northernmost areas. For example, cultivation of

legumes may become more common and augment increasing protein self-sufficiency in domestic

production of feed and food. On the other hand, the currently grown cultivars may no longer be

suitable and need to be replaced. Milder climate may also pave the way for new pests and pathogens to

invade the region. For example, virus and phytoplasma diseases might become more common if the

changing climate supports their vectors better than the current conditions. Soilborne plant diseases are

also likely to become more severe. These challenges should be overcome by efficient use of

biotechnological approaches that can enhance plant breeding, pathogen diagnostics, control of insect

pests and nematodes, and monitoring environmental changes, e.g., in soil microbial communities.

Biotechnology also allows making use of the latest scientific discoveries, such as those related to

molecular defence and disease resistance, molecules and signalling networks controlling plant-

microbe interactions, and the molecular basis of plant development and their responses to the

environment.

12

O2

Effect of climate change and management on plant-associated microbial communities

G. Berg, A. Krikovtseva, M. Cardinale, H. Müller, C. Zachow

Graz University of Technology, Environmental Biotechnology, Petersgasse 12, 8010 Graz

All plant species are colonised by specific microbial communities [2]. While the structure is of these

communities well-investigated, their functional diversity is only partly understood. Bryophytes as the

phlylogenetically oldest land plants are interesting model plants to study the function [4]. They

harboured specific bacteria especially inside their cells. Beside their effect on plant growth they are

also involved in bryophyte health and carbon storage. To study the impact of climate change on plant-

associated microbial communities in terrestrial ecosystem is an important issue. Europe's soils contain

an estimated 73 to 79 billion tonnes of carbon. Almost 50% of this carbon is sequestered in the peat

bogs consisting of Sphagnum bryophytes.

Soil management practices have a considerable impact on carbon stocks. Agricultural practices can be

improved to minimise carbon losses, at the level of the crop and the crop residues, and by ensuring

that soils are protected against water and rain with a permanent vegetation cover, less intrusive

ploughing techniques and less machinery. Such practices could sequester between 50 and 100 million

tonnes of carbon annually in European soils. To use micro-organisms to control plant pathogens, to

enhance plant growth and protect them against abiotic stress are environmentally friendly alternatives

in agriculture [1]. Although originating from plant-associated microenvironments themselves,

beneficial bacteria, if applied to plants in adequate numbers, may perturb indigenous microbial

populations and the important ecological functions associated therewith. Therefore, possible non-

target effects of the applied antagonists on ecologically important soil-microbes need to be considered.

Methods as well as examples from literature and own research are summarized [3,5]. As the analysed

biological control agents belong to different microbial groups like grampositive (HRO-71) and

gramnegative (HRO-C48, L13-6-12, 3Re2-7, 3Re4-18) bacteria or the ascomycota (G1/8, G3/2) and

originated from different microhabitats like the rhizosphere or the endorhiza, general conclusion could

be drawn from our results. After BCA treatment we did not observe any long-term effect on the plant-

associated microbes in any tested pathosystem. Therefore, no sustainable risks could be seen for the

indigenous micro-organisms. Our new findings may help to improve the development as well as the

registration procedures of future microbial inoculants.

References:

[1] Berg, G. 2009. Plant-microbe interactions promoting plant growth and health: perspectives for

controlled use of microorganisms in agriculture. J. Appl. Microbiol. Biotechnol. 84: 11-8.

[2] Berg, G, & Smalla, K. 2009. Plant species and soil type cooperatively shape the structure and

function of microbial communities in the rhizosphere. FEMS Microbiol. Ecol. 68: 1-13.

[3] Grosch, R., Scherwinski, K., Lottmann, J. and G. Berg. 2007. Fungal antagonists towards the plant

pathogen Rhizoctonia solani: selection, control of the pathogen on host plants and influence on the

indigenous microbial community. Mycol. Res. 110: 1464-1474.

[4] Opelt, K., Chobot, V., Hadacek, F., Schönemann, S., Eberl, L. & Berg, G. 2007. Investigations of

the structure and function of bacterial communities associated with Sphagnum mosses. Environ.

Microbiol. 9: 2795–2809.

[5] Scherwinski, K., Grosch, R. & Berg, G. 2008. Effect of bacterial antagonists on lettuce: active

biocontrol of Rhizoctonia solani and negligible, short-term effect on non-target microbes. FEMS

Microb. Ecol. 64: 106–116.

13

O3

Rhizobial Molecules that Control Nodulation of Legumes

William J. Deakin, Silvia Ardissone, Olivier Schumpp and William J. Broughton.

LBMPS, Université Genève, 30 quai Ernest-Ansermet, 1211 Genève, Switzerland

Rhizobia are soil bacteria with several different lifestyles. In the saprophytic form, rhizobia live in the

vicinity of roots of various plants that fulfil their nutritional needs. Amongst the many compounds

generally secreted, phenolic compounds (especially flavonoids) emanating from legume roots are

sensed by rhizobia. By interacting with the environmental sensor which is either an activator of

transcription in its own right, or is associated with a transcriptional activator, flavonoids up-regulate

genes that are directly involved in nodule formation. Directly or indirectly, these signals are

responsible for the conversion of rhizobia into an endo-symbiotic form. Families of lipo-chito-oligo-

saccharidic nodulation signals (called Nod-factors) are synthesised and released back to the root where

they provoke deformation and curling of the root-hairs. As a result, rhizobia enter roots centripetally

and eventually fill cortical cells with a differentiated form of the bacterium that permits nitrogen-

fixation. Signal exchange between both partners modulates all stages of nodule development. Here, the

current knowledge of how these diverse signals are synthesised, the pathways that regulate their

synthesis and how they are perceived will be discussed.

14

O4

Genetic integration of plants and microbes into the beneficial symbiotic systems

Igor A. Tikhonovich

All-Russia Research Institute for Agricultural Microbiology, Podbelsky Sh. 3, St.-Petersburg,

Pushkin-8, 196608, Russia

Beneficial plant-microbe symbioses are based on the functional or even structural integration of

genetic material from the diverse organisms resulted in formation of the super-organism genomes

(symbiogenomes). Analysis of their development, functions and evolution provides a basis for

fundamental and applied research of the genetic control over different (symbiotic, biocenotic) types of

biotic interactions [1]. The functional integrity of symbiogenomes provides a general strategy for the

symbiosis development which involves: signaling interactions of partners, morphogenesis of novel

structures and the partners‘ metabolic interactions. In N2-fixing legume-rhizobia symbiosis, these

processes are based on the partners‘ feedbacks which are mostly negative at the early stages of

interactions (at the molecular/cellular level these feedbacks have much in common to pathogenic

processes based on the ―gene-for-gene‖ interactions) but at the late stages they are transformed into

the positive feedbacks (based on the formation of joint plant-bacterial C/N metabolic pathways).

Formation of symbiogenome improved the integrity of plant-microbe systems at the structural level

(culminated in the intra-cellular symbiosis and irreversible bacteroid differentiation), biochemical

level (exchange of nutrients and signals) and ecological level (cross-operation of selective pressures in

the partners populations resulted in correlated shifts of frequencies in the beneficially interacting plant

and bacterial genotypes). The symbiogenome analysis provides the genetic and molecular approaches

for dissecting the more complicated, multi-component genetic systems: microbiomes (the integral

genomes of microbial communities residing in an individual plant or animal hosts) and metagenomes

(the integral genomes of microbial communities residing in the defined biotopes). In ecology and

agrobiology, the structural/functional dissection of symbiogenomes is indispensable for optimizing the

interactions between plants and beneficial microbes to be used in the ecosystem management and in

the sustainable crop production in which the hazardous fertilizers and pesticides are replaced by the

environmentally friendly microbial inoculants.

Reference:

1. Tikhonovich I.A., Provorov N.A. (2009). Ann. Appl. Biol. 154: 341-350.

http://www3.interscience.wiley.com/journal/121683128/journal/122376617/issue

15

O5

Multipartite symbiotic systems in legumes: genetics, natural history and the use in sustainable

agriculture

O.Y. Shtark1, A.Y. Borisov

1, V.A. Zhukov

1, G.A. Akhtemova

1, E.S. Ovchinnikova

1, T.A. Nemankin

1,

T.N. Danilova1, Y.G. Borisov

1, M.V. Barbashov

2, T.S. Naumkina

2, V.R. Chebotar

3 and Tikhonovich,

I.A.1

1All-Russia Research Institute for Agricultural Microbiology, St.-Petersburg, Russia

2Institute of Grain Legumes and Groat Crops, Orel, Russia

3BisolbiInter Ltd, St.-Petersburg, Russia

Higher plant provides ecological niches for different types of beneficial microbes, which are able to

colonize roots and use photosynthates. Some of them form plant root symbioses, which are of interest

of sustainable agriculture, such as: arbuscular mycorrhiza (AM) with fungi of a phylum

Glomeromycota, root nodule symbiosis (RNS) of legumes with rhizobia bacteria and root associations

with plant growth promoting bacteria (PGPB). The symbioses are beneficial for plant and environment

supplying plant with nutrients, protecting it from pathogens and raising soil fertility. The potential of

microbial synergisms suggests that the prospects for establishing multipartite symbioses which

improve different agronomic traits in leguminous crops are high (1, 2).

In spite of the fact that all the above-mentioned symbioses have a lot of differences in their

morphology, specificity and functions in plant, there are similar molecular and cellular mechanisms,

which are involved in their development and functioning (3, 4). Particularly, there are common

legume genes implicated in development of AM, RNS and PGPB which belong to the ―common

symbiotic pathway‖ (CSP) (3, 4).

The AM is most widespread symbiosis through the plant kingdom: more than 80% of land

plants are able to form it (5). The symbiosis has originated at the same time when the terrestrial plants

have appeared (400 millions years ago) and is supposed to be ―the mother of the root symbioses‖ (3)

being a source of plant pre-adaptations for their development (4, 6).

It was demonstrated for pea that it is possible and necessary to carry out plant breeding to

improve its symbiotic effectiveness using complex microbial inoculants composed of AM fungi,

rhizobia and PGPB (2, 4, 7, 8).

Summarizing all these knowledge permits the development of a new conception of plant-

microbe interactions exploitation in sustainable agriculture (2): (I) plant is an organizer of multipartite

plant-microbe system; (II) plant possesses indivisible genetic system controlling its development; (III)

therefore plant should be bred to improve its symbiotic effectiveness and grown under multi-microbial

inoculation.

The work was supported by the grants of RFBR (09-04-13895, 09-04-91054, 09-04-91293,

10-04-00961, 10-04-01146), Russian Ministry of Science and Education (3440.2010.4,

02.512.11.2280, 02.740.11.0276), NWO (047.018.001).

References:

1. Barea J.M., Pozo M.J., Azcon R., Azcon-Aguilar C. 2005. J. Exp. Botany 56:1761–1778.

2. Shtark O.Y, Borisov A.Y., Zhukov V.A., et al. 2010. Intimate associations of beneficial soil

microbes with host plants. In: Soil Microbiology and Sustainable Crop Production, G.R. Dixon & E.

Tilston (eds.) Springer Science +Business Media B.V., Dordrecht, The Netherlands.

3. Parniske M. 2008. Nature Rev. Microbiol. 6:763–775.

4. Provorov N.A., Shtark O.Y., Zhukov V.A., et al. 2010. Developmental genetics of plant-microbe

symbioses. Nova Science Publishers, NY, USA.

5. Smith, S.E. & Read, D.J. 2008. Mycorrhizal Symbiosis (3nd

ed.). London: Academic Press.

6. Provorov, N.A., Vorobyov, N.I., 2010. Evolutionary genetics of plant-microbe symbioses, I.A.

Tikhonovich (ed.) NOVA Science Publishers, NY.

7. Jacobi L.M., Kukalev A.S., Ushakov K.V., et al. 1999. Pisum Genet. 31:44–45.

8. Shtark O.Y., Danilova T.N., Naumkina T.S., et al. 2006. Ecological genetics ("Ekologicheskaja

genetika") 4(2):22–28 (in Russian).

16

O6

A new group of bacteria from Dickeya genus - an emerging risk factor for potato crops in

Poland

Ewa Lojkowska, Marta Potrykus, Monika Slawiak

Department of Plant Protection and Biotechnology, Intercollegiate Faculty of Biotechnology

University of Gdansk & Medical University of Gdansk, Poland

Pectinolytic bacteria from the Pectobacterium carotovorum genus (syn. Erwinia carotovora) cause

potato crop damage and high economic loss in former years in Poland. However, recently the bacteria

from Dickeya genus were found to be able to cause black leg and soft rot on potato in Poland. These

recent findings suggest that Dickeya strains can effectively infect the potato plant and cause disease

symtoms also in temperate climate (Laurila et al 2008). Dickeya strains isolated from Poland in 2005

(Slawiak et al 2009a) and 2009 shared high level of similarity to the isolates from Finland, the

Netherlands and Israel but did not belong do any known Dickeya species thus it has been postulated to

establish a new species, Dickeya solani (Slawiak et al. 2009) Only in case of one infected plant the

isolated Dickeya strains indicated characteristics of Dickeya dianthicola.

The performed molecular characteristics (e.g. ERIC, BOX, REP patterns) indicated that strains

isolated from single plants share high similarity to each other. Dickeya strains isolated from one plant

in 2005 and four plants in 2009 exhibited a similar band pattern. All tested strains, exept of those

asigned to D. dianthicola indicated 100% homology of the dnaX and recA gene sequences.

A representative Dickeya strain, belonging to the postulated D. solani species indicate high

pathogenicity on chicory leaves and potato tubers. Results suggest that newly isolated strains are

highly virulent and cause massive tissue maceration.

The obtained results indicated that Dickeya sp. is an emerging risk factor for potato crops in country

with temperate climate. Dickeya strains in favourable conditions are expected to cause economic loss

to potato in Poland.

References:

1. Laurila, J., Ahola, V., Lehtinen, A., Joutsjoki, T., Hannukkala, A., Rahkonen, A., Pirhonen, M.

2008. Characterization of Dickeya strainsisolated from potato and river water samples in Finland.

European Journal of Plant Pathology 122(2): 213-225.

2. Slawiak M., Lojkowska E., van der Wolf J.M. First report of bacterial soft rot on potato caused by

Dickeya spp. (syn. Erwinia chrysanthemi) in Poland. Plant Pathology 2009, 58(4): 794.

3. Slawiak, M., van Beckhoven,J.R.C.M., Speksnijder, A.G.C.L., Czajkowski, R., Grabe, G., van der

Volf, J.M. 2009b. Biochemical and genetic analysisreveal a new clade of biovar 3 Dickeyaspp. strains

isolated from potatoin Europe. European Journal of Plant Pathology 125(2): 245-261.

17

O7

An extracellular and a membrane glycopolymer of the bacterium Herbaspirillum seropedicae

Z78

Shishonkova N.S., Smol´kina O.N., Ignatov V.V

Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of

Sciences, Saratov, Russia

Bacteria of the genus Herbaspirillum, which belong to the β-subclass of the Proteobacteria, are

endophytic associative nitrogen fixers that promote plant growth (1). An important role in microbial

contacts with other biological systems is played by lipopolysaccharide (LPS) – a constructive

biopolymer of the outer membrane. There are no data that LPS can be found as an extracellular

endotoxin in the culture medium as well. There are no data in the literature concerning the

composition and properties of Herbaspirillum extracellular LPS (ELPS) and O antigens. In

connection with this, our aim was to isolate and characterize the glycopolymers of

H. seropedicae Z78. The bacterial culture was grown in glucose-supplemented liquid malate medium

until the end of the exponential phase of growth. A polysaccharide (EPS) was precipitated from the

culture liquid with alcohol. The yield of EPS was 1.48 % of cell dry weight. The polysaccharide was

ultracentrifuged at 45,000×g for 6 h without using a concentration gradient. This procedure yielded a

transparent gellike sediment (ELPS) and a supernatant liquid containing pure EPS (not studied). The

yield of ELPS was 2.7 % of the EPS sample. LPS was extracted from the encapsulated cells with 95 %

phenol with separation of layers. The aqueous and phenolic portions of the extract were treated with

proteinase K and were ultracentrifuged. The yield of LPSap and LPSphp were 2.0 and 1.8 %. A study of

biopolymer composition showed the presence of all components characteristic of LPS, namely

carbohydrates, 2-keto-3-deoxyoctulosonic acid (KDO), and fatty acids (FAs) of lipid A. The

carbohydrate content in ELPS, LPSap, and LPSphp was 66.1, 12.3, and 6.6 %, respectively; the protein

content, 3.9, 0.6, and 0.5 %, respectively; the phosphorus content, 1.6, 3.0, and 1.5 %, respectively;

and the KDO content, 0.2, 1.0, and 0.4 % respectively. The low protein content in LPSap and LPSphp

attests to the high purity of the preparation. After methanolysis, the preparations were examined by

GLC for the presence of FAs. The preparations were found to contain acids with a chain length of C12

to C24. C14:0, 3-OH-C12:0, and C16:0 predominated. Monosaccharide composition was examined by TLC

and GLC as polyol acetates. Man, Gal, and Glc were identified as a part of the samples. SDS-PAGE of

the preparations showed most of the macromolecules to be concentrated in the lower part of the

electrophoretic track, indicating the predominance of R-form molecules in the pool of the membrane

LPS. For the ELPS sample, we also found one broad band in the center part of the electrophoretic

profile.

References:

1. Baldani J.I., Baldani V.L.D., Seldin L., Dobereiner J. 1986. Characterization of Herbaspirillum

seropedicae gen., nov., sp. nov., a root-associated nitrogen-fixing bacterium. Int. J. Syst. Bacteriol. 36:

86–93.

18

O8

Legumes in the agricultural and food context: Nitrogen fixation, climate change, and local food

and feed supplies

F.L. Stoddard

Department of Agricultural Sciences, University of Helsinki, PL 27 (Latokartanonkaari 5), 00014

University of Helsinki, Finland

Rotations in the Nordic and Baltic region are, as elsewhere in Europe, heavily biased towards cereals.

Broadleaved crops in general, and grain legumes in particular, offer a range of environmental and

agricultural benefits that are inadequately exploited in this region.

Legumes are a key part of the ecological intensification (1) of agriculture. They provide symbiotically

fixed nitrogen, so not only are they free of the need for nitrogen fertilizer, they also contribute to the

nitrogen nutrition of the following crop. A grass-free legume crop breaks soil-borne cereal disease

cycles. Legume root exudates, and hydrogen released from root nodules during nitrogen fixation,

enhance the growth of many beneficial soil organisms (2). Grain and forage legumes can be locally

grown to provide stockfeed rich in protein, energy and bioactive compounds, without the need for

long-distance shipment. Legumes add to the options available to the farmer, improving farm viability;

they increase regional biodiversity and landscape diversity, and they support pollinating bee

populations. The net release of nitrous oxide (N2O, a powerful greenhouse gas) in two years of a

legume-supported crop rotation was only 54% of that of a continuous cereal rotation in central North

America (3). In spite of these benefits and in the context of agricultural intensification, the cultivation

of legume crops has gone through a long decline in Europe, leading to a dependence on protein

imports (4). Legumes represent a tiny proportion of the agricultural production of the Nordic-Baltic

region and thus appear in the average field only once every several hundred years. Most farmers grow

familiar crops, and if a grain legume is not familiar, it does not get grown. There are few species and

within them, cultivars, that are tolerably well adapted to the region‘s growing conditions. Recent

increases in many agricultural commodity prices, including fertilizer and soybean meal, have

rekindled grower and processor interest in both grain and pasture legumes. Grain legumes can be

incorporated, with benefits to both profit and sustainability, into crop rotations from the Mediterranean

to Denmark every 3-6 years (5). A new EU-funded project, ―Legume Futures‖, is extending this

investigation into Northern and Eastern Europe and into a broader range of rotations.

References:

1 Cassman, K.G. 1999. Ecological intensification of cereal production systems: Yield potential, soil

quality, and precision agriculture. Proceedings of the National Academy of Sciences of the United

States of America 96: 5952-5959.

2 Dean, C.A., Sun, W., Dong, Z. & Caldwell, C.D. 2006. Soybean nodule hydrogen metabolism

affects soil hydrogen uptake and growth of rotation crops. Canadian Journal of Plant Science 86:

1355-1359.

3 Dusenbury, M.P., Engel, R.E., Miller, R.P., Lemke, R.L. & Wallender, R. 2008. Nitrous oxide

emissions from a northern Great Plains soil as influenced by nitrogen management and cropping

system. Journal of Environmental Quality 37: 542-550.

4 Stoddard, F.L., Hovinen, S., Kontturi, M., Lindström, K. & Nykänen, N. 2009. Legumes in Finnish

agriculture: History, present status and future prospects. Agricultural and Food Science 18: 191-205.

5 Nemecek, T., Richthofen, J.S. von, Dubois, G., Casta, P., Charles, R., & Pahl, H. 2008.

Environmental impact of introducing grain legumes into European crop rotations. Eur. J. Agronomy

28: 380–393.

19

O9

Sequencing the pea (Pisum sativum L.) regulatory symbiotic genes controlling nodulation

V. Zhukov1, T. Rychagova

1, V. Titov

1, E. Ovchinnikova

1,2, J.-M. Couzigou

3, E. Limpens

2, S.

Radutoiu4, L. H. Madsen

4, A. Borisov

1, P. Ratet

3, T. Bisseling

2, J. Stougaard

4, I. Tikhonovich

1.

1All-Russia Research Institute for Agricultural Microbiology, St-Petersburg – Pushkin, Russia;

2Wageningen University, Wageningen, The Netherlands;

3Institut des Sciences du Végétal, CNRS, Gif

sur Yvette, France; 4Centre for Carbohydrate Recognition and Signalling, Department of Molecular

Biology, University of Aarhus, Aarhus, Denmark.

In pea, which is important crop plant, several genes playing role in symbiotic interactions with nodule

bacteria and arbuscular mycorrhizal fungi had been identified by means of experimental mutagenesis

(1). Identification of primary structure of regulatory genes will help to understand how symbiotic

systems in pea function at molecular level and, probably, how pea differs from other legumes.

If mutated gene of interest is mapped in pea genome, one can find probable homologs among already

cloned symbiotic genes of model legumes, based on syntenic genome location and similar mutant

phenotype. In this way, sequences of PsSym35 and PsSym37 genes had been identified on the base of

proposed homology with cloned genes LjNIN and LjNFR1, respectively, of Lotus japonicus (Regel.)

Larsen (2, 3). As well, in present work, the sequence of PsSym33 was determined based on homology

with MtIPD3 of Medicago truncatula Gaertn.

Results of mapping pea genes may also facilitate cloning genes tagged by multiple transposon

insertions in M. truncatula. The gene MtNOOT was identified as one of insertions and probably

homologous pea gene PsCochleata were mapped in syntenic regions. Then, by homology with

MtNOOT, PsCochleata had been also sequenced.

Search for pea mutants carrying genes homologous to known genes of model legumes can also be

performed based on decrease of expression level of target gene by nonsense-mediated RNA decay (4).

Using this approach, mutated pea gene homologous to LjSST1 had been found in pea line RisFixN.

Identification of primary structure of regulatory genes will help to understand how symbiotic system

in pea functions at molecular level. Sequencing these genes could also be useful for breeding as allelic

state of symbiotic genes of legumes is supposed to have influence upon effectiveness of interactions

with soil microorganisms.

The work was supported by the grants of RFBR (09-04-13895, 09-04-91054, 09-04-91293, 10-04-

00961, 10-04-01146), Russian Ministry of Science and Education (3440.2010.4, 02.512.11.2280,

02.740.11.0276), NWO (047.018.001).

1. Borisov, A.Y., Vasil'chikov, A.G., Voroshilova, V.A., Danilova, T.N., Zhernakov, A.I., Zhukov,

V.A., Koroleva T.A., Kuznetsova, E.V., Madsen, L., Mofett, M., Naumkina, T.S., Nemankin, T.A.,

Ovchinnikova, E.S., Pavlova, Z.B., Petrova, N.E., Pinaev, A.G., Radutoiu, S., Rozov, S.M.,

Rychagova, T.S., Solovov, I.I., Stougaard, J., Topunov, A.F., Weeden, N.F., Tsyganov, V.E., Shtark,

O.Y. & Tikhonovich, I.A. 2007. Regulatory genes of garden pea (Pisum sativum L.) controlling the

development of nitrogen-fixing nodules and arbuscular mycorrhiza: a review of basic and applied

aspects. Prikl Biokhim Mikrobiol. 43: 265-271.

2. Borisov, A.Y., Madsen, L.H., Tsyganov, V.E., Umehara, Y., Voroshilova, V.A., Batagov, A.O.,

Sandal, N., Mortensen, A., Schauser, L., Ellis, N., Tikhonovich, I.A. & Stougaard, J. 2003. The Sym35

gene required for root nodule development in pea is an ortholog of Nin from Lotus japonicus. Plant

Physiol. 131: 1009-1017.

3. Zhukov, V., Radutoiu, S., Madsen, L.H., Rychagova, T., Ovchinnikova, E., Borisov, A.,

Tikhonovich, I. & Stougaard, J. 2008. The pea Sym37 receptor kinase gene controls infection-thread

initiation and nodule development. Mol Plant Microbe Interact. 21: 1600-1608.

4. Hori, K. & Watanabe, Y. 2008. Chapter 8. In vivo analysis of plant nonsense-mediated mRNA

decay. Methods Enzymol. 449:165-176.

20

O10

Use of rhizobial Nod factors as biofertilizers for legumes

D. Kidaj1, J. Wielbo

1, M. Marek-Kozaczuk

1 and A. Skorupska

1

1Department of Genetics and Microbiology, M. Curie-Sklodowska University, Lublin, Poland

Rhizobia are soil bacteria, which have ability to induce root nodules on legume plants of

agricultural importance and provide these plants with reduced nitrogen, enabling them to grow in

nitrogen-limited agricultural soils. Rhizobial strains vary in effectiveness and competitiveness. Thus,

selection of highly effective strains that successfully compete with less effective ones is fully justified

when developing legume inoculants. Several factors influence the symbiotic interactions, including the

effective exchange of plant and bacterial signaling molecules, such as flavonoids and nodulation factor

(Nod factor). Flavonoids released by host plant are recognized by bacterial NodD protein and nod

genes transcription is activated. Nod factor initiates nodule primordium in plant roots. Inside the

nodules rhizobia fix dinitrogen into ammonia utilized by host plant (1,2). Beneficial responses of

legumes to rhizobia employed as inoculants, as well as different manipulations of symbiotic signaling

factors, allow increasing their potential value as biofertilizers for sustainable agriculture to promote

growth and nodulation of legume plants.

In our study, response of rhizobia to flavonoids as factors that potentially influence their

competitivenes and symbiotic activity was assessed. Using Rhizobium leguminosarum nodule isolates,

We found that competitive strains more readily responded to a wide range of synthetic and natural

flavonoids present in seed exudates in comparison to uncompetitive strains (3).

We used two competitive strains: R. leguminosarum bv. trifolii KO17 (4) and R.

leguminosarum bv. viciae GR09 as inoculants for clover and pea. We assessed the effect of pea and

clover seeds treatment with specific LCOs on germination, growth and nodulation ability of the

inoculant strains under soil conditions. Significant increase in the germination of seeds and growth of

plant seedlings was observed after LCOs application. In addition, statistically significant increase of

plants fresh masses and percentage of nodule numbers as the most important effect of LCOs seed

treatment was demonstrated. We did not observe any effect of LCOs treatment on the competitiveness

of KO17 and GR09 inoculant strains. In conclusion, purified LCOs could be use as effective

biofertilizers for legumes. Our ―green fertilizers‖ inducing several important physiological changes in

leguminous plants make possible the indigenous rhizobia to invade faster the legume roots.

References:

1. Perret, X., Staehelin, C. & Broughton, W.J. 2000. Molecular basis of symbiotic promiscuity.

Microbiol. Mol. Biol. Rev. 64:180-201.

2. Spaink H.P. 2000. Root nodulation and infection factors produced by rhizobial bacteria. Annu. Rev.

Microbiol. 54:25-288.

3. Maj D., Wielbo J., Marek-Kozaczuk M. & Skorupska A. 2010. Response to flavonoids as a factor

influencing competitiveness and symbiotic activity of Rhizobium leguminosarum. Microbiological

Research 165:50-60.

4. Maj D., Wielbo J., Marek-Kozaczuk M., Skorupska A. 2009. Pretreatment of clover seeds with Nod

factors improves growth and nodulation of Trifolium pratense. J Chem Ecol 35:479-487.

21

O11

Climate change, salinization and soil microbial community adaptive evolution

Andronov E.E.

All-Russia Institute for Agricultural Microbiology, St-Petersburg, Pushkin, RUSSIA

Microbial community is a part of biosphere with the highest potential of adaptivity. Climate changes

often resulted in aridity and salinization (1). Total area of salinised lands on the Earth is more than

3∙108 ha and it is comparable with India‘s area. Thus, the salinity is one of the most important factors

affecting biosphere. Moreover accordingly to last metagenomic studies (2) salinity is an ecological

factor which has extremely deep impact on the soil microbial community structure. During last years

ARRIAM undertook several expeditions to salt affected areas in the former USSR areas and as a result

a representative collection of microorganisms (basically Rhizobium) and soil samples collected in

these areas was created. All the microorganisms collected were studied with number of molecular

techniques including genome fingerprinting, microarray based genome comparative studies,

sequencing, Real-time PCR expression analyses etc. Total DNAs were isolated from soil samples and

studied with cultivation-independent molecular approaches allowing microbial quantification

(separately for bacteria, achaea and fungi) and microbial community structure studying (T-RFLP).

Analyses of all the data obtained showed a spectrum of adaptive modifications in microbial

communities on different levels of organization: 1) genetic modification of a particular locus in

microbial genome affecting the expression of adjacent genes; 2) genetic modifications of long

segments of genomes by genome rearrangements; 3) modifications of population structure of a

particular microbial specie; 4) modification of the metapopulation structure comprising all groups of

microorganisms (bacteria, archaea and fungi). All the data obtained point to extremely high adaptive

potential of the microbial world and demonstrate extremely broad repertoire of genetic variability

mechanisms on the all levels from the single cell to the whole metacommunity.

The work was supported by RFBR grant 09-04-00386a

References:

1. Noyes P.D. et al. The toxicology of climate change: environmental contaminants in a warming

world. Environ Int. 2009 Aug;35(6):971-86. Epub 2009 Apr 16.

2. Lozupone C.A., Knight R. Global patterns in bacterial diversity. PNAS. 2007 vol. 104. No. 27. P.

11436 – 11440.

22

O12

Insight into microbial world – molecular biology research in environmental microbiology

Aleksandra Ziembińska

The Silesian University of Technology, Environmental Biotechnology Department, Akademicka 2,

44-100 Gliwice, Poland

The lack of the proper tools was the reason why we were not able to study the diversity of the

microbial world for a long time. But during last 30 years the research techniques based on the genetic

material have been developed and now they are the most commonly used methods not only in the area

of microbial research. The studies at the molecular level enable the precise microorganisms

identification, describing metabolic pathways due to the usage of functional enzyme coding genes and

showing the possibilities of these bacteria usage in medicine, industry and environmental protection.

From the ecological point of view molecular techniques enable the investigation of pure microbial

cultures and complex biocenosis composition and dynamics. It should be mentioned, that without the

development of molecular biology techniques the insight into complex microbial groups, such as

biocenosis of soil, activated sludge or water wouldn‘t be possible. The most frequently used and the

most effective tools for such investigations are denaturing gradient gel electrophoresis (DGGE) and

fluorescent in situ hybridization (FISH). Both techniques are used on DNA analysis and its results

complete one another, due to the different methodological aspect studied. DGGE is based on PCR-

amplified genetic material and shows the biocenosis composition [1, 2], while FISH is based on

microscopic imaging and presents the microbial group composition and mutual relationships [2, 3].

References:

1. Heuer H. & Smalla K., 1997, Application of denaturing gradient gel electrophoresis (DGGE) and

temperature gradient gel electrophoresis (TGGE) for studying soil microbial communities, in: van

Elsas J.D., Trevors, J. T., Wellington E. M. H., (ed.) Modern Soil Microbiology, Marcel Dekker, New

York.

2. Muyzer G., 1999, Genetic fingerprinting of microbial communities – present status and future

perspectives, Microbial Biosystems: New Frontiers, Proceedings of 8th International Symposium on

Microbial Ecology, Bell C. R., Brylinsky M., Jonson-Green P. (ed.), Atlantic Canada Society for

Microbial Ecology, Halifax, Kanada.

3. Thimm T. & Tebbe C. C., 2003, Protocol for Rapid Fluorescence In Situ Hybridization of Bacteria in

Cryosections of Microarthropods, Applied and Environmental Microbiology, 69/5: 2875-2878.

23

O13

LSD1 is the Darwinian Fitness Regulator in Arabidopsis

Wituszyńska W ¹*, Ślesak I ¹*, Vanderauwera S ²*, Kornaś A ³, Choo KS ¹, Mühlenbock P ², Van

Breusegem F ², Karpiński S ¹

¹ Department of Plant Genetics, Breeding and Biotechnology, Faculty of Horticulture and Landscape

Architecture, Warsaw University of Life Sciences, Warszawa, Poland

² Department of Plant Systems Biology, Flanders Institute for Biotechnology (VIB), Ghent, Belgium

and Department of Plant Biotechnology and Genetics, Ghent University, Ghent, Belgium

³Institute of Biology, Cracow Pedagogical University, Kraków, Poland

* These authors equally participated in this work

LSD1 (LESION SIMULATING DISEASE 1) was characterized as a negative regulator of

salicylic acid (SA) dependent hypersensitive disease response (HR) (Jabs et al., 1996). The defence

regulators EDS1 (ENHANCED DISEASE SUSCEPTIBILITY 1) and PAD4 (PHYTOALEXIN

DEFICIENT 4) were also identified as components of SA signalling in HR and proven to be regulated

by LSD1 (Rusterucci et al., 2001). In addition, we have demonstrated that LSD1 regulates ethylene and

reactive oxygen species (ROS) dependent light and root hypoxia acclimatory responses, retrograde

chloroplast to nucleus signalling and photorespiration (Mühlenbock et al., 2007 and 2008).

Under controlled laboratory conditions, lsd1 plants display a strongly reduced Darwinian

fitness (interpreted as the ability of a plant to reproduce), which is markedly improved when plants are

subjected to controlled drought stress conditions. This prompted us to investigate the Darwinian

fitness of lsd1 plants in natural, multivariable environmental conditions. In several independent field

experiments performed on different locations, a significantly improved Darwinian fitness of lsd1

plants compared to wild type plants was observed. Transcriptomic profiling of lsd1, eds1, pad4,

lsd1/eds1 and lsd1/pad4 mutated plants grown under laboratory or field conditions demonstrates that

LSD1 expression is suppressed by multiple stresses and reveals specific LSD1, EDS1 and PAD4

regulons, involved in different signalling and metabolic pathways, important for Darwinian fitness

regulation. This, and our previous findings cast new light on the current system used for defining gene

functions in Arabidopsis.

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Vanderauwera%20S%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Choo%20KS%22%5BAuthor%5D

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Van%20Breusegem%20F%22%5BAuthor%5D


24

O14

Nitric oxide as a key regulator of induced resistance in potato leaves to Phytophthora infestans

D. Abramowski1, Ł. Janus

1, J. Floryszak-Wieczorek

1

1Department of Plant Physiology, Poznań University of Life Sciences, Poznań, Poland

As it was well documented, plants following a primary stress occasionally acquire resistance to a

much stronger secondary response. This phenomenon in relation to pathogenic microorganisms is

usually described as systemic acquired resistance (SAR) or induced systemic resistance (ISR).

Defense can be non-specifically induced in susceptible plants. In our study high systemic protection of

susceptible potato leaves cv. ‘Bintje‘ – against late blight disease caused by Phytophthora infestans

was provoked by local pre-treatment leaves with the mentioned pathogen or with the following

inducers: 2,6-dichloroisonicotinic acid and β-amino-n-butyric acid1. Among endogenous signals,

which can potentially modulate defense responses in the course of resistance acquisition, a special role

is played by the nitric oxide (NO). This important molecule, which synthesis increases rapidly during

stress, may both cooperate with other signals and intracellular hormones as well as participate in the

transduction of stress information, e.g. via nitrosation or S-nitrosylation of signalling proteins and

protein transcription factors2. Therefore we analyzed the intensity and the kinetics of NO burst. We

assessed the importance of NO in triggering defense responses in a potato tissue challenged by

haemibiotrophic pathogen - Phytophthora infestans or pre-treated with SAR inducers. We attempted

to elucidate how NO has been linked to reactive oxygen species generation and the activation of other

signalling compounds leading to resistance. Seeing that potato late blight caused by Phytophthora

infestans, ranks as the world‘s agriculture most destructive disease (according to the International

Potato Centre – approx. 10 billions of dollars of losses per year) all efforts leading to the recognition

of the role of NO in plant-host defence responses and invader metabolism could provide new tools for

rational crop controlling in order to improve potato disease resistance. Moreover, the climate changes

observed as the global warming have resulted in facilitating the evolution of new races of P. infestans.

The changes in the frequencies of virulence alleles increase the infection potential and strongly affect

potato plants in the Baltic Sea region3.

References:

1. Andreu, A.B., Guevara, M.G., Wolski, E.A., Daleo, G.R., Caldiz, D.O. 2006. Enhancment of

natural disease resistance in potatos by chemicals. Pest Manag. Sci 62:162-170

2. Arasimowicz, M., Floryszak-Wieczorek, J. 2007. Nitric oxide as a bioactive signalling molecule in

plant stress responses. Plant Science, 172: 876-887.

3. Gianessi L., Sankula S., Reigner, N. 2003. Plant Biotechnology: Potential Impact for Improving,

Pest Management in European Agriculture, A Summary of Nine Case Studies, NCFAP

25

O15

A high-efficiency medium-free technology for monocot transformation by genes increasing

water deficit, salinization and resistance to plant diseases

M.I.Chumakov1,2

1

Laboratory of Genetic Engineering, Science and Education Center of Biotechnology, Saratov State

University; Saratov, 410035, Russia; 2

Laboratory of Bioingeneering, Institute of Biochemistry and

Physiology of Plants and Microorganisms, RAS; Saratov, 410049, Russia

The research is directed to obtaining plant lines with increased expression of genes that ensure all-

round resistance to unfavorable conditions (water deficit, salinization, plant diseases) for subsequent

reproduction using new bioengineering approach. This review describes methods of Agrobacterium-

mediated T-DNA transfer to plant vegetative and generative cells under in planta conditions, including

designing and testing methods that do not require the in vitro cultivation of transgenic plant cells and

tissues [1]. A high-efficiency medium-free new technology for monocot transformation will be

presented. The marker genes highly efficiently integrated into the germ cells of different maize,

sorghum lines. A possible ways of T-DNA delivery into embryo sac cells using pollen tubes will be

discussed. Practical application of new technology for corn transformation for transfer genes required

for adaptation to water deficit, salinization and plant virus resistance will be presented. Particularly,

investigating of the possibility of Agrobacterium-mediated T-DNA transfer antisense suppressor of

the proline dehydrogenase and extracellular ribonuclease genes to maize gametophyte cells was

conducted.

References:

1. Chumakov, M.I. 2007. Agrobacterium-mediated plant transformation under in planta conditions.

Transgenic Plant Journal. 1: 60-65.

26

O16

The environmental role of associations of cyanobacterium and actinomycetes – the components

of plant symbioses

E. A. Ivanova and E.O. Omarova

Lomonosov Moscow State University, Moscow, Russia

Nowadays a new line in the investigation of symbiosis - an associative symbiology presupposing the

presence of several associative symbionts along with the macrosymbiont and a dominant

microsymbiont - is being successfully developed.1 In order to reveal the interaction and mutual

influence of microsymbionts in natural symbioses it is necessary to study their physiological

characteristics in the experimental association.

For this purpose, the model associations composed of cyanobacterium Anabaena variabilis ATCC

29413 and actinomycetes, isolated from apogeotropic roots of Stangeria eriopus and identified by 16

S rRNA gene sequencing as Streptomyces pluricolorescens str.1 and Streptomyces cyaneofuscatus str

1 ' were constructed.

The stimulation of the experimental associations partners' growth, the change in the kinetics of

photosynthesis and the increase in the nitrogen-fixing activity of cyanobacterium in the association

were found with the latter being correlated with an increase in the proportion of heterocyst in the

filaments of cyanobacterium, which was a part of the experimental associations as compared to a

monoculture.

The strengthening of antibiotic activity in association in comparison with monocultures of its

components was revealed. The use of nuclear magnetic resonance technique2 allowed to determine the

presence of free water in the thallus of experimental association. In the monocultures of streptomycete

and cyanobacterium water was absent. The transformative effect of cyanobacterial actinomycetes

associations on the structure of clay minerals - kaolinite, vermiculite, montmorillonite, micas and

hydrous micas - was observed.

The detection of functional properties of microorganisms can be successfully applied in the screening

of new antibiotics and bioremediation of contaminated soils. The methodological techniques of the

creation of the artificial associations can be used in order to develop the technology of biological

products. Apart from that, the role of microorganisms in the process of destruction of the structure of

silicate minerals is closely related to the formation process of weathering crusts.

This work was financially supported by the Russian Federal Targeted Programme "Scientific and

scientific-pedagogical personnel" (2009 - 2013). State contract number P307

References:

1. Bukharin O.V., Lobakova E.S., Nemtseva N.V., Cherkasov S.V. 2007. Assosiative symbiosis. 263 p.

2. Lundin F.G., Fedin E.I. 1986. NMR spectroscopy. 222 p.

27

O17

New approaches for the control of common scab

L. Hiltunen1,2

and J. Valkonen2

1MTT Agrifood Research Finland, Biotechnology and Food Research, FI-92400 Ruukki, Finland;

2Department of Agricultural Sciences, PO Box 27, FI-00014 University of Helsinki, Finland

Climate change factors such as warming, precipitation and atmospheric CO2 are likely to interact and

modify ecosystem properties and processes. However, the effects on the soil microbial communities

are poorly studied and difficult to predict (1). Common scab is a soil-borne disease of potato caused by

a number of Streptomyces spp., in Finland by S. scabies and S. turgidiscabies (3). Management of this

disease is difficult because many environmental factors influence the disease outbreak. Furthermore,

different pathogen species seem to have different optima for the environmental factors. The climate

change scenarios for Finland project the annual mean temperature and precipitation to rise (4). It is

envisaged that these changes may increase the general occurrence of common scab and favor the

Streptomyces species or strains that are able to cause common scab under high soil moisture

conditions. Therefore, new methods for the control of common scab are needed. The aim of this work

was to study the potential of two new approaches for the control of common scab. These approaches

are based on i) use of thaxtomins, the dipeptide phytotoxins produced by Streptomyces, for screening

potato genotypes for their differences in sensitivity to common scab (2), and ii) enhancing soil

suppressiveness caused by non-pathogenic Streptomyces species to common scab.

F1 potato progeny from a single cross were screened in vitro by exposing the seedlings to

thaxtomin added in the culture medium. Genotypes were selected based on their sensitivity to

thaxtomin, multiplied in vitro, and tested for resistance to common scab caused by S. turgidiscabies

and S. scabies in the field. The relative shoot height in vitro and the scab index in the field correlated

significantly. These results suggest that the in vitro bioassay could be a time- and cost-effective

method to reject scab-susceptible genotypes at early stages in a potato breeding program.

To investigate the possibility to enhance the development of soil suppressiveness to common

scab, long-term field experiments were set up. The applications of non-pathogenic Streptomyces spp.

(strain 272 and K61) known to be antagonistic to common scab pathogens are repeated annually. In

the first year, both non-pathogenic Streptomyces spp. reduced the incidence and severity of common

scab indicating effective antagonistic action against pathogenic Streptomyces spp. in field conditions.

However, several years‘ results are required to confirm the build up of suppressiveness in soil.

References:

1. Castro, H.F., Classen, A. T., Austin, E.E., Norby, R.J. & Schadt, C.W. 2010. Soil microbial

community responses to multiple experimental climate change drivers. Applied and Environmental

Microbiology 76: 999-1007.

2. Hiltunen, L.H., Laakso, I., Chobot, V., Hakala, K., Weckman, A. & Valkonen, J.P.T. 2006. The

influence of thaxtomins in different combinations and concentrations on growth of micropropagated

potato shoot cultures. Journal of Agricultural and Food Chemistry 54, 3372-3379.

3. Hiltunen, L.H., Ojanperä, T., Kortemaa, H., Richter, E., Lehtonen, M.J. & Valkonen, J.P.T. 2009.

Interactions and biocontrol of pathogenic Streptomyces strains co-occurring in potato scab lesions.

Journal of Applied Microbiology 106, 199-212.

4. Jylhä, K., Tuomenvirta, H. & Ruosteenoja, K. 2004. Climate change projections for Finland during

the 21st century. Boreal Environment Research 9: 127-152.

28

O18

Expression analysis of genes involved in scopoletin biosynthesis in Arabidopsis thaliana wild

accessions

J. Siwińska and A. Ihnatowicz

Department of Biotechnology, Intercollegiate Faculty of Biotechnology of University of Gdańsk and

Medical University of Gdańsk, Kładki 24, 80-822 Gdańsk, Poland

Accessions of Arabidopsis thaliana originating from different parts of the world exhibit natural

variation in a range of traits. This great phenotypic variation reflects selection for adaptation to a wide

distribution of Arabidopsis through the north hemisphere, providing genetic variation in responses to

local environmental factors. Nowadays natural genetic variation is becoming a very powerful tool in

plant biology and is currently being used in the fields of genomics, metabolomics and transcriptomics

[5]. Recently, many data have showed that Arabidopsis produces a numerous and diverse secondary

metabolites [2]. Moreover, it was discovered that Arabidopsis synthesizes coumarins in roots and

shoots – scopoletin, scopolin, skimmine and esculin – as a response to biotic and abiotic stresses [1,

3 ,4]. Importantly, coumarins have multi-pharmacological properties, which are being used in

medicine. Selected Arabidopsis accessions were investigated in order to find a possible correlation

between the level of accumulation of coumarins (in particular scopoletin) and differentially expressed

genes involved in their biosynthesis. Variation in the accumulation of detected coumarins among 28

Arabidopsis accessions was observed. Interestingly, the application of different elicitors clearly

induced changes in scopoletin content in roots, particularly after treatment with 2,4-

dichlorophenoxyacetic acid. On the basis of given results accessions for further gene expression

analysis were selected. In the near future it is planned to transform Ruta graveolens (well-known

coumarins ―accumulator‖ plants) with isolated Arabidopsis alleles.

References:

1. Bednarek, P., Schneider, B., Svatos, A., Oldham, N.J. & Hahlbrock, K. 2005. Structural Complexity,

Differential Response to Infection, and Tissue Specificity of Indolic and Phenylpropanoid Secondary

Metabolism in Arabidopsis Roots. Plant Physiol 138: 1058-1070.

2. D‘Auria, J.C. & Gershenzon, J. 2005. The secondary metabolism of Arabidopsis thaliana: growing

like a weed. Curr Opin Plant Biol 8: 308-316.

3. Kai, K., Shimizu, B., Mizutani, M., Watanabe, K. & Sakata, K. 2006. Accumulation of coumarins

in Arabidopsis thaliana. Phytochemistry 67: 379-386.

4. Kai, K., Mizutani, M., Kawamura, N., Yamamoto, R., Tamai, M., Yamaguchi, H., Sakata, K. &

Shimizu, B. 2008. Scopoletin is biosynthesized via ortho-hydroxylation of feruloyl CoA by a 2-

oxoglutarate-dependent dioxygenase in Arabidopsis thaliana. Plant J. 55: 989-999.

5. Koornneef, M., Alonso-Blanco, C. & Vreugdenhil, D. 2004. Naturally occurring genetic variation in

Arabidopsis thaliana. Annu Rev Plant Biol 55: 141-172.

This research is supported by grants from the Foundation for Polish Science (HOMING Programme), the University of

Gdańsk (Gdańsk University Grant BW/B051-5-0290-9) and the German Research Foundation (GZ: RE 3013/1-1).

29

O19

Elaboration of a vaccine protecting wild animals from borreliosis

A. Urbanowicz

Institute of Bioorganic Chemistry, Polish Academy of Sciences

The recently observed climate changes strongly enhance the expansion of zoonotic diseases, tick-

borne diseases among others1. During this decade, the morbidity of borreliosis, also called Lyme

disease, has increased tenfold in Poland2. The major reservoir of Borrelia burgdorferi in Poland are

wild rodents, in a lesser degree other wild mammals and birds, while hard tick Ixodes ricinus is the

vector spreading the infection. The goal of the research is to elaborate an oral vaccine against Lyme

disease for wild animals. The reduction of reservoir of bacteria in wild animals would decrease the

number of new infected ticks and the occurrence of Lyme disease in humans. The vaccine will be

based on bacterial OspA and OspC surface proteins and their tick receptors, TROSPA and Salp15.

Because of the high variability of bacterial DNA, usage of genetic material isolated from Ixodes

ricinus ticks collected in Poland is crucial for high effectiveness of the vaccine. The four proteins are

being produced via bacterial and plant transient expression systems.

References:

1. Gilbert L. Altitudinal patterns of tick and host abundance: a potential role for climate change in

regulating tick-borne diseases? Oecologia. 2010 Jan;162(1):217-25. Epub 2009 Aug 15.

2. Epidemiological reports. National Institute of Public Health, National Institute of Hygiene, Poland.

30

O20

Distribution, detection and genetic variability of Potato mop-top virus

Johanna Santala,1 Satu Latvala-Kilby,

2 Asko Hannukkala,

2 and Jari P.T. Valkonen

1

1 Plant Pathology Laboratory, Department of Applied Biology, PO Box 27, FIN-00014 University of

Helsinki, Finland; 2 MTT Agrifood Research Finland, Plant Production Research, FIN-31600

Jokioinen, Finland.

Potato mop-top virus (PMTV, genus Pomovirus) induces necrotic spraing symptoms in potato tubers

and causes severe quality problems in potato production in the Nordic countries. However, occurrence

of PMTV in other countries in the Baltic Sea region has not been known. Therefore, a joint research

programme (MOP-TOP, 2005-2008) was carried out to study PMTV distribution in Baltic Sea region

and to develop methods to prevent PMTV from spreading to new areas. In Finland, long-term field

trials revealed a high incidence of symptomless PMTV infections in many potato cultivars

emphasizing the insufficiency of visual inspections in PMTV detection. Surveys using harmonized

virus-specific detection procedures revealed that the non-Nordic countries of Baltic Sea region are

mainly free of PMTV whereas the potato growing areas of the Nordic countries are widely

contaminated.

The genetic variability of PMTV in Finland was also studied and two distinguishable types of RNA2

and RNA3 were detected, each showing only little genetic variability. Sequencing and restrictotype

analysis of PCR amplicons from tubers indicated that the majority of PMTV isolates comprised

RNA2-II and RNA3-B. However, a few tubers were infected with both types of RNA2 (I and II) and

one type of RNA3, one type of RNA2 and both types of RNA3 (A and B), or both types of both RNAs.

No specific type of RNA2 or RNA3 or their combination was associated with tuber symptoms or lack

of symptoms.

References:

Latvala-Kilby, S., Aura, J.M., Pupola, N., Hannukkala A. & Valkonen, J. P. T. 2009. Detection of

Potato mop-top virus in potato tubers and sprouts: combination of RNA2 and RNA3 variants and

incidence of symptomless infections. Phytopathology 99: 519-531.

Santala, J., Samuilova, O., Hannukkala, A., et al. 2010. Detection, distribution and control of Potato

mop-top virus, a soilborne virus, in northern Europe. Accepted to Annals of Applied Biology.

31

O21

PCR technologies as tools for diagnostic and identification of plant pathogens

S.K. Zavriev

M.M. Shemyakin & Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of

Sciences, Moscow, Russia

Methods for the detection of plant viruses, as well as many other pathogens commonly involve

serological means. Such methods have been popular for several reasons viz., relative ease of use,

availability of antibodies and reasonable cost. Serological methods of detection, however, have their

limitations: sensitivity, specificity and the high mutation rate of target antigens which may render

them non-reactive to certain antibodies. An alternative to serological methods of detection that can

overcome such limitations is polymerase chain reaction (PCR). Several examples of PCR-based

technologies applications for different goals of plant pathogen diagnostic and identification are

discussed.

The steps of the development of PCR-based virus group tests which allow detect known and

identify new members of the almost 20 plant virus groups including unknown isolates are described.

These tests are developed using primers based on regions that are highly conserved among members

of a group (1). In this detection system PCR results are analyzed by gel electrophoresis. With the

conditions optimized for the respective reactions, this approach eliminates the need for running

numerous specific tests to ensure that a plant sample is truly free of viruses in a specific group.

One of the approaches to make a rapid and sensitive diagnostic and identification of the pathogens,

are PCR modifications with the detection of fluorescence during the amplification (real-time PCR) or

after its termination (FLASH–PCR, Fluorescent Amplification-based Specific Hybridization). These

approaches enable to avoid electrophoresis analysis of the results significantly decreasing a risk of

working space contamination by the amplification products. Based on FLASH- and real-time PCR

technologies the diagnostic and identification systems of a broad range of different plant pathogens

including important quarantine pathogens, potato viruses (2), toxigenic Fusarium fungi (3) and other

were created.

References:

1. Maaron C.J.M. and Zavriev S.K. 2002. PCR-based tests for detection of Tobamoviruses and

Carlaviruses. Acta Horticulturae. 598: 117-122

2. Ryazantsev, D.Yu., Abramova, S.L., Evstratova, S.V., Gagkaeva, T.Yu., & Zavriev, S.K. 2008.

FLASH-PCR diagnostics of toxigenic fungi of the genus Fusarium. Bioorganicheskaia khimiia. 34 :

716-724.

3. Ryazantsev, D. Yu., & Zavriev, S.K. 2009. Efficient approach for potato viral pathogen sensitive

diagnostics and identification. Molekuliarnaia biologiia. 6: 558-567.

32

O22

Application of FTA®Cards to Sample Microbial Plant Pathogens for PCR and RT-PCR

Elisabeth Grund, Omar Darissa and Günter Adam

Department of Biology, Biocenter Klein Flottbek, University of Hamburg, Germany

The first attempt of a tissue print from Plum pox virusinfected leaves on filter paper for RT-PCR

detection of the virus was published 1996 (1). The described method was further deve-loped into FTA

cards where the paper was impregnated with several chemicals to assist in lysing cells and protect the

released nucleic acids from degradation. It is asserted that at least DNA survives storage on dry cards

for several years at room temperature (2), whereas RNA needs storage at minus 20 – 80°C (3). Besides

the advantage of improved stability, another advantage is the loss of infectivity of the adsorbed

material, making transportation of samples, still suitable for nucleic acid based detection, easier and

safer (4).

The status for usage of FTA cards with plants and their pathogens can be described as follows. The

proof of principle has been published by Ndunguru (5). They were able to recover viral DNA or RNA

from several plant tissues pressed on FTA cards. The recovered NA provided efficient template to start

up several molecular methods such as PCR, cloning and sequencing. The viruses used in their study

include ssDNA geminiviruses, tobacco mosaic virus (TMV) and two potyviruses. FTA cards were

employed as well in a relatively large-scale field sampling of ssDNA virus-infected plant tissues (6).

The authors showed that NA recovered from FTA cards were comparable to those obtained by other

classical methods like CTAB. They also experi-menttally proved that NA is stable during 8–9 months

storage on the FTA cards at RT. Roy and Nassuth (3) described application of FTA cards for RNA

viruses, mRNA from plants as well as plant genes, and were the first to describe work on DNA, RNA

and RNA virus from the same sample successfully. Suzuki et al. (7) have shown that NA recovered

from FTA cards could be used in genotyping assays as well as for the detection of NA from higher

fungi. In this report, we have tested the suitability of FTA cards for the extraction of NA from a

broader range of microbial plant pathogens and for the first time for a genomic dsRNA virus and a

viroid.

References:

1. Olmos A, Dasi MA, Candresse T, Cambra M. (1996) Print-capture PCR: a simple and highly sensitive

method for the detection of Plum pox virus (PPV) in plant tissues. Nucleic Acids Res 24:2192–2193.

2. Smith LM, Burgoyne LA. (2004) Collecting, archiving and processing DNA from wildlife samples

using FTA_ databasing paper. BMC Ecol 4:4.

3. Roy Y, Nassuth A. (2005) Detection of Plant genes, gene expression and viral RNA from tissue prints

on FTA cards. Plant Mol Biol 23:383–395.

4. Moscoso H, Raybon EO, Thayer SG, Hofacre CL. (2005) Molecular detection and serotyping of

infectious bronchitis virus from FTA filter paper. Avian Dis 49:24–29.

5. Ndunguru J, Taylor NJ, Yadav J, Aly H, Legg JP, Aveling T, Thompson G, Fauquet CM. (2005)

Application of FTA technology for sampling, recovery and molecular characterization of viral

pathogens and virus-derived transgenes from plant tissues. Virology J 2:45.

6. Owor BE, Martin PM, Shepherd DN, Edema R, Monjane AL, Rybicki EP, Thomson JA, Varsani A.

(2007) Genetic analysis of maize streak virus isolates from Uganda reveals widespread distribution of

a recombinant variant. J Gen Virol 88:3154–3165.

7. Suzuki S, Taketani H, Kusumoto K-I, Kashiwagi Y. (2006) Highthroughput genotyping of filamentous

fungus Aspergillus oryzae based on colony direct polymerase chain reaction. J Biosci Bioeng

102:572–574.

33

O23

siRNA sequencing: reconstructing viral genomes from defence molecules

J.F. Kreuze, W. Cuellar

International Potato Center (CIP), Lima, Peru

Novel and emerging plant viruses, driven by pathogen evolution, global trade, crop intensification and

potentially climate change, pose a key threat to agriculture worldwide. Even apparently symptomless

virus infections can cause considerable yield losses, which can be further exacerbated by synergistic

interactions with other viruses. RNA silencing constitutes a fundamental antiviral defence mechanism

in plants in which host enzymes cut viral RNA into pieces of 20-24 nt. When isolated, sequenced en

mass and properly aligned these virus-derived small RNA (sRNA) sequences can reconstitute genomic

sequence information from the viruses being targeted in the plant. This approach is independent of the

ability to culture or purify the virus and does not require any specific amplification or enrichment of

viral nucleic acids as it automatically enriches for small RNAs of viral origin by tapping into a natural

antiviral defence mechanism. Two groups have recently shown the applicability of this relatively

straight forward but powerful technique in plants (1) and invertebrate cell lines (2), and Kreuze et al (1)

was even able to assemble an entire genome sequence de novo at great depth. Besides the

identification of expected viruses from the experimentally infected samples, surprisingly, both groups

also identified several previously unknown and apparently symptomless viruses in experimental plants

and insect cell lines. Together the two studies identified seven new viruses from six separate families

including single and double stranded RNA, DNA and reverse transcribing viruses, emphasizing the

power and generic nature of the technique. Our subsequent experiments have further confirmed the

applicability of the technique in identifying and sequencing novel plant viruses from various host

species, even if it is limited by the ability to find similarity to sequences in databases by BLAST

searches. On the other hand it has also provided an important reminder that there may be many more

viruses infecting our crop plants than we have previously been aware of, and their potential impacts on

agricultural productivity remain to be understood.

References:

1. Kreuze, J.F., Perez, A., Untiveros, M., Quispe, D., Fuentes, S., Barker, I. & Simon, R. 2009. Complete

viral genome sequence and discovery of novel viruses by deep sequencing of small RNAs: a generic

method for diagnosis, discovery and sequencing of viruses. Virology 388: 1-7

2. Wu, Q., Luo, Y., Lu, R., Lau, N., Lai, E.C., Li, W.-X. & Ding, S.-W. 2010. Virus discovery by deep

sequencing and assembly of virus-derived small silencing RNAs. Proceedings of the National

Academy of Sciences of the United States of America 107, 1606–1611.

34

35

Poster presentation abstracts

P1

The selection of arbuscular mycorrhizal fungi for symbiotic system: Bradyrhizobium japonicum

– soybean plants

S.F. Abdurashitov1, S.V. Didovich

1, V.V. Volkogon

2

1Southeren Experimental Station of Institute of Agricultural Microbiology, Gvardeyskoe, Ukraine;

2Institute of Аgricultural Мicrobiology, Chernigov, Ukraine.

The development of agriculture in climate change and population growth of the Earth leads to

replacement of traditional cultivars and introduction of previously rarely used rotations of crops and

technologies of their cultivation. The potential mutualistic symbioses of plants with rhizobia and

arbuscular mycorrhizal fungi should be used to increasing productivity and adaptability of soybean as

the main legume in Ukraine. In the Southeren Experimental Station of IAM carried out significant

work to select strains of Bradyrhizobium japonicum for soybean ( 2). High effective strains passed to

the Collection of useful microorganisms, which has the status of national heritage of Ukraine. The B.

japonicum strain 36 - one of the high effective strain, which they use as the basis for a biological

product for inoculation of soybean. However, selection for high productivity isolates of arbuscular

mycorrhizal fungi is not carried out in Ukraine more than 15 years ( 1). Therefore the aim of our

research is isolation of AM fungi association from natural cenoses and agrocenoses, which are

effective for the symbiotic system of soybean plants - nodule bacteria. New associations of AM fungi

have bean isolated from agrocenoses of soybean, alfalfa, sainfoin, beans and natural phytocenoses of

Fabaceae, Asteraceae, Lamiaceae, Gramineae in 2008. It is established, that the effectiveness of

soybean mycorrhization by AM fungi associations, which was isolated from natural soil microbial

cenoses, was on 20% higher than mycorrhization by the agricultural soils associations. It is revealed,

high effective AM fungi associations S5, P3, P5 for the soybean plants – nodule bacteria symbiotic

system. They should be used to create of productive mutual plant-microbe systems.

References:

1. Косенко Н.М., Андреева Н.О., Патика В.П. 1995. Эндомікоризні гриби у ґрунтах півдня

України. Землеробство70: 91-95

2. Толкачов М.З., Дідовіч С.В., Турін Є.М., Шабанов Е.А., Щігорцова О.Л. 2004. Селекція

бульбочкових бактерій на високоефективний симбіоз з сучасними сортами зернобобових

культур. Х з’їзд Товариства мікробіологів України: Тези доповідей 15-17 вересня 2004 р.:

247.

36

P2

The influence of meteorological factors for sanitary condition in birch stands in Lithuania

A. Bagdziunaite

Institute of Forestry, Lithuanian Research Centre for Agriculture and Forestry, Lithuania

The sanitary condition of birch trees was studied in the 50-80 years old birch (Betula pendula Roth)

stands in autumn in 2005-2008. The birch trees were observed in the pure and mixed stands in two

forest sites (with temporarily overmoistured soils and soils with normal moisture). At all the 13 birch

stands (100 trees in everyone) in all territory of Lithuania were estimated. The main attention was paid

to the visually detected damages made on the stems of the trees. The purpose of this study was to

estimate the variety composition, distribution of wood decay fungi colonizing birch stems; and to

evaluate the meteorological parameters influence for fungi development in birch stands.

Meteorological data (mean summer weather temperature and amount of precipitation) were received

from bulletins of Lithuanian Hydrometeorogical Service. The dependence between wood decay fungi

and meteorological factors was revealed by the correlation analysis. It was estimated that the wood

decaying fungi were the most common damages from visually identified damages on birch trees in the

studied stands in 2005-2008. Damages made by insects, mechanical and other were made less than on

6% of birches. In total, 16 species of Basidiomycetes were identified on birch trees. The most frequent

species of decay fungi in living birches were: Inonotus obliquus (Ach. ex Pers.) Pilát, Pholiota

aurivella (Batsch) Fr.; in dead birches: Piptoporus betulinus (Bull.) P. Karst., Fomes fomentarius (L.)

J.J. Kickx, Armillaria mellea (Vahl) P. Kumm. Correlation analysis between number of infected trees

and meteorological factors showed that the dependence was weak. The dependence between birch

trees damaged by wood decay fungi and mean summer weather temperature was higher in the forest

sites with normal moisture soils than in the sites with temporarily overmoistured soils. The correlation

between trees damaged by wood decay fungi and mean summer precipitation was very low in the both

forest sites.

Keywords: birch stands, sanitary condition, wood decay fungi, meteorological factors, correlation.

37

P3

Comparative genome hybridization of native Sinorhizobium spp. strains from distinct alfalfa

gene-centers differ from soil climate conditions.

Belova V. S.1, K. Lindstrom

2, A. Becker

3, B. V. Simarov

1, and M. L. Roumiantseva

1

1Research Institute for Agricultural Microbiology, St.Petersburg, Russia;

2Biocenter, University of

Helsinki, Finland; 3 Institute of Biology,

University of Freiburg, Germany

Oligo based microarrays constructed from the genome of S. meliloti 1021 was applied to comparative

genome hybridisations (CGH) with DNA isolated from four Sinorhizobium spp. agriculturally

important inoculants for alfalfa applied at stressful environment. Originally these isolates were

originated from arid and semi arid saline regions of Kazakhstan, Algeria and Russia. One more isolate

S.arboris HAMBI1552 from Prosopis chilensis native to drought salinised area of Sudan was applied.

This species is taxonomically closest related to S. meliloti (1), with a host range different from the two

S. meliloti strains but with similar inoculant properties, i.e. good performance in drought and heat

stress in the field (2). The main observation of present research was to understand the genetic basis for

stress tolerance among the members of this group of salt tolerant isolates (IST) and to search whether

taxonomic relatedness was reflected in theirs genome composition. In order to infer gene sets related

to IST phenotype the data produced by CGH analysis with Sm6kOli for each isolate were processed

across database for ‗ancestral genome of α-proteobacterial species‘ (3) by computational approaches.

In total 2385 ORFs at SMc were assigned to ‗environmental genome‘ and 382 ORFs at SMa and 1053

at SMb to an auxiliary genome. The products of these genes are represented by 17 different functional

categories (COG-groups), mainly related to cellular processes and signaling, information storage and

processing, metabolism. Remarkable that the numerous amount of genes poorly characterized up to

now were marked. The flux of rhizobia gene sets related to stress environment was quantified and

evaluated along branches of α-proteobacterium.

1. Martens M, Delaere M, Coopman R, De Vos P, Gillis M, Willems A. 2007. Multilocus sequence

analysis of Ensifer and related taxa. Int J Syst Evol Microbiol. 57(Pt 3):489-503.

2. Nick G, de Lajudie P, Eardly BD, Suomalainen S, Paulin L, Zhang X, Gillis M, Lindström K. 1999.

Sinorhizobium arboris sp. nov. and Sinorhizobium kostiense sp. nov., isolated from leguminous trees

in Sudan and Kenya. Int J Syst Bacteriol 4:1359-68.

3. Boussau B, Karlberg EO, Frank AC, Legault BA, Andersson SG. 2004. Computational inference of

scenarios for alpha-proteobacterial genome evolution. PNAS 101: 9722–9727.

38

P4

Application of food industry wastes to produce ecologically safe biofuel

E.V. Bolotnik, E.I. Kolomiec, G.I. Novik., I.N. Ananeva

Institute of Microbiology, Minsk, Republic of Belarus

Nowadays, one of the most promising trends in applied biotechnology is the production of

ecologically safe motor biofuel from renewable raw materials. However, problems related to possible

consequences of land malmanagement during cultivation of crops – potential sources of biofuel are

gradually arising. Food safety threat and some ecological challenges provoke interest to second-

generation biofuel - butyl alcohol, not requiring specially grown cultivars for its production and hence

not competing with food industry. Solventogenic bacterial strains of genus Clostridium are used for

manufacturing biobutanol. According to numerous literature reports, accumulation of butanol during

fermentation of C. acetobutylicum strains isolated from natural sources on conventional food

substrates, like rye flour, corn meal, potatoes, glucose, etc. averages 7-12 g/l [1, 2]. Butanol yield

following strain culture on domestic organic refuse, food and agricultural wastes ranges from 6 to 11

g/l [3-5]. Completed studies demonstrated that a promising substrate for biobutanol production is corn

cake - corn-starch manufacturing waste. Maximal amount of solvents produced by selected strain C.

acetobutylicum on mixture of corn cake hydrolyzate and rye flour (at 40/60 ratio) equaled 22.0 g/l,

with butanol share 14.7 g/l, and these values exceed by 20-60 % average literature parameters. Further

studies will be aimed at increasing the portion of supplemented waste substrate – corn cake, which

will enable to cheapen significantly acetone-butanol fermentation process and to enhance ecological

safety of starch fabrication due to utilization of multitonnage wastes. So, currently joint efforts of

researchers from Institute of Physical-Chemical Problems, Belarus State University, Institute of

Microbiology, Belarus National Academy of Sciences, Mogilev State Food University, Belarus

National Technical University are centered on development of technology for producing fuel butanol

from corn-starch manufacturing waste. Search for other cheap substrates as an alternative to food

resources is currently under way. Nutrient medium composition was defined for production of C.

acetobutylicum industrial inoculums. Effects of growth-promoting supplements and method of

conditioning starter cultures on activity of solventogenic bacteria were studied. Physiologically active

24-h cultures of 2d generation at active sporulation stage containing vegetative cell concentration at

least 109 CFU/cm

3 were recommended as starter cultures. Laboratory bioreactor for cultivation of C.

acetobutylicum bacteria comprising fermenter ANCUM-3M equipped with controlling and measuring

apparatus was developed. Experiments were performed to increase fermentation efficiency by

immobilization of cells-producers using fibrous carriers. In collaboration with the Institute of

Physical-Chemical Problems we are studying efficient methods of recovery and purification of end

products from cultural liquid.

References:

1. Durre, P. 1998. New insights and novel developments in clostridial

acetone/butanol/isopropanol fermentation. Appl. Microbiol. and Biotechnol 49: 639–648.

2. Ramey, D. 2004. Production of Butyric Acid and Butanol from Biomass. Work Performed Under:

Contract No.: DE-F-G02-00ER86106 For: U.S. Department of Energy Morgantown.

3. Zverlov, V.V., Berezina, O., Velikodvorskaya, G.A., Schwarz, W.H. 2006. Industrial biosynthesis

of acetone and butanol by bacteria in the Soviet Union: use of hydrolyzed agricultural waste for

biological treatment. Appl. Microbiol. and Biotechnol. 71: 587–597.

4. Claassen P.A., Budde M.A., Lopez-Contresas A.M. 2000. Acetone, butanol and ethanol production

from domestic organic waste by solventogenic clostridia J. Mol. Microbiol. Bitechnol. 2: 39–44.

5. Schoutens G.H., Groot W.J. 1985. Economic feasibility of the production of isopropanol-butanol-

ethanol fuels from whey permeate Process Biochem. 20: 117–121.

39

P5

Occurrence of Tomato torrado virus (ToTV) - dangerous pathogen originated from hot climate

regions in Polish greenhouse cultivation of tomato plants

M. Budziszewska1, P. Wieczorek

1, H. Pospieszny

2, A. Obrępalska-Stęplowska

1

1Interdepartmental Laboratory of Molecular Biology, Institute of Plant Protection-National Research

Institute, Poznan, Poland, 2Department of Virology and Bacteriology, Institute of Plant Protection-

National Research Institute, Poznan, Poland.

ToTV was reported in many countries in the world. Recently it was classified to the new

genus Torradovirus, in Sequviridae family, among picorna-like viruses (1). ToTV is transmitted by

whiteflies but the preliminary results have shown that it could be transmitted with tomato‘s seeds

(personal communication). The natural host is Solanum lycopersicum, but the virus may also infect

weeds. ToTV causes local necrotic spots on the leaves, fruits, and in effect necrosis of whole plants

leading to permanent withering, resulting in significant crops reduction (2).

Our goal was biological and molecular characterization of Polish ToTV isolates (Wal, Kra,

Ros). Using cDNA synthesis and PCR, or RT-PCR, followed by molecular cloning, whole genome of

analyzed isolates were sequenced and characterized. On the basis of nucleotide and amino acid

sequences two genomic +ssRNA strands were described. Obtained sequencing data was analyzed

subsequently in phylogenetic study. Results showed high sequence similarity with Spanish isolates,

indicating that ToTV is highly conservative within coding genome parts (3). However, 3` end of

RNA1 characterizes high variability between analyzed isolates as well as within the same isolate.

Comparative analyses of 3ÙTR RNA1 and RNA2 showed 100% similarity of its central region

sequence. Moreover, symptom observation, and preliminary real-time PCR analysis indicated that Kra

may accumulate in plant at higher concentration suggesting its higher virulence. Further research will

be focused on the determination of potential impact of nucleotide differences in 3ÙTR on the

virulence, indicating also plants proteins that may interact with aforementioned regions, become

crucial for disease development.

ToTV is virulent pathogen appearing mainly in hot climate. Its origin is probably South Europe

with favorable weather conditions. In Poland, so far ToTV was reported only in glasshouse cultivation.

Its appearance in control conditions may be a result of economy globalization (trading of tomatoes

seedlings and infected seeds) that facilitates virus spreading in the world, becoming a real danger for

countries producing tomatoes. Moreover, ToTV is RNA type virus known to have high ability to

mutate. Therefore, there are real possibilities for appearance of new mutated populations of identified

isolates that will turn into more virulent for susceptible tomatoes cultivation.

References:

(1) Sanfacon H., Wellink J., Le Gall O., Karasew A., van der Vlugt R, Wetzel T., 2009. Secoviridae: a

proposed family of plant viruses within the order Picornavirales that combines the families

Sequiviridae and Comoviridae, the unassigned genera Cheravirus and Sadwavirus, and the proposed

genus Torradovirus. Archives of Virology 154:899-907.

(2) Verbeek M., Dullemans AM., van den Heuvel J.F.J.M., Maris PC., van der Vlugt R., 2007.

Identification and characterization of tomato torrado virus, a new plant picorna-like virus from tomato.

Archives of Virology 152:881-890.

(3) Budziszewska M., Obrępalska-Stęplowska A., Wieczorek P., Pospieszny H., 2008. The nucleotide

sequence of a Polish isolate of Tomato torrado virus. Virus Genes 37:400-406.

40

P6

Genetic sources of resistance to golden potato cyst nematode Globodera rostochiensis Woll.

among wild potato species

Chalaya N. A., Kiru S. D.

N.I. Vavilov All-Russian Research Institute of Plant Industry, St. Petersburg, Russia

One of the causes of yield decreasing of potato is the affection of its plants by golden potato

cyst nematode. This wrecker cause a significant damage to potato in many countries. The German and

Dutch scientists (1) distinguished 5 physiological pathotypes (Ro1, Ro2, Ro3,Ro4,Ro5) of Globodera

rostochiensis Woll. and three pathotypes of Globodera pallida (Pa1,Pa2, Pa3). Both species are object

of internal and external quarantine in the Russian Federation.

The most effective protection of a potato from golden potato cyst nematode is use

cultivars, possessing by resistance to it. One of the most actual tasks of breeders in the Russian

Federation - increase the number of resistant cultivars. For this purpose the investigations on search

new sources of genetic resistance as initial material for breeding are necessary

Studying of a genetic diversity of wild species from VIR Potato Collection and singling out

new genotypes, resistant to G. rostochiensis, allows to carrying out more effective and purposeful

breeding work on creation of new varieties, possessing by resistance to various pathotypes of this

species.

As a result of long-term studying of potato germplasm, stored in Russia, Germany, Netherlands

and USA, accessions, resistant to different pathotypes of G. rostochiensis were found out among more,

than 40 wild and two cultivated potato species.

The screening of accessions of wild potato species was carried out in the greenhouse using

method of Stelter and Rauber (2). The plastic pots by 500 sm³ volume were filled with the ground

infected by larva and cysts of G.rostochiensis, pathotype Ro. Average concentration of larva was 3,5 -

4 thousand per 100 sm ³ ground. For evaluation of plant resistance 30 seedlings of each accession were

planted in the individual pots. As the standard were used the tuber plants of susceptible and resistant

varieties.

Screening was carried out trough two months after planting seedlings when on the roots of a

susceptible variety have appeared the cysts with golden-yellow color. A resistant plants had no cysts.

For 4 years of screening of 76 accessions concerning 38 wild potato species, including 32

South-American and 6 to North American was carried out. Accessions of 13 species were screened for

the first time. In result of screening at such species, as S. alandiae, S. doddsii, S.dolichostigma, S.

yungasense and S.multiinterruptum were singled out a resistant plants.

As a result of investigation at 16 species a resistant genotypes are revealed. These species are S.

yungasense S.dolichostigma, S.sparsipilum S.sucrense S. doddsii S.famatinae, S.kurtzianum S.vernei

S.gourlayi, S.leptophyes, S.multidissectum S.multiinterruptum S. alandiaeS.tarijensе S.berthaultii,

S.microdontum f.gigantophyllum.The analysis of screening results has shown, that the absolute

majority of resistant genotypes are revealed at South-American species.

References:

1. Kort J., Ross H., Rumpenhorst H.J., Stone A.R. An internationalscheme for identifying and

classifying pathotypes of potato cyst-nematodes Globodera rostochiensis and Globodera pallida//

Nematologica, 1977.- c 333-339.

2. Stelter H., Rauber A. Untersuchunden uber den Kartoffelnematoden (Heterodera rostochiensis

Woll.) V. Die veranderung einer Nematodenpopulation unter dem Einfluβ widerstand sjchider und

anfalliger Kartoffelvoranderung in einjahriden Topf versuchen. Z.Pflanzenkrankh – 1959.- № 9.-

P.572-582.

41

P7

Characterization of bacteria isolated from rotten potato tissue exposing antagonistic activity

towards “Dickeya solani”

Czajkowski R.1,2

, Van Veen J. A.2,3

, Van der Wolf, J.M.,1

1Plant Research International, P. O. Box 16, 6700 AA, Wageningen, The Netherlands

2Dutch Institute of Ecology, Dutch Royal Academy of Art and Science (NIOO-KNAW),

Boterhoeksestraat 48, 6666 GA Heteren, The Netherlands 3Institute of Biology, University of Leiden, Sylviusweg 72, 2333 BE Leiden, The Netherlands

Potato blackleg caused by pectinolytic bacteria Pectobacterium and Dickeya species is a worldwide

disease of potato. In Europe, Dickeya spp. are causing increasing losses in seed potato production,

mainly due to declassification and rejection of seed tubers.

A new unclassified genetic clade of Dickeya belonging to biovar 3, temporarily called ―D. solani‖

appeared in Europe. ―D. solani” was isolated from potato in Finland, Poland, Israel, France, Belgium

and in United Kingdom. Increased losses in seed potato production due to blackleg are related to the

presence of this new strain.

Control of blackleg is hampered by the absence of effective procedures. Blackleg can be controlled

only by the integrated control strategy, which comprises on the use of pathogen-free seed tubers,

hygienic procedures during planting, harvest, grading and storage and disinfection of equipment. Our

study aimed to explore the potential of biocontrol of blackleg causing agents, including the use of

antagonistic bacteria.

Bacteria were isolated from rotten potato tissue by plating of rotten potato tuber extracts on agar media.

Six hundred forty nine isolates were screened for antibiosis against ―D. solani” or production of iron

ions-binding siderophores. Forty one strains (6.4%) produced antibiotics and 112 strains (17.3%)

produced siderophores in plate assays. A selection of 41 antibiotic-producing strains and 41

siderophore-producing strains were tested in a potato slice assay for control of “D. solani”. Strains

that were able to reduce rotting of potato tuber tissue to at least 50% of the control were selected.

Strains were characterized by 16S rDNA sequencing as Bacillus, Pseudomonas, Rhodococcus,

Serratia, Obesumbacterium and Lysinibacillus species. In a greenhouse experiment, twelve

antagonistic strains were tested in planta using minitubers of cv. Kondor co-inoculated by vacuum

infiltration with an antagonist and a GFP-tagged ―D. solani”. Antagonistic strains were found able to

reduce pre-emergence rot, blackleg development and colonization of stems by ―D. solani‖. Some of

the tested antagonists showed even a complete protection of potato plants against blackleg.

42

P8

Phylogenetic analysis and REP-PCR profiling of lupine's bacterial wet rot − “P. xanthoclora”

L. A. Dankevych

Zabolotny Institute of Microbiology and Virology of the National Academy of Sciences of Ukraine,

department of phytopatogenic bacteria, Kyiv, Ukraine

The advent of molecular biology has caused a significant shift in the types of approaches used to

characterize and identify plant pathogens and devise disease management strategies. Multiple

strategies have been adopted to characterize microorganisms based on features of their ribosomal

DNA. Genus – specific rDNA sequences among phytobacteria are well documented, and primers have

been developed for Pseudomonas, Xanthomonas and so on. The rRNA genes have functioned as

targets for highly sensitive detection of target DNA, but the level of discriminatory power lies at the

species or genus levels. Recently methods called „genomic fingerprints methods‖ have been applied.

They generate a collection of genomic fragments via PCR, which are resolved as banding patterns that

provide a high level of taxonomic resolution. One of these methods is rep-PCR analyze. It was

developed on the observed occurrence of specific conserved repetitive sequences (REP, ERIC and

BOX) distributed in the genomes of diverse bacteria. That is why three primers sets are commonly

used for rep-PCR genomic fingerprinting analysis, corresponding to REP, ERIC and BOX sequences.

The primers are designed to amplify intervening DNA between two adjacent repetitive elements. A

complex array of 10 to 30 or more PCR fragments is generated per genome, ranging in size from less

than 200 bp to more than 6 kb [1]. Scientists have noticed that this method is very effective in a case

of accurate identification of phytopathogens on species, patovar and strain level that is especially true

in the case of closely related pathogens that can strike a wide range of plants such as P. syringae pv.

syringae, P. marginalis pv. marginalis [1]. Therefore at our investigation, the application of

phylogenetic analysis and rep-PCR to determinate the species and patovar status of agent of lupine‘s

bacterial wet rot of a current concern. The data of full-size DNA replica of 16S rRNA sequence

confirmed relatedness the majority of investigated strains ―P. xanthoclora‖ with species P. marginalis

and P. fluorescens. All investigated strains have high level (97-99 %) of nucleotide‘s chain homology

with both species. Using REP-PCR it has been determined that REP - profiles of all collection "P.

xanthochlora" strains, P. marginalis 8572 strain and typical P. marginalis pv. marginalis 9175 strain

are related. The collection "P. xanthochlora" 8540 and P.marginalis 8572 strains are a little bit

different from others strains on basic of ERIC and BOX-profile. Although in this group of bacteria

observed some heterogeneity on ERIC and BOX- profiles the detailed analyzing of products of this

amplification showed the predominating of common with typical P. marginalis pv.marginalis 9175

strain products, which confirms their affinity. Instead, REP, ERIC and BOX-profile of typical P.

fluorescens B-17т and P. syringae pv. syringae В1027

т strains differ from other which have been

testified. That confirmed the absence of relationship of these strains with all investigated strains. So,

on the results of phylogenetic analysis and REP – profiling, it has been shown that agent of lupine‘s

bacterial wet rot "P. xanthochlora" is not a separate species and belongs to species P. marginalis and

pathovar marginalis. As a result, we can use this database for the quickly and accurately

distinguishing of the agent of lupine‘s bacterial wet rot from other pathogens, which strikes a wide

rang of plants.

References:

1. Louws F.J., Radenmaker J.L.W. and Bruijn F.J. 1999.The three Ds of PCR-based genomic analysis

of phytobacteria: diversity, detection and disease diagnosis. Annu. Rev. Phytopathol.37:81–125

43

P9

Application of Tobacco Rattle Virus-Based Gene Silencing in Gerbera hybrid

X. Deng, T. Hytönen, J. P.T. Valkonen, P. Elomaa and T. H. Teeri


Gerbera hybrida, belonging to the large sunflower family, has attracted considerable attention as a

new model plant for flower development study because of its particular flower organization in the

inflorescence. As a typical member of Asteraceae, gerbera tissues are rich in two glucosidic lactones

gerberin and parasorboside, and some other secondary metabolites such as 5-methylcoumarins,

chromenes and benzofurans, which are very important for the plants to protect against microbial attack

and insect herbivores. During the recent years, high throughput genetic methods, such as EST

sequencing and microarrays, have been adopted for identification of hundreds of candidate genes

affecting both gerbera flower development and secondary metabolism. In this highly heterozygous

species, the functional studies need to be conducted through reverse genetic methods by producing

transgenic lines. To facilitate these studies, our aim is to adapt virus-induced gene silencing (VIGS)

method for gerbera. Tobacco rattle virus (TRV) has almost the broadest host range among plant

viruses including gerbera. VIGS vectors based on TRV has been successfully used on silencing

endogenous genes in multiple Solanaceae genera, as well as in Arabidopsis and Papaver somniferum.

Our initial studies showed that TRV based vectors can be used to induce gene silencing in gerbera leaf

and flower tissues. Screening of 21 different gerbera genotypes showed large differences in VIGS

response. Out of these varieties, 6 most sensitive gerbera genotypes were selected for further studies.

Among different inoculation methods, vacuum inoculation induced most intensive silencing on

gerbera leaf tissues, and stem wound scratching was more practical for inducing gene silencing on

flowers. The silencing of gerbera phytoene desaturase gene (GPDS) and gerbera chalcone synthase

(GCHS) gene induced typical gene knock-out symptoms on host gerbera plants.

44

P10

Application of pharmacological approaches for control of diseases in higher plants

Isabel Weinheimer and Jari Valkonen

Department of Agricultural Sciences, University of Helsinki, Latokartanonkaari 7, 00014 Helsinki,

Finland

Climate change suggests that the weather variability is likely to increase leading to extreme weather

events. Productivity in agriculture is endangered by exposing crops to increased risks of droughts,

rains or other unfavourable conditions which could include also a new range of diseases. Susceptibility

to newly emerging disease agents into the cultivated areas will result in high losses of annual crop

production.

RNA silencing is one of the plants´ natural resistance mechanisms to counteract viral infections.

Viruses have evolved different strategies to overcome RNA silencing and the production of silencing

suppressor proteins appears to be the most successful. In addition, also applications of conventional

breeding methods to increase the resistance in plants and disease control either failed or the resistance

was rapidly overcome by the infecting pathogen. These strategies are often very time-consuming and

natural resistance in germplasm may not be found. Recent advances in molecular biotechnology have

made it possible to obtain and to modify genes useful for generating disease-resistant crops.

Expression of pathogen-derived sequences or anti-pathogen agents has been developed to improve

resistance in transgenic plants. Novel strategies comprise the expression of pathogen-specific

recombinant antibodies, antibody fragments or antibody fusion proteins. Transgenic expression of a

dominant interfering target-specific peptide áptamer` showed a promising novel strategy for

engineering virus-resistant plants [Rudolph et al. 2003]. Aptamers can specifically inhibit the

functioning of the target protein in vivo or inhibit viral replication, independent of sequence homology

but dependent on structurally and functionally conserved domains.

To improve virus resistance in plants a specific small-molecule (e.g. peptide)-based inhibition of viral

silencing suppressor protein actions could be applied. Catalytic sites, DNA and RNA binding domains,

regions involved in protein-protein interaction or homo-/hetero-dimerization could be used as targets

for the inhibition. This strategy could also gain more importance if applied against viruses that failed

to be controlled by other approaches which did not involve specific aptamer expression. Setup of

novel strategies to generate virus-resistant plants is an economical relevant challenge. Implementation

of 3D structure prediction and drug-design coupled with functional analysis of the silencing

suppressor proteins can give new inputs for the development of novel resistance strategies especially

when conventional ones fail and a less controversial solution than the application of viral transgenes is

required.

Here an overview about small-molecules strategies which were successfully applied to improve

resistance in plants is presented.

Reference:

Rudolph, C., Schreier, P. H., and J. F. Uhrig. 2003. Peptide-mediated broad-spectrum plant resistance

to tosposviruses. PNAS 100: 4429-4434.

45

P11

Antagonistic potential of selected isolates and synthetic peptide CAMEL towards plant

pathogen Pseudomonas syringae.

M. Golanowska and S. Jafra

Laboratory of Plant Protection and Biotechnology , Department of Biotechnology, Intercollegiate

Faculty of Biotechnology University of Gdansk and Medical University of Gdansk, Gdansk, Poland

Pseudomonas syringae is known as a plant pathogenic bacterium that may infect various plants. Genus

Pseudomonas syringae is divided into many pathovars that differ by host plant. Pathovars syringae

and morsprunorum are serious pathogens of stonefruit. Nowadays, in stonefruit plant protection,

copper compounds are used which are detrimental not only for plants during fruitification but also for

humans. Finding a biocontrol agent seems to be promising tool in future plant protection.

Twelve bacterial isolates, out of 100, were selected on the basis of antibiosis towards five pathogenic

strains of P. syringae pv. syringae (3 strains) and pv. morsprunorum (2 strains). These isolates were

then identified and subjected to evaluation of siderophore and biosurfactants production, swimming

and swarming motility. Minimal bactericidal concentration (MBC) of synthetic peptide CAMEL was

determined towards pathogenic strains.

Twelve tested isolates were classified to Pseudomonas fluorescens (4 strains), Pseudomonas putida.(6

strains), Delftia sp. (1 strain) and Pantoea sp. (1 strain). The isolate of P. fluorescens 660 specifically

inhibits growth of P. syringae pv. morsprunorum strains but not those of pv. syringae. The remaining

of P. fluorescens and P. putida isolates inhibit growth of all tested pathogenic strains at the similar

level. The analysis of such features as motility and siderophore production indicated that P. putida

strains exhibit the better swimming ability on the medium surface and the higher level of siderophore

production comparing to P. fluorescens isolates. MBC value for all tested pathogenic strains was

evaluated at 1,6 µM concentration. Pathogenic strains exhibit very low motile ability and do not

produce siderophores. However, these pathogens produce similar amount of biosurfactants to selected

isolates.

P. putida and P. fluorescens isolates due to their high motility, siderophore production and ability to

inhibit pathogens growth in vitro can be competitive in inhabitating the same biological niche as

pathogens. Study of pathogenicity inhibition on fruitlets and synergistic activity of CAMEL with

selected isolates will be conducted in the future.

References:

1. Kamysz, W., Krolicka, A,. Bogucka, K., Ossowski, T., Lukasiak, J. and E. Lojkowska 2005.

Antibacterial Activity of Synthetic Peptides Against Plant Pathogenic Pectobacterium Species.

Journal of Phytopathology 153: 313-317.

46

P12

Analysis of the development and functioning of nitrogen-fixing pea nodules using transformed

strains of Rhizobium leguminosarum bv viciae.

Grishina О.А.1,2

, Akhtemova G.А.1, Shtark О.Y.

1, Zhukov V.А.

1, Borisov А.Y.

1, Tikhonovich I.А.

1,2

1All-Russia Research Institute for Agricultural microbiology, Saint-Petersburg, Pushkin, Russia;

2Saint-Petersburg State University, Saint-Petersburg, Russia.

Legume plants have an ability to establish two symbioses with nodule bacteria collectively named

rhizobia and arbuscular mycorrhizal fungi (Glomales) that play an important role in mineral nutrition

of host-plants. High symbiotic effectiveness of these endosymbiotic systems is provided by

development of specialized symbiotic structures and even new plant organs – root nodules (1). The

process of development of those structures is very important for understanding mechanisms of

interactions between partners. Legume-rhizobial symbiosis is one of the most interesting form of

interactions as it is an example of biological nitrogen fixation (2). With the purpose of studying

molecular and genetic mechanisms of legume-rhizobial symbioses we carry out analysis of plant and

bacterial mutants that unable to form the normal nitrogen-fixing nodules (3). Rhizobial strains that

carry reporter genes including gene of β-glucuronidase (gusA) were used for visualisation of bacteria

on various stages of nodule development in plant symbiotic mutants. Strains of Rhizobium.

leguminosarum bv viciae that carry reporter gene gusA were obtained by three-parental crossing

method. They were used for investigation of nodule development on pea (Pisum sativum L.) symbiotic

mutants. It was shown that inoculation of pea mutant line RisFixV (Pssym42) with those strains is

resulted in formation of faulty nodules (4). These nodules were characterized by early senescence and

violation of histological differentiation. It was supposed that pea gene Pssym42 could control plant

cell differentiation in pea nodules.

References:

1. Gianinazzi-Pearson V. 1996. Plant cell responses to arbuscular mycorrhizal fungi: getting to the

roots of the symbiosis // Plant Cell. 8: 1871–1883.

2. Ann M. Hirsch., Haber F., Bosch C. 2001. What makes the Rhizobium-Legume Symbiosis so

special? // Plant Phisiol. 127: 1484–1492.

3. Borisov A.Y., Barmicheva E.M.., Jacobi L.M. et al. 2000. Pea (Pisum sativum L.) mendelian

genes controlling development of nitrogen-fixing nodules and arbuscular mycorrhiza // Czech J.

Genet. Plant Breed. 36: 106–100.

4. Borisov A.Y., Danilova T.N., Koroleva T.A., et al. 2007. Regulatory genes of garden pea (Pisum

sativum L.) controlling the development of nitrogen-fixing nodules and arbuscular mycorrhiza: A

review of basic and applied aspects. APPLIED BIOCHEMISTRY AND MICROBIOLOGY.

43(3):237-243

47

P13

The digestive enzyme inhibitors as an effective tool in restriction of the population size of the

pest Western corn rootworm (Diabrotica virgifera virgifera)

J. Guzik, K. Michalczyk, K. Rozpędek, M. Nakonieczny

University of Silesia, Faculty of Biology and Environmental Protection, Katowice, Poland

Using chemicals in pest control on crops is associated with high economic costs, growing

resistance of the insects to higher and higher doses of chemicals and also with negative effect on other

organisms. Hence, a survey of other natural herbicides is carried out. One of the agents successfully

applied in agriculture, is Bt toxin. The newest trends in scientific investigations of pest control are

usage of natural and synthetic digestive enzyme inhibitors, mainly inhibitors of proteases (PIs).

A new expansive pest of maize crops - Western corn rootworm (Diabrotica virgifera virgifera)

has been noted in Poland since 2005. Only in 2008 in the south-eastern part of Poland more than

30000 individuals of this beetle were collected.

So far the available data on D. virgifera biology are based on the research carried out in North

America, where Diabrotica species are one of the most harmful crop pests. It is related both with great

adaptation ability of Chrysomelids beetles to new environmental conditions, and rapidly appearing

resistance towards new generations of classic herbicides. Obviously those features do not make pest

control in Europe easy. Assuming that to be true that D. virgifera was brought into the Old Continent

the once its population here is highly homogeneous in contrast with populations in North America.

Therefore, the biology of D. virgifera in European conditions should be well recognized, what would

allow to find the most effective methods of controlling this pest in Europe and prevent economical

loss.

One of the factors which determines insects survival in the environment is ability to effective

assimilation of food. Hence, knowledge about specific enzyme activity of D. virgifera midgut in

conditions of feeding in European maize varieties or substitute native plants in absence of the main

host plant is very important.

So far in the midgut of examined insect species confirmed presence of many isoenzymes of

particular proteases which differ in specificity and reactivity to substrate. In order to protease

inhibitors give the highest inhibitory effects they should be selected for particular pest species, also for

D. virgifera. Using PIs might be one of the most promising modern methods of pest control in

agriculture.

References:

1. Bown, D. P., Wilkinson, H. S., Jongsma, M. A., & Gatehouse, J. A. (2004). Characterisation of

cysteine proteinases responsible for digestive proteolysis in guts of larval western corn rootworm

(Diabrotica virgifera) by expression in the yeast Pichia pastoris. Enzyme, 34, 305-320. doi:

10.1016/j.ibmb.2003.11.005.

2. Fabrick, J., Behnke, C., Czapla, T., Bala, K., Reeck, G. R., Rao, A. G., et al. (2002). Effects of a

potato cysteine proteinase inhibitor on midgut proteolytic enzyme activity and growth of the southern

corn rootworm, Diabrotica undecimpunctata howardi (Coleoptera: Chrysomelidae). Insect

Biochemistry and Molecular Biology, 32, 405-415.

3. Wolfson J.L., Murdock L.L. (1990). Diversity in digestive proteinase activity among insects. J.

Chem. Ecol., 16, 1089-1102.

4. Fan S.G., Wu G.J. (2005). Characteristics of plant proteinase inhibitors and their applications in

combating phytophagous insects. Botanical Bulletin of Academia Sinica, 46, 273-292.

48

P14

Biological control for plant protection against soft rot bacteria from the Pectobacterium

carotovorum and Dickeya sp. species.

Jafra S1., Przysowa J

1., Polonis K

1., van der Wolf J

2., Łojkowska E

1.

1Laboratory of Plant Protection and Biotechnology, Intercollegiate Faculty of Biotechnology of

University of Gdansk and Medical University of Gdansk, Gdansk, Poland 2Plant Research International, Wageningen, The Netherlands

Pectinolytic plant pathogenic bacteria belonging to the Dickeya sp. and Pectobacteria genera cause

diseases symptoms on a wide range of economically important vegetable and ornamental crops

worldwide (1). Sspread and development of the disease can be controlled by the use of pathogen-free

propagation material, sanitation of machines and equipments. The use of chemical, physical and

biological control agents may also help to reduce soft rot but no effective preparations have been

found so far.

This study aimed to select and characterize the bacterial isolates from natural environment, capable of

inhibiting the pathogenic activity of plant pathogens from: Pectobacterium atrosepticum (Pa),

Pectobacterium carotovorum subsp. carotovorum (Pcc) and Dickeya sp. (Dsp) species. Selected

bacteria were characterized for their ability to inhibit the pathogens growth – antibiosis; to compete for

iron - siderophore production and to degrade N-acyl homoserine lactones (AHLs), signal molecules

involved in quorum sensing. The in planta pathogenicity assay was performed either on potato tuber

slices or hyacinth bulb tissue discs. The inhibition of tissue maceration caused by Pc or Dsp in the

presence of selected antagonists was evaluated.

Selected isolates were able to inhibit the growth of at least one strain of the tested pathogens in in vitro

assay (50 isolates) or degrade AHL signal molecules important for regulation of pathogenicity

determinant production in Pcc and Pa cells (20 isolates). Most of the isolates inhibiting the growth of

the tested pathogens indicated also a strong siderophore production (35 isolates). The isolates able to

protect the potato tuber tissue against tested pathogens were classified to Pseudomonas and Bacillus

genera and those protecting the hyacinth bulb tissue were classified as Rhanella aquatilis and Erwinia

persicinus species (2). For most of the selected isolates from Pseudomonas, Bacillus and Rahnella

species the antibiosis is a major mechanism involved in antagonism, however in the case of E.

persicinus siderophore production and possibly quorum quenching play an important role. Further

genetic studies are required to unravel the mechanism of the observed antagonism.

We can conclude, that selected isolates could be potentially useful for the control of soft rot disease

caused by bacteria from Pectobacterium and Dickeya genera in potato tuber and hyacinth bulb

production.

Reference

1. Ma, B., Hibbing M.E., Kim H.S., Reedy R.M., Yedidia I., Breuer J., Breuer J., Glasner J.D., Perna

N.T., Kelman A. & Charkowski A.O. 2007. Host range and molecular phylogenies of the soft rot

enterobacterial genera Pectobacterium and Dickeya. Phytopathology 97:1150-1163.

2. Jafra S., Przysowa J., Gwizdek-Wiśniewska A.& van der Wolf J.M.. 2009. Potential of bulb-

associated bacteria for biocontrol of hyacinth soft rot caused by Dickeya zeae. Journal of Applied

Microbiology 106:269-277.

49

P15

The effects of alloxydim on lipoxygenase and acy hydrolases/ phospholipases activity in root tips

of wheat

Katarzyna Jasieniecka, * Walentyna Banas, Antoni Banas

Intercollegiate Faculty of Biotechnology UG-MUG, Kladki 24, 80-822 Gdansk, Poland; * Institute of

Biology, University of Podlasie, Prusa 12, 08-110 Siedlce, Poland

Alloxydim belongs to graminicides, the herbicides active against most of annual and perennial grasses.

Mechanism of action of these herbicides was most intensively studied in the last two decades of the

twentieth century, however it is still controversial how these herbicides annihilate plants. Two

competitive hypotheses have been formulated. The first claims that graminicides inhibits de novo

syntheses of fatty acids and that this is primarily the reason to their phytotoxicity (1). The second one

claim that catabolic reactions are stimulated (2).

Different parts of plant are not equally sensitive to graminicides. Inhibitory effects are primarily and

with highest intensity observed in meristematic and young developing tissues (3). In the presented

study on the effect of alloxydim on lipoxygenase and acyl hydrolases/ phospholipases activity we

chose the root tips of sensitive wheat plant.

For the determination of lipoxygenase activity the crude homogenate of 1-cm root tips were used. The

studied substrates were linoleic (18:2) and linolenic (18:3) acids. The fatty acid hydroperoxide formed

during the incubation time were quantified spectrophotometricaly.

The acyl hydrolases activity was determined in the microsomal preparation of wheat root tips.

Phosphatidylcholine (PC) with radioactive fatty acids ([14

C]18:1, [14

C]18:2, [14

C]18:3, [14

C]ricinoleic

acid) in position sn-2 was added to the microsomal fractions and it was dispersed by sonication in the

presence of CHAPS. After incubation the radioactive products were analysed with LSC.

The activity of lipoxygenase in wheat roots treated with alloxydim was higher than in the control roots

in the period of 12-48 hrs after addition of the herbicides. The highest increase of lipoxygenase

activity towards 18:2 substrate was at the beginning of the treatment (12 and 24 hrs) while towards

18:3 was similarly high during all of the time of the treatment.

The activity of acyl hydrolases/ phospholipases was also increased in the alloxydim treated roots. The

percentage of 18:1, 18:2, 18:3 and ricinoleic acid released from position sn-2 of PC was about 1,5

times higher in the assays with microsomes of alloxydim treated plants compared to the control ones.

The obtained results indicate that in alloxydim treated plants the catabolism of phospholipids could be

increased.

References:

1. Lichtenthaler, H.K. 1990. Mode of action of herbicides affecting acetyl-CoA carboxylase and fatty

acid biosynthesis. Z. Naturforsch., 45c: 521-528.

2.Shimabukuro, R.H., Hoffer, B.L. 1996. Induction of ethylen as an indicator of senescence in the

mode of action of diclofop/methyl. Pestic. Biochem. Physiol., 54: 146-158.

3. Banas, A., Johansson, I., Stenlid, G., Stymne, S. 1993. The effect of haloxyfop and alloxydim on

growth and fatty acid composition of wheat roots. Swedish J. agric. Res. 23: 55-65.

50

P16

Evaluation of metabolites variation in St. John's wort (Hypericum perforatum L.) and rapeseed

(Brassica napus L.) plants

I. Jonuńkienė, S. Sikorskaitė, S. Kuusienė, V. Mickevičius

Kaunas University of Technology, Faculty of Chemical technology, Department of Organic Chemistry,

Radvilėnų pl. 19, LT-50254, Kaunas, Lithuania

Biotechnology is expected to help meet the most urgent global challenges – growing and ageing

populations, limited resources of raw materials, energy and water, the threat of global warming – by

facilitating the development of a sustainable economy built on biobased industrial processes.

Combined with advanced bioprocess engineering the development of high performance crop plants is

the key to this vision becoming reality. Crops will serve as factories for enzymes, amino acids,

pharmaceuticals, polymers and fibres, and will be used as renewable industrial feedstock to produce

biofuels, biopolymers and chemicals [1]. In recent years plant biotechnology has made a tremendous

progress in Lithuania and in the world.

The main goal of research was to optimize suspension cultures growth of St. Joh‗s wort

(Hypericum perforatum L.) and rapeseed (Brassica napus L.) and to increase the amount of

metabolites in plant cultures.

St. Joh‗s wort (Hypericum perforatum L.) is a well known medicinal plant, but the studies of its

pharmocological properties and the corresponding compounds still continues [2, 3]. Plant

biotechnology techniques have been extensively applied to rapeseed (Brassica napus L.) for

improving fodder quality, as well as resistance to diseases and pests.

It was determined, that the most intensive callus genesis of Hypericum perforatum was on

Murashige – Skoog (MS) medium with BAP (2.5 mg/l) and NAA (0.5 mg/l). The amount of

flavonoids (according hyperozide) of Hypericum perforatum callus cultures in MS medium with BAP

(0.2 mg/l) increased 2.7 times in comparison with their amount in MS medium with BAP (2.5 mg/l)

and NAA (0.5 mg/l). The highest amount of nontannin phenolics was in Hypericum perforatum

suspension MS with BAP (2.5 mg/l) and NAA (0.5 mg/l) and jasmonic acid (150 µM). The highest

amount of total phenolics was in Hypericum perforatum suspension MS with BAP (2.5 mg/l) and

NAA (0.5 mg/l) and methyl jasmonate (150 µM).

The highest amount of nontannin phenolics was in Hypericum perforatum suspension MS with

BAP (2.5 mg/l) and NAA (0.5 mg/l) and 1-[4-({3-[4-(4-carboxy-2-oxopyrrolidin-1-yl)phenoxy]-1.4-

dioxo-1.4-dihydronaphtalen-2-il}oxy)phenyl]-5-oxopyrrolidin-3-carboxyacid (100 µM). The highest

total amount of phenolics was in Hypericum perforatum suspension MS with BAP (2.5 mg/l) and

NAA (0.5 mg/l) and N-acetyl-N-(3-chlor-1.4-naphtoquinon-2-yl)-β-alanine (150 µM).

It was determined, that the most intensive callus genesis of rapeseed was on MS medium with

BAP (2 mg/l) and 2.4-D (0.2 mg/l). The highest rapeseed callus biomass was in rapeseed MS

suspension with BAP (0.2 mg/l) and N-acetyl-N-(3-chlor-1.4-naphtoquinon-2-yl)-β-alanine (150 µM).

The highest amount of nontannin phenolics was in rapeseed suspension MS with BAP (0.2 mg/l) and

N-acetyl-N-(3-chlor-1.4-naphtoquinon-2-yl)-β-alanine (150 µM).

References:

1. En Route to the knowledge based bioeconomy 2007. Cologne paper.

2. Gadzovska S., Maury S., Delaunay A. 2007. Jasmonic acid elicitation of Hypericum

perforatum L. cell suspensions and effects on the production of phenylpropanoids and

naphtodianthrones. Plant Cell Tissue Organ Culture 89: 1–13.

3. Pasqua G., Avato P., Monacelli B. 2003. Metabolites in cell suspension cultures, calli, and in

vitro regenerated organs of Hypericum perforatum cv. Topas. Plant Science 165: 977–982.

51

P17

The analysis of geographical populations of Synchytrium endobioticum (Schilb.) Perc. for

virulence and DNA-markers.

A. Khyutti, N. Mironenko and O. Afanasenko

All-Russian research institute for plant protection (SSE VIZR), St. Petersburg-Pushkin, Russia

Synchytrium endobioticum (Schilb.) Perc. is the causal agent of potato wart disease. S. endobioticum

is an obligate plant pathogen that can infect stems, stolons and tubers by means of motile zoospores.

The typical symptom of potato wart disease is the galls (warts). S. endobioticum infects plant tissue

via zoospores and survives as thick-walled sporangia (resting sporangia). Resting sporangia of the

fungus can survive in the soil for up to 40 years. Yield losses can reach 50-100 %.

The purpose of our research was to study the geographical populations of potato wart for virulence and

DNA-markers. Identification of the pathotypes structure for four geographical populations (from

Moscow region, Leningrad region, Belarus and Ukraine) of potato wart on a set consisting of 20

differential cultivars (Alma, Lorkh, Polesskii Rozovyi, Svitanok Kievskii, Lvovskii Belyi, Prolisok,

Lugovskoi, Antares, Ora (Mira), Apollo, Giewont, Fontana, Kardula, Volovetskii, Nezabudka,

Spadshchina, Barbara, Resurs, Temp and Bozhedar) was conducted. All population samples belonged

to the 1(D1) pathotype. Despite this, the different geographical populations had different levels of

aggressiveness to susceptible cultivars. Only the Moscow population infected all susceptible cultivars

(Liza, Lorkh, Polesskii Rozovyi, Tulunskii, Alma and Svitanok Kievskii) more than 80%.

Aggressiveness of the Belarus and Ukrainian populations was less to susceptible cultivars Alma and

Svitanok Kievskii.

Differences between samples of population from Leningrad and Moscow regions, Belarus and Ukraine

were also determined on DNA markers. Genetic variability of four populations of Synchytrium

endobioticum from Moscow and Leningrad regions, Belarus and Ukraine was studied by RAPD and

UP-PCR analyses. There were selected three random and one universal primers which can distinguish

DNA of potato warts and healthy potato. In spite of the fact, that all population samples of S.

endobioticum were tested as pathotype 1D, we found genotypic variability inside and between

populations of pathogen.

The research was supported by a grant from Russian Fund for Basic Research (No. 08-04-00447).

52

P18

Study on molecular basis of pollen beetle’s (Meligethes aeneus) pyrethroid resistance.

D. Kierzek1, P. Wieczorek

1, K. Nowaczyk

1, P. Węgorek

1, J. Zamojska

1, A. Obrępalska-Stęplowska

1

1Institute of Plant Protection, Poznan, Poland

The pyrethroids constitute neurotoxins acting on sodium channels (VDSC) in membranes of neurons

of insect. The toxin keeps the channels in their open state, the nerves cannot de-excite resulting in

paralysis of an insect. The efficiency of pyrethroids is greater when they are applied together with

inhibitor of oxidases piperonyl butoxide (PBO), that weakens detoxification mechanisms (1). Because

incest acquire resistance toward pyrethroids, their affectivity of them become lower every year.

Nowadays are indicated many resistant populations of Meligethes aeneus, the economically important

rape pathogen. In our experiments, we used M. aeneus populations collected from four regions in

Poland. Insects were treated with pyrethroid (esfenvalerate) alone or together with PBO (1, 4).

Afterwards, DNA and RNA were isolated and used in further analyses. Because VDSC are the target

for pyrethroids, we decided to examine the fragment of its gene sequence and its expression level. We

have identified several point mutations. This polymorphism is individually specific rather than

characteristic for populations we can not draw a general conclusion. Furthermore, we have not

observed increase in sodium channels expression that might functionally compensate blocked VDSC.

On the basis of biochemical data, we hypothesised that the oxidase enzymes might take a part in

acquisition of resistance (2, 3) We decided to analyse mutations and changes in expression of

mitochondrial cytochrome oxidase subunits genes (mtCOI, mtCOII). The point mutations in the

sequences seem to not affect protein conformation, but the regulation of expression level of mtCOI

and mtCOII can be important because it increases after addition of insecticide. However, our recent

results show that cytochrome oxidases might not be the only one factor responsible for this

mechanisms The varying crop cultivars as well as the applied insecticides profiles, migration of

insects, and climate changes exert selection pressure on M. aeneus and have an impact for acquisition

of resistance. Observed resistance development seems to be caused by many factors and to be a

dynamic process connected with quick changes related with circumstances mentioned above together

with environmental conditions.

References:

1. Kasai, S. 2004. Role of Cytochrome P450 in Mechanism of Pyrethroid Resistance. J. Pestic. Sci.

29(3):220–221.

2. Wheelock, E. C., Shan, G., Ottea, J. 2005. Overview of Carboxylesterases and Their Role in the

Metabolism of Insecticides. J. Pestic. Sci. 30(2): 75–83.

3. Tan, J., Liu, Z., Nomura, Y., Goldin, A. L., Dong, K. 2002. Alternative Splicing of an Insect

Sodium Channel Gene Generates Pharmacologically Distinct Sodium Channels. The Journal of

Neuroscience 22(13):5300–5309.

3. Pridgeon, J. W., Zhang, L., Liu, N. 2003. Overexspression of CYP4G19 associated with a

pyrethroid-resistant strain of the German cockroach, Blattella germanica (L.). Gene 314: 157– 163.

53

P19

Microtubular rearrangements during cell differentiation in root nodules of Pisum sativum L.

Kitaeva A.B., Tsyganov V.E.

All-Russia Research Institute for Agricultural Microbiology, Saint-Petersburg, Russia

Legumes are characterised by unique ability to establish symbiotic relationships with Rhizobium

bacteria. As a result a new organ, symbiotic nodule, is formed. In this nodule bacteria fix atmosphere

nitrogen and reduce it to ammonia, a well-adapted form for plants.

The object of our research is cell differentiation during nodule development. In response to infection

by Rhizobium bacteria plant cells undergo many cellular changes. We are interested in cytoskeleton

rearrangements during the process of nodule cell differentiation. Our studies are based on genetically

characterised collection of pea symbiotic mutants. We used wild type line SGE and mutant line

SGEFix-2 (sym33), characterised by lack of bacteria endocytosis into the nodule cells.

To study microtubule organosation in nodule cells immunofluorescence for laser scanning confocal

microscopy was used.

In wild type nodule we observed thick and randomly oriented cortical microtubules (MTs) in

meristematic cells. In recently infected cells cytoplasmic MTs are diffuse with short strands. Cortical

MTs are randomly oriented. Long MTs along infection threads (ITs) are observed. Older infected cells

are characterised by long and straight MTs, most part of which are randomly oriented and criss-cross.

Cytoplasmic MTs are short and thick. In uninfected cells MTs are limited to the cortex of the cell, they

are long and transverse to the long cell axis.

In SGEFix-2 (sym 33) nodules in meristematic cells MT organisation was similar to wild type. Central

nodule cells containing thick ITs are characterised with thin and long cortical MTs, MT fluorescence

around the IT is diffuse. MTs of cells containing thin IT are long and think, randomly oriented, some

of them surround IT. Uninfected cells have only long cortex MTs with oblique orientation.

54

P20

Effect of PGPR strains on the nutrients supply into rhizosphere of tomato

I.I. Kostyuk, A.I. Shaposhnikov, L.V. Kravchenko

All-Russia Research Institute for Agricultural Microbiology RAAS

The main goal of our research was to investigate mechanism for adaptation PGPR and plants which

intensify the mobilization partner's genetics resources. The effect of PGPR on the metabolism of

plants resulted by increase the provision of rhizosphere soil organic matter (1). The distinguishing

features of root exudates composition (organic acids, sugars, amino acids) for vegetables (tomato) and

wheat (hexaploid genotypes) under inoculation by Pseudomonas strains were studied. It was shown

that, although active growth of Pseudomonas in rhizosphere, remain levels of organic acids and sugars

in tomato root exudates determined by chromatography were high and in some cases reach amounts

that equivalent sterile plants, but in wheat's root exudates their 100% utilization was shown. Root

exudation of organic acids and sugars of tomatoes in the presence of rhizobacterial strains of

P.fluorescens and P. chlororaphis should be 1.9-8.7 times higher than in sterile plants. As was shown

via chromatography analysis, different groups of amino acids dominated in root exudates of tomato

and wheat. The most differences were between tomato and hexaploid wheat genotype. The growth of

rhizobacteria in rhizosphere induce increasing of amino acids amounts in root exudates. The

specificity in changes of various amino acids exudation that was related with plant genotype and

strain-inoculant was shown.The maintenance of nutrients pool in rhizosphere is one of the factors for

successful metabolic integration between plants and rhizobacteria. In this case high competition ability

of Pseudomonas will serve as advantage that will maintain of high amounts of rhizobacteria and

activity their biocontrol and phytostimulation mechanisms and potential of adaptation for plant-

microbe system will be increased.These findings may indicate that in plant-microbe system metabolic

integration of partners established, and it defined by regulation of the rhizosphere pool of nutrient

compounds due rhizobacteria impact on the system of carbon exudation by plants. This effect may

help preserve fertility and productivity of agrophytocenoses.

References:

1. Broeckling, C. D., Broz, A. K., Bergelson, J., Manter, D.K., & Vivanco, J.M., 2008. Root

exudates regulate soil fungal community composition and diversity. Applied and

Environmental Microbiology, Feb. 2008:p. 738 – 744.

55

P21

The occurrence, characteristic and diagnostic of bacterial diseases of maize in Poland

K. Krawczyk, A. Zwolinska, J. Kamasa and H. Pospieszny

Institute of Plant Protection – National Research Institute, Poznan, Poland

Climate warming that we can observe in the last few years is the reason of warmer and mild winters

and more humid and rainy summers in Poland. Such environmental conditions are favourable for

bacterial infections of plants in general. In the last decade the acreage and agricultural meaning of

maize as crop in Poland is increasing. Maize is grown for production of grain, fodder and energetic

purposes. In Polish scientific literature bacterial maize diseases are not described yet. There is only

one publication concerning bacterial diseases of maize and one EPPO report concerning the presence

of bacteria on maize plants. Field observations for occurrence of maize bacterial pathogens are

continued in Virology and Bacteriology Department since 2007. Maize plant samples are being

collected every summer (from June till August) from different localizations in Poland. A collection of

170 isolates fulfilling Koch`s postulates (including reisolation) was founded and characterized using

pathogenicity, biochemical, serological and molecular tests. Three maize pathogenic bacteria species,

not described in the Polish scientific literature were isolated, characterized and identified as: Pantoea

ananatis (leaf spot disease) (3), Enterobacter cloaceae subsp. dissolvens (bacterial stalk rot) (1) and

Pantoea agglomerans (leaf blight and vascular wilt) (2). In the tested plant samples we also detected

the presence of bacteria not described in the scientific literature as maize pathogens however being a

pathogens of other plants: Pseudomonas fluorescens (tomato), Erwinia cypripedii (many hosts),

Erwinia pyrifoliae (pear tree).Classification and identification of bacteria is often a hard and

complicated process. That is why experiments concerning developing faster, more specific and reliable

protocols for molecular detection methods of mentioned pathogens are in progress.

References:

1. Hoffmann H., Stindl S., Ludwig W., Stump A., Mehlen A., Heesemann J., Monget D., Schleifer K.,

Roggenkamp A. 2005. Reassignment of Enterobacter dissolvens to Enterobacter cloacae as E.

cloacae subspecies dissolvens comb. nov. and emended description of Enterobacter asburiae and

Enterobacter kobei. Systematic and Applied Microbiology 28:196–205.

2. Morales-Valenzuela G., Silva-Rojas H.V., Ochoa-Martínez D., Valadez-Moctezuma E., Alarcón-

Zúńiga B., Zelaya-Molina L.X., Córdova-Téllez L., Mendoza-Onofre L., Vaquera-Huerta H.,

Carballo-Carballo A., Farfán-Gómez A., Ávila-Quezada G. 2007. First Report of Pantoea

agglomerans Causing Leaf Blight and Vascular Wilt in Maize and Sorghum in Mexico. Plant Dis.

91:1365. doi:10.1094/PDIS-91-10-1365A.

3. Paccola-Meirelles L.D., Ferrera A.S., Meirelles W. F. Marriel I.E., Casela C.R. 2001. Detection of a

bacterium associated with a leaf spot disease of maize in Brazil. Journal of Phytopathology 149:275-

279.

56

P22

Sphagnum-associated microbial communities: biodiversity as key to the ecological role

A. Krikovtseva1, M. Cardinale

1, C. Berg

2, I. Tikhonovich

3, W. Chebotar

3, and G. Berg

1

1Graz University of Technology, Environmental Biotechnology, Petersgasse 12, 8010 Graz, Austria,

email: [email protected] 2Institute of Plant Sciences, Karl-Franzens-University, Holteigasse 6, 8010 Graz, Austria

3All-Russia Research Institute for Agricultural Microbiology, Shossee Podbelskogo 3, St. Petersburg,

Russia

The bryophyte genus Sphagnum is a dominant component of wetlands vegetation being of high

importance for our world climate. Recently it was shown that Sphagnum mosses are colonized in high

abundances with special microbial communities, which carry out important functions for the moss

plantlets as well as for the whole bog ecosystem [1, 2, 3]. To make an insight into the ecological role

of Sphagnum-associated microbial communities, our research was focused on phylogenetic diversity,

spatial structure and host-specificity of them. Gametophytes of Sphagnum fallax and Sphagnum

magellanicum were collected from three different peat bogs in Austria. The Bacteria community

composition was investigated by reconstruction of 16S rRNA gene clone libraries. Up to 97% of all

cloned sequences were affiliated to uncultivable microorganisms. Taxonomic position and ratio of the

most abundant groups differ between moss species. Bacterial clones and closely related

microorganisms from the databases represent remarkable physiological functions, especially, nitrogen

fixation, and adaptation to acidic environments. Fluorescent rRNA-targeted probes specific for

Alfaproteobacteria (ALFA968) and Planctomycetes (PLA46 and PLA886), presented in both moss

species, were used to detect microorganisms in situ. Confocal laser scanning microscopy (CLSM)

revealed colonization of hyaline cells of branch and stem leaves as well as outer cortex of stems by

microcolonies and chains of coccoid or rod-shaped bacterial cells. Bacteria were also found to be

associated with fungal hyphae and algal cells. For study of communities` specificity we performed a

single strand conformational polymorphism (SSCP) fingerprinting with universal primers. SSCP gels

showed high diversity of 16S rRNA fragments, meanwhile computer-assisted analysis elicited host-

specificity of the community content. To conclude, Sphagnum-associated microbial communities

represent a unique pool of high diverse and host-specific uncultivable microorganisms, habituating

appropriate for water and nutrient supply moss tissues and possessing complex metabolic relationships

within the communities.

References:

[1] Opelt, K., Chobot, V., Hadacek, F., Schönemann, S., Eberl, L. & Berg, G. 2007. Investigations of

the structure and function of bacterial communities associated with Sphagnum mosses. Environ.

Microbiol. 9: 2795–2809.

[2] Opelt, K., Berg, C., Schönmann, S., Eberl, L. & G. Berg. 2007. High specificity but contrasting

biodiversity of Sphagnum-associated bacterial and plant communities in bog ecosystems independent

of the geographical region. The ISME J. 1: 502-516.

[3] Opelt, K., Berg, C. and G. Berg. 2007. The bryophyte genus Sphagnum is a reservoir for powerful

and extraordinary antagonists and potentially facultative human pathogens. FEMS Microb. Ecol. 61:

38-53.

57

P23

Molecular markers employment for identification of oat hybrids (Avena sativa L. × Avena fatua

L.) that vary in grain and groat characteristics

Katarzyna Kruk

University of Life Sciences in Lublin, Institute of Plants Genetics, Breeding and Biotechnology,

Poland

Oat is a cereal of a unique composition of nutritional components, it is also used as a fodder or as an

ingredient for production non-dietary products, for example cosmetics (1). One of methods of creating

new varieties is crossing oat (Avena sativa L.) with wild oat species and this may lead to generating

hybrids with new, desirable features (2) but selection and backcrossing must also take place in order to

dispose hybrids of undesirable traits (3). In this work 4 Polish oat varieties were crossed with 4 wild

Avena fatua L. accessions and six F2 hybrid combinations were received. RAPD and ISSR markers

were utilized to identify whether these combinations were real hybrids. Every polymorphic band

detected on gels for hybrid and

A. fatua accession but not for A. sativa variety confirmed that the real hybrids were obtained.

21 RAPD bands received with 15 primers and 23 ISSR bands received with 13 primers satisfied these

requirements. Grain and groat characteristics of all hybrids and both parental components were

evaluated. Two hybrids ‗German‘ × AVE 2804 and ‗Hetman‘ × AVE 1322 had significantly higher

number of spiklets and kernels per main panicle than their maternal forms. These two combinations

had also higher weight of groat per main panicle compared with maternal varieties but the difference

was not significant for this feature. All hybrids but ‗Rajtar‘ × AVE 2804 had significantly smaller

weight of 1000 kernels than maternal components but also significantly higher than paternal

components, which means that hybrid kernels were smaller than these of varieties but greater than

those of A. fatua accessions. The hybrids did not differ significantly from A. sativa for hull content but

it was significantly lower compared with paternal forms. Because spiklet fertility was similar for all

parental components, the hybrids did not differ from each other and from their parents. All A. fatua

accessions are distinguished by higher grain protein and fat percentage than A. sativa and all hybrid

combinations had more of these elements than oat varieties but less than wild oat accessions, but the

differences were not always significant. The observed large diversity of traits is of great advantage

when selection of valuable forms for oat breeding is made.

References:

1. Hoffman L.A. 1996. World production and use of oats. The Oat Crop. Editor: Welch R.W.

Chapman & Hall, London: 34-61.

2. Loskutov I.G. 2001. Interspecific crosses in the genus Avena L. Russian Journal of Genetics 37:

467-475.

3. Luby J.J., Stuthman D.D. 1983. Evaluation of Avena sativa L./A. fatua L. progenies for agronomic

and grain quality characters. Crop Science 23: 1047-1052.

58

P24

Assesment of biocontrol potencial of Bacillus sp. rhizosphere isolates against plant soft rot

pathogens from genera Dickeya and Pectobacterium

M. Potrykus1, S. Jafra

1 and D. Krzyzanowska

1

1 Intercollegiate Faculty of Biotechnology University of Gdansk-Medical University of Gdansk,

Laboratory of Plant Protection and Biotechnology, Gdansk, Poland

Bacterial plant pathogens from genus Pectobacterium are well documented causatives of black leg and

soft rot disease of potato in worldwide (Ma et al., 2007). In recent years however pectinolytic bacteria

from genus Dickeya, formerly problematic only in warmer climate, were detected in symptomatic

potato plants in in many European countries including Poland (Slawiak et al., 2008). Taking into

account the present and the potential losses in potato crops, we have screened bacterial isolates from

the rhizosphere of tomato and beet root in search of potential biocontrol agents against Pectobacterium

atrosepticum (Pba), Pectobacterium carotovorum subsp. carotovorum (Pcc) and Dickeya sp. (Dsp).

The research was focused on bacteria from genus Bacillus, which already used in agriculture as

antifungal agents. Heat resistant, spore forming isolates were screened for their ability to either inhibit

the growth of tested pathogens or inactivate signal molecules involved in the production of their

virulence determinants. One growth inhibitory strain identified as B. subtilis and six N-hexanoyl-

homoserine lactone (C6-HSL) degrading strains belonging to Bacillus cereus group were chosen for

further study. The ability of selected isolates to inhibit maceration of plant tissue was assessed using

potato slice assay. Traits potentially responsible for their ability to compete with plant pathogens were

investigated, including motility, production of siderophores, biosurfactants and presence of aiiA gene

encoding for the AHL lactonase. All Bacillus sp. isolates deprived of the ability to inhibit pathogens‘

growth, yet capable of attenuating soft rot symptoms caused by either Pba, Pcc or Dsp, possess the

aiiA gene and are highly motile. AiiA was lately suggested to play more complex than AHL

degradation role in the metabolism of Bacillus thuringensis (Park et al., 2008). Further research needs

to be conducted to confirm the role of the protein in obtained Bacillus sp. isolates and to asses the

contribution of other environmental fitness-related factors to their soft rot attenuating potential. As the

efficiency of biocontrol agents may vary depending on the experimental conditions used, in vitro and

tissue maceration assays need to be verified by greenhouse experiments.

References:

1. Ma, B., Hibbing M.E., Kim H.S., Reedy R.M., Yedidia I., Breuer J., Breuer J., Glasner J.D., Perna

N.T., Kelman A. & Charkowski A.O. 2007. Host range and molecular phylogenies of the soft rot

enterobacterial genera Pectobacterium and Dickeya. Phytopathology 97:1150-1163.

2. Park S.J., Park S.Y., Ryu C.M., Park S.H. & Lee,J.K. 2008. The role of AiiA, a quorum-quenching

enzyme from Bacillus thuringiensis,on the rhizosphere competence. Journal of Microbiology and

Biotechnology 18: 1518-1521.

3. Slawiak M., Lojkowska E. & van der Wolf J.M. 2008. First report of bacterial soft rot on potato

caused by Dickeya sp. (syn. Erwinia chrysanthemi) in Poland. New Disease Reports 18.

59

P25

The main factors making play on hybrid aspen microshoots adaptation ex vitro on a mass scale

clones production

S. Kuusienė¹², J. Ņiauka¹², V. Grunskis², S. Lenortavičiūtė² and M.Ńilininkas²

¹Laboratory of forest plant biotechnology, forest research institute, Agro and forest research centre,

Liepu 1, Girionys, Kauno reg. Lithuania

² Forest biotechnology enterprise ― Euromediena ― , Trilińkės, Kaińiadorių reg., Lithuania

Micropropagation has been extensively used for rapid multiplication of many plant species including

forest trees too. Tissue culture has been investigated for rapid propagation of superior trees as well as

uniform planting material. Micropropagation of hybrid aspen (P. tremuloides X P. tremula) is

achieved through the establishment of explants, their initial growth in tissue culture and regeneration

to microshoots. After ex vitro transfer, microshoots might easily be impaired by sudden changes in

environmental conditions and so need a period of adaptation. The successful ex vitro acclimatization

of micropropagated plants determine the quality of the end –product- seedlings. Desirable result can

be achieved by careful environmental control during acclimatization. The goal of this study is to

determine the influence of the main factors as temperature, pH of watering solution, concentration of

growth regulators and spectrum of lighting on microshoots growth during adaptation period. The

hardening microcuttings of hybrid aspen in low temperature + 4°C for 12 h before transplantation to

acclimatization conditions had a positive effect during adaptation ex vitro. The reaction of genotypes

to various temperature slightly differed. The nutritional solutions which pH 5,5 -6,0 had the best effect

on plantlets development because pH similar to pH of medium for microshoots regeneration in vitro.

The growth regulators PBZ and ABA had positive effect on adaptable microshoots ex vitro too. LEDs

spectrum and light had special effects on microcuttings rooting and survival. The results of

experiments are valuable information on mass scale multiplication clones of hybrid aspen different

genotypes.

References:

1. Pospisilova J., Ticha I., Kadlecek P., Haisel D., d Plzakova S. 1999. acclimatization of

micropropagated plants to ex vitro conditions. Biologia plantarum 42 (4): 481 – 497.

2. Preece, J.E., Sutter, E.G.: Acclimatization of micropropagated plants to the greenhouse and

field. - In: Debergh, P.C., Zimmerman, R.H. (ed.): Micropropagation. Technology and

Application. Pp. 71-93. Kluwer Academic Publishers, Dordrecht - Boston - London 1991.

3. Yu Q. (2001) Selection and propagation of hybrid aspen clones for growth and fibre quality.

Academic dissertation. University of Helsinki, Department of Applied Biology.

4. 4.Ņiauka J., Kuusienė S. 2009. Different inhibitors of the gibberellin biosynthesis pathway

elicit varied responses during in vitro culture of aspen (Populus tremula

L.)http://www.springerlink.com/content/0t1001ut187nj747/; DOI 10. 1007/s11240-010-9725-

5

60

P26

Combined bioaugmentation and biostimulation to treatment wastewater contaminated with high

concentrations of FOGs (Fats, Oils and Greases)

A. Wiechetek, K. Miksch

The Silesian University of Technology, Gliwice, Poland

Fats, oils and greases (FOGs) are typically contained in wastewater generated from diaries,

slaughterhouses, kitchens and restaurants. High concentration of these compounds in wastewater often

causes a major problem in biological wastewater treatment processes. They tend to clump together and

clog drain lines. FOGs can form oil films on the surface of activated sludge flocs, preventing from the

diffusion of oxygen. Furthermore, lipids promote growth of filamentous microorganisms, which cause

bulking and foaming (1, 2).

FOGs can be removed from wastewater by physical or chemical methods . However, lipids can pass

through physicochemical treatment processes contribute to the levels of BOD and COD

(biological/chemical oxygen demand) in the effluents. Therefore, biological treatment processes and

their improvements, like bioaugmentation and biostimulation, are commonly used to remove FOGs

from lipid-rich wastewater (3, 4).

Bioaugmentation is a method for enhancing biodegradation of lipids by addition of microorganisms

(indigenous or genetically modified) or enzyme supplements for treatment of wastewater. Whereas,

biostimulation involves the modification of the environment to stimulate existing bacteria capable of

hydrolyzing the lipids.

This research focused on the influence of bioaugmentation and biostimulation on FOGs removal from

wastewater. Two bioreactors were used: without (A) and with (B) microbial supplement (PBA

7002.052, APB Environnement, Fontenay le Vicomte, France). Polysorbate 80 (commercially also

known as Tween 80), a nonionic surfactant and emulsifier, was used for biostimulation. The reactors

were fed with a synthetic medium and the activated sludge was adapted for the removal of high

concentration of lipids (0.25 - 1 g l-1

rapeseed oil; 85 - 500 mg COD VSS-1

d-1

).

The study demonstrated no significant difference in COD removal efficiency between the two reactors.

Efficiency of COD removal depended on lipids load to the reactors and was within the range of 56-97%

and 63-96% for reactor A and B, respectively.

To provide a maximum surface for hydrolytic cleavage by lipases or phospholipases, solid fats, oils or

greases must be emulsified (5). The addition of surfactant (Tween 80) caused an increase of oil

dispersion (5 – 10 μm particle size). Controlling the oil particle size could be positive influence on the

wastewater treatment process.

References:

1. Dueholm, T.E., Andreasen, K.H., Nielsen, P.H. 2001. Transformation of lipids in sctivated sludge.

Water Science of Technology 43:165–172.

2. Martins, A.M.P., Pagilla, K., Heijnen, J.J., van Loosdrecht, M.C.M. 2004. Filamentous bulking

sludge – a critical review. Water Research 38:793–817.

3. Chipasa, K.B., Mędrzycka, K. 2006. Behavior of lipids in biological wastewater treatment

processes. Journal of Industrial Microbiology and Biotechnology, 33:635–645.

4. Brooksband, A.M., Latchford, J.W., Mudge, S.M. 2007. Degradation and modification of fats, oils

and grease by commercial microbial supplements. World J. Microbiol. Biotechnol 23:977–985.

5. Matsui, T., Miura, A., Iiyama, T., Shinzato, N., Matsuda, H., Furuhashi, K. 2005. Effect of fatty oil

dispersion on oil-containing wastewater treatment, Journal of Hazardous Materials B118:225–258

Acknowledgements: The research was supported by the Polish Ministry of Science and Higher

Education, Grant. No. N N523 410735

61

P27

Genetic variability in the physiological responses of Andean lupin to drought stress

Clara Lizarazo, Frederick Stoddard, Pirjo Mäkelä and Arja Santanen

Department of Agricultural Sciences, PL 27 (Latokartanonkaari 5), 00014 University of Helsinki

Drought is one of the most important abiotic stresses that causes significant reductions in crop yield,

and thus hinders the food security of the growing world population. In consequence, it is urgent to

select crops able to resist drought, maintain high yield and have a good nutritive content. The purpose

of this project was to evaluate the responses of different accessions of Andean lupin to drought stress,

and identify if there are significant differences in their physiological responses.

To identify germplasm for further investigation, thirty accessions of Lupinus mutabilis Sweet. and one

accession of L. albococcineus Hort. were screened in two sets, A of 15 and B of 16 accessions. From

these sets, four lines were chosen on the basis of extreme values in the measurements, and this set of

four was investigated in depth (set C). Water stress consisted of controlled water loss from the soil (at

2% per day). The screening techniques used were: leaf temperature, stomatal conductance, relative

water content, water potential, ion membrane leakage, and shoot dry weight were measured and

transpiration efficiency was calculated. In addition, carbon isotope discrimination, root length, root dry

matter, proline content and soluble sugar content were included in only set C.

Based on the results from set A and set B, accessions PI 457981, PI 457972, and AC 2792 were

considered as being drought resistant and PI 510572 was considered as drought sensitive. However,

the results from set C showed that accession PI 510572 is able to adapt its cell membrane to the

drought stress so that its ion leakage is less than in the other accessions, and in addition it has the

ability to accumulate higher concentrations of osmoprotectants.

Thus, this survey identified that some accessions of Andean lupin were able to avoid drought stress

through stomatal traits and root traits, and others that were able to tolerate drought through the

accumulation of osmotically active substances. Consequently, there are good prospects for breeding of

Andean lupin to improve its drought resistance.

uld assist in biodiesel/bioenergy production at a regional level. This study is relevant to other

European countries that cultivate similar crops and like Ireland, are facing multiple challenges to their

tillage sector in the near future.

62

P28

Seasonal fluctuations of bacterial communities and their influence on cyanobacterial blooms in

inland water reservoirs

W. Mazur 1, J. Barylski

1, B. Messyasz

2, M. Gabka

2, A. Rybak

2, L. Burchardt

2, A. Gozdzicka-

Jozefiak 1

1 Department of Molecular Virology, Institute of Experimental Biology, Faculty of Biology, Adam

Mickiewicz University, Umultowska 89, 61-614 Poznan, Poland 2 Department of Hydrobiology, Institute of Environmental Biology, Faculty of Biology, Adam

Mickiewicz University, Umultowska 89, 61-614 Poznan, Poland

The organic matter cycle and its decomposition in water is the most important function of the

microbial community in lakes. Most bacteria are not only the nutriment for other organisms in the

water reservoir, but also are the contamination indicators.

Therefore, the study of bacterial composition and diversity is important to understand the community

structure and interactions with cyanobacterial blooms.

The aim of the study was to explore and characterize bacterial community composition and estimate

relations between groups of microorganisms and its correlation with cyanobacterial blooms in

Goreckie Lake.

Water samples were taken in vertical profile of pelagic zone and horizontal profile in littoral zone

from January to December 2009. Tests taken from horizontal profile were collected at eight stations

along the southern shore.

The different bacteria strains were isolated on MacConkey medium and Water Plate Count Agar

Medium inoculated with water samples.

Each isolate was purified by sub-culturing onto Lysogeny Broth medium and analyzed by biochemical

Analytical Profile Index test and sequencing of PCR-amplified 16 SrRNA with universal bacterial

primer set.

The antagonism between cyanobacteria and chosen species of bacteria was researched by inoculating

the Cylindrospermopsis raciborskii culture with different a concentration of bacteria Hafnia alvei

culture. After the 12-hour incubation the chlorophyll level was measured in respect of control sample.

In those samples of Cylindrospermopsis raciborskii culture where the concentration of added

Hafnia alvei culture was higher, the decrease in the chlorophyll level was observed when compared to

the control sample. Probably the growth of Cylindrospermopsis raciborskii was slowed by substances

produced by Hafnia alvei. However, this influence can be short-termed, and the cyanobacteria can

activate defense reaction, effecting its mass expansion.

That is why the relations between the bacteria and cyanobacteria should be thoroughly investigated.

63

P29

Increased effectiveness of chickpea (Cicer arietinum L.) symbiotic systems

M. Naumkina

All-Russia Research Institute of Legumes and Groat Crops, Orel, Russia

Development and application of environmental friendly systems of agriculture, obtaining of

ecologically clean food products is one of the most important trends of contemporary agriculture.

Decrease of application of chemical fertilizers evokes interest for use of biopreparations in agricultural

technologies that in complex with other methods can provide for realization of genetically conditioned

potential of chickpea genotypes (Cicer arietinum L.). At the same time preference must be given to the

preparations, capable of influence of efficiency of symbiosis, causing visible changes in growth and

development of plants (1). Application of new forms of microbiologic preparations on the basis of

rhizobia strains and arbuscular mycorrhiza (AM) will afford to decrease costs for application of

fertilizers. This measure will decrease costs of chickpea grain production and will make it ecologically

clean.

The goal of the present work was research of influence of new complex of microbiologic preparations

on efficiency of chickpea plants without application of fertilizers on dark grey forest soils.

Research object were 9 chickpea genotypes, obtained from Vavilov‘s All-Russia Research Institute

(St-Petersburg): k-494 (Cuba); k-526 (Columbia); k-997, k-998, k-1507 (India); k-1029 (Ethiopia); k-

1189 (Russia); k-1684, k-1786 (Pakistan). Complex microbiologic preparations produced by the firm

BisolbiInter (St-Petersburg): BisolbiSun and BisolbiMix were used in the experiments. Scheme of

testing included the following variants: control (without application of fertilizers and microbiological

preparations); BisolbiSun (seed inoculation in ratio 0,2 l/t of seeds); BisolbiMix (pre-sowing

application – 500 kg/ha); BisolbiSun (seed inoculation in ratio 0,2 l/t of seeds)+ BisolbiMix (pre-

sowing application – 500 kg/ha); half of dose of fertilizer; complete dose of fertilizer.

It was determined that inclusion of complex microbiologic preparations into technology of cultivation

of chikpea (Cicer arietinum L.) is not only a very effective way of increase of its productivity, but also

a very important source of improvement of grain quality. Preparations BisolbiSun and BisolbiMix

increase field germination rate of chickpea on 6,2…11,4%, and survival rate of plants – on 5,0…8,9%.

Treatment of chickpea seeds with preparation BisolbiSun (0,2 l/t) and pre-sowing application of

BisolbiMix into soil (500 kg/ha) extends vegetation period of plants in comparison to control on 5…7

days. Application of complex microbiologic preparations increases foliar apparatus and root system of

chickpea on 6,3…14,1% and 5,8…12,1% towards control. Pre-sowing application of BisolbiMix into

soil affords to increase biologic fixation of atmospheric nitrogen of chickpea plants on 18…24% due

to increase of number of active root rhizobia. Complex microbiologic praparations BisolbiSun and

BisolbiMix increase productivity of chickpea plants in comparison to standard on 0,5…0,8t/hа.

References:

1. Chebotar, V.K. 2007. Efficiency of application of biopreparations Extrasol 320:16

64

P30

Cloning of symbiotic genes of Pisum sativum L. Sym40 and Sym42, involved in symbiosome

formation and maintenance

T. Nemankin1,2

, E. Ovchinnikova1,2

, V. Zhukov1, A. Borisov

1, E. Limpens

2, R. Geurts

2, I.

Tikhonovich1

1 Laboratory of Genetics of Plant-Microbe Interactions, All-Russia Research Institute for Agricultural

Microbiology RAAS, St.-Petersburg, Russia,

2 Department of Molecular Biology, Wageningen University, Wageningen, The Netherlands.

Research on the molecular-genetic basics of plant-microbial interaction during legume-rhizobium

nitrogen-fixing symbiosis development is going on for decades, but the mechanisms of intracellular

accommodation of Rhizobium bacteria as nitrogen-fixing organelles, called symbiosomes, inside plant

root nodule cells are still poorly understood. Application of forward genetics approaches allowed the

identification of about 40 pea genes (more than 150 independently obtained mutants) affected in

endosymbioses development and functioning. It is currently the most extensive and best-characterized

(at morphological level) set of mutants disturbed in nitrogen symbiosis development in all legumes,

including different stages of symbiosome formation and maintenance. Mutants in two pea genes,

Sym40 and Sym42, impaired in uptake of the bacteria into the nodule cells and maintenance of

symbiosomes, respectively, have been obtained by EMS-mutagenesis and characterized at the

morphological level (1,2).

However, map-based cloning strategies in pea are hampered by the large genome-size and lack of

genomics tools. Making use of synteny between crop and model legumes is the key approach for

cloning pea genes. To exploit genome synteny, it is necessary to localize genes of interest precisely on

the genetic map of pea and then try to find candidate genes in the syntenic region of a model legume.

In this work, a set of gene-based molecular cross-species markers has been created, destined for

cloning pea genes on the basis of synteny between pea and the model legume Medicago truncatula.

This set of markers is being used to map the pea genes Sym40 and Sym42, involved in pea genetic

control of endosymbioses development and functioning. Further studies will lead to the cloning of

these genes and identification of their function at the molecular level.

This work was supported by the grants of RFBR (09-04-00907, 09-04-13895, 09-04-91054, 09-04-

91293, 10-04-00961, 10-04-01146), NWO 047.018.001, Grant to support leading Russian science

school 3440.2010.4, Governmental contracts for research with Russian Federal Agency of Science and

Innovations (02.512.11.2280, 02.740.11.0276).

References:

1. Tsyganov V.E., Morzhina E.V., Stefanov S.Y., Borisov A.Y., Lebsky V.K., Tikhonovich I.A. 1998.

The pea ( Pisum sativum L.) genes sym33 and sym40 control infection thread formation and root

nodule function . Mol Gen Genet 259:491-503.

2. Morzhina E.V., Tsyganov V.E., Borisov A.Y., Lebsky V.K., Tikhonovich I.A. 2000. Four

developmental stages identified by genetic dissection of pea (Pisum sativum L.) root nodule

morphogenesis. Plant Science 155:75–83.

65

P31

Ecosystem services of soil microbial communities – developing sustainable cultivation methods

for agriculture

A. Palojärvi1, T. Sipilä

2, K. Yrjälä

2, A. Hatakka

3 and L. Alakukku

4

1MTT Agrifood Research Finland, Plant Production Research, Jokioinen, Finland;

2Department

of Biosciences, University of Helsinki, Finland; 3Department of Food and Environmental

Sciences, University of Helsinki, Finland; 4Department of Agricultural Sciences, University of

Helsinki, Finland

Agriculture is facing major environmental challenges due to climate change. Erosion and nutrient

loading to waterways is increasing, if autumns get wetter and winters milder as predicted. Crop cover

protects the soil surface reducing the risk of erosion and nutrient leaching. The remaining crop

residues may, however, hamper agriculture and create optimal conditions for soil and residue born

crop pathogens. Tillage, crop rotation and practices to increase organic matter in soils are shown to be

beneficial for microbes suppressive on pathogenic fungi, but detailed mechanisms and the relationship

between general disease suppression and agrotechnological practices are not fully understood.

The aim of the project is to develop innovative and feasible agro-environmental technology for

improvement of environmental sustainability of cultivation methods. This will be achieved by

increasing soil crop cover taking advantage of ecosystem services of soil microbes to suppress crop

pathogens as an alternative system for chemical control, and to optimal degradation of crop residues.

Multidisciplinary research consortium consisting of researchers from MTT and the University of

Helsinki in environmental technology in agriculture, environmental biotechnology, biology and

microbiology will exploit long term field experiments of MTT on tillage intensity and crop rotations.

Automatic techniques with continuous real-time measurement of soil and weather parameters all year

round will be combined with cultivation method and microbiological studies outside growing season.

Microbial communities in agricultural soils will be evaluated by massive parallel sequencing. A

physical based model will be constructed and used to examine the effects of crop cover on conditions

in soil-air –interface. A system‘s approach is needed to find out the best agrotechnological possibilities

to enhance the occurrence and performance of soil microbes capable of suppressing soil and crop

residue born plant pathogens. Results can be used to develop sustainable cultivation methods and

commercial microbial products, which both enhance the adaptation of agriculture to the climate

change. The presentation will give an overview of the project.

66

P32

Effects of sugar beet chitinase IV on root-associated fungal community of transgenic silver birch

in a field trial

H-L. Pasonen1, J. Lu

1, A-M. Niskanen

1, S-K. Seppänen

1, A. Rytkönen

2, J. Raunio

3, A. Pappinen

4, R.

Kasanen1 and S. Timonen

5

1Department of Forest Sciences, P.O. Box 27, FIN-00014 University of Helsinki, Finland

2Finnish Forest Research Institute, P.O. Box 18, FIN-01301 Vantaa, Finland

3Water and Environment Association of the River Kymi, Tapiontie 2 C, FIN-45160 Kouvola, Finland

4University of Joensuu, Faculty of Forest Sciences, P.O. Box 111, FIN-80101 Joensuu, Finland

5Department of Biological and Environmental Sciences, P.O. Box 56, FIN-00014 University of

Helsinki, Finland

Heterogenous chitinases have been introduced in many plant species with the aim to increase the

resistance of plants to fungal diseases. We studied the effects of the heterologous expression of sugar

beet chitinase IV on the intensity of ectomycorrhizal (ECM) colonization and the structure of fungal

communities in the field trial of fifteen transgenic and eight wild-type silver birch (Betula pendula

Roth) genotypes. Fungal sequences were separated in denaturing gradient gel electrophoresis (DGGE)

and identified by sequencing the ITS1 region to reveal the operational taxonomic units (OTUs). ECM

colonization was less intense in seven out of fifteen transgenic lines than in the corresponding non-

transgenic control plants but the slight decrease in overall ECM colonization in transgenic lines could

not be related to sugar beet chitinase IV expression or total endochitinase activity. One transgenic line

showing fairly weak sugar beet chitinase IV expression without significantly increased total

endochitinase activity differed significantly from the non-transgenic controls in the structure of fungal

community. Five sequences belonging to three different fungal genera (Hebeloma, Inocybe, Laccaria)

were indicative of wild-type genotypes, and one sequence (Lactarius) indicated one transgenic line. In

cluster analysis, the non-transgenic control grouped together with the transgenic lines indicating that

genotype was a more important factor determining the structure of fungal communities than the

transgenic status of the plants. With the tested birch lines, no clear evidence for the effect of the

heterologous expression of sugar beet chitinase IV on ECM colonization or the structure of fungal

community was found.

67

P33

Study of the regenerative ability of plants of the genera, which were found sequences

homologous to the T-DNA of agrobacteria.

O.A. Pavlova, T.V. Matveeva and L.A. Lutova

Saint-Petersburg State University, Saint-Petersburg, Russia.

To date it is known that some species of the genus Nicotiana (White et al, 1984) contain in their

genomes sequences homologous to the fragment of the Agrobacterium strain, termed T-DNA. In our

laboratory was show that some species of the genus Linaria contain such sequences too. Agrobacteria

have ability to transfer T-DNA into the plant genome due to the agrotransformation. T-DNA-like

sequences appears to be integrated into the Nicotiana and Linaria genomes and maintaned there after

agroinfection during evolution of these genera. Plants ability to form shoots-regenerants after

agrotransformation should played important role for maintaining introduced sequences. Moreover, it is

important, which morphogenetic reactions predominate during regeneration and in addition whether is

there a relationship between the presence of T-DNA-like sequences in the plant genome and its ability

to regenerate.

Based on this statement, we had made an attempt to characterize the ability to regenerate of Nicotiana,

Linaria species carrying T-DNA-like sequences, and without these sequences.

We had analized regeneration ability of species L.purpurea (L.) Mill., L. aeruginea (Gouan) Cav., L.

maroccana (Hook.), L. vulgaris L., N. tabacum L., N. gossei Domin , N. suaveolens Lehm., N. rustica

L., N. langsdorffii Weinm. in vitro on Murashige-Skoog media with the addition of synthetic

phytohormones: naphthylacetic acid and benzylaminopurine. We observed morphogenetic processes

formation of calli, shoots and roots during cultivation explatns on media with phytohormones.

All studied Nicotiana and Linaria species were shown to have high regenerative properties and had not

reveal a single pattern in development of morphogenetic processes.

Summarizing obtained results, we can conclude that at this time revealed no strong relationship

between the presence of T-DNA-like sequences in the genome of plants and processes predominate

during regeneration. However, it should be noted that all the studied species have a high regenerative

properties. Probably due to these plant features agrobacterial sequences were able to maintain in some

species of genera Nicotiana and Linaria after the act of transformation.

References:

1. White F.F., Garfinkel D.J., Huffman G.A., et al. Sequence homologous to Agrobacterium

rhizogenes T-DNA in the genome of uninfected plants. // Nature. 1983, V. 301 (5898) p. 348-350.

68

P34

Looking for the better way in studying soil microbial diversity

E. Pershina 1 and E. Andronov

1

1All Russia Research Institute for Agricultural Microbiology, Russia

The main feature of natural microbial communities is an extremely high level of biodiversity. It causes

some problems connected with the resolving capacity of molecular biology techniques such as 16S

rRNA gene cloning and T-RFLP-analyses. The first of them is primer bias that bases on incomplete

correspondence between primer and PCR-matrix (1). As a result some variants of 16S rRNA genes are

insufficiently presented in final sample. The second problem is the complexity of T-RFLP peaks that

disable us to find out the taxonomic characteristics of microorganisms (2). Keeping in mind these two

problems there was made an attempt to create an appropriate algorithm for describing the soil

microbial biodiversity. In this respect for sequencing data analyses as well as for T-RFLP results the

multidimensional scaling was successfully used. For T-RFLP data this theoretical scheme generally

presented as PCA is very common. Especially it became very useful for soil, because of extremely

high levels of biodiversity. Comparison of T-RFLP-profiles with sequencing data showed that almost

all of the obtained peaks had a complex nature. Concerning soil biodiversity, multidimensional scaling

is the only way for analyzing T-RFLP data. We consider T-RFLP to be indispensable for the express

and comprehensive analysis of the microbial community. For primer bias calculation three clone

libraries according to three primer pairs were constructed. Sequencing results were presented using

conception of «taxonomic space». This multidimensional mathematical space is based on the analysis

of phylogenetic distances between the nucleotide sequences of experimental data and of selective

members of each microbial phylum. Consequently two determinants for primer bias measuring were

found. The first is size of the region occupied by clone library in taxonomic space and the second is

the location of its central point. According to suggested scheme among three tested primers the most

appropriate one was chosen. As a result of our complex research we propose that application of

multidimensional conceptions opens to us new horizons in describing of true microbial biodiversity

and reserves more attention in future.

This work was supported by RFBR grant 09-04-00386a.

References:

1. Suzuki M.T., Giovannoni S.J. 1996. Bias caused by template annealing in the amplification of

mixtures of 16S rRNA genes. Applied and environmental microbiology 62:625–630.

2. Dunbar J., Ticknor L.O., Kuske C.R. 2000. Assessment of microbial diversity in four United States

soils by 16S rRNA gene terminal restriction fragment analysis. Applied and environmental

microbiology 66:P. 2943–2950.

69

P35

Dickeya sp. as emerging risk factor for potato crops in temperate climate – molecular

characterisation and pathogenicity of strains isolated recently in Poland

M. Potrykus, M. Sławiak, E. Lojkowska

Department of Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdansk and

Medical University of Gdansk, Kladki 24, 80-822 Gdansk, Poland

One of the major crops in Poland is the potato (Solanum tuberosum) with a value of 4 billion PLN in

2008. Poland, bringing in 18,4% of total European potato production (2007), is a leader in the

European Union. Pectinolytic bacteria from the Pectobacterium (syn. Erwinia carotovora) and

Dickeya (syn. Erwinia chrysanthemi) genera are casual agents of blackleg and soft rot diseases and

cause potato crops damage and high economic loss. Dickeya strains were responsible for rot in

numerous crops, formerly, in warm and tropical climate. However, recently Dickeya strains have been

isolated from diseased plants in Poland and the Netherlands and from plants and river water in Finland

(Laurila et al., 2008, Sławiak et al., 2009). These recent findings suggest that Dickeya strains can

effectively infect the potato plant and cause disease symtoms in temperate climate.

In 2009, approximately 150 potato stems and tubers with symptoms of blackleg and soft rot were

collected from the 16 regions in Poland. One thousand pectinolytic bacterial strains were isolated from

the received samples. The same procedure was applied previously in 1996 and 2005. In 1996, all the

collected isolates were assigned to the Pectobacterium genus, in 2005, surprisingly three Dickeya

strains were isolated in the region of Podlaskie in Poland. In 2009, 31 Dickeya strains were isolated

from 6 plants with blackleg symptoms collected in 4 different regions of Poland. The performed

molecular characteristics (e.g. ERIC patterns) indicated that strains isolated from single plants share

high similarity to each other. The Dickeya strains isolated from four plants in 2009 and the Dickeya

strains isolated in 2005 exhibited a similar band pattern.

A representative Dickeya strain isolated from each of the diseased plants was chosen and

pathogenicity tests on chicory leaves and potato tubers have been conducted. Results suggest that the

newly isolated strains are highly virulent.

The obtained results indicated that Dickeya strains have spread further in Poland. Most of the newly

isolated strains share similarity with strains isolated in 2005. Dickeya strains showed to be highly

virulent, thus in favourable condictions are expected to cause high economic loss to potato crops.

References:

1. Laurila, J., Ahola, V., Lehtinen, A., Joutsjoki, T., Hannukkala, A., Rahkonen, A., Pirhonen, M.

2008. Characterization of Dickeya strains isolated from potato and river water samples in Finland.

European Journal of Plant Pathology 122(2): 213-225.

2. Sławiak, M., van Beckhoven, J.R.C.M., Speksnijder, A.G.C.L., Czajkowski, R., Grabe, G., van der

Volf, J.M. 2009. Biochemical and genetic analysis reveal a new clade of biovar 3 Dickeya spp. strains

isolated from potato in Europe. European Journal of Plant Pathology 125(2): 245-261.

70

P36

Microorganisms associated with Sphagnum mosses as perspective objects for agricultural

microbiology

Shcherbakov A.1, Kuzmina E.

2, Chebotar V.

1

1 All Russia Research Institute for Agricultural Microbiology, St. Petersburg, Russia

2 Komarov Botanical Institute RAS, St. Petersburg, Russia

e-mail: [email protected]

This work aim to decision of fundamental scientific problem – interaction between microorganisms

and plants and creating high-effective plant-bacterial systems with focus to their practical usage in the

conditions of shortage of fertilizer and for plant protection. It was shown previously that Sphagnum

mosses associated with unique microorganisms which have important functions for Sphagnum mosses

and for the marsh ecosystem as a whole. The purpose of work include the investigations of the

properties of these organisms, selection of perspective strains and development of new high-effective

biofertilizers for agriculture. During work mosses of two species which play a different ecological role

in the ecosystem of marshes have been sampled in different geographical areas of the Leningrad

Region (Russia) and Austria. The spatial localization of endophytic bacteria within the cells of moss

was defined using methods FISH and CSLM. About 150 strains of bacteria were isolated from the

tissues of plants, carried out a preliminary genetic analysis to identify identical isolates as well as

cultural and morphological properties of the isolates have been studied. It was investigated the

antagonistic properties of the isolated strains against a number of phytopatogenical fungi and bacteria.

It was shown that more than 60% of all isolates have strong antifungal properties (in vitro). Identified

strains of bacteria capable to plant-growth promotion, phosphate solublillizing and the growth of non-

nitrogen media. Thus, selected strains can be recommended for further study and development of

technology for their cultivation in the laboratory and production conditions.

This work was supported by grants RFBR 09-04-01007-ANF_a, 09-04-13782-ofi_c

71

P37

Optimization of HandyGun for inoculation with Potato virus A (PVA)

Sidona Sikorskaite1, Anssi L. Vuorinen

1, Minna-Liisa Rajamäki

1, Arto Nieminen

2, Victor Gaba

3, Jari

P.T. Valkonen1

1Department of Agricultural Sciences, PO Box 27, FIN-00014, University of Helsinki, Finland;

2The

Instrument Center, PO Box 27, FIN-00014, University of Helsinki, Finland; 3Department of Plant

Pathology and Weed Science, Agricultural Research Organization, The Volcani Center, PO Box 6, Bet

Dagan 50250, Israel

In recent years, a large number of gene transfer methods have been investigated, thus there are many

different gene delivery systems developed for various types of cells and tissues. The biolistic process

appears to be something of a universal gene delivery mechanism, available in a very diverse range of

organisms and of a broad utility. The targets of bombardment include plant, fungal, algal and animal

cells. To date it is uniquely suitable for transformation of totipotent tissues, including pollen, embryos

and meristem. The aim of this study was to optimise the easily constructed HandGun setup (1) for

inoculation with Potato virus A (PVA). Because our version for pratctical reasons was no longer a

handheld device, we renamed it HandyGun. Physical parameters for delivering a full-length cDNA

clone of PVA, chimeric pBUIII (2) into Nicotiana tabacum, Nicotiana benthamiana and potato clone

‗A6‘ plants has been successfully established. The following parameters were investigated: DNA

precipitation method on gold particles, helium pressure, cDNA concentration and amount of gold

particles. Different DNA precipitation methods on gold particles have been tested and precipitation

with sodium acetate/polyvinylpyrrolidone (NaAc/PVP) was shown superior over calcium nitrate

(Ca(NO3)2). High efficiency of PVA infection was achieved with pressures higher than 2 bars,

however, frail leaves of N. benthamiana were damaged at 4-5 bars, thus 3 bars were considered as the

optimal. DNA amounts of 0.5 and 1.0 µg pBUIII per shot were recommended to use, though all tested

DNA amounts resulted in similar numbers of NLLs on inoculated ‗A6‘ leaves. High numbers of NLLs

were achieved using 0.10 mg microprojectiles per shot, thus it was five-fold less than it was previously

recommended for inoculation with PVA by shooting with Bio-Rad Helios Gene Gun (3).

References:

1. Gaba, V., Gal-On, A. 2005. Inoculation of plants using bombardment. In: Simon, A., Kowalik, T.,

Quarles, J. (Eds.), Current Protocols in Microbiology. Wiley Interscience, pp. 16B.3.1–16B.3.14.

2. Paalme, V., Gammelgård, E., Järvekülg, L., Valkonen, J.P.T. 2004. In vitro recombinants of two

nearly identical potyviral isolates express novel virulence and symptom phenotypes in plants. J. Gen.

Virol. 85: 739–747.

3. Kekarainen, T., Valkonen, J.P.T. 2001. Inoculation of viral RNA and cDNA to potato and tobacco

plants using the Helios Gene Gun. Bio-Rad Bulletin #2531.

72

P38

Real-time PCR-based assay for the detection of potential mycotoxin-producing Fusarium species.

A.A. Stakheev1, D.Y. Ryazantsev

1, T.Y.Gagkaeva

2 and S.K.Zavriev

1

1Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia;

2 All-Russian Institute

of Plant Protection, St. Petersburg, Russia.

The Fusarium head blight (FHB) is one of the most worldwide dangerous diseases of agriculturally

important plants in different geographical areas of Europe, including Russia. Several species of

Fusarium cause head blight of small grain cereals, thus leading to significant harvest losses and grain

quality decrease, as well as to accumulation of mycotoxins, hazardous to human and animal health.

The major groups of Fusarium toxins are trichothecenes, fumonisins enniatins, and also zearalenone

and moniliformin. Taking into account a worldwide distribution of FHB agents and their danger, a

continual monitoring of the plants, grain and their products is important. One of the simplest

approaches to rapid and sensitive diagnosis and identification of pathogens is PCR modification with

the detection of fluorescence during the amplification (real-time PCR). The real-time PCR-based

system has been developed for the detection and quantification of the most common Fusarium species

with the similar profiles of mycotoxins they can potentially produce. Corresponding group-specific

primers for various Fusarium fungi have been designed based on the translation elongation factor 1α

(tef1α) gene sequences. The specificity of the selected primers and TaqMan probes has been tested

with 26 single-spore isolates of seven most common Fusarium species and 21 wheat and barley grain

samples. PCR results were analyzed relative to the most common mycotoxin deoxynivalenol (DON)

content in infected grain samples. The developed system provides accurate, rapid, sensitive detection

and quantification of common toxigenic Fusarium species in cultures and in the infected grain

decreasing the number of the tests to be done. Furthermore, the developed test systems can be of use

for quantitative analysis of the pathogen content in grain. The results described seem promising to

create the corresponding PCR kits, which will minimize a routine diagnostics of FHB agents.

References:

1 Kristensen, R., Torp, M., Kosiak, B., & Holst-Jensen, A. 2005. Phylogeny and toxigenic potential is

correlated in Fusarium species as revealed by partial translation elongation factor 1 alpha gene

sequences. Mycological Research. 109, 173-186.

2. Moss, M.O., & Thrane, U. 2004. Fusarium taxonomy with relation to trichothecene formation.

Toxycology Letters. 153, 23-28.

3. Niessen, L. 2007. PCR-based diagnosis and quantification of mycotoxin producing fungi.

International Journal of Food Microbiology. 119, 38-46.

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Diversification of genomic organization of Rhizobium leguminosarum bv. trifolii nodule isolates

originated from single soil environment

G.T. Stasiak, A. Mazur, J. Wielbo and A. Skorupska

Department of Genetics and Microbiology, Institute of Microbiology and Biotechnology, University

of Maria Curie Skłodowska, Lublin, Poland

Soil bacteria, commonly known as rhizobia, possess ability to induce root nodules on

legumes and provide plants with fixed nitrogen, enabling them to grow in nitrogen-limited soils.

Rhizobial genomes are large and composed of chromosome and plasmids, which comprise up to 35%

of total genome. Genes involved in nodulation and N2-fixation are clustered on symbiotic plasmid

(pSym), or incorporated into the chromosome as symbiotic islands. The complex and diverse genomic

architecture may directly underlie the great genetic and physiological variation of rhizobial strains,

resulting in large diversity of populations (1).

The main aim of this study was identification of genetic determinants of adaptive

capacity of Rhizobium leguminosarum bv. trifolii (Rlt) to the symbiotic and soil environment by

finding correlation between the variable organization of the bacterial genomes and the efficiency of

symbiosis. Variability of the genomes organization was seen primarily as a diversity of

extrachromosomal pool of rhizobia, which represent endosymbionts of ten plants growing in their

immediate vicinity, by PFGE analyses.

Studies of genomic organization and diversity of isolates was conducted on the level of

chromosomes and plasmid pool. The assays involved identification and localization of selected genes

in the different genomic compartments (chromosome, non-symbiotic and symbiotic plasmids) in

several strains, using Southern hybridization with molecular probes based on genome of RtTA1

laboratory strain (2). Despite the large diversity of analysed genomes in terms of number and size of

plasmids, relative stability of the markers location on each of the replicons was found, especially in

chromosomes of Rlt isolates. To further examine genetic differentiation of isolates, 12 markers

(chromosomal- and plasmid-borne) of the selected pool of strains were sequenced. Discrimination

analyses of sequences showed that genes belonging to different genome compartment differed

substantially with respect to frequency of codon usage and codon adaptation index (CAI), indicating

different degree of genes adaptation to particular compartment in a host genome.

References:

1. Palacios R, Flores M (2005) Genome dynamics in Rhizobial organisms. In: Palacios R, Newton WE

(eds) Genomes and genomics of nitrogen-fixing organisms. Springer, Dordrecht, pp 183-2005.

2. Król JE, Mazur A, Marczak M, Skorupska A. (2008) Application of physical and genetic map of

Rhizobium leguminosarum bv. trifolii TA1 to comparison of three closely related rhizobial genomes.

Mol Genet Genomics 279, 107-121.

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Influence of Dionaea muscipula extract on bacterial responses depending on quorum sensing

mechanism

R. Olszewski, A. Szpitter, E. Łojkowska

Department of Biotechnology, Intercollegiate Faculty of Biotechnology of University of Gdańsk and

Medical University of Gdańsk, Gdańsk, Poland

Bacterial quorum sensing (QS) mechanism is a signaling network employing small molecular

compounds that act as messengers enabling bacteria to sense many characteristics of their environment.

As physiological responses influenced by QS include major pathogenicity traits such as biofilm

formation and virulence factors production, the interference in the QS network has been recognized as

an interesting target for fighting plant and human bacterial pathogens (1). Extracts from carnivorous

plant Dionaea muscipula (Droseraceae) have been used in traditional medicine due to their

antibacterial, immunomodulatory and anticancer properties (2). The aim of this work was to evaluate

the influence of D. muscipula extract on the bacterial QS mechanism and related processes.

Pseudomonas aeruginosa - an opportunistic human pathogen occasionally causing infections in plants

has been chosen as a model organism for evaluation of plant secondary metabolite impact on the

complex bacterial QS network. Chloroform extract from in vitro grown D. muscipula plants was

obtained using Soxhlet apparatus and subsequently divided into seven fractions by HPLC. Both extract

and fractions, as well as several plant-derived and synthetic compounds were tested for their ability to

decrease indicator strain response to bacterial signaling molecules. P. aeruginosa cultures were grown

together with examined compounds and tested for the presence of signaling molecules and virulence

factors (enzymes, siderophores). Additionally, the ability of D. muscipula extract to impede bacteria

motility was tested. The QS molecules were extracted using ethyl acetate and subsequently their

quantity was evaluated using indicator strains. Iron chelators (siderophores) were isolated using solid

phase extraction and separated by TLC. In addition, the capability of P. aeruginosa supernatants to

chelate iron was measured. The obtained results showed that D. muscipula extract and plumbagin - a

naphthoquinone present in the preparation - decreased P. aeruginosa motility. Moreover, their

addition to the culture medium resulted in the reduced level of signaling compound belonging to 4-

quinolones and some of the P. aeruginosa virulence determinants. Establishing the molecular basis for

D. muscipula metabolite action on the bacterial QS mechanism can lead to the discovery of a new

class of antimicrobial agents with potential application against both plant and human pathogenic

bacteria.

References:

1. Rasmussen T.B. & Givskov M. 2006. Quorum sensing inhibitors: a bargain of effects. Microbiology,

152:895–904.

2. Juniper B.E., Robins R.J. & Joel D.M. 1989. The carnivorous plants. Academic Press, inc. Harcourt

Brace Jovanovich, Publishers.

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The current PVY population affecting potatoes in Finland

Yanping Tian1, Sascha Kirchner

1, and Jari P.T. Valkonen

1

1Department of Agricultural Sciences, University of Helsinki, Finland

Potato virus Y (PVY) is one of the most common viruses in potato. It causes serious problems in the

production and quality of potatoes. To investigate the PVY population in potatoes in Finland, we

collected 21 PVY isolates from seed potatoes and seven additional isolates from potato fields in 2006

and 2007. Biological, serological and molecular methods were applied to characterize the isolates.

Results showed that the isolates fell into two strain groups (PVYN and PVY

O) according to the

responses on potato cultivars Pentland Crown (Ny:nc), P. Ivory (Ny:Nc), King Edward (ny:Nc) and

Nicotiana tabacum cv. Samsun. Interestingly, several isolates which caused necrosis on N. tabacum

Samsun also induced necrotic blotches on locally and systemically infected leaves of P. Ivory and/or P.

Crown which contains the Ny resistance gene specific to PVYO, but the symptoms differed clearly

from the necrotic local lesions and no systemic infection caused by PVYO. Serological results were

consistent with the biological assays with one exception: isolate 182-14 caused veinal necrosis on

tobacco but was detected with a PVYO–specific monoclonal antibody (Mab1129), which is similar to

the PVYNW

strain group. The 5‘- and 3‘-proximal sequences of the PVY genome including 5‘UTR, P1,

HC-Pro, CP and 3‘UTR were determined in the PVY isolates. In 14 out of 21 isolates from seed

potatoes and five out of seven isolates from other potato crops were found to contain recombination

events which have been previously reported in PVYNTN

strain group, within the CP encoding sequence.

One isolate was tested on many potato cultivars and found to induce tuber necrosis symptoms in

cultivars Nicola and Annabelle. Only the isolate 182-14 was found to contain a recombination event in

the P1 encoding region. Phylogenetic analysis indicated that 182-14 has a PVYN-like HC-Pro but

PVYO-like CP. In conclusion, PVY

N strain group was the predominant one in seed potatoes in Finland

and hence likely to be also most common in ware and industrial potato crops; only four isolates

belonged to PVYO strain group.

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Genetic mapping pea (Pisum sativum L.) gene Crt, controlling root system morphogenesis

V.S. Titov¹´² V.A. Zhukov¹ A.I. Zhernakov¹ A.Y. Borisov¹ I. A. Tikhonovich¹

¹All-Russia Research Institute for Agricultural Microbiology, St. Petersburg, Pushkin 8, Russia;

²Department of Genetics and Breeding, St. Petersburg State University, St. Petersburg, Russia

Using ethyl methane sulfonate (EMS) treatment of the line SGE seeds, a new mutant of pea (Pisum

sativum L.) with alterations in root development was obtained. The mutant phenotype depends on the

density of the growth substrate: on sand (a high density substrate) the mutant forms a small compact

curly root system whereas on vermiculite (a low density substrate) differences between the root

systems of the mutant and wild type plants are less pronounced. Also mutant has the ethylene-sensitive

phenotype. Thus gene Crt controlling morphogenesis of root system would take part in ethylene

signaling. Gene Crt has been originally localized in pea linkage group V relatively morphological

markers r, tl. For sequencing Crt it is necessary to map this gene using comparative genomic

methodology of pea (Pisum sativum L.) and lucerne (Medicago truncatula Gaertn.), which genome is

practically sequenced. At the first stage CAPS (cleaved amplified polymorphic sequence) molecular

markers developed on the basis of the pea genes located in V linkage group were used. In F2 (SGEcrt

× NGB1238) sample frame the inheritance of mutant crt phenotype and given molecular markers

RPL24, Enol, Paal2 were analyzed. The obtained results allowed more precise Crt mapping and two

possible regions of gene-homologue localization in lucerne genome revealing. New gene-specific

molecular markers were developed on the basis of lucerne genes located in the revealed regions. In the

same regions 2 genes-candidate, homologues to Arabidopsis (Arabidopsis thaliana L.) genes EOL1

and ERS1, participating in ethylene signaling were revealed. The analysis of lucerne chromosome 1

markers including EOL1 has shown the absence of coupling with Crt, on the contrary, chromosome 7

markers has coupled Crt in pea genome. The obtained results show that the Crt gene-homologue

located in lucerne chromosome 7 is the homologue to ERS1 gene of Arabidopsis (A. thaliana). With

the purpose of gene coding part mutation searching the further new molecular markers developing,

genes-candidates in lucerne genome's sinteny region revealing and corresponding genes in pea lines

SGE and SGEcrt sequencing are planned.

References:

1. Tsyganov V.E., Borisov A.Y., Rozov S.M., Tikhonovich I.A. New symbiotic mutants of pea

obtained after mutagenesis of line SGE // Pisum Genet. 1994. V. 26. P. 36-37.

77

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Conservation of plants genetic resources by cryopreservation – efficient method of liquid

nitrogen preservation of Gentiana tissue cultures and assessment of the quality and uniformity

of recovered plants

A. Mikuła, K. Tomiczak and J.J. Rybczyński

Laboratory of Plant Biotechnology, Botanical Garden – Center for Biological Diversity Conservation,

Polish Academy of Sciences, Warsaw, Poland

There are no doubts, that humans have impacted biodiversity loss – directly through the

destruction of habitats, and indirectly through climate change. In the era of rapid global warming,

species have three basic alternatives – they can: (i) migrate to appropriate environmental conditions;

(ii) adapt to the new environmental conditions; or (iii) become extinct. In a changing environment, conservative species with specific habitat requirements or long generation times are more prone to the

threat of extinction. At present an estimated one-quarter of vascular plant species are under threat in

the wild (1). The best and most cost-effective way of protecting plants genetic resources is in situ,

however it is often necessary to conserve a variety of unique and rare species also ex situ – outside

their natural habitat. Among different ex situ methods cryopreservation manifests possibility of long-

time preservation of plant material with maximum stability of phenotypic and genotypic

characteristics and reduced susceptibility to diseases, mutations and environmental conditions at a

minimal storage space.

Biotechnology and tissue culture techniques accompanied by germplasm storage in ultra-low

temperatures provide a mean to propagate and preserve valuable genotypes, cultivars and cell lines of

Gentiana – rare or endemic plants of great importance in ethnobotany, pharmacology and horticulture

(3). In our laboratory we have developed efficient cryopreservation protocols for preservation of

viability, regrowth and embryogenic capacity of G. tibetica (2, 3), G. cruciata (3) and G. kurroo cell

suspension cultures (4). For the assessment of the quality and uniformity of plants recovered in post-

thawing culture nuclear DNA content measurements by flow cytometry and methylation sensitive

AFLP analysis were routinely applied. Examination of the genetic stability at the genome size, DNA

sequence and methylation levels showed that cryoregenerants were true-to-type.

References:

1. Hawkins, B., Sharrock, S. & Havens, K. 2008. Plants and climate change: which future? Botanic

Gardens Conservation International, Richmond, UK, http://www.bgci.org.

2. Mikuła, A. 2006. Comparison of three techniques for cryopreservation and reestablishment of long-

term Gentiana tibetica suspension culture. CryoLetters 27(5):269–282.

3. Mikuła, A., Olas, A., Sliwinska, E. & Rybczyński, J.J. 2008. Cryopreservation by encapsulation of

Gentiana spp. cell suspensions maintains regrowth, embryogenic competence and DNA content.

CryoLetters 29(5):409–418.

4. Mikuła, A., Tomiczak, K., Wójcik, A. & Rybczyński J.J. 2010. Encapsulation-dehydration method

of cryopreservation elevates embryogenic abilities of Gentiana kurroo cell suspension and carrying on

genetic stability of its regenerants. Acta Horticulturae (in press)

78

P44 .

Creation of adjuvant plants producing bovine gamma-interferon

V.E. Tvorogova, N.V. Savelieva, V.V. Yemelyanov and L.A. Lutova

Department of genetics and breeding, Saint-Petersburg University, Russia

At present transgenic plants as producers of pharmaceutical proteins are used ever more actively in

medicine and veterinary. However, since such producers are created not long ago, there is a lot of

unsolved problems and questions which concern this field of biotechnology (3).

Therefore the study of transgene's behavior in plant is one of the important stages of work when we

create the line of plants-producers of heterologous protein.

Our laboratory carries out the experiments for making plants-producers of bovine gamma-interferon.

This protein is widely used in veterinary as immunotherapeutic and antiviral agent. By now lines of

transgenic Nicotiana tabacum and Pisum sativum which produce gamma-interferon are created.

The aim of this research is creation and analysis of thansgenic Arabidopsis thaliana plants, producing

bovine interferon gamma.

The plasmid pART27INT6 was used for transformation. This plasmid contains the gene of bovine

gamma-interferon and two selective markers — kanamycin and

spectinomycin / streptomycin resistance genes — for plants and for bacteria, respectively (1).

We used the method of vacuum agrobacterial transformation (2): flower buds of plants were dipped

into suspension of bacteria in the medium for infiltration and plants were placed in vacuum for 15

minutes. Then we harvested the seeds from these plants and sowed them on selective medium, which

contained kanamycin.

As a result, we obtained three kanamycin-resistant plants, whose DNA harbours inset of gene of

bovine gamma-interferon. The progeny of one of this plants contained achlorophyllic germs (about a

quarter of total amount), and we drew a conclusion that this plant contain the inset of heterologous

gene in one of essential genes. This plant was withdrawn from further investigations.

The RT-PCR, carried out with total RNA isolated from two other plants, showed them expressing the

heterologous gene of bovine gamma-interferon.

Our plans for further investigation include the study of transgene‘s inheritance from generation to

generation, the measuring of level of gene expression by using real-time PCR and also the analysis of

protein spectrum of transgenic plants.

References:

1. Gleave, A. 1992. A versatile binary vector system with a T-DNA organisational structure conducive

to efficient integration of cloned DNA into the plant genome. Plant Molecular Biology 20: 1203-1207.

2. Bechtold, N., Ellis, J., Pelletier, G. 1998. In planta Agrobacterium mediated gene transfer by

infiltration of adult Arabidopsis thaliana plants. Methods in molecular biology 82: 259-266.

3. Ma, J.K., Barros, E., Bock, R., Christou, P., Dale, P.J., Dix, P.J., Fischer, R., Irwin, J., Mahoney, R.,

Pezzotti, M., Schillberg, S., Sparrow, P., Stoger, E., Twyman, R.M. 2005. Molecular farming for new

drugs and vaccines. Current perspectives on the production of pharmaceuticals in transgenic plants.

EMBO reports 6: 593-599.

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Agrobacterium – mediated transfer of the antisense suppressior of the proline dehigrogenase

gene in the maize genome by an in planta method

E.M.Mamontova2 , V.A.Velikov

1,2 , I.V.Volokhina

2, M.I.Chumakov

1,2

1

Laboratory of Genetic Engineering, Science and Education Center of Biotechnology, Saratov State

University; Saratov, 410035, Russia; 2

Laboratory of Bioingeneering, Institute of Biochemistry and

Physiology of Plants and Microorganisms, RAS; Saratov, 410049, Russia;

This study was focused on investigating of the possibility of Agrobacterium-mediated T-DNA transfer

antisense suppressor of the proline dehydrogenase gene (ASPG) to male and female gametophyte cells

of maize plants. A cell suspension of Agrobacterium tumefaciens carrying activated vir genes was

applied onto previously isolated pistil filaments, which were afterwards pollinated with the pollen of

the same cultivar [1]. Integration of T-DNA into the maize female gametophyte cells was confirmed

by PCR using primer for ASPG. Amplification of the ASPG showed the presence of about 0.9% of

PCR-positive plants out of the total number of seedlings examined.

References

1. Mamontova, E.M., Velikov, V.A., Volokhina I.V., & Chumakov M.I. 2010.

Agrobacterium-mediated in planta transformation of maize germ cells. Rus. J. Genetics. 46: 501–504.

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Preliminary analysis on appearance of Potato mop-top virus (PMTV) in Poland

Przemysław Wieczorek1, Marta Budziszewska

1, Henryk Pospieszny

2 and Aleksandra Obrępalska-

Stęplowska1

1Interdepartmental Laboratory of Molecular Biology, Institute of Plant Protection- National Research

Institute, Poznań, Poland; 2Departmnet of Virology and Bacteriology, Institute of Plant Protection-

National Research Institute, Poznań, Poland

PMTV (Potato mop-top virus) is a rod-shaped tripartite single-stranded RNA virus

belonging to the genus Pomovirus infecting mainly Solanum tuberosum (1). PMTV causes a great

loses in potato production in Europe mostly in Nordic countries: Norway, Denmark, Finland and

Sweden (2, 3). Plants infected with the pathogen develop characteristic symptoms that manifest with

necrotic lines, arcs or circles within tuber flesh (―spraing‖ symptoms) that dramatically decrease

quality of the tubers. Transmission of PMTV to Solanum tuberosum occurs only via its vector

Spongospora subterranea f.sp. subterranean (4) a soil-borne obligate pathogen that causes powdery

scab of potato. PMTV, as well as its vector, is related to the same wet and relatively cold climate that

are optimal for life cycle of both pathogens (5).

Since 2005 wide range of performed analyses (near 18,000 tubers from Western,

South-Western, Central and North-Eastern regions of Poland) and application of highly sensitive and

specific serological and molecular methods (DAS-ELISA, RT-PCR, IC-RT-PCR and real-time PCR)

did not indicate PMTV positive samples. In 2008 we detected the mop-top virus in one potato tuber, in

the Łódź province. Using specific primers CP region was amplified and sequenced showing high

sequence similarity as well as high phylogenetic correlation to Canadian and European isolates of

PMTV. In analysed tuber, the relative PMTV concentration was about 260 times lower in comparison

to reference sample. The first identification of PMTV in Poland in 2008, however incidental, could be

connected with local weather conditions in Łódź province in 2007 favourable for development of

Spongospora and occurrence of PMTV. This could have been sufficient for mop-top virus emergence,

maintenance and subsequent spreading in Poland potato‘s fields. Further analyses performed in

2009/2010 focused on the province, where the positive PMTV sample was first found in 2008,

indicated several tubers of three cultivars with mop-top virus infections.

Significance of the climatic conditions for the mop-top virus appearance, and then maintenance are

difficult to determine. However, it is a fact that virus was first reported in the country during

favourable weather conditions similar to those observed usually in Nordic countries.

References:

1. Scott, K.P., Kashiwazaki, S., Reavy, B. & Harrison, B.D. 1994. The nucleotide sequence of Potato

mop-top virus RNA 2: a novel type of genome organization for a furovirus. Journal of General

Virology 75, 3561-3568.

2. Nielsen, S.L. & Mølgaard J.P. 1997. Incidence, appearance and development of potato mop-top

furovirus-induced spraing in potato cultivars and the influence on yield, distribution in Denmark and

detection of the virus in tubers by ELISA. Potato Research 40, 101-110.

3. Rydén, K., Lövgren, L. & Sandgren, M. 1989. Investigations on potato mop-top furovirus in

Sweden. EPPO Bulletin 19, 579-583.

4. Jones, R.A.C. & Harrison, B.D. 1969. The behaviour of potato mop-top virus in soil, and evidence

for its transmission by Spongospora subterranea (Wallr.) Lagerh. Annals of Applied Biology 63, 1-17.

5. Carnegie, S. F., Davey, T. & Saddler, G. S. 2010. Effect of temperature on the transmission of

Potato mop-top virus from seed tuber and by its vector, Spongospora subterranean. Plant Pathology

59, 22-30.

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Monitoring of TRV and PMTV in Poland

Z. Yin, K. Michalak, E. Zimnoch-Guzowska

Plant Breeding and Acclimatization Institute (IHAR), Młochów Research Centre, Poland

Monitoring the occurrence of viruses causing spraing symptoms on potato tuber including TRV and

PMTV has been a sustained research objective of the Młochów Research Centre of IHAR since 2004.

A combination detection methods including inspection of symptom, DAS-ELISA and virus-specific

RT-PCR were used in the survey. Previously, in five subsequent years (2004-2008), a total of 17300

tubers from more than 60 different cultivars were inspected (1). TRV was detected in the spraing-

expressing tubers of 7 cultivars from 4 out 13 locations checked and in the soil samples from one

location. No PMTV was detected in any tuber or soil samples tested. In the present study, a set of 4228

tubers from 40 cultivars collected in 2008 was inspected visually and spraing symptoms were detected

in 331 tubers of 38 cultivars. In 2009, among 3387 tubers of 30 cultivars inspected, 411 tubers of all

the tested cultivars displayed spraing symptoms. The regions with the observed tuber spraing

symptoms cover 10 locations from northern and central Poland and three locations from eastern

Poland. TRV was detected in all the spraing-expressing tubers as well as in some necroses-expressing

ones of 5 cultivars from Zamarte, Lębork and Tucze tested by TRV-specific RT-PCR targeting RNA1

(the 16K gene) of the TRV genome. TRV was also detected in the sprouts of the TRV-infected tubers

of cv. Innovator, but the virus was not detectable in the leaves of the mature plants derived from the

respecting TRV-infected tubers. In addition, TRV was detected in soil samples of three locations from

northern Poland using tobacco bait plants by RT-PCR. TRV isolate from Dębnica Kaszubska can

successfully infect N. tabacum by a sap inoculation. PMTV-specific RT-PCR revealed that PMTV

was not present in any tuber or soil samples tested. Our investigation indicates that TRV is the causal

agent of the observed spraing symptom in potato tubers. Although no PMTV was detected by our

study so far, continued monitoring this virus is essential.

References:

1. Santala, J. et al. (corresponding by Valkonen, J.) 2010. Detection, distribution and control of Potato

mop-top virus, a soilborne virus, in Northern Europe. Annals of Applied Biology (in peer review).

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454 pyrosequencing increase understanding of root inhabiting fungal diversity in pea plant

Lingling Yu, Mogens Nicolaisen and Sabine Ravnskov

Dept. of Integrated Pest Management, Faculty of Agricultural Sciences, Aarhus University, Research

Centre Flakkebjerg, Denmark

Plant roots harbor a high diversity of microorganisms and the diversity of root inhabiting fungal

communities can determine plant community structure and ecosystem productivity. In addition, plant

root age has a strong effect on composition of mycorrhizal fungal community and also influence

general fungal diversity and microbe activity (1, 2). These root inhabiting microbes play an important

role in ecosystem functioning. However, knowledge of unculturable fungal diversity and function is

still limited. As studying the fungal communities remains a challenge because of this high taxonomic

and ecological diversity. Our primary goals are to identify diversity of pea root inhabiting fungal

communities in healthy and diseased soils, and to determine the dominant root fungal communities in

three growth stages of pea plant.

To better understand the knowledge of the fungal diversity and community composition, a high

throughput 454 pyrosequencing is applied in this study. 454 pyrosequencing is a new generation

sequencing (3) method which allows to obtain the fungal community diversity without need the pre-

cultivating of the microbes in the laboratory. It is more rapid and cost-effective than traditional

cloning-sanger sequencing. With developments in sequencing techniques and DNA databases, the

nuclear ribosomal internal transcribed spacer (ITS) region is now used as a DNA barcode marker for

the identification (4), the use of bar-coded primers can sequencing a large number of samples in a

single run. In this study, pea plants were grown in a climate chamber, 6 samples from growth,

flowering and withering stage were analysed separately. Total DNA was extracted, using primer

ITS1F and ITS4 for amplifying a gene fragment of 550 bp of the SSU rRNA of the fungal

communities. The fungal diversity in three different plant growth stages using by tag-encoded 454

pyrosequencing, no less than 200 000 ITS reads were obtained from all samples. Bioinformatics on

sequence data is performed now.

References:

1. Lupwayi N.Z., Rice W.A., Clayton G.W. 1998. Soil microbial diversity and commuinty structure

under wheat as influenced by tillage and crop rotation. Soil Biol Biochem 30:1733–1741.

2. Yang C.H., Crowley D.E., 2000. Rhizosphere microbial community structure in relation to root

location and plant iron nutritional status. Appl Environ Microbiol 66: 335–351.

3. Margulies M., Egholm M., Altman W.E., Attiya Said. 2005. Genome sequencing in microfabricated

high-density picolitre reactors. Nature 437: 376-380.

4. Seifert KA. 2008. Integrating DNA barcoding into the mycological sciences. Persoonia 21: 162-166.

83

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The observation of genetical mechanisms of plant tumor development. The role of meristem-

specific genes in tumor formation in inbred lines of radish

E.Yurlova, I.E. Dodueva, M.A Osipova

Department of Genetics and breeding, Saint-Petersburg State University, Saint-Petersburg, Russia.

Plant tumors are known to be a model for studying various genes participation in organism control.

We use tumors that develop on inbred lines from radish genetic collections as such model. According

to our data tumor development an increased level of expression of genes, which normally involved in

meristem development regulation takes place. CycD3, KNOX (KNAT1), WOX (WOX5) genes are

among them.

To pass the local expression analysis we make transformation of radish lines (with tumor growth and

without) by the reporter construction pWOX5:GUS. The expression of meristem genes in the tumors

makes sense of the same regulatory mechanisms of normal and abnormal meristems. The next stage of

our work is to study the local expression KNAT1 and CycD3 genes during the tumor formation in

radish lines.

The work was supported by NSh 7623.2006.04, RFBR 08-04-00537a, СRDF ST- 012 and СRDF

BP2M12 grants.

References:

Mishra, P.K., Tewari, J.P., Clear, R.M. & Turkington, T.K. 2004. Molecular genetic variation and

geographical structuring in Fusarium graminearum. Annals of Applied Biology 145:299–307.

Dodueva, I.E., Frolova, N.V., Lutova L.A. 2007. Plant Tumorigenesis: Different Ways for Shifting

Systemic Control of Plant Cell Division and Differentiation. Transgenic Plant Journal. Global Science

Books.

Terpstra I., Heidstra R.. 2009. Stem cells : The root of all cells Seminars in Cell & Developmental

Biology 20: 1089–1096.

84

P50

Abscisic acid influence on aspen (Populus tremula L.) root growth in vitro under varied

conditions

J. Ņiauka and S. Kuusienė

Institute of Forestry, Lithuanian Research Centre for Agriculture and Forestry, Kaunas distr.,

Lithuania

Plant hormones ethylene and abscisic acid (ABA) are traditionally considered as stress hormones

which, if applied exogenously, negatively affect plant growth. However, these stress hormones were

found to be antagonistic to each other and able to stimulate the growth of different plant organs under

particular circumstances (1, 2). In this study, the influence of ABA was investigated using aspen

(Populus tremula L.) shoot cultures established in vitro. ABA was applied to the nutrient medium at

the concentrations ranging from 1 µM to 5 µM, and its effect was compared to the effects of the

respective concentrations of ethylene precursors ethephon and 1-aminocyclopropane-1-carboxylic-

acid (ACC). The morphometric characteristics of developing plants, such as shoot and root lengths,

were evaluated under different cultivation conditions: aspen shoot segments were cultured either in

test tubes (150 × 20 mm) or in Petri dishes (15 × 55 mm; both vertical plant growth and the space for

gas exchange were largely restricted, as compared to test tubes). Both ABA and ACC (ethylene

precursor) significantly decreased shoot length both in test tubes and Petri dishes. However, ABA, in

contrast to ACC, strongly increased root length in test tubes. In Petri dishes, this positive effect of

ABA on root growth was lost, and ABA decreased root length even more effectively than ACC which

proved a strong repressor of root elongation in test tubes. Since ethephon was also found to be a

repressor of root growth in test tubes, the conclusion can be made that ABA and ethylene may have

totally different effects on aspen root growth under particular circumstances.

References:

1. Sharp, R.E. & LeNoble, M.E. 2002. ABA, ethylene and the control of shoot and root growth under

water stress. Journal of Experimental Botany 53: 33-37.

2. Benschop, J.J., Jackson, M.B., Gühl, K., Vreeburg, R.A.M., Croker, S.J., Peeters, A.J.M. &

Voesenek, L.A.C.J. 2005. Contrasting interactions between ethylene and abscisic acid in Rumex

species differing in submergence tolerance. 2005. The Plant Journal 44: 756-768.

http://en.wikipedia.org/w/index.php?title=1-aminocyclopropane-1-carboxylic-acid&action=edit&redlink=1

http://en.wikipedia.org/w/index.php?title=1-aminocyclopropane-1-carboxylic-acid&action=edit&redlink=1

85

P51

Oilseed rape (Brassica napus) in vitro cultures as testing systems for newly synthesized chemical

compounds with potential growth regulating activity

D. Ņiaukienė, V. Mickevičius, I. Jonuńkienė

Department of Organic Chemistry, Kaunas University of Technology, Kaunas, Lithuania

Oilseed rape (Brassica napus L.) is an important worldwide grown crop for oil production, and

various growth regulators are applied to increase the harvest of B. napus in the fields. The search for

new, more effective growth regulators is also continued. The requirement for growth regulators that

could be used in practise is not only to have a significant positive effect on the final harvest but also

not to change plant growth characteristics too much, since such hormone-type effects could have

hardly predicted consequences in the fields. In the area of chemistry, a large variety of new

compounds is synthesized continuously that could be tested for their biological activity. However, to

test various concentrations of various newly synthesized compounds in the field trials would be a very

costly work. Therefore in vitro cultures of plants can be applied for the primary evaluation of newly

synthesized compounds. In this report, the example is given of the primary evaluation of four chemical

compounds synthesized in the Department of Organic Chemistry (Kaunas University of Technology)

for their biological activity in B. napus seedling culture in vitro. The tested concentrations of chemical

compounds in nutrient medium (Murashige and Skoog was taken as a basal medium) ranged from 1

mg*l-1

to 10 mg*l-1

(slightly over the usual level of plant hormones that is applied to tissue cultures).

Morphological parameters of developing B. napus seedlings, such as hypocotyl length and root length,

were evaluated. In most cases, the newly synthesized compounds increased hypocotyl length and

decreased root length. Thus the tendency was revealed for these compounds to increase the above-

ground part of plant at the cost of the under-ground part. Consequently, it could be predicted that B.

napus plants treated with these chemical compounds would have problems establishing themselves in

the soil. However, one of the four tested compounds, while significantly increasing hypocotyl length,

had no negative effect on root length, thus suggesting that it would be less risky to test it in the field

trials than the other in vitro tested compounds.

86

P52

Occurrence of 1-aminocyclopropane-1-carboxylate deaminase genes (acdS) in symbiotic nodule

bacteria

N. Y. Zinovkina, V.I. Safronova, A. P. Chizhevskaya and A. A. Belimov

All-Russia Research Institute for Agricultural Microbiology, Saint-Petersburg, Russian Federation

It is known that 1-aminocyclopropane-1-carboxylate (ACC) deaminase of rhizosphere bacteria breaks

down ACC, the immediate precursor of phytohormone ethylene, decreases ethylene evolution in plants

and thereby increases tolerance of plants to abiotic stresses such as drought, salinity, flooding and

heavy metals (1). However much less is known about distribution and the role of ACC deaminase

genes (acdS) presented in rhizobia, the bacteria nodulating legume plants (2). The aim of the present

study was to find acdS genes in various taxonomic groups of nodule bacteria and to obtain acdS

knockout mutants for further investigation of the role of ACC deaminase in development of nitrogen-

fixing symbiosis under stressful environmental conditions. For this purposes, a PCR technique was

tuned using a set of known universal or originally designed specific primers. As a result, acdS genes

were found in 35 strains of Rhizobium leguminosarum bv. viciae , Sinorhizobium meliloti and

Mesorhizobium sp. strains originated from the Russian Collection of Agricultural Microorganisms

(RCAM, St.-Petersburg). Positive PCR reaction was evident for 71 % of the strains tested, suggesting

that ACC deaminase is of widespread occurrence throughout rhizobia. The results also pointed to the

presence of differences in the nucleotide sequences of the end-regions of acdS genes (i) among

rhizobial species or genera tested and (ii) between rhizobia and other ACC deaminase containing

bacteria available in the NCBI database. Several ACC deaminase minus mutants were obtained using

site specific mutagenesis through construction and transformation of plasmid pK18acdS into R.

leguminosarum bv. viciae. Future study will be concentrated on the molecular characterization of

rhizobial acdS genes and on the interactions between the obtain mutants and legume plants. We

propose that the obtained results are of importance in view of investigation of adaptation of legume-

rhizobia symbiosis to unfavourable environmental conditions and climate changes.

This work was supported by the RFBR grant 09-04-01614-a.

References:

1. Glick, B.R., Cheng, Z., Czarny, J. & Duan, J. 2007. Promotion of plant growth by ACC deaminase-

producing soil bacteria. European Journal of Plant Pathology 119:329–339.

2. Musarrat, J., Al-Khedhairy, A.A., Al-Arifi, S. & Khan, M.S. 2009. Role of 1-Aminocyclopropane-

1-carboxylate deaminase in Rhizobium–Legume Symbiosis. M.S. Khan et al. (eds.), Microbial

Strategies for Crop Improvement, Springer Verlag, Berlin Heidelberg, 63-83.

87

P53

Elaboration and use of new approach to species and strain identification of phytopatological and

nitrogen-fixing bacteria

V.S. Zotov1,2

, N.V. Punina3, A.N. Ignatov

1, E.V. Matveeva

1, A.F. Topunov

2, N.W. Schaad

4

1Institute of Phytopatology, Bolshie Vyazemy, Moscow region, Russia;

2A.N. Bach Institute of

Biochemistry, Moscow, Russia; 3Medical-Genetic Scientific Center, Moscow, Russia;

4Agricultural

Research Service, USDA, Fort Detrick, MD, USA

Complex of phenotypic and genetic methods is now used for classification and identification of

different bacteria. The most perspective for diagnostics are methods of molecular biology. We tried to

design fast and reliable method for study of bacteria biodiversity using preferences of RFLP (1, 2) and

AFLP (3) methods. We named the method elaborated as single adapter AFLP (saAFLP).

Phytopatogenic bacteria of Xanthomonas genus were used as model objects. This genus has

complicated taxonomy which is permanently changing and was not determined using standard

methods. Features and advantages of the method were shown on 7 Xanthomonas strains with

sequenced genomes (Xcc528T, Xcc8004, XccB100, Xca373, Xeuv56, Xo.pv.oryzae5233,

Xo.pv.oryzicola5234). Strains were also studied using methods REP-PCR, DIR-PCR, microsatellite

PCR and MLSA. Obtained saAFLP fingerprints exactly divided strains for species and subspecies

levels. Each species had the unique set of fragments which sequences confirmed belonging bacteria to

genera. REP- and AP-PCR made with saAFLP semiproducts authentically differentiated strains of

subspecies. High correlation of data obtained and Xanthomonas strains host specialization was shown.

Heterogeneity of bacteria populations on each host plant (Oryza sativa, Cucurbita pepo, Solanum

lycopersicum, Capsicum annuum, Sinapsis alba, Brassica nigra, Brassica oleracea, Helianthus

annuus, Secale cereale, Pisum sativum) after their infection with typical Xanthomonas strains was

revealed. It possibly indicated the polyphyletic character of the populations. saAFLP fingerprints

formation during bacteria adaptation to new hosts was also studied. We tested 30 Xanthomonas strains

infecting different cultivars of Brassica nigra and Brassica napus in Moscow and North Caucasus

regions. Strains were analysed using MLSA (4), saAFLP and Box-PCR (5) techniques which

confirmed their taxonomic positions. saAFLP divided studied strains to 4 groups differed in infecting

activity for various hosts. One group infected Brassica napus did not belong to X. campestris species.

Strains of the each group were genetically monotonous.

It is considered now that nodulating nitrogen-fixing bacteria are belonged to different genera:

Azorhizobium, Bradyrhizobium, Mezorhizobium, Rhizobium and Sinorhizobium (6). We verified such

systematization using several methods. Computer analysis of sequenced genomes of 13 nodulating

bacteria based on distribution of octamer palindrome repeats confirmed validity of this division. Only

strain Rhizobium sp. NGR234 was the exception. According to our data it has to be most probably

attributed to Sinorhizobium than to Rhizobium genera. In-silico results were supported with

experimental saAFLP data.

The work was supported by International Science and Technology Center (grant 3431) and

Russian Foundation for Basic Research (grant 09-04-01674).

References:

1. J. H. Graham, J. S. Hartung, R. E. Stall, A. R. Chase. 1990. Phytopathology 80: 829-836.

2. G. R. Lazo, R Roffey, D. W. Gabriel. 1987. Int. J. Syst. Bacteriol. 37: 214-221.

3. P. H. M. Savelkoul, H. J. M. Aarts, J. de Haas, L. Dijkshoorn, B. Duim, M. Otsen, J. L. W.

Rademaker, L. Schouls, J. A. Lenstra. 1999. J. Clinical Microbiol. 37: 3083–3091.

4. J.M. Young, D.-C.Park, H.M.Shearman, E.Fargier. 2008. Syst. Appl. Microbiol. 31: 366–377.

5. J.L.W. Rademaker, B. Hoste, F.J. Louws, K. Kersters, J. Swings, L. Vauterin, P. Vauterin, F.J. de

Bruijn. 2000. Int. J. Syst. Evol. Microbiol. 50, 665–677.

6. Bergey‘s Manual of Determinative Bacteriology. 1994. Williams & Wilkins: Baltimore, etc.

88

P54

Effect of heat and drought stresses on crop production, and introduction of new plants as a

short-term solutions

Ling Zou


Temperature rise and inadequate water supply, caused by climate change and extreme weather in some

regions, expose plants to heat and drought stresses and have been shown to cause yield loss,

unsatisfactory product quality in wheat, oilseed rape, and other crops.

In the field, it is quite common that heat and drought stresses happen simultaneously. Plants under heat

and drought stresses, In general, showed activated senescence or even failure to finish life cycle.

Several studies on rice, wheat, cotton have shown that photosynthesis was inhibited due to

deactivation of photosynthesis-related enzymes and respiration rate was increased and abortion of

flowers of canola was observed under heat and drought stresses. Consequently, yield loss was resultant

and cause the crop production not profitable.

Yield loss has been showed to be attributed to the reduction of all seed components. Carbohydrates,

protein content were shown to be reduced under heat stress in wheat, rice andean lupin. Oil content of

canola was also decreased significantly. Quality of the products made of the flour of heat-stressed

seeds could be altered due to the increase of protein content. It has been proved that increase of protein

content in flour was not caused by heat stress but protein content was proportionally reduced less than

other components such as carbohydrates. This may affect the end product quality and some characters.

The negative impacts caused by heat and drought stresses have pressed the society to find solutions.

Breeding tolerant cultivars has been emphasized and a lot of efforts in seeking for tolerant genotypes

have been made globally in recent years. However, breeding is known to be long-term solution,

requiring long time to release tolerant cultivar and enormous financial support. In short-term solutions,

introducing some new plants would be better and faster in combat against those negative impacts.

Especially, in Nordic region where the agricultural products are mainly from barley, wheat, rye,

oilseed rape, so introduction of new plants can diversify agricultural production, besides, some

rotation benefit, such as decreased pest incidence, improvement of soil physicochemical traits, can be

expected. For instance, blue lupin has very strong tap root system which can reduce the soil hardening

and avoid moderate drought stress, and its high protein-containing seed can be regarded as an

important protein source. To conclude, introduction of new plants is an efficient and short-term

method to better adapt to the problems caused by climate change.

89

List of participants with oral presentation

Presenter Talk Co-authors

Abramowski Dariusz O14 Janus Ł, Floryszak-Wieczorek J

Adam Guenter O22 Grund E, Darissa O

Andronov Evgeny O11

Berg Gabrielle O2 Krikovtseva A, Cardinale M, Müller H, Zachow C

Broughton William O3 Deakin W J, Ardissone S, Schumpp O

Chumakov Mikhail O15

Hiltunen Lea O17 Valkonen J P T

Ivanova

Ekaterina

O16 Omarova E O

Karpiński Stanislaw O13 Wituszyńska W, Ślesak I, Vanderauwera S, Kornaś A, Choo K

S, Mühlenbock P, Van Breusegem F

Kidaj Dominika O10 Wielbo J, Marek-Kozaczuk M, Skorupska A

Kreuze Jan O23 Cuellar W

Lojkowska Ewa O6 Potrykus M, Slawiak M

Santala Johanna O20 Latvala-Kilby S, Hannukkala A, Valkonen J P T

Shishonkova Natalya O7 Smol´kina O N, Ignatov V V

Shtark Oksana

O5 Borisov A Y, Zhukov V A, Akhtemova G A, Ovchinnikova E S,

Nemankin T A, Danilova T N, Borisov Y G, Barbashov M V,

Naumkina T S, Chebotar V R, Tikhonovich I A

Siwińska Joanna O18 Ihnatowicz A

Stoddard Fred O8

Tikhonovich Igor O4

Urbanowicz Anna O19

Valkonen Jari O1

Zavriev Sergey O21

Zhukov Vladimir

O9 Rychagova T, Titov V, Ovchinnikova E, Couzigou J-M, Limpens

E, Radutoiu S, Madsen L H, Borisov A, Ratet P, Bisseling T,

Stougaard J, Tikhonovich I A

Ziembińska Aleksandra O12

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Vanderauwera%20S%22%5BAuthor%5D




90

List of participants with poster presentation

Presenter Poster Co-authors

Abdurashitov

Suleyman

P1 Didovich S V, Volkogon V V

Bagdziunaite Aiste P2

Belova Victoria P3 Lindstrom K, Becker A, Simarov B V, Roumiantseva M L

Bolotnik Elena P4 Kolomiec E I, Novik G I, Ananeva I N

Budziszewska Marta P5 Wieczorek P, Pospieszny H, Obrępalska-Stęplowska A

Chalaya Nadezhda P6 Kiru S D

Czajkowski Robert P7 Van Veen J A, Van der Wolf J M

Dankevych Liudmyla P8

Deng Xianbao P9 Hytönen T, Valkonen J P T, Elomaa P, Teeri T H

Golanowska

Malgorzata

P11 Jafra S

Grishina Оlga P12 Akhtemova G А, Shtark О Y, Zhukov V А, Borisov А Y,

Tikhonovich I А

Guzik Joanna P13 Michalczyk K, Rozpędek K, Nakonieczny M

Jafra Sylwia P14 Przysowa J, Polonis K, van der Wolf J, Łojkowska E

Jasieniecka Katarzyna P15 Banas W, Banas A

Jonuńkienė Ilona P16 Sikorskaitė S, Kuusienė S, Mickevičius V

Khyutti Alexander P17 Mironenko N, Afanasenko O

Kierzek Dorota P18 Wieczorek P, Nowaczyk K, Węgorek P, Zamojska J,

Obrępalska-Stęplowska A

Kitaeva Anna P19 Tsyganov V E

Kostyuk Irina P20 Shaposhnikov A I, Kravchenko L V

Krawczyk Krzysztof P21 Zwolinska A, Kamasa J, Pospieszny H

Krikovtseva Anastasia P22 Cardinale M, Berg C, Tikhonovich I, Chebotar W, Berg G

Kruk Katarzyna P23

Krzyzanowska Dorota P24 Potrykus M, Jafra S

Kuusiene Sigute P25 Ņiauka J, Grunskis V, Lenortavičiūtė S, Ńilininkas M

Lizarazo Clara P27 Stoddard F, Mäkelä P, Santanen A

Mazur Weronica P28 Barylski J, Messyasz B, Gabka M, Rybak A, Burchardt L,

Gozdzicka-Jozefiak A

Naumkina Maria P29

Nemankin

Timofey

P30 Ovchinnikova E, Zhukov V, Borisov A, Limpens E, Geurts

R, Tikhonovich I

Palojärvi Ansa P31 Sipilä T, Yrjälä K, Hatakka A, Alakukku L

Pasonen Hanna P32 Lu J, Niskanen A-M, Seppänen S-K, Rytkönen A, Raunio J,

Pappinen A, Kasanen R, Timonen S

Pavlova Olga P33 Matveeva T V, Lutova L A

Pershina Elizabeth P34 Andronov E

91

Potrykus Marta P35 Sławiak M, Lojkowska E

Shcherbakov Andrey P35 Kuzmina E, Chebotar V

Sikorskaite Sidona P37 Vuorinen A L, Rajamäki M-L, Nieminen A, Gaba V, Valkonen J

Stakheev Aleksandr P38 Ryazantsev D Y, Gagkaeva T Y, Zavriev S K

Stasiak Grazyna P39 Mazur A, Wielbo J, Skorupska A

Szpitter Anna P40 Olszewski R , Łojkowska E

Tian Yanping P41 Kirchner S, Valkonen J P T

Titov Vyacheslav P42 Zhukov V A, Zhernakov A I, Borisov A Y, Tikhonovich I A

Tomiczak Karolina P43 Mikuła A, Rybczyński J J

Tvorogova Varvara P44 Savelieva N V, Yemelyanov V V, Lutova L A

Volokhina Irina P45 Mamontova E M , Velikov V A, Chumakov M I

Wegrzyn Anna P26 Miksch K

Weinheimer Isabel P10 Valkonen J P T

Wieczorek Przemyslaw P46 Budziszewska M, Pospieszny H, Obrępalska-Stęplowska A

Yin Zhimin P47 Michalak K, Zimnoch-Guzowska E

Yu Lingling P48 Nicolaisen M, Ravnskov S

Yuorlova Elena P49 Dodueva I E, Osipova M A

Ņiauka Jonas P50 Kuusienė S

Ņiaukienė Dovilé P51 Mickevičius V, Jonuńkienė I

Zinovkina Nadezda P52 Safronova V I, Chizhevskaya A P, Belimov A A

Zotov Vasily P53 Punina N V, Ignatov A N, Matveeva E V, Topunov AF, Schaad

N W

Zou Ling P54

92

List of all participants

Participant

Home institute Country Email and address

Abdurashitov

Suleyman

Institute of Agricultural Microbiology of

Ukrainian Academy of Agricultural

Sciences

UKRAINE AR Crimea, region Simferopol,

village Gvardeyskoe, street K.

Marksa, 107, 97513

Abramowski

Dariusz

Poznań University of Life Sciences,

Department of Plant Physiology

POLAND 35 Wolyńska st., Poznan, 60-637

Adam Guenter University of Hamburg, Biozentrum

Klein Flottbek

GERMANY 22609 Hamburg, Ohnhorststr. 18

Andronov Evgeny All-Russia Research Institute for

Agricultural Microbiology

RUSSIA Podbelskogo Sh., 3, Pushkin-8,

196608, St.-Petersburg

Bagdziunaite Aiste Institute of Forestry, Lithuanian

Research Centre fo Agriculture, Kaunas

LITHUANIA Kaunas distr.

Belova Victoria All-Russian Research Institute of

Agricultural Microbiology (ARRIAM)



Berg Gabriele TU Graz, Institute for Environmental

Biotechnology

AUSTRIA Petersgasse 12, 8010 Graz

Bolotnik Elena Institute of Microbiology, BELARUS

National Academy of Sciences

BELARUS Minsk, Kuprevich str. 2

Broughton William Laboratory of Higher Plant Molecular

Biology University of Geneva

SWITZERLA

ND

30 quai Ernest-Ansermet, 1211

Genève

Budziszewska

Marta

Plant Protection Institute-National

Research Institute

POLAND Wl. Wegorka 20 street

Chalaya Nadezhda N. Vavilov Institute of Plant Industry,

St. Petersburg

RUSSIA 42-44 B. Morskaya Street, 190000

St. Petersburg

Chumakov Mikhail Institute of Biochemistry and Physiology

of Plants and Microorganisms, Russian

Academy of Sciences

RUSSIA 13 Prospect Entuziastov, Saratov

410049

Czajkowski Robert Plant Research International NETHERLAN

DS

6708 AA P.O. Box. 16

Wageningen

Dankevych

Liudmyla

Zabolotny institute of microbiology and

virology of the National academy of

sciences of Ukraine (IMV NASU)

UKRAINE Kyiv, Zabolotnogo str., 154,

Д03680

Deng Xianbao University of Helsinki, Department of

Agricultural Sciences

FINLAND PO Box 27, FIN-00014, University

of Helsinki

Golanowska

Malgorzata

Intercollegiate Faculty of Biotechnology

UG-MUG

POLAND Kładki 24, 80-822 Gdańsk

Grishina Оlga All-Russia Research Institute for




Guzik Joanna University of Silesia, Department of

Animal Physiology and Ecotoxicology,

Faculty of Biology and Environmental

Protection

POLAND Bankowa 9, 40 - 007 Katowice

Hiltunen Lea MTT Agrifood Research Finland, Ruukki FINLAND Tutkimusasemantie 15, 92400

Ruukki

Ivanova Ekaterina Moscow State University RUSSIA 119454, Lobachevskogo street,

92 (1) -175, Moscow

93

Jafra Sylwia Intercollegiate Faculty of Biotechnology

UG-MUG


Jasieniecka

Katarzyna


UG-MUG


Jonuńkienė Ilona Kaunas University of Technology,

Faculty of Chemical Technology, Kaunas

LITHUANIA Radvilėnų pl. 19, LT-50254,

Kaunas

Karpiński Stansilaw Warsaw University of Life Sciences,

Department of Genetics

POLAND Nowouyrsynowska st. 159, 02-

776 Warszawa Khyutti Alexander All-Russian Research Institute for plant

protection

RUSSIA 3, Podbelsky shosse, St.

Petersburg, 196608

Kidaj Dominika Department of Genetics and

Microbiology, M. Curie-Sklodowska

University

POLAND Akademicka 19, 20-033 Lublin

Kierzek Dorota Interdepartmental Laboratory of

Molecular Biology, Institute of Plant

Protection

POLAND Władysława Węgorka 20, 60-318

Poznań

Kitaeva Anna All-Russia Research Institute for


RUSSIA Podbelsky chaussee 3, St.

Petersburg, Pushkin 8, 196608

Kostyuk Irina All-Russia Research Institute for

Agricultural Microbiology RAAS



Krawczyk Krzysztof Institute of Plant Protection – National

Research Institute

POLAND Wladyslawa Wegorka 20, 60-318

Poznan

Kreuze Jan International Potato Center, Lima PERU Apartado 1558, Lima 12, Peru

Krikovtseva

Anastasia

TU Graz, Institute for Environmental

Biotechnology

SWITZERLA

ND

Petersgasse 12, 8010 Graz

Kruk Katarzyna University of Life Sciences in Lublin POLAND ul. Akademicka 15, 20-950 Lublin

Krzyzanowska

Dorota


University of Gdańsk - Medical

University of Gdańsk

POLAND ul. Kładki 24, 80-822 Gdańsk

Kuusiene Sigute Lithuanian Forest Research Institute,

Kaunas

LITHUANIA Liepu 1, Girionys, Kauno reg.

Lielmane Irena Plant Protection Service of Latvia,

National Phytosanitary Laboratorio, Riga

LATVIA Latvia, Riga, Lielvardes iela

36/38, LV-1006

Lizarazo Clara University of Helsinki, Department of



of Helsinki

Lojkowska Ewa Intercollegiate Faculty of Biotechnology

UG-MUG


Lutova Liudmila Professor of Saint-Petersburg State

University Department of Genetics and

Breeding

RUSSIA 7-9, Universitetskaya Emb., St.

Petersburg, 199034

Mazur Weronika Department of Molecular Virology,

Institute of Experimental Biology and

Department of Hydrobiology, Institute

of Environmental Biology; Faculty of

Biology, Adam Mickiewicz University

POLAND ul. Umultowska 89, 61-614

Poznań

Mihalova Jana Plant Protection Service of Latvia,

National Phytosanitary Laboratorio, Riga

LATVIA Riga, Lielvardes iela 36/38, LV-

1006

Naumkina Maria All- Russia Research Institute of

Legumes and Groat Crops

RUSSIA p/b Streletskoye, Orel, 302502

Nemankin Timofey

All Russia Research Institute for

Agricultural Microbiology RAAS

RUSSIA 196608, St. Petersburg, Puskin,

Shosse Podelskgo 3

94

Palojärvi Ansa MTT Agrifood Research Finland,

Jokioinen

FINLAND FI-31600 Jokioinen

Pasonen Hanna University of Helsinki, Department of

Forest Sciences


of Helsinki

Pavlova Olga Saint-Petersburg State University RUSSIA 7-9, Universitetskaya Emb., St.

Petersburg, 199034

Pershina Elizabeth All Russia Research Institute for




Potrykus Marta Department of Plant Protection and

Biotechnology, Intercollegiate Faculty of

Biotechnology University of Gdansk and

Medical University of Gdansk


Santala Johanna Department of Agricultural Sciences,



of Helsinki

Shcherbakov

Andrey

All-Russia Research Institute for


RUSSIA Podbelskogo Sh., 3, Pushkin-


Shishonkova

Natalya

Institute of Biochemistry and Physiology


Academy of Sciences

RUSSIA Entyziastov,13, 410049, Saratov

Shtark Oksana All-Russia Research Institute for




Sikorskaite Sidona Kaunas University of Technology,

Marijampole

LITHUANIA PO Box 27, FIN-00014, University

of Helsinki

Siwińska Joanna Intercollegiate Faculty of Biotechnology

UG-MUG


Stakheev Aleksandr M.M. Shemyakin & Yu.A. Ovchinnikov

Institute of Bioorganic Chemistry of the

Russian Academy of Sciences

RUSSIA Ul. Miklukho-Maklaya, 16/10,

117997 GSP, Moscow V-437

Stasiak Grazyna Department of Genetics and

Microbiology, Institute of Microbiology

and Biotechnology, University of Maria

Curie Skłodowska

POLAND 20-033 Lublin, Akademicka 19 st

Stoddard Fred University of Helsinki, Dept. of



of Helsinki

Szpitter Anna Department of Biotechnology,


of University of Gdańsk and Medical

University of Gdańsk


Tian Yanping University of Helsinki, Dept. of



of Helsinki

Tikhonovich Igor All-Russia Research Institute for




Titov Vyacheslav All-Russia Research Institute for




Tomiczak Karolina Botanical Garden – Center for Biological

Diversity Conservation, Polish Academy

of Sciences

POLAND Prawdziwka 2, 02-973 Warsaw

Tvorogova Varvara Saint-Petersburg State University

RUSSIA 7-9, Universitetskaya Emb., St.

Petersburg, 199034

Urbanowicz Anna Institute of Bioorganic Chemistry Polish

Academy of Sciences

POLAND Nosowkiego str. 12/14

95

Valkonen Jari Department of Agricultural Sciences,



of Helsinki

Volokhina Irina Institute of Biochemistry and Physiology


Academy of Sciences

RUSSIA Saratov, 410049

Wegrzyn Anna The Silesian University of Technology POLAND Akademicka 2, 44-100 Gliwice

Weinheimer Isabel Department of Agricultural Sciences,



of Helsinki

Wieczorek

Przemyslaw

Interdepartmental Laboratory of

Molecular Biology, Institute of Plant

Protection – National Research Institute

POLAND Władysława Węgorka 20, 60-318

Poznań

Yin Zhimin Research Division Młochów, Plant

Breeding and Acclimatization Institute

POLAND Str. Platanowa 19, PL-05-831

Młochów

Yu Lingling Aarhus University, Faculty of


DENMARK Forsogsvej 1, 4200 Slagelse

Yuorlova Elena Saint-Petersburg State University RUSSIA 7-9, Universitetskaya Emb.,

199034, St. Petersburg

Zavriev Sergey Ovchinnikov Institute of Bioorganic

Chemistry, Russian Academy of

Sciences

RUSSIA Ul. Miklukho-Maklaya, 16/10,

117997 GSP, Moscow V-437

Zhukov Vladimir All-Russia Research Institute for




Ņiauka Jonas Institute of Forestry, Lithuanian

Research Centre for Agriculture and

Forestry

LITHUANIA Kaunas distr.

Ņiaukienė Dovilé Kaunas University of Technology,

Faculty of Chemical Technology, Kaunas

LITHUANIA Radvilėnų pl. 19, LT-50254,

Kaunas

Ziembińska

Aleksandra

The Silesian University of Technology,

Environmental Biotechnology

Department

POLAND Akademicka 2 str., 44-100 Gliwice

Zinovkina Nadezda All-Russia Research Institute for




Zotov Vasily Moscow Technological Institute of Food

Production, Department of Biochemistry

RUSSIA Volokolamskoe shosse 11,

Moscow 125080

Zou Ling University of Helsinki, Department of


FINLAND PO Box 331, FIN-00014,


96

The joint 5th Postgraduate Course and Minisymposium of AB-RMS

(Agro-Biotechnology focused on Root-Microbe Systems Baltic Sea region network)

and

The 16th Biotechnology Summer School of the University of Gdansk, Poland,

and

The 2nd Workshop of The Polish Academy of Sciences (PAS) and The Russian Academy of

Agricultural Sciences (RAAS) on Plant Molecular Biotechnology

ISBN 978-952-10-6368-8

http://fi.wikipedia.org/wiki/Tiedosto:Etel%C3%A4-Savo.vaakuna.svg

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