Biologisk markkartering

Transkript

1 Biologisk markkartering Jordburna patogener kan orsaka stora skördeförluster. Klumprotsjuka och ärtrotröta är exempel på sjukdomar som producerar vilsporer med lång överlevnad, år, i marken. Inom BioSoM (Biological Soil Mapping), som är ett tematiskt forskningsprogram vid NL-fakulteten på SLU, är målet är att utveckla ny service till lantbruket för att bestämma förekomsten av jordburna patogener. SLU ansvarar för det vetenskapliga arbetet och utveckling av råd för att optimera växtföljd och odlingsåtgärder i samarbete med ett flera intressenter från näringslivet. Provtagning Representativ provtagning är förutsättningen för bra kartering av markburna patogener. Bestämning med PCR Artspecifika DNA sekvenser används för att bestämma förekomst och mängd av patogen av markburna patogener förekomst och mängd av patogen. kan integreras med precisionsodlings- Karta och management Resultaten presenteras på en karta så att åtgärder kan varieras inom fält. Kartdatan kan integreras med precisionsodlings teknik för platsspecifika insatser styrda med GPS. Jord och växtstudier Biotest och fältförsök utförs för att fastställa sambandet mellan förekomst av patogen och skada på gröda. Biologisk grundkunskap Säker bestämning och effektiv bekämpning kräver kunskap om patogenens biologi, exempelvis hur vilsporer fungerar. Markkemi och patogen Markkemiska förhållanden kan påverka förekomsten och sjukdomsutvecklingen. P. brassicae fg/ g soil P. brassicae versus ph ,4 5,6 5,8 6,0 6,2 6,4 ph-value 6,6 Klumprotsjuka Ärtrotröta Bomullsmögel Kransmögel Phytophthora pisi (SaraHosseini) Rotbrand, sockerbeta Kontaktpersoner Finansiärer och näringslivsintressenter Programansvarig Anders Jonsson Anders.Jonsson@slu.se +46 (0) Precisionsodling och pedometri Institutionen för mark och miljö SLU, Box Skara Forskare i BioSoM Ann-Charlotte Wallenhammar Arne Schwelm Christina Dixelius Charlotta Almquist Fredrik Heyman Katarzyna Marzec-Schmidt Mats Söderström Kontakt via mail: namn@slu.se ( SL-Stiftelsen

2 Biologisk markkartering Integrerad analys av jordbundna växtsjukdomar och markkemi (BioSoM I ) Det övergripande syftet med är att bygga upp grundläggande kunskap om jordburna patogener och de faktorer som påverkar deras utveckling och skadepåverkan. Slutmålet, visionen, är karteringstjänst som kan nyttjas av lantbrukare för att optimera sin platsspecifika växtodling, en sk Biologisk markkartering. Forskningsprogrammet Biologisk markkartering är ett sk TEMA-forskningsprogram vid NL-fakulteten, SLU. Avsikten med programmet var att det skulle pågå i 3+3 år efter en utvärdering efter den första tre-årsperioden. Fas två av BioSoM är nu också beviljat och löper under Traditionellt har diagnos och prognos av jordburna växtpatogener, dvs mikroorganismer som utnyttjar jorden för överlevnad i väntan på lämplig växt att angripa, har varit mycket begränsade i Sverige och i övriga världen. Den har främst bestått av resurskrävande biotester i växthus. I Australien har emellertid under de senaste 10 åren utvecklats en världsunik service där man med DNA-teknik bestämmer förekomsten av några för Australien viktiga jordbundna patogener i jordprov. De jordburna växtpatogenerna sprids vanligtvis med sk sporer eller sklerotier eller mycel i jorden. De infekterar roten och tillväxer till inuti växten, vilken i många fall försvårar en korrekt diagnos. Många överlever i fält med sk vilokroppar/vilostrukturer (sklerotier) som har olika grad av överlevnad beroende omständigheter i marken som jordart, grödval och väderlek. I Sverige har vi avsevärda problem med främst klumprotsjuka och kransmögel i oljeväxter, rotröta hos ärter och i rödklöver. Problemen kan vara stora och är främst kopplade till olämplig växtföljd. De kan endast reduceras genom att utesluta mottagliga växter under 5-15 år. I framtidens växtodling, ställs stora krav på att effektiv fånga solljus till höga skördar med rätt kvalitet. Kombinationen av precisionsodlingsteknik, (GPS-styrning och geografiska informationssystem, GIS) samt rimligt dyra DNA-baserade analyser av markburna patogener öppnar ett nytt kunskapsfält, som vi valt att kalla Biologisk markkartering. Utifrån en bestämning av förekomst av jordburna patogener kan förbättra valet av gröda, odlingsåtgärder och avsevärt öka den platsspecifika anpassningen i växtodlingen. Eftersom det är känt att förekomsten av patogener kan variera i fält beroende på biotiska och abiotiska faktorer och att flertalet av patogenerna är långlivade i marken är målet att efter en kartering bygga modeller och prognoser som visar när och var det åter är aktuellt att odla en mottaglig gröda eller när en resistent sort måste användas samt hur näringstillförseln skall optimeras för att reducera angrepp. Extra problematiskt med dessa sjukdomar nu när intresset för och behovet av inhemskt producerad biodrivmedel och proteinfoder ökar i EU. Trots att odlingen varit liten under 2000-talets inledning så uppmärksammas redan nu avsevärda problem med sjukdomar kopplade till växtföljd i våra oljeväxter och ärter. För oljeväxteret gäller det bomullsmögel (Sclerotinia sclerotiorum), kransmögel (Verticillium longisporum) och klumprotsjuka (Plasmodiophora brassicae). Dessa sjukdomar kan även angripa andra växter inklusive vissa ogräs förutom att de kan överleva i marken i väntan på nästa mottagliga gröda För att nå EUs mål att öka självförsörjningsgraden av protein ligger det i dagsläget närmast tills hands att försöka öka ärtodlingen i Sverige. Uppförökning av markburna växtföljdsjukdomar som ärtrotröta (Aphanomyces euteiches) är redan ett stort problem för

3 konservärtsindustrin och många ekologiska odlare. Andra patogener angriper också ärtgrödan t.ex. Fusarium oxysporum f. sp pisi som orsakar vissnesjuka respektive F. solani f. sp pisi som orsakar rotröta samt Pythium spp. Den senare orsakar skador på rot och groddplanta. En rutinmässig kartering skulle förenkla identifieringen av lämpliga fält för oljeväxtodling liksom för konservärts- och foderärtsodling såväl för konventionella som ekologisk. Rödklöver är den viktigaste baljväxten i svensk vallfoderproduktion och den intar en central roll i odlingssystemet genom den biologiska kvävefixeringen. Uthålligheten är sviktande och bestånden blir ofta angripna av rotröta redan insåningsåret. Rotröta orsakas av ett komplex av patogener; Fusarium avenaceum, F. culmorum, Cylindrocarpon destructans och Phoma exigua. Rödklöverns uthållighet påverkas också av klöverröta (Sclerotinia trifoliorum). Senare tidsforskning i bland annat BioSoM visar att stora skador orsakas av rotrötesvamparna! En kartering av förekomst av patogener i grödor som vall, oljeväxt och ärt blir också ett viktigt verktyg för att följa effekterna av förändringar i klimatet. I de förväntade klimatscenariosen för Norden med mildare och fuktigare klimat, längre vegetationsperioder och reducerade perioder av frost och tjäle finns förväntningar på förändringar i patogentryck och sammansättning (se exempelvis Fanan, En framtida återkommande mark-biologisk kartering blir alltså viktig för att följa vad som händer och ställa prognoser och den borde kunna bli en viktig del i den platsspecifika produktionen Målsättningen med BioSoM-program var alltså att lägga den vetenskapliga grunden för en praktisk användbar biologisk markkarteringstjänst som skall kunna utnyttjas av lantbrukare för att optimera sin växtodling. Detta har skett genom: - att utveckla och validera DNA-baserade metoder för effektiv ras- och artbestämning samt kvantitativ bestämning av markburna patogener i svensk jord. Främst för klumprotsjuka och ärtrotröta samt bomullsmögel. Analysen av DNA från organismen som orsakar klumprotsjuka, Plasmodiphora brassicae, är nu 2013 kommersiellt tillgänglig sedan några år. - att öka den biologiska kunskapen om bildandet av vilosporer/vilokroppar och deras överlevnad samt genom att inleda arbetet med sekvensering av hela P. brassicae genetiska material. Hösten 2012 blev vi först i världen med att kunna presentera ett första förslag till alla gener och DNA i Plasmodiphora brassicae!! - att utarbeta och validera provtagningsrutiner och innefattande bland annat en aseptisk homogenisering av jordprover inför DNA-extraktion. Det finns en de facto rutin för hantering av prover, från aseptisk provtagning till PCR-protokoll. En film som beskriver den praktiska provtagningen har också lagts ut på Youtube. - att utvärdera samband och påverkan mellan förekomst av infektiösa patogener och markparametrar som ph, innehåll av makro- och mikronäringsämnen, lerhalt mm. Det har bland annat visats att bor har hämmande effekt på ärtrotröta i ärtor och klumprotssjuka i raps och att förekomsten i enskilda fält samvariera med ph-värdet i marken. - att ett arbete har inletts med en pilotstudie i samverkan med Högsklan i Skövde för att öka förståelsen av utformningen av presentationsrutiner för markbiologiska parametrar betydelse för implementering och upptag hos kunder (rådgivare/lantbrukare m fl) och validera lämpliga till exempel pedagogiska GIS-kartor på gårds, fält eller

4 regionnivå. Samarbetet har kommit gång mycket väl. En gemensam forskarstuderande och ansökan till FORMAs för fördjupning och utveckling av nya rådgivningskoncept har inletts under BioSoM I och kommer att utvecklas under fas 2 av BioSoM Vidare har kunskapen om en ny patogenen på ärt (och åkerböna) fördjupats och en ny metod för att bedöma angrep av rotdödare har utveckalts. Mycket intressant observationer har gjorts kring förloppet när bomullsmögel infekterar en rapsgröda. Angrepp av rotröta i klöver har för första gången dokumenterats med hjälp av DNA-metoder och kunskapen om de gener som är inblandade i sklerotiebildning hos Verticillium har fördjupats liksom förståelsen av resistens hos sockerbetor mot rhizoctonia-svampen. För ytterligare detaljer hänvisas till ( där också en pdf-version av slutrapporten för BioSoM I kan laddas ner.

5 Partnerskap Alnarp Jordbruks- & Trädgårdskonferensen BioSoM Tema framtida möjligheter inom de gröna näringarna Anders Källström, VD LRF: Vilken är LRFs roll i framtida produktionsmöjligheter. Lisa Sennerby Forsse, Rektor SLU: SLUs möjligheter att utveckla inriktningen mot Framtidens Lantbruk. Anders Hildeman, Global Forestry Manager IKEA: IKEAS väg från skogsråvara till heminredningsdetaljer. Fredrik Javensköld, Skånemejerier: Skånemejeriers strategi mot en hållbar livsmedelsproduktion. Rune Andersson, professor SLU: Tankar kring Framtidens Lantbruk står på flera ben. map the soil-borne pathogens «Läs mer om samtliga programpunkter och Prediction anmäl & Interaction dig senast 28 februari via QR-koden till vänster Disease severity eller webbsidan: Crop management Detection & Biology qpcr STORA AULAN, ALNARPSGÅRDEN PathotypesTORSDAGEN DEN 3 MARS Sclerotia Implementation GIS and extension Precision agriculture Sampling Program report phase I

6 BioSoM map the soil-borne pathogens BioSoM (Biological Soil Mapping) is a thematic research programme that aims to provide scientific support to a new soil-borne pathogen detection service for farmers. This in turn would improve cropping reliability and could form an essential part of management schemes to optimise crop production. Soil-borne plant pathogens can be a major limitation in the production of marketable yields and are adapted to grow and survive in the bulk soil, causing root disease. These pathogens include fungi, protists (supergroup Rhizaria), bacteria and nematodes. At the onset of the BioSoM project, the practical use of analyses to detect the presence of soil-borne pathogens was restricted to bioassay tests for a few pathogens. Pathogens survive in the soil by resistant propagules such as sclerotia, chlamydospores, resting spores, thick-walled conidia or hyphae, or survive in plant roots and crop residues. Most soil-borne fungi and protists attack young juvenile roots as opposed to secondary roots. After the roots have been killed, the pathogen reproduces and forms spores within the root tissue. Mycelium can continue to spread up the root, internally or externally, or can spread to other roots. Late above ground symptoms These diseases are difficult to diagnose from aboveground symptoms, since they may be vague and similar to symptoms caused by drought, stress and lack of nutrients. As a consequence of multiplication and reproduction of pathogens, the diseases increase over time. The lifetime of resting spores is often long, years, and the time of survival depends on soil conditions, crop rotation etc. Soil-borne pathogens are confined within the soil, although some pathogens on infected crop debris or soil can be spread by wind. Some soil-borne pathogens produce airborne sexual spores that are spread by the wind. Pathogens can also move above the ground with irrigation water or rain runoff. The long persistence of pathogens and the distribution pattern make it profitable and relevant to develop mapping of the distribution of disease infestation for several soil-borne pathogens. In Sweden we have considerable problems with pathogens such as club root in cruciferous plants, root rot in pea, Verticillium wilt and Sclerotinia stem rot in oilseed rape and root rot in red clover. New technology breakthroughs In future agriculture with high expectations for increasing yields of good quality in a changing climate, the level of pathogen infestation must be effectively monitored in order to employ adequate crop management schemes. With technologies used in Precision Agriculture such as Global Positioning System (GPS) and Geographic Information Systems (GIS), it is possible to keep track of and present infestation levels of soil-borne pathogens in a way that is easy to grasp. New detection methods based on DNA technologies make it possible to specifically estimate infestation levels of organisms in the soil. The rapidly growing possibilities to map DNA sequences in the genome of organisms might also lead to new ways of management of soil and breeding of crops to reduce the effects of pathogens. Linked to such progress 2

7 in knowledge and technology are steps of implementation into practical use. The chain of innovation must not be broken from laboratory to farmer if the potential of the new tools is to be exploited. Objectives of the programme The overall objectives of the programme are to provide scientific background and to design procedures for a mapping routine of soilborne pathogens that is of use in optimising crop rotations in Sweden. The work is divided into the following areas: Development and validation of PCRbased detection methods for soil-borne pathogens; Development of sampling methods and routines for presentation of the distribution of pathogen infestation. Correlation of infestation level with soil characteristics: Increased knowledge of pathogen biology, forecasting economic impact of disease, and investigations of the effect of crop management on plant pathogens: Initiation and advancement of the implementation process in agriculture. The programme has during phase I been divided into five work packages (WP) WP 1a Sampling and detection WP 1b Phytophthora sp. on pea WP 2 Forecast - based on bioassay and field trials WP 3a Pathogen biology resting structures and applied genomics WP 3b Beta vulgaris Rhizoctonia solani interaction WP 4 WP 5 Soil characteristics and infestation of pathogen Implementation and use in R&D and practice Programme management and funding The programme is funded by the Faculty of Natural Resources and Agricultural Sciences, SLU (50% of funding), and by stakeholders and their foundations: Foundation of Swedish Farmers Research (SLF), Västsvenska Lantmännen (VL) and Skånska Lantmännen (SL) Foundations, Swedish Seed and Oilseed Growers Research Foundation (SSO), Eurofins Food and Agro Testing Sweden AB (Lidköping), Scanbi Diagnostics AB (Alnarp), Findus R&D (Bjuv), Rural Economy and Agricultural Society/ HS Konsult AB (Örebro) and in later phases during 2011 and 2012 Lantmännen SWSeed, Nordic Beet Research (NBR) at Borgeby, Skåne, and Syngenta Seed AB, Landskrona. The large number of stakeholders reflects the extraordinary interest in soil-borne pathogens and associated research questions highlighted in the programme. A Steering Committee has been appointed to ensure that the programme is carried out in accordance with the intentions in the programme plan and the policy guidelines for NL Faculty thematic programmes. NL Faculty and each external stakeholder are entitled to appoint one committee member each. The Programme Director has appointed a Programme Management Group, responsible for assisting in managing the programme. 3

8 A broad scope of pathogens The first phase of the Programme has run from May 2009 to end of Pathogens that were studied were: Plasmodiophora brassicae (clubroot), Verticillium longisporum (Verticillium wilt), Sclerotinia sclerotiorum (stem rot), Phytophthora species on pea (pea rot), Aphanomyces euteiches (pea rot root), Fusarium avenaceum/phoma exigua/cylindrocarpon destructans (clover root rot), Gaeumannomyces graminis on wheat (take-all), Aphanomyces cochlioides (rot root sugar beet), Rhizoctonia solani (Rhizoctonia root rot, sugar beet) (Table 1). This programme builds on new collaborations, and the majority of the work in the workpackages started from scratch with the recruitment of new personnel. Thus, some of intended outcomes will be visible first in phase II, although one of the methods, detection of P. brassicae has been introduced in practice by one of the stakeholders. (Table 1) Sampling and detection A new method to mill and homogenise soil samples has been developed. A soil sample of approximately kg is poured into a plastic tin together with iron balls and screw nuts, and processed in a Skandex paintmixer. The sample is then passed through a 2-mm sieve before being stored. This method reduces the risk of contamination at milling. A de facto standard operating procedure (SOP) has been proposed to be used for soil sampling in practice, including routines for W-pattern and point sampling in the field, storage of samples and withdrawal for DNA extraction (Handledning för provtagning vid biologisk markkartering). In 2012 the proposed sampling routine with W-pattern was tested on 23 fields on the test farm Bjertorp in Västergötland. A very high variation in infestation was observed between the field-averages and fields unsuitable for oil seed rape were identified. Next step, an additional fixed point sampling will take place during spring 2013 to identify sites with high and low level of infestation for testing of resistant cultivars and treatment with nutrients. For the development of field sampling and a better understanding of the variation in field, collaboration has been initiated with Prof. Kim Esbensen at De Nationale Geologiske Undersøgelser for Danmark og Grønland, GEUS, in Copenhagen. He is an expert in the theory of sampling and the aim of the work is to improve the representativeness in our field sampling. A joint project did run for three months, January-March Most of the soil samples analysed during phase I have been saved and stored. A collection of more than 600 soils with different levels of infestation is now available. Pathogen P. brassicae Club root V. longisporum Verticillium wilt S. sclerotiorum Sclerotina stem root Phytopphthora sp New rot i Pea A. euteiches Rot root of Pea F. avenaceum P. exigua C. destructans Clover root rot G. graminis Take all A cochiloides Rot root sugar beet Biological Basic Science DNA Detection qpcr BioAssay Field Trials BioSom Map, Farm Service (PCR/GIS) Table 1. Pathogens given priority in the BioSoM programme, phase I. Pathogens, activities and knowledge. Filled squares = knowledge status, unfilled squares = knowledge gaps left to be filled in. 4

9 Available qpcr methods Real-time PCR methods for quantification of P. brassicae (club root), S. sclerotiorum (stem rot) and A. euteiches (pea root rot) have been developed and used to analyse field samples and plants. The detection limit in case of clubroot is below resting spores g-1 soil, which is the generally accepted limit for risk of disease development. Interestingly, stem rot sclerotia (often >4-5 mm) can be detected already at sieving of the soil samples, which gives a simple method for screening samples for presence of high amounts of S. sclerotiorum. Sieving is included in the SOP for biological soil mapping. For pea root rot, the qpcr protocol can detect one single oospore in a PCR tube and gives good detection at extraction of 0.35 g soil from soils with a high DSI (Disease Severity Index 0 100) based on a bioassay of 500 g soil. The problem is that soils with low DSI (<20 30) seem to have very low levels of oospores per gram of soil, probably less than a few oospores g -1. At very low oospore densities, the qpcr assay with 0.35 g soil gives too many false negative signals, and the method needs further development at this stage. The real-time PCR assay for quantification of A. cochlioides, causing rot root in sugar beet, has been developed and initial tests show that varying levels of soil infestation can be detected (Figure 1). A larger set of soil samples with known disease severity indices (~ 50 samples) will be analysed early in phase II as well as a soil dilution series, artificially infested with A. cochlioides oospores. (Fig1) DNA extraction of large soil samples The detection limit in soils can be improved by extracting larger amounts of soil and changing the extraction conditions. The development of a method for DNA extraction from large Disease Severity Index Target copies / g soil y=0,0106x + 33,633 R 2 = 0,84008 Figure 1. Analysis of A. cochlioides in soil samples. Relationship between qpcr (target copies/g soil) and disease severity index (DSI) based on naturally infested soil samples from seven Swedish fields. amounts of soil has been performed together with ScanBi Diagnostics AB. A protocol for extraction of 5 g soil instead of 0.35 g, followed by ball milling, has been developed and tested for detection of A. euteiches in soils with low levels of infestation. The first results were encouraging, as 1 2 oospores in 5 g soil were detected. Repetitions are planned to take place in the early part of phase II. A new commercial kit that extracts DNA from 5 10 g soil will be available from 2013 (FastDNA 50ml SPIN Kit for Soil, MP Biomedicals). Extracting DNA from larger soil samples will hopefully lower the detection limits of our diagnostic methods, but also make the sub sampling more representative. International collaboration Take-all Gaeumannomyces graminis (take-all) is an important soil-borne pathogen in cereals. Earlier experience and problems of developing a qpcr in our own laboratory and contacts with Dr. Mckay, Plant and Soil Health Laboratory at SARDI in Australia, have led to collaboration where qpcr analysis of takeall is performed at SARDI. The first results from January 2012 indicate that their methods and oligo-primers are working well for 5

10 Wheat cv. Dacke and Apogee, grown in the artificially infested commercial soil with Ggt Average Disease Severity Index 6,0 5,0 4,0 3,0 2,0 1,0 0,0 2-weeks 3-weeks 5-weeks Apogee-1 Apogee-0,25 Apogee-0,063 C-Apogee-1 Dacke-1 Dacke-0.25 Dacke C-Dacke-1 Figure. 2. Average disease severity index (DSI) in wheat cv. Dacke and Apogee which were grown in an artificially infested commercial soil at inoculum concentrations of 0.063, 0.25 and 1%. our Swedish strains of G. graminis. The goal for the take-all disease project was also to develop a reliable and reproducible climate chamber bioassay for the assessment of G. graminis var. tritici (Ggt) infection levels in different soils and to be used for validation of qpcr-based methods. A fast bioassay was developed for screening of aggressivity of G. graminis isolates and in climate chamber, the pathogenicity of selected Ggt isolates was established on spring wheat cv. Dacke and the fast growing wheat USU- Apogee (Fig. 2). The unique possibility with this new assay using the cultivar USU-Apogee is that its entire life cycle can be completed within 65 days under optimal condition. It can be used for assessment of yield loss in naturally infested soil within a relatively short period of time, when such analyses are needed. qpcr under development Quantitative PCR methods for pathogens causing root rot of red clover (Fusarium avenaceum, F. culmorum, Cylindrocarpon destructans and Phoma exigua) have been developed and will be used to monitor the development of these pathogens in roots and soil samples from Swedish red clover fields and field experiments where a range of cultivars are being investigated. The main challenge in WP1 has been the development of qpcr for Verticillium. We have investigated 10 DNA loci of V. longisporum in 82 experiments comprising 32 primers in 21 combinations. Of these new PCR primers were 17 designed by us, and additionally 15 were found in the literature. We have also designed four Taq-Man probes. In our work we have used 14 fungal isolates (seven V. longisporum, two V. dahliae, four V. albo-atrum and one V. tricorpus). We have continued the work with optimising the β-tubulin assay by using two qpcr machines, three different enzyme kits, numerous additive concentrations and time/temperature conditions. The assay has at this moment sometimes faulty amplification efficiency and sometimes it works. The cause for this inconsistency is lack of high quality sequence data on V. longisporum and related species. That and the rather close phylogenetic relationship between the Verticillium species make it difficult to optimise qpcrs and advance the sub-project. Therefore, all sequence data that are generated in WP3 will also be used in the following development. New information on Verticillium genomes was generated at the end of 2012 outside the programme. These new data forming the basis for new primer-sets to be evaluated 2013 will finalize the work on Verticillium at this level. Phthor sp. on pea and faba bean A new Phytophthora species causing root rot of pea and faba bean has been characterised including molecular phylogenetic analysis, 6

11 and an isolate collection has been deposited in international culture collections. Isolation protocols and bioassay procedures for pathogenicity tests on different host species and genotypes have been established. Germplasm of pea, faba bean, lentil and chickpea have been screened for resistance, and strong variation in resistance among cultivars has been observed. Selective PCR primers for detection and quantification in soil and in plant roots are under development. A dry inoculum formulation for soil and growth substrate inoculation has been developed, and is infective at rates down to 80 oospores per ml soil. This is a key prerequisite for both accurate screening of resistance with equal infection pressure in every pot, and realistic soil inoculation experiments, for example with the purpose of identifying disease-suppressive soils. A four-year field trial at three field sites naturally infected with P. pisi and A. euteiches has been completed (Figure 3). Significant effects of the different pre-crops on disease severity and yield were found. (Table 2) The cooperation with Findus AB to search for P. pisi in their routine root rot bioassays yielded valuable information on Phytophthora root rot incidence and distribution in Skåne and Hallands län during three years. Overall, P. pisi was found in the entire area in which Findus AB grow peas, but at a lower incidence (2-5%) compared to Aphanomyces root rot (23-27%). The two pathogens often coexist in the same sample, indicating that they are both favoured by intensive pea cultivation. Table 2. Design of field trials where effects of P. pisi and A. euteiches are studied (4 blocks x 3 fields). Typical symtoms of club root caused by P. Brassicae on white mustard. Photo: Ann-Charlotte Wallenhammar Reports and knowledge dissemination Standard Operation Protocol for sampling, handling, milling and storage of soil samples. In Swedish: Handledning för provtagning och hantering av jord vid biologisk markkartering. BioSoM Report, first draft. 7

12 Forecast based on bioassay and field trials Detection of clubroot in field experiments Soils from 31 field experimental sites of variety trials in winter oilseed rape and summer oilseed rape were analysed for the presence of DNA from P. brassicae. The pathogen was detected in 14.6% of the fields distributed in south and central Sweden. The detected amount of DNA ranged from 1 to 323 fg g -1 soil. Sampling techniques in an infested field were evaluated by qpcr. The results show that sampling along a diagonal as well as in a W-pattern is adequate for getting an indication of the inoculum level in this field. Detection of clubroot in infested fields A field with a high population density of P. brassicae was identified in 2010, and the presence of P. brassicae DNA has been determined by qpcr at 24 individual sampling points chosen by GPS. The sampling was repeated in 2011, and a great reduction in DNA was shown. Continuous data from this field will give us a unique possibility to study the decline in pathogen DNA. By using qpcr we will be able to get a more accurate measure in different cropping schemes. Pathotype testing of P. brassicae The knowledge of different pathotypes prevalent in Swedish soils has been improved. The term pathotype is used in place of race. Two sets of differential hosts, including Williams and the European Clubroot Differentials (ECD), were used for pathotype differentiation. A root dip method according to the original protocol (Buzcaki et al., 1975) was used for infection. A total of 11 P. brassicae populations were selected, representing fields from various parts of Sweden. Four isolates were repeated three times. The isolate from Hallsberg was aggressive and, while the other isolates did not show satisfactory infection for the susceptible cultivars in the ECD set. qpcr was used to analyse diffuse symptoms on three selected isolates. The results confirmed the results according to the bioassay. A set of bio-assays were also performed by Lantmännen SW Seed. Further, seven different cultivars of Raphanus sativus ssp. oleifera were tested for susceptibility at the request of Agortus AB. Airborne inoculum of S. sclerotiorum The real-time PCR method developed in preceding projects was used for detecting S. sclerotiorum DNA on petals and leaves at different levels of the plant at different points of time starting prior to early bloom in field experiments. A Burkhard 7-day volumetric spore trap was introduced in 2009 for sampling of airborne spores. This was done in order to better understand the correlation between sclerotia in the soil, airborne spores and actual infection. For analysis of DNA in airborne samples, a new type of DNA extraction was developed, and the protocol modified for analysis of DNA. Validation of the methods developed, including sampling and preparation of petals and leaves as well as corresponding reading of disease incidence in the field, was undertaken in 10 fields in Soils were also sampled. A disease support 8

13 system will be developed based on predictive tests, field data and local climate. Rapid diagnosis of patogens of red clover in soil and roots Red clover is the most important legume crop in Swedish forage production, however, the persistence is poor. Real time PCR has been developed to quantify the dominating soil-borne pathogens of red clover, Fusarium avenaceum, F. culmorum, Cylindrocarpon destructans and Phoma exigua in root and soil. The effects on germination, seed borne pathogens and field germination of a Thermoseed treatment of clover seeds was also investigated in green house, agar plates and field experiments. The methods developed will facilitate analysis on the importance of different sources of infection, effects on crop measures and choice of cultivars. The epidemiology of the pathogens and sustainability of the plants are investigated in a field experiment in selected future cultivars of red clover. Papers: Heyman, F., Blair, J.E., Persson, L., and Wikström, M Root rot of pea and faba bean in southern Sweden caused by Phytophthora pisi, sp. nov. Plant Disease, dx.doi.org/ /pdis re. (only published online at the time of writing). Heyman, F., Almquist, C., Jonsson, A., Wallenhammar, A-C., Lindahl, B., Stenlid J Evaluation of a quantitative PCR method for detection and quantification of Aphanomyces euteiches in soil samples. Developed and altered manuscript from dissertation by F. Heyman 2008, SLU. Hosseini, S., Karlsson, M., Funk Jensen, D., Heyman, F Quantification of Phytophthora pisi DNA and RNA transcripts during in planta infection of pea. Eur J Plant Pathol. 132: Wallenhammar, A-C., Almquist, C., Söderström, K., Jonsson, A In-field distribution of Plasmodiophora brassicae measured using quantitative real-time PCR. Plant Pathology 61: Reports and knowledge dissemination Studier av infektionsprocessen av Sclerotinia sclerotiorum i vårraps och vidareutveckling av modell för riskbedömning Slutrapport av projekt H Stiftelsen Lantbruksforskning. Oral and poster presentations Heyman, F A new Phytophthora species causing root rot in pea and other legumes. 9th conference of the European Foundation for Plant Pathology; Evora, Portugal, Nov (poster) Jonsson.A, Wallenhammar A-C and Dixelius C Long-term soil data sets reveals shifts in population of Plasmodiophora brassica. 6th International Symposium on Brassica and 18th Crucifer Genetics Workshop,; 12th-16th November 2012, Catania, Italy. (oral) Wallenhammar. A-C, Almquist C., Söderström M., Jonsson A In-field distribution of Plasmodiophora brassicae measured using quantative real-time PCR and the influence of soil physiochemical parameters on disease development. 6th International Symposium on Brassica and 18th Crucifer Genetics Workshop,; 12th-16th November 2012, Catania, Italy. ( poster) Wallenhammar, A., Almquist, C., Söderström, M., Jonsson, A In-field distribution and quantification of Plasmodiophora brassicae in soil samples measured using real-time PCR. In: Proc 13th International Rapeseed Congress, June 5-9, Prague, Czech Republic, p Wallenhammar, A-C., Almqvist, C., Redner, A Development of disease support systems of Sclerotinia stem rot in oilseed rape using real-time PCR. In: Proc. 13th International Rapeseed Congress, June 5-9, Prague, Czech Republic, p

14 Pathogen biology resting structures and applied genomics Gene sequencing of P. brassicae A method to obtain pure P. brassicae in sufficient quality and quantity for de novo whole genome sequencing has been developed. DNA of a single spore isolate has been used to create two different insert size Illumina libraries. A sequencing data set, based on a 200 bp pair-end library was sequenced at Scilife Lab in Stockholm while a 5 kb insert library was sequenced at BGI Hong Kong. The obtained datasets have been combined, and a draft assembly of the first P. brassicae genome has been established. The assembled genome has a total length of 24Mb with an average coverage of 200 times and 50% of the contigs are 273.8kb or longer, with the longest contig spanning 814kb. An approximately 100kb long contig appears to represent the mitochondrial genome. Interestingly, the DNA sequences obtained show very low similarity to DNA sequences in the public databases, suggesting that P. brassicae genes might be quite unique compared with known genes of other species. To identify and annotate genes it was therefore crucial to obtain RNA data. RNA of P. brassicae infected Chinese cabbage was used to obtain transcriptome data, sequenced at BGI, Hong Kong. The data sets were filtered by mapping the reads to the P. brassicae genome draft or to the host genome B. rapa and assembled. Our first analyses revealed approximately 6500 gene models. Those gene models can now be exploited in future studies for their biological functions, i.e. in virulence and life stage specific events. The genome and transcriptome data sets generated for P. brassicae will shed light on the biology of this poorly understood protist species, and it s life cycle which is not yet completely described. The data will also substantially improve our knowledge associated to phylogeny and evolution of Rhizaria organisms, a species-rich supergroup of unicellular eukaryotes with only very limited information on genome data to date. Initial testing of Swedish isolates as well as international isolates from Canada, Germany and New Zealand for the variation in the rdna sequence published for a Japanese P. brassicae population, revealed that all tested P. brassica have an identical rdna sequence which differs from the sequences. Verticillium sclerotia biology In silico comparative analyses of the published genome data for Verticillium dahliae with sclerotia forming and non-sclerotia forming fungi revealed a total of 205 putative candidate genes that may be involved in sclerotia in V. dahliae. Literature research and putative functions of blastp hits of the candidate list were used to narrow down the list to 21 candidates that were selected for further analysis, i.e. proteins with similarities to proteins involved in blood clotting or freezing tolerance. Gene knock-outs of 7 candidates (verified by PCR) have been achieved up to now and are currently analysed for phenotypes and virulence. Analyses were complicated due to the low quality of existing V. dahliae genome data. To improve the genome data of Verticillium species and to further understand V. longisporum species, the major Brassica 10

15 pathogen in Sweden, DNA and RNA material for sequence analyses are in the pipeline, financed outside of BioSoM. This will provide insights both on the pathogen, which is suggested to have evolved from a hybrid of V. dahliae and an unknown fungal species, and on the host response using the Brassica rapa genome. The sequence data for V. longisporum might also explain the different DNA content detected in V. longisporum isolates and help to understand the preference of V. longisporum for Brassica hosts. High-quality sequence data will also support advances on qpcr for detection of Verticillium in soil. Beta vulgaris Rhizoctonia solani interaction In a PhD project, Rhizoctonia solani on sugar beet will be studied. The first year has largely been committed to establish protocols and materials for next generation sequencing analysis. We started to establish materials for RNA-sequencing analysis with the aim to identify highly activated genes in the pathogen and in the plant host that are important for this specific interaction. Thus, materials have been prepared, RNA isolated from 36 samples and sent to the sequencing platform of Syngenta, in North Carolina, USA. In parallel, fungal RNA has been prepared and samples fused with a project on similar analysis on other R. solani isolates at Bielefeld University, Germany. In order to shed light on R. solani taxonomy and anastomosis groupings, collection of isolates from various plant hosts and soils was initiated In next step their DNA sequences will be amplified to generate information for extensive phylogenetic analysis including mating type associated genes. This workpackage will after phase I be transferred to a specific VR industry grant outside BioSoM. Genome and transcriptome data sets generated in this workpackage will in phase II deepen the understanding of disease cycles, plant host interactions and diagnostic tools of the soil-borne pathogens studied. Papers: Martin, T., Schwelm, A., Dixelius, C Genome-wide comparative analysis reveals insight into maize fungal pathogens. (Manuscript in T. Martin s PhD thesis 2011:66). Roos, J., Hopkins, R., Kvarnheden, A., Dixelius, C The impact of global warming on plant diseases and insect vectors in Sweden. Eur J Plant Pathol 129:9-19. web version roos_j_etal_ pdf Reports and knowledge dissemination PCR primer sequences related to Verticillium and qpcr protocols delivered to Scanbi AB PM BioSoM. Oral and poster presentations Roos, J., Schwelm, A., Dixelius, C Plant defence to Verticillium wilt and fungal sclerotia biology. In: Proc. 13th International Rapeseed Congress, June 5-9, Prague, Czech Republic, p Schwelm, A Fogelqvist. J and Dixelius C First glance at the genome of the club root pathogen Plasmodiophora brassicae. 6th International Symposium on Brassica and 18th Crucifer Genetics Workshop,; 12th-16th November 2012, Catania, Italy. (oral) Schwelm, A Fogelqvist. J and Dixelius C First glance at the genome of the club root pathogen Plasmodiophora brassiae. Joint ISOP and ISEP meeting Protist2012, 29th July-3rd August; Oslo, Norway (poster) Schwelm, A Fogelqvist. J and Dixelius C First glance at the genome of the club root pathogen Plasmodiophora brassiae.at the MPMI Conference, Kyoto, Japan 29 July-2 August (poster) 11

16 Soil characteristics and Infestation of pathogens Boron enhances P. brassicae tolerance The effects of selected nutrients on the development of club root on Chinese cabbage were tested in pot experiments in three locations, the greenhouse at Uddetorp Agricultural College (Skara), the growth chamber at University in Skövde and in growth chamber at SLU, Skara. There were no significant effects of different doses of N, S and Mn on disease severity. However, in the case of boron (B) a significant decrease in clubroot disease severity was observed. During the first experiment four doses of B were tested (11.5 to 69 kg B/ha). Despite high infestation level (5x10 6 spores/g soil), DSI decreased from 64 in non-treated control to 35 in plants treated with 11.5 kg B/ha. This result was confirmed by a second experiment (2.5 to 23 kg B/ha). Field experiments During 2012 two field trials using boron (Bortrac 150, Yara) were conducted. Field experiments were established in spring oilseed rape at Bjertorp and in winter oilseed rape at Lanna Research Station. Experimental plots were sprayed with water solution containing 0, 2.5 or 5 kg B/ha. Because of a rainy spring and location of plots at Bjertorp, disease severity was quite high in all plots (DSI 30 40), regardless of treatment and despite a moderate inoculum load prior to growing season (2.3x10 5 spores/g soil). On the other hand, almost no infected plants (2 out of 300) were found during symptom assessment in the winter oilseed rape trial, even though initial infestation of this field was higher 4x10 6 spores/g soil. The field trials will be followed during phase II, also in cooperation with Yara AB. Micro-bioassay Traditional bioassay to test the infection level of P. brassicae is time and space consuming, as disease symptoms are evaluated after six to eight weeks of growing Chinese cabbage plants in pots in greenhouse or growth-chamber. A micro-scale bioassay was developed to provide a more efficient and quick (3 4 weeks) tool to investigate the influence of different treatments on club root severity. Possibilities of further reducing the time of the experiment will be tested in phase II with microscope observations of roots, real-time PCR technique for assessing the amount and development stage of pathogen in the roots, as well as the background of boron-plant-pathogen. Nutrient treatments against A. euteiches pea root rot The influence of chosen nutrients on pea root rot was studied in pot experiments in the greenhouse at Findus R&D, Bjuv. No welldefined pattern in the effect of phosphorus, copper, manganese and sulphur on disease severity was found. However, as in the case of club root, treatment with boron (5.5, 11.5, 23 kg B/ha) reduced pea rot root severity in all of three tested naturally infested field soils with different infestation levels. The second experiment confirmed positive influence of boron on decreasing pea root rot severity. During plant assessment slight toxic effects of boron was observed on the lowest leaves, but there was no decrease in fresh and dry weight in plants with toxicity symptoms. 12

17 Figure 3. A field with probe points positioned and pathogen count. Moreover, on roots of plants treated with B more large nodules were observed. A small pilot experiment with pea seed dressing was conducted. Seed was treated with 1% of boron solution. Plants germinating from seeds treated with B were more resistant to A. euteiches, when compared to water or non-treated seeds. Further experiments with more replications and other concentrations will be done during phase II in cooperation with Findus R&D. S. sclerotiorum The possibilities of using cotyledon assay as a down-scaled bioassay for S. sclerotiorum have been tested. The first results have been promising, indicating that this type of assay can work for testing the effects of nutrients on the establishment of pathogens. Further testing is not included in phase II. Papers Stoltz, E., Wallenhammar, A-C Micronutrients reduces root rot development in red clover (Trifolium pratense). Journal of Plant Diseases and Protection, 119 (3), Wallenhammar, A-C., Almquist, C., Söderström, K., Jonsson, A In-field distribution of Plasmodiophora brassicae measured using quantitative real-time PCR. Plant Pathology 61:

18 Implementation and use in R&D and practice The focus of implementation during the first years was on presenting the BioSoM programme to farmers, advisors and researchers through posters and presentations at different meetings and exhibitions, to start using the available qpcr methods in some field trials and to screen some fields for the presence of soilborne pathogens. Sampling for biological soil mapping has been performed at Lanna Research Station SLU, Bjertorp, a farm managed by Lantmännen SWSeed, at Uddetorp Agricultural College, Skara, and on individual fields at Åkerby, Örebro, and Stockagården, Källby. The clubroot analysis is on-going and some preliminary maps have been produced. Analysis of samples from long-term fertility experiments established over fifty years ago and sampled every 4 or 7 years show how the population of clubroot has varied over time at experimental sites in Skåne as well as in western Sweden (Figure 4). The crop rotation includes oilseed rape every fourth year in Skåne and every sixth year in the rest of Sweden. A similar development of P. brassicae was observed at 4 of 5 sites in Skåne. The soil at the site Fjärdingslöv has a high content of limestone and a high ph-value compared to the other sites. The evaluation of these experiments will continue in phase II. Since the dissemination of the results from BioSoM is a very important task, a communication plan for the programme was developed by the Steering Committee during A collaboration has also been initiated with researchers at the University of Skövde working with cognition and machine/man interaction. The aim is to identify possibilities to use knowledge from decision-making and from game development to improve dissemination and use of soil mapping and biological soil mapping in particular. In 2012, a pilot project, DEMIPROF (Decision Making in Professional agriculture), was started. The project aims to study farmers decision-making, a process that is influenced by many different factors, and requires competence in many specialized areas. Farmers decision-making is characterized by a very complex situation in which biological, technical, economic, ethical and social factors are integrated. Very little previous research has been done where farmers decision- making is looked upon from an overall perspective, which made this project extra important. In the project the farmers decision-making is studied in their natural environment, so-called naturalistic decision making (NDM). The cognitive theoretical framework is the so called distributed cognition. This highlights cognition as a complex dynamic system between people and technology, where decision-making and problem solving are prominent cognitive processes and cognition is studied in the wild, i.e. in its natural environment. The project s implementers were three postdoctoral researchers from the University of Skövde and a project leader from precision agriculture and pedometrics, Department of Soil and Environment SLU, Skara. The first phase is characterized by description of the working situation and the need for advice and IT support. Each performer meets a farmer and his advisor through regular visits to the farm over a 14-month period. The idea is to follow the farmers during an entire growing season. The visits are made both when the adviser and the farmer meet and when the farmer is alone. The experiences so far have been that the farmers has great confidence in the advisor and that the farmer s situation is characterised by a great mix of levels of decision-making strate- 14

19 gic, tactic and operative. Farmers were selected by the Agricultural Society, and all advisors in the project are employed by that organization. In 2013, the project will deliver its final report and the results will be presented at a conference focusing on extension science. The value of the wide competencies in the programme This programme covers aspects from gene/genome levels to field and soil management and on to advisory services to farmers. The wide competencies of the partners in the programme and the stakeholders are of the utmost importance in achieving the ambitious goals set. This initiative is also part of the work to meet the requirements of EU Directive 2009/128 on integrated pest management, to be implemented in practice from January The possibility to quantify the status of soilborne pathogens would enable farmers to improve management, choose more appropriate cultivars (resistant if available), reduce unnecessary use of plant protection (fungicides) and use some of the precision agriculture tools now available for crop production. In order to meet new climate-related challenges and regulatory frameworks, there is an urgent need to strengthen cooperation and to develop methods for improved implementation of new knowledge between academia and agricultural-associated stakeholders Furthermore, it is essential to improve communication to farmers and advisors and to find new possibilities for communication using all the available information technologies through collaboration with other universities. DNA (fg/g soil) Ekebo DNA (fg/g soil Figure 4. Change in level of infestation of Plasmodiophora brassicae in soil (ie DNA fg/g soil) from long-term fertility experiment at Ekebo, Transfer of knowledge Transfer of the knowledge generated in the programme is secured through a working network, i.e. a network with many ways of interaction and communication between stakeholders, BioSoM scientists and end-users such as farmers and colleagues working with R&D. The most important are: - Meetings of Steering Committee. - PhD students from industry, from stakeholder companies such as Syngenta and Eurofins Food and Agro AB. - Stakeholder participation in workpackages, both in kind and discharged. - Workshops with all personnel in connection with meetings of the Steering Committee. - Newsletter. - Video-link meetings every second month, connecting Uppsala-Alnarp-Skara for discussion of activities and problems in the WP projects etc. Popular science articles Wallenhammar, A-C. & Jonsson, A Biologisk markkartering ger koll på sjukdomar. Arvensis 5, 11. Wallenhammar, A-C DNA på kronbladen avslöjar bekämpningsbehovet. Arvensis 2, 13. Wallenhammar, A-C Rapsens fotboja. Arvensis 4, Netterlund, H. & Wallenhammar, A-C Rätt Rättika. Arvensis 3, Jonsson A Biologiska markanalys kan minska sjukdomsrisker. Idetidskriften C. Årgång 24, nr 2. Nilsson S-Å, Engdahl Axelsson C Kontrollera risken för klumprotsjuka. Svensk Frötidning 3, Wallenhammar A-C Konsten att hantera klumprosjuka. Svensk Frötidning 3,