Startup Meeting Clermont Ferrand
Participants:
Christoph Germeier (P0: coordinator and leader of WP1 and WP10),
Zofia Bulinska- Radomska (P1: leader of WP2),
Matthias Herrmann (P3: leader of WP 4),
Jean Koenig (P5: leader of WP 6),
Audrey Didier, Gerard Branlard and Agnes Piquet-Pissaloux (P5),
Rita Redaelli (P6: leader of WP 7),
Lena Dimberg (P7: leader of WP 8),
ValeriaTerzi (P8: leader WP 9),
Danela Murariu and Daniela Placinta (P9: leader of WP 3),
Nadeshda Antonova (P10),
Külli Annamaa (P11),
Malgorzata Cyran (P12),
Lenka Nedomova (P14),
Ivana Polisenska (P15 and leader of WP 5).
Additional consulting experts financed by ECPGR:
A.Katsiotis, chair of the ECP/GR Avena Working Group
I. Loskutov, head of the Cereal Department at the N.I. Vavilov Institute, Russia.
Representative of the EU AGRI GENRES Team:
Olivier Diana
Welcome addresses
The president of the INRA station in Clermont Ferrand (Michel Beckert) gave an introduction
to INRA and the meeting place. Research areas in INRA are the environment, product quality,
basic research on living organism (genomics), the integration of genomics and biology, farming practice and production systems. With 2654 publications during the last five years INRA
ranks second after the USDA. INRA also has numerous industrial partnerships.
Further introduction to research in the station was given by the president of the research unit
Gilles Charmet. Research focus is changing into genetics and diversity of cereals, mainly
wheat and triticale, while breeding programs for barley, sunflower and fungi have been
closed. Important resources are a genotyping platform and mutagenesis populations as functional validation tools. They are targeted to research on the structural and functional organisation of the genome, evolutionary biology, physical mapping of chromosomes, especially the
largest chromosome of wheat and phenotyping for decreasing protein content in grain. Integrative and functional biology, ecophysiological modelling and quantitative genetics are used
to improve stability of yield and quality.
Francoise Belfourier gave an example on the analysis of a genetic resources collection of
bread wheat, which could be interesting also for the European oat collection. The total of the
French bread wheat collection was evaluated in two sites in 2000-2002 and the results related
to geographic distribution, especially latitude, of origin. Genotypes of Nordic countries tend
to be late, South European origins early. Correlated with year of registration a decrease in
plant height of 60 cm can be observed. NIRS absorbance curves are used for describing genetic diversity. Using 23 biochemical traits it was possible to discriminate old and new cultivars
by cluster analysis. A location map of 42 SSR markers was applied on 4000 accessions to
evaluate within and between country diversity on a European and world-wide scale. Clear
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bottleneck effects were indicated in the 1960s as result of the green revolution. Geographical
analysis revealed a separation of types from Northwest, Southeast and the Mediterranean
(Spain, Italy). The results indicate different pathways of the spread of bread wheat from the
Fertile Crescent to Europe.
It was indicated that a core collection of 372 accessions, based on phenotyping, marker analysis, SNP and the analysis of chromosome 38 represented 98% of the diversity in 4000 accessions from more than 70 countries. He explained a so-called N-Strategy by calculating a total
number of alleles and a number of rare alleles.
The results also revealed that pedigree information very often is false, probably in about 30%
of cases given. For new cultivars pedigree information is normally not available at all.
Introduction of the project
Christoph Germeier gave an introduction of the work plan, timetable of deliverables of the
project. The project group consists of 15 partners from nine countries between Sweden and
Italy, France and Romnania. Besides the European Union (programme (EC) No 870/2004 as
main sponsor, three private sponsors (Peter Koelln KGaA, Elmshorn, Germany, Emco Ltd.,
Praha, Czech Republic, Gemeinschaft zur Förderung der privaten deutschen Pflanzenzüchtung e.V. (GFP), Bonn, Germany) are involved in funding of the project. The project consists
of ten work packages: project management and coordination, selection and multiplication of
the project working collection, field experiments and sampling seeds for quality analysis, field
experiments with artificial Fusarium inoculation, Fusarium and mycotoxin analysis, analysis
for protein and fat and minerals, analysis for ß- glucan, analysis for antioxidants, analysis for
cold tolerance, project documentation and internet portal. A first address was given to the way
of samples between the project partners and the logistic implications.
Milestones and deliverables for the work packages and questions to be discussed during the
meeting were listed.
A further presentation was given by the project coordinator on the logistics of the project. As
15 partners will have to be provided with seed and samples for analysis, logistics is a considerable enterprise. In the first and second year genebank accessions will have to be acquired
for three multiplication sites and after the harvest 13 field experiments in nine countries and a
laboratory test for cold tolerance will have to be provided with seed from the multiplication
sites. Seed harvested in the field experiments will have to be provided in sufficient quantities
to eight analytical labs for analysis of various quality traits and mycotoxin contamination.
Items to be discussed and decided are needed quantity by each partner and exact ways of distribution. Possibilities would be to have a central distribution or a partner by partner distribution. Orthogonality in seed and sample sources will have to be considered.
Olivier Diana outlined some aspects of the programme (EC) No 870/2004 and expectations of
the EC towards the projects. Of 72 proposals, which have been submitted, 65 were eligible for
approval: 41 of these covered plant topics, 20 animal topics, 4 microorganism or tree topics
and three covered plants and animals. 17 of these have been approved, 10 covering plants, 5
animals and two trees. 15 are targeted actions, 1 concerted and 1 accompanying action. The
total financial volume in the programme is 8.917.717. 66% of it goes into plant projects, 12%
into tree and 22% into animal projects. Olivier Diana presented a list of all supported projects.
The total number of beneficiaries is 179 in 23 member states, on average 10 partners per project. Involved, but not funded are the most recent member states Bulgaria and Romania and
12 non EU countries.
Some explanation was given on definitions and eligibility criteria in the regulation. A difficult
issue is the avoidance of overlapping with other international initiatives, e.g. the Framework
Programmes. Important aspects of project management are rules of the grant agreement to be
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respected, sound financial management, financial and technical reports in time, a dialogue
between coordinators and the commission, publicity and dissemination of the results of the
projects, demonstration for financial and technical reporting, auditing and cost statements, and
publication and copyright issues. AGRI GENRES itself runs a communication policy, which
consists in the preparation of a GENRES brochure summarizing the outcomes of 20 cofunded projects under the former Council Regulation (EC) N° 1467/1994, meetings and on
spot missions. A GENRES Committee is to develop synergies between GENRES actions having complementary objectives and GENRES actions and other initiatives like EUFGISEUFORGEN, conferences, meetings on forest and other genetic resources.
Web sites are operated by DG AGRI at http://ec.europa.eu/agriculture/envir/index_en.htm and
http://ec.europa.eu/agriculture/funding/index2_en.htm.
In contrary to other programs, e.g. the rural development programme, this programme is completely decentralised. The future after this programme is open. It is considered to merge it into
the rural development programme.
Some explanations were given to reporting procedures. For financial reporting each partner
has to fill preformed financial forms and send the filled tables to the coordinator. The Excel
application programmes will validate the entries and indicate inconsistencies. For transfer in
other currencies than EURO monthly updated exchange rates provided by the EU web site are
to be used.
Several questions from project partners were asked to Mr. Diana. In several cases Mr. Diana
will have to forward the questions to the financial department and later on write the answer to
the coordinator.
One question related to seasonal workers, which are employees of the institutes, but work for
the by additional contracts after their official working time. Rules of contracting, as outlined
in the in Article II.9 have to be followed. Especially important is avoidance of conflicts of
interest. All work by partners needs to be documented in time sheets. As a minimum every
month recording work in working days or parts of working days, according to WPs should be
made.
Another question was the eligibility of VAT. Generally VAT is not eligible, but there are regional differences, which have to be determined by financial authorities.
Eligibility of purchase cost of equipment has been questioned. It was outlined that only the
proportion of depreciation corresponding to the period of the project are eligible.
The report has to be submitted in three hard copies and on a CD, annual interim reports
not later than two months after the anniversary. The technical and financial report should be
submitted until 30. April. The final report must be submitted three months after closing of the
project at 31. May 2011. All financial statements have to be audited. 45 days are foreseen for
the technical assessment and additional 45 days for the financial assessment. Most important
is the comparison what has been foreseen, and what has been achieved.
In case tasks have not been achieved, explanations could be given in the paragraph technical
problems encountered. The maximum duration for expenses is 48 months. Before and after
this time no expenses are eligible. If changes of the action are needed, they must be reported
to the commission. The reports will also include graphical display of distribution of work between coordinator and lead partners
The web site is very important, as it is an accurate way to disseminate results. Technical reports should be available in the web site. The paragraph next steps for the new reporting period could also relate to delayed tasks. Annexes may contain inputs of each partner.
Some aspects of communication policies were outlined. A call for expression of interests is
out for finding independent experts for revising the reports. Regarding dissemination of results it has to be kept in mind that the preparation of scientific papers takes about a year. The
content of papers lies in the sole responsibility of the author as outlined in article II.5 of the
grant agreement.
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In case of delayed tasks full consistency between technical report and financial statement is
important. Flexibility in cost transfers between different items is possible in the margin of
10% at the level of the project (not each participant). It is recommended to use this flexibility
not already in the beginning of the project. A question was raised about possibility of changing the number of people for distribution of the personnel budget. Also this question will be
forwarded to the financial department.
Introduction to the work packages
Christoph Germeier presented aspects of the project management and coordination work
package. Most important aspects are the technical and financial reporting. Reports have to be
compiled by the coordinator. He should be supported by reports from the work package leaders, who request reports on respective activities from paretners. Normal exchange of documents will be by Email. For large documents including images a web platform is available on
the project web site at http://eadb.bafz.de/aveq by the link document downloads. All important documents, including presentations given in the project meetings will be available
there. The financial implementation of the project will be coordinated by the administration of
the Federal Centre of Breeding Research, now belonging to the Federal Research Centre for
Cultivated Plants – Julius Kuehn Institute. Mrs. A. Schulz is the officer responsible there for
international projects. The scientific coordination of the implementation of the action will be
to ensure working of the logistics, ensure orthogonality of field and laboratory work, to ensure
inclusion of the results into an online accessible database, the statistical analysis and the generation of knowledge from the data. These targets shall be achieved with the help of web applications to be used by the project partners. Web applications will be available for:
- Generation of field plans according an agreed randomization design: it was suggested to
use an augmented randomized block design, which allows for augmentation of randomized blocks of standard cultivars with un-replicated target genotypes.
- Generation of downloadable pdf and MS Excel sheets for data acquisition in the field including a description of the observation methodology to use.
- Downloadable lab protocols including methodology description and sample lists.
- Upload or online input of project results including pictures.
Information was presented on requirements for the technical activity reports, which had been
sent as a draft by DG AGRI GENRES. It will clarify the contents and the maximal number of
pages expected for each issue. The report should contain an executive summary, a concise
statement of the tasks undertaken, a comparison of foreseen and achieved deliverables, a description of the management of the action and a concise statement of the activities carried out
for dissemination of interim results.
The financial statements should list eligible costs actually incurred and paid drawn up individually from each beneficiary for the respective annual period with certified, complete and
numbered list of paid salary expenses, invoices and totals, external audit reports and an update
of the action’s budget.
Zofia Bulinska Radomska, leader of work package 2 concerned with the multiplication of the
working collection explained some practical aspects of this task. In total 563 accessions have
been requested by European genebanks starting from project begin in March. 450 accessions
have been received, 352 have been sown at the multiplication site Radzikow. Two accessions
of wild species have been sown in a tunnel. Additional accessions, not listed in the order lists
have been received from some genebanks and some modern cultivars from breeders.
Due to late arrival of some accessions, they were sown on several dates. Sowing in generally
nine rows per plot as well as harvesting was done manually. Fertilizer was applied two times.
Weeds were controlled by herbicides and hand weeding. Some accessions suffered from herb4
icide damage. Especially A.strigosa did poor, and seed shedding was observed, while obsolete
varieties and landraces did very well. 13 accessions of wild species were received. Two of
them have been grown in a tunnel from pre-germinated seed. Treatment with fungicides
proved not successful.
Following issues have been raised: The list of accessions for next sowings will have to be
revised list. It has to be decided what to do with the extra accessions received. The methodology for multiplication, especially for winter forms should be considered. Considering the use
of the harvest mixing of samples from the different multiplication sites would be preferred.
The question was raised, whether to use harmonized taxonomy or stick with determination of
holding genebanks. The methodology for germination and Fusarium testing in the harvest will
have to be decided. Germination tests according to ISTA rules were suggested and additionally visual assessment and determination of seed damage.
Audrey Didier reported, that in Clermont-Ferrand 387 accessions have been sown. Accessions
which appeared as mixtures, where not harvested. Growth habit, grows class (winter, intermediate, summer, days to heading, lemma colour, harvest weight and thousand grain weight
were determined.
Danela Murariu, as leader of WP3 mentioned, that field experiments will take place in very
different conditions. Descriptors for taking notes in the field will have to be agreed on.
For WP4 Matthias Hermann explained that Fusarium infection and mycotoxin production are
different traits and not necessarily very correlated. They are quantitatively inherited. Field
experiments will be carried out under fixed conditions with 300 accessions per year and 10
standards in five replications. Plot size and descriptors for observations in the field will have
to be agreed on. He mentioned the following suggested descriptors:
Heading date as the date when 50% of the panicles are emerged; Panicle density, which has a
low heritability; Crop height; Distance between panicle and flag leaf; Panicle shape and panicle length; Lodging at harvest, while lodging should be prevented; Test weight; Fusarium
infection;
WP5 was introduced by Ivana Polisenska: With ELISA tests only one mycotoxin per analysis
can be determined. Thus it will be necessary to agree on the toxins to be analyzed. This also
relates to the question, which Fusarium species should be considered for inoculation. European law deals with Deoxynivanelol, ZEA, T-2 and HT-2. These are currently in the spot light,
and currently discussed in EU commission especially for oat products. For the HT-2 toxin an
ELISA test is currently not available.
She raised the question, which Fusarium species will be used in the differently located field
experiments, whether Fusarium mycelia could be quantified and a relationship between mycotoxin production and Fusarium mycelium could be established. She also raised the question
about natural or artificial infection. In Czech Republic Fusarium poae is the main T-2 producer. Further selection of samples for analysis, standards and controls has to be considered.
Often great variability in replications is observed.
Harmonizing of analysis methods between the contributing labs will be necessary. Thus it
should be agreed on, which analysis kits to use and about the sample amount per analysis. She
suggested, that sample preparation (milling) should be done in one lab.
Jean Koenig introduced WP6 with micronutrients, avenin patterns and NIRS analyses calibrated mainly for protein and oil content. Participating partners are P5, P9 and P11.
NIRS analysis will be performed on all samples harvested in the quality field experiments by
P5 and P11. NIRS calibration will be done for protein using the Kjeldahl method, for fat with
the Soxhlet method by P9 and P11 on 10% of the samples. The main decision to be taken now
is the quantity of seeds needed for the analysis.
Avenin patterns will be determined for 600 accessions. They are not subject to environmental
influence and can be determined with harvest from the French site – already from the multiplication plots. Thus it would be possible to start this activity already before the indicated start
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of WP6 in the proposal (month 24 means March 2009). Minerals (major and trace elements)
will be determined from 200 accessions harvested at the French site.
Work will start after harvest of the first year’s quality experiments at the end of 2008 or beginning of 2009 with the receipt of the first 300 accessions from field plots by technical labs.
NIRS will determine the most diverse samples, which will be used to calibrate for protein and
oil content and for selection of the first 100 samples for micronutrients analysis. A similar
work plan will be followed with harvests in 2009/2010.
Gerard Branlard gave a short introduction into Avenins, which are, like glutenins alcohol soluble storage proteins. They are coded by a clustered block of loci with several alleles. Avenin
patterns will be determined for 600 accessions. Agnese ?? introduced analysis for micronutrients and mineral quality. She raised questions about number of accessions, agricultural environments, sampling procedures and number of replications. Mineral analysis will be done by
AAS and 20 g samples will be necessary for this analysis.
Rita Redaelli gave an introduction to WP7. It will characterise carbohydrates (starch, dietary
fibre and ß-glucan) in the working collection,.when grown under contrasting growing conditions all over Europe from the Nordic countries to South (Italy) and South East (Bulgaria,
Romania) in relation to the structure of the collection. P6, P2 and P12 will be involved. She
also introduced acting persons at the partner labs and listed the milestones for the work package as outlined in the proposal.
A main issue will be the analysis for mixed-linked (1-3),(1-4)-ß-D-glucans, which are frequently present as soluble and insoluble ß-glucans in endosperm cell walls of cereals. Contents in wheat average 1%, in rye 2%. Relatively high contents, on average 3.6% (2-10%) are
found in barley and on average 3.4% (2-6%) in oats. While in cereal products like malt, wheat
and rye bran ß-glucan contents are reduced, in oat bran they are alleviated up to 8.3%. In oats
also the soluble fraction is larger, up to 80%, of total ß-glucan. Both genetic and environmental factors affect the content of total ß-glucan.
Physiological effects, like lowering the cholesterol levels in blood, and reducing the postprandial increase of glucose with effects on the glucose metabolism are recognized and lead
in 1997 to the establishment of a “health claim” by the US Food and Drug Administration
stating that a 3 g daily intake of oat soluble fibre (ß-glucan) can reduce the risks of cardiovascular diseases. This claim was confirmed in 2002.
glucan
Methodological details of analytical methods were given in this presentation: analysis of total
ß- will be done by P2 according to Analytica-EBC, Method 3.10.2, involving α-amylase digestion at 100°C using a thermostable enzyme, hydrolyses in 0.004M H2S04 and fluorometric
determination of released glucose using Calcofluor and by P6 with AOAC reference method
995.16 involving hydrolysis with lychenase and ß-glucosidase and the spectrophotometric
determination (λ=510 nm) of released glucose with a glucose oxidase and peroxidise reagent.
Soluble ß-glucan will be determined by P6 with the same method as difference after extracting for 120 min in distilled water at 38°C.
Starch content in oat ranges from 40 to 60%. Significant genotypic and environmental effects
have been observed in the content of total starch. Oat starch is difficult to separate from other
grain components. Especially it is associated with a larger lipid content (1-3%) than the starch
of other cereals. The average size of individual oat starch granules varies from 3 to 10 μm.
Analysis of total starch will be done by P12 with the Amyloglucosidase/α-Amylase method,
which is available as a Megazyme Assay Procedure, and has been described as AOAC Method 996.11, AACC Method 76.13, and ICC Standard Method No. 168. It involves extraction of
low molecular weight components, incubation at 60°C with thermostable α-(1→4)-amylase
and (1→6) amyloglucosidase at 50ºC, overnight and spectrophotometric determination of
released glucose with the glucose oxidase and peroxidise reagent.
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Dietary fibre is the edible part of plants or analogous carbohydrates that are resistant to digestion and absorption in the human small intestine with complete or partial fermentation in the
large intestine. It includes polysaccharides, oligosaccharides, lignin and associated plant substances. Dietary fibres promote beneficial physiological effects including laxation, blood cholesterol or glucose attenuation. Analysis of total dietary fiber will be done by P12 using the so
called Uppsala Method (Theander et al., 1995). It involves the determination of Klason lignin
with a gravimetric method after Theander et al. (1995), boiling with DMSO for 1h, incubation
with α-amylase, and with amylo-glucosidase and proteinases at 40°C overnight, hydrolysis of
polymers in H2SO4 for 1 h and determination of uronic acids by a colorimetric method after
Scott (1979) and of quantitative analysis by GC of derivated monosugars after Englyst and
Cummings (1984). As issues for discussion reproducibility of β-glucan measurement with
different methodologies used by P2 and P6 and necessary de-hulling of samples were raised.
Lena Dimberg introduced antioxidants and their analysis in WP8. Autooxidation of lipids
generates free radicals, which cause cell damage and spoilage of foods. The main function of
antioxidants lies in retarding lipid oxidation with different mechanisms, which may protect
food from rancidity and the body from free radical damages. In cereals tocopherols and tocotrienols (Vitamin E) are the most potent antioxidants and membrane stabilizers, further hydrocynnamic acids. Tocopherols are located in the germ, tocotrienols in the endosperm and
are positively correlated with the oil content. Contents of both underlie genetic and environmental variation. One NP-HPLC analysis for tocols detects eight compounds. A range of 1967 mg/kg has been found for tocol contents in oat.
Avenanthramides are antioxidants unique to oats, which have been first described in the
1980s. 30-40 different compounds are known. Their antioxidant activity is less effective compared to tocopherols, but acts for a longer time. They have been attributed with antiinflammatory and anti-artherosclerotic activity and are chemically very much similar to tranilast, an antiallergic drug. Their formation is induced in young leaves in response to incompatible crown rust infection and not related to the oil content. In the field they are always
found in leaves and grain, mainly in the bran. They increase during germination and from
heading to maturity. They are resistant to storage and heat. High variation with site and year
has been observed. Effect of cultivation methods are of interest. Regarding nitrogen an optimum relationship has been observed. Higher contents in crown rust resistant cultivars are assumed.
The analysis for cold tolerance (WP9) was introduced by Valeria Terzi. Field tests at two sites
(Fiorenzuola d’Arda in North Italy and Suceava Genebank in Romania) over two years in
nurseries of small plots (1 m2) will explore the genetic variability for cold tolerance in winter
(and spring) oats and identify genotypes with superior frost tolerance. If considered as a winter cereal, oat is the most frost sensitive and its insufficient level of winterhardiness is the
main factor limiting sowing and yield of winter oat in Northern Italy and Central Europe.
From experiments in Fiorenzuola d’Arda with 62 genotypes of different origin (Europe, North
America) during 1996-’97 a broad genetic variability for winterhardiness in a range between
2.5 and 8.59 could be shown in oat. Cold resistant cultivars sown in winter realize a higher
yield potential. Frost resistance is an inducible process, promoted by cold acclimation (hardening) at low non-freezing temperature and associated with a number of biochemical and molecular changes
The methodology for the measurement of hardening effects is based on the observation, that
low temperature and light intensity affect the photosynthesis system (PSII excitation pressure), observable in chlorophyll fluorescence responses. An experimental unit (PAM 2000) is
available for these measurements. For the standard treatment one week old plants are hardened at 3-1°C (day/night) and 200mE m-2 s-1 for 4 weeks. Then they are subjected to a freezing treatment (–12°C, –13°C). The ratio of variable to maximal fluorescence (Fv/Fm) in darkadapted state is measured. In a test with 16 cultivars four maintained a value close to 0.8 after
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-12°C, three after -13°C. Accumulation of a certain substance (COR14) was found to be associated with the hardening.
In a comparison with other cereal species oat behaved similar to winter resistant barley, suggesting that it should be possible to improve the resistance of winter oat to a level similar to
winter barley. This would lay the basis for a possible winter sowing in oat and for breeding of
winter oats. The freezing evaluation test represents a useful tool for the evaluation of frost
tolerance. The AVEQ project is a unique opportunity to evaluate biodiversity for this trait.
Igor Loskutov remarked that there are different categories involved. Cold resistance and frost
resistance can be affiliated also with spring types. Winter hardiness is a complex of traits,
which will be needed by a winter type. The winter type itself is determined by the vernalization requirement.
Presentation and discussion of work package session results and decisions on next steps
In work package sessions for WP2 state of compilation of the working collection, conditions
and field designs for multiplication of accessions were discussed. Not all requested have been
received and new orders will have to be made especially at those genebanks, which had not
yet sent any samples (United Kingdom, Greece and Spain). Some harmonization of taxonomy
will be used for the working collection, mainly oriented at the taxonomy used in the Vavilov
institute and proposed by Igor Loskutov.
Modern commercial cultivars should form the replicated core of the augmented design which
is constant over sites and years. Further they should allow comparing traits in the genebank
material with the current state of modern breeding. Different agro-ecological conditions in the
regions participating with field experiments in the project should be represented by the standard cultivars. Thus 2-3 standards from each region should be selected.
Following standards were finally selected by the project group:
Representing the Nordic region (Estonia, Sweden): Belinda: breeder SW-Seeds (Swaloef
Weibull, Sweden): It represents the mid-late varieties in this region and is currently the main
variety in grown in Sweden, Norway and Finland, but is also listed in the Baltic countries.
Jaak: breeder Jogeva, Estonia: It is one of the Estonian latest-bred varieties and two to three
days earlier than the other Estonian varieties.
Representing Western Europe (France): Auteuil: breeder Serasem, France: It is the most cultivated spring cultivar in France and has been already used as a standard cultivar in the previous project. Its lemma is brown to black. Evora: breeder Serasem, France: It is the second
most cultivated winter cultivar in France and has been already used as a standard cultivar in
the previous project. It has dark lemma colour as well.
Representing Central Europe (Czech Republic, Germany, and Poland): Central Europe is represented by three countries within the project. Thus it was decided to have this region been
represented also by three standard cultivars: Ivory: breeder Nordsaat, Germany: it has been
reported to accumulate less DON because of low hull content. Krezus: breeder Hodowla
Roślin, Poland, released in 2005: It is grown all over Poland as a semi-early cultivar with medium-sized plants, is very good in lodging resistance, yield, tolerance to soil acidity and resistance to stem rust and Helminthosporiosis. It has yellow lemma, low fat content and a high
TGW. Saul: breeder SELGEN, Czech Republic: a naked variety with low fat.
Representing Southern Europe (Italy): Argentina: breeder SIS, Italy: most cultivated cultivar
in the past. Genziana: breeder ISC, Italy: very new variety.
Representing South East Europe (Bulgaria, Romania): Mures: breeder Dr. Vasile Moldovan,
Romania. Mina: naked cultivar, Bulgaria.
It was decided to use seed directly acquired from breeders for the standard cultivars. Partners
from respective countries (Alf Ceplitis, Külli Anammaa, Jean Koenig, Christoph Germeier,
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Zofia Bulinska, Lenka Nedomova, Rita Redaelli, Danela Murariu, Nadeshda Antonova) will
be responsible for distribution of standard cultivars.
The fieldplan generator and augmented designs should be used also for multiplication fields.
Standards will not have to be multiplied, as seed directly provided from breeders will be used.
Yet they could be used to make observations in the multiplication experiments comparable.
A special problem in acquisition and multiplication are the wild species. It has to be checked,
what number of accessions will be available at which quantity after all multiplication results
are available. I.Loskutov characterised especially the diploid species A.pilosa, A.clauda,
A.ventricosa, and the tetraploid A.insularis as difficult to handle. A.longiglumis, A.hirtula,
A.wiestii, A.barbata, A.magna, A.vaviloviana and all hexaploids would be more easy to use
for the project. The question was raised, whether to multiply all wild species in tunnels. As
there was good experience also with having them outside with bagged panicles, it was decided
to leave method of protection from lodging and seed shattering up to the partners. Preparation
of germination already in February was recommended. Dormancy is to be expected in wild
species, several of them behave like winter types (e.g. A.fatua)
To maximize multiplication rates low stand densities with 25 cm row distance, and 5 cm seed
distance in row were recommended in all multiplication plots.
During seed multiplication in the field following descriptors have been recorded by P0:
Emergence in 1 m row, growth habit in youth stage, BYDV, visual score (%plot and score on
one (diseased) plant (1-9 dead ) and ELISA on single plants, heading date, mildew (1-9), panicles: orientation of lateral branches, spikelet attitude, and morphological classification, lodging, grain shedding, awnedness, awn type, grains per spikelet, crop height (plant length) and
panicle length. P0 also made already photo documentation in the multiplication plots. Following phenological stages were recommended as interesting for photo documentation: 1.) Before
tillering (stage 15-20) to demonstrate erect or prostrate types, 2.) Before heading 3.) After
heading, the inflorescence. I. Loskutov recommended spikelets as primary target for photo
documentation.
To test germinability in the material to be sown following an ISTA procedure with breaking
dormancy (e.g. by chilling) was recommended. Zofia Bulinska suggested three times 50 or 25
seeds depending on availability to use for tests on filter paper. Percentage of germination and
mean days of germination should be recorded. It was considered desirable to use germination
tests also for getting indication on Fusarium infection of the seed, e.g. by additionally recording mycelium growth and mycelium identifiable as Fusarium. It was decided to acquire exact
information on ISTA procedures for germination tests and breaking of dormancy in oats and
possibilities to combine it with Fusarium observation.
Heading date should be observed to identify winter types. It would be useful to sow these earlier. A further issue was distribution of seeds from multiplication sites to field experiments.
As there are three multiplication sites and three work packages performing field experiments
(quality field experiments, field experiments with Fusarium inoculation, frost tolerance), the
most straightforward procedure would be, that each multiplication site distributes seed to one
field work package. WP2 with the largest number of experiments should get seed from the
site with largest harvests.
For WP3 it was suggested to have all winter types in the second year. Logistics in distribution
of seed to the analytical work packages was a major issue for discussion. Tab.1 shows requirements from WP6 to WP3. Dehusking will be necessary for about 3300 samples in quality
analysis. It was decided that in total 180g of each accession needs to be available to P5 for
distribution within WP6, 220g to P6 for distribution in WP7 and WP8. Experiments could be
made in advance to check necessity of dehulling, but it is anticipated, that it will be generally
necessary for quality analysis. For Fusarium and mycotoxin analysis samples will be generally not dehusked. Another question raised was, where milling of samples should take place and
9
how they should be maintained. Especially maintenance of samples with high oil content is
problematic.
Table 1) Logistics of seed distribution from WP3 to WP6 after harvest 2008
Number of Condition
of
Partner
Activity
accessions
sample needed
P5
NIRS
3300
Dehusked
P11
NIRS
500
Dehusked
P11
Protein Clibration
300
Dehusked
P9
Protein Clibration
50
Dehusked
P11
Fat Clibration
50
Dehusked
P9
Fat Clibration
300
Dehusked
P5*
Minerals
100
Dehusked
* Mineral analysis only from Clermont- Ferrand samples
Quantity needed [g]
minimal
optimal
20
50
20
50
15
20
15
20
15
20
15
20
20
It was discussed how to make agronomic measures comparable over the sites. It was suggested to keep to local practices as close as possible. Row distance, sowing density and sowing
time are the main variables. Row distances of 12.5-15 cm and sowing density of 200-400 kernels m-2 according to local practices were recommended. A sowing density of 400 kernels m-2
would require 1000 seeds distributed to each field experiment. For Southern European partners seeds would be needed already at the end of January.
Further treatments, which have to be considered, are fertilization, plant protection and lodging
prevention. Fertilization should keep to low input condition. Especially no nitrogen should be
applied for lodging prevention. Herbicides will have to be applied, insecticides and fungicides
only if necessary. Generally the whole plot will be used for harvest.
Following descriptors were agreed to be recorded in the quality field experiments:
A) Obligatory descriptors, to be determined in all quality field experiments:
Days to heading, days to maturity, crop height [cm], lodging, panicle shape, occurrence of
diseases (assessment keys of James 1971), colour of lemma, hull content (done by partners
P2, P3 with dehulling equipment), yield, thousand kernel weight and test weight.
B) Optional descriptors, to be determined, if possible:
Emergence (number of emerged plants in one meter in two neighbouring rows per plot), number of fertile tillers (in one meter in two neighbouring rows per plot) and number of seeds per
panicle (determined on panicles in one meter of two neighbouring rows)
Further details in observation methodology will have to be made available for discussion by
the web application
A main issue for discussion in WP4 was composition and preparation of the Fusarium inoculums and the inoculation methodology to be used for field experiments 2008. Field experiments with inoculation will be set up at three locations (P3, P9, and P14). It was agreed, that
the inoculum should consist of a mixture of different local strains of four Fusarium species
selected according to their toxin production. Possible competition between the Fusarium species in a compound inoculum will be neglected. P3 will produce inoculum for P3 and P9,
while P15 will produce inoculum for Czech Republic. P8 will test only winter oats without
inoculation within the frost tolerance field screening.
It was discussed to group accessions of different phenology (heading dates) and plant heights
into different experiments to facilitate inoculation timing and procedures. But finally it was
decided to inoculate on three different dates to catch all accessions at flowering. On three inoculation dates 20 ml m-2 of a defined inoculum with 1 Mio. spores ml-1 should be applied. In
total this will require 60 l inoculum solution. Inoculation should be done on three dates before
and after flowering in the evening and supported by irrigation, if possible. Daily weather rec10
ords should be taken. If possible, maize should be used as a precrop as it is conducive to mycotoxin accumulation. An augmented design with 2.5 m2 plots (2x1.25 m2) will be used. One
summer wheat should be included as additional standard to demonstrate inoculum pressure
and a comparison of susceptibility of wheat and oat.
All experimental measures (herbicides, fungicides etc.) need to be documented. The harvest
should be used for analysis without cleaning not to loose flowers without seed set. The selection of samples for analysis should be discussed in the next meeting based on results of field
observations.
Following descriptors were agreed to be recorded in the Fusarium inoculation experiments:
Days to heading, number of panicles (one meter in two neighbouring rows per plot), crop
height, panicle shape, panicle length [cm], infection of panicles with Fusarium scored from 1
(no symptoms) – 9 (severe symptoms in all spikelets), score of other diseases (assessment
keys of James 1971), lodging before harvest should be recorded and avoided, thousand kernel
weight, test weight, lemma colour, percentage of Fusarium infected kernels (1 = healthy, 2=
with grey tips, 3= damaged - small, thin, discolored), Fusarium infection of seeds, hull content of standards (inoculated / non inoculated).
Cross cutting issues in field experiments by WP3 and WP4 were discussed in a separate session involving partners of both work packages. Besides aspects of descriptors to be used in
both work packages this was mainly treatment of wild species. It was put forward, that wild
species should not be present in all field experiments. Their experimental management is difficult and costly and genotype site interactions for them are not interesting. Rather it is interesting, on what level they compare with cultivated material in the respective traits. For this it
would be sufficient to have them on few sites. Volunteers for including wild species into their
experiments were looked for. For the quality field experiments P9 (Romania) and P11 (Estonia) agreed to integrate them. For the Fusarium inoculated experiments all three partners
agreed to include wild species.
For WP5 main issues for discussion were the toxins top be analyzed, the Fusarium species
related to these, which have to be included into the inoculum by WP4, requirements from
WP4 regarding sample size and logistics, and analytical methods to use for samples produced
in 2008. Based on the EU limits for contaminants in foodstuffs, laid down in Commission
Regulation (EC) 1881/2006 Deoxynivalenol (DON) and T-2 toxin are the main targets. DON
and Zearaleone (ZEA) are produced by F.graminearum and F.culmorum, T-2 and HT-2 by
F.sporotrichoides, F.poae and F.langsethiae. Higher levels of T-2 and HT-2 are often found
in oats. Proposed limits of these toxins, shown in Tab. 2 were presented
Table 2. Proposed limits [ppb] for Fusarium mycotoxins in cereal food stuff
cereals unprocessed
oats unprocessed
cereals intended for direct human consumption,
cereal flour…
*oat products
11
DON
1250
1750
750
ZEA
100
100
75
T-2 + HT-2
100
500
200*
P8 will receive all strains to be used for preparation of inoculum (F.avenaceum,
F.graminearum, F.culmorum and F.sporotrichoides) till the end 2007 from all of the WP4
partners and will check the PCR identification and quantification of the amount of mycelia.
For PCR analysis standards from all experiments with Fusarium inoculation will be used.
Calculations made up for sample logistics are shown in Tab.3
Table 3: Calculations for sample logistics in WP5 for 2008-05-04
Samples produced in WP5
Sample type
accessions - inoculated (I)
11 standards x 5 replications – I
11 standards x 2 replications - non-inoculated (NI)
1 wheat control (I + NI)
Total per site
x 3 sites (Germany, Romania, Czech Republic)
Available ELISA tests for 2 mycotoxins
P6 (Italy)
P8 (Italy)
P15 (Czech Republic)
Number
300
55
22
2
379
1137
50
250
250
550
100
LC-MS/MS
-> samples must be selected
It was still unclear, how many additional winter types from an Italian not inoculated field experiment (WP9) are to be expected.
A total of 550 samples selected into WP5 will be analysed by ELISA for DON and T-2 toxin.
100 LC-MS/MS tests will be performed for verification of the ELISA results, a comparative
study on T-2 and HT-2 and the analyses for other mycotoxins.
Following aspects should be considered for sample selection:
- Is it necessary to analyse all standard replications?
- It is clear, that obviously heavily infected genotypes should be omitted from analysis. The
main question remains, whether Fusarium head blight symptoms will be readily observable on panicles in the field.
Sample selection will have to be discussed during the next project meeting based on the results of the field trials in WP4.
Methodological issues are harmonisation of the different labs involved in ELISA analysis by
the following measures: 1.) Use of reference samples, 2.) Use of identical ELISA kits.
ELISA kits will have to be selected according to the observed level of infection. It was discussed, whether it will be possible to make use of different prices of the kits in different countries.
According to European rules (EC 1881/2006) non-dehulled samples will be used for analyses.
An important issue will be preparation of homogeneous samples. From incremental samples,
by milling more than 100g ca. 20g will be used for analysis. It will be necessary to keep additional seed for possible re-analyses or more precise analyses (LC-MS/MS, HPLC). These will
be based on ELISA results
The work packages dealing with quality analysis (WP6, WP7, WP8) faced very similar issues
and had a great deal of sessions together. Standard methodology is mainly used in these work
packages and sample logistics was the main issue to be discussed. In WP6 preliminary paral12
lel NIRS measurements of P5 and P11 should indicate consistency of results between both
labs. A minimum of 30g sample will be necessary for NIRS, an optimal sample size would be
50g. Flour will be sent by the NIRS labs to the calibration partners. There was a discussion,
whether Avenin analysis could be already started in 2008. For the analysis of minerals, which
will be restricted to a set of accessions harvested by P5 in France, 20g samples are necessary.
As P6 is represented in WP7 and WP8, this partner will take up the roll of a central distribution hub for seeds in both work packages. Only part of the material produced in the field experiments can be analyzed in these work packages. Thus all samples from only three contrasting sites, representing cold wet Nordic conditions, the hot and dry Southern conditions
and the temperate Central European conditions, will go into these work packages each year.
In the first year this will be P2 (Sweden), P1 (Poland), and P10 (Bulgaria), in the second year
P11 (Estonia), P8 (Italy), and P9 (Romania). Changes may be necessary in case of insufficient
harvest or other problems in the field experiments. De-hulled seeds will be necessary for all
analysis. P6 will collect de-hulled, but not milled groat from the field experiments of P1, P2
and P10 and re-distribute it to the other partners. Following quantities will be needed: 50 g for
total ß-glucan; 70 g for soluble ß-glucan and tocols, 50 g for starch and fibre on 200 accessions; 80 g for Avenanthramides on 75 accessions; The total amount needed for all analysis
would be 250 g. A further reduction (selection) of number of accessions to 200 each year will
be necessary for analysis of tocols, fibre, starch and soluble ß-glucan. Content of total ßglucan will be the main criterion for this selection.
Dehulling will be a great deal of work, which can be done only by two partners (P2, P3), who
have the necessary equipment. Table 4 gives a summary of total sample size needed from
each quality field experiment, which is also the amount needed to be de-hulled. De-hulling
will be a major issue for discussion in the next meeting.
Table 4 Summary of sample logistics for quality analysis
P1 (Poland)
P2 (Sweden)
P5 (France)
P8 (Italy)
P9 (Romania)
P10 (Bulgaria)
P11 (Estonia)
P5 for redistribution in WP6
180 g
180 g
180 g
180 g
180 g
180 g
180 g
2008
P6 for redistribution
in WP7 and WP8
250 g
250 g
250 g
2009
P5 for redistriP6 -> WP7
bution in WP6
and WP8
180 g
180 g
180 g
180 g
250 g
180 g
250 g
180 g
180 g
250 g
Analysis of Avenanthramides will be very restricted, because of high analysis costs. As
Avenenthramides are phytoalexins induced by pathogen infection, analysis as well from
quality experiments as from Fusarium inoculated experiments would be of interest. A following preliminary sample strategy for analysis of Avenathramides was suggested, as shown in
table 5
Three standard cultivars inoculated and not inoculated will be analysed from the experiments
with Fusarium inoculation to find out the influence of Fusarium on formation of Avenanthramides. A restricted set of 25 accessions will be analysed from the three quality experiments selected for WP7 and WP8. Two replications of standard cultivars from one site could
be included as a control in two replications.
Table 5 Preliminary sample strategy for the analysis of Avenanthramides
13
2009
Accessions
Standards
2010
Accessions
Standards
P1 (Poland)
25
2x2
25
2x2
WP 3
P2
(Sweden)
25
25
P10 (Bulgaria)
25
P3 (Germany)
WP4
P14
(Czech Republic)
P9 (Romania)
3x2x2
3x2x2
3x2x2
3x2x2
3x2x2
3x2x2
25
Three partners (P8, Italy, P9, Romania and P10, Bulgaria) were involved in discussions on
WP9. They will perform field trials in 2008 and 2009 and evaluate 500 accessions (excluding
wild species). Different types of screening should be done: 1.) Screening of spring types for
frost resistance (P9, P10), 1.) Screening of spring types for winter hardiness (P10), 3.) Screening of winter types for winter hardiness (P8, P9, and P10). Screening for frost resistance will
involve sowing in early spring (January, February), screening for winter hardiness sowing in
autumn (September). Information about growth type (spring or winter) will be needed to distribute accessions to these types of screening.
Plots of 2 m2 will be sown with 1000 seeds (about 60 g) for each accession. 300 g seed will be
needed for five replications of the standards. Each partner will record meteorological data.
Frost injury in the field will be visually estimated on a 0-9 scale according to Rizza et al.
(1994). After beginning of the stable vegetation period the survived plants m-2 will be determined.
Based on data from the field screening 100 accessions will be selected for the chlorophyll
fluorescence evaluation test: 1 week-old plants are hardened at 3-1°C (day/night) and 200mE
m-2 s-1 for 4 weeks and subjected to a freezing treatment (–12°C). The Romanian partner will
perform cold resistance tests with two regimes of -10° C and -12°C.
The web platform and web applications developed in WP10 were presented in two Power
Point presentations with screen shots and some practicing was offered to introduce login procedures and generation of field plans, which will be the first task to do online for all project
partners. Acquisition of harvest data has been already successfully used by the three multiplication partners.
It was agreed, that the next meeting should be held not before completion of the harvest of
field experiments. All field data need to be available in the project database for being able to
make detailed plans for the analytical work packages. It was anticipated, that this will not be
before the first half of December 2008. Thus the meeting in Quedlinburg will be planned for
first half of December or begin of 2009 (January or February).
The coordinator thanked all participants for their valuable contributions and especially the
representatives of the hosting institute (INRA, UMR Amélioration et Santé des Plantes, Clermont-Ferrand, France) for the excellent organisation of the meeting.
14