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BioSoM map the soil-borne

pathogens

Sampling

Detection & Biology• qPCR• Pathotypes• Sclerotia

Prediction & Interaction• Disease severity• Crop management

Implementation• GIS and extension• Precision agriculture

Program report phase I2009 – 2012

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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 manage-ment 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 pre-sence of soil-borne pathogens was restricted to bioassay tests for a few pathogens. Patho-gens survive in the soil by resistant propagu-les 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 symptomsThese 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 reproduc-tion of pathogens, the diseases increase over time. The lifetime of resting spores is often long, 10–20 years, and the time of survival

BioSoM – map the soil-borne pathogens

depends on soil conditions, crop rotation etc.Soil-borne pathogens are confined within

the soil, although some pathogens on infec-ted 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 re-levant 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, Verticil-lium wilt and Sclerotinia stem rot in oilseed rape and root rot in red clover.

new technology breakthroughsIn future agriculture with high expectations for increasing yields of good quality in a chan-ging climate, the level of pathogen infesta-tion must be effectively monitored in order to employ adequate crop management schemes. With technologies used in Precision Agricul-ture such as Global Positioning System (GPS) and Geographic Information Systems (GIS), it is possible to keep track of and present infesta-tion 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 ef-fects of pathogens. Linked to such progress

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in knowledge and technology are steps of im-plementation into practical use. The chain of innovation must not be broken from labora-tory to farmer if the potential of the new tools is to be exploited.

objectives of the programmeThe overall objectives of the programme are to provide scientific background and to de-sign procedures for a mapping routine of soil-borne pathogens that is of use in optimising crop rotations in Sweden. The work is divided into the following areas:

• Development and validation of PCR- based detection methods for soil-borne pathogens;

• Development of sampling methods and routines for presentation of the distribution of pathogen infestation. Correlation of in-festation level with soil characteristics:

• Increased knowledge of pathogen biology, fo-recasting economic impact of disease, and in-vestigations of the effect of crop management on plant pathogens:

• Initiation and advancement of the imple-mentation process in agriculture.

The programme has during phase I been divi-ded into five work packages (WP)WP 1a Sampling and detectionWP 1b Phytophthora sp. on peaWP 2 Forecast - based on bioassay and field trialsWP 3a Pathogen biology – resting structures and applied genomicsWP 3b Beta vulgaris – Rhizoctonia solani interaction

WP 4 Soil characteristics and infestation of pathogenWP 5 Implementation and use in R&D and practice

Programme management and fundingThe programme is funded by the Faculty of Natural Resources and Agricultural Sci-ences, SLU (50% of funding), and by sta-keholders and their foundations: Founda-tion of Swedish Farmers Research (SLF), Västsvenska Lantmännen (VL) and Skånska Lantmännen (SL) Foundations, Swedish Seed and Oilseed Growers Research Foun-dation (SSO), Eurofins Food and Agro Tes-ting Sweden AB (Lidköping), Scanbi Diag-nostics 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 pat-hogens and associated research questions highlighted in the programme.

A Steering Committee has been appoin-ted 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 mem-ber each. The Programme Director has ap-pointed a Programme Management Group, responsible for assisting in managing the programme.

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The fIrST PhaSe of The PrograMMe has run from May 2009 to end of 2012.

Pathogens that were studied were: Plasmo-diophora brassicae (clubroot), Verticillium longisporum (Verticillium wilt), Sclerotinia sclerotiorum (stem rot), Phytophthora species on pea (pea rot), Aphanomyces euteiches (pea rot root), Fusarium avenaceum/Phoma exi-gua/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 collabo-rations, 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 detectionA new method to mill and homogenise soil samples has been developed. A soil sample of approximately 0.5–1.0 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 redu-ces 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

A bRoAD SCoPe of PAthoGenS

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 ad-ditional fixed point sampling will take place during spring 2013 to identify sites with high and low level of infestation for testing of resis-tant 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 2012.

Most of the soil samples analysed during phase I have been saved and stored. A collec-tion of more than 600 soils with different le-vels of infestation is now available.

Table 1. Pathogens given priority in the BioSoM programme, phase I. Pathogens, activities and knowledge. Filled squares = knowledge status, ­unfilled­squares­=­knowledge­gaps­left­to­be­filled­in.

Pathogenbiological ”basic Science”

DnA Detection qPCR

bioAs-say

field trials

bioSom Map, farm Service (PCR/GIS)

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

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Pathogenbiological ”basic Science”

DnA Detection qPCR

bioAs-say

field trials

bioSom Map, farm Service (PCR/GIS)

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

available qPcr methodsReal-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 clu-broot is below 1 000 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 inclu-ded in the SOP for biological soil mapping.

For pea root rot, the qPCR protocol can de-tect one single oospore in a PCR tube and gi-ves 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 oospo-res 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 samp-les with known disease severity indices (~ 50 samples) will be analysed early in phase II as well as a soil dilution series, artificially in-fested with A. cochlioides oospores. (Fig1)

Dna extraction of large soil samplesThe detection limit in soils can be improved by extracting larger amounts of soil and chang-ing the extraction conditions. The development of a method for DNA extraction from large

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 de-veloped 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 Biome-dicals). Extracting DNA from larger soil samp-les will hopefully lower the detection limits of our diagnostic methods, but also make the sub sampling more representative.

International collaboration Take-allGaeumannomyces graminis (take-all) is an important soil-borne pathogen in cereals. Earlier experience and problems of develo-ping a qPCR in our own laboratory and con-tacts with Dr. Mckay, Plant and Soil Health Laboratory at SARDI in Australia, have led to collaboration where qPCR analysis of take-all is performed at SARDI. The first results from January 2012 indicate that their met-hods and oligo-primers are working well for

Figure­1.­Analysis­of­A.­cochlioides­in­soil­samples.­Relationship between qPCR (target copies/g soil) and disease severity index (DSI) based on naturally infested­soil­samples­from­seven­Swedish­fields.

Dis

ease

Sev

erit

y In

dex 90

80706050403020100

0 1000 2000 3000 4000 5000

Target copies / g soil

y=0,0106x + 33,633R2 = 0,84008

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our ‘Swedish’ strains of G. graminis.The goal for the take-all disease project was

also to develop a reliable and reproducible cli-mate 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 en-tire life cycle can be completed within 65 days under optimal condition. It can be used for as-sessment of yield loss in naturally infested soil within a relatively short period of time, when such analyses are needed.

qPcr under developmentQuantitative PCR methods for pathogens cau-sing root rot of red clover (Fusarium avena-

ceum, F. culmorum, Cylindrocarpon destruc-tans 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 de-velopment 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 con-tinued the work with optimising the β-tubulin assay by using two qPCR machines, three dif-ferent enzyme kits, numerous additive concen-trations 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. longis-porum and related species. That and the rather close phylogenetic relationship between the Verticillium species make it difficult to opti-mise qPCRs and advance the sub-project. The-refore, all sequence data that are generated in WP3 will also be used in the following deve-lopment.

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 beanA new Phytophthora species causing root rot of pea and faba bean has been characterised including molecular phylogenetic analysis,

Figure. 2. Average disease severity index (DSI) in wheat cv. ‘Dacke’ and ‘Apogee’ which were grown in­an­artificially­infested­commercial­soil­at­ino-culum­concentrations­of­0.063,­0.25­and­1%.

Wheat cv. Dacke and apogee, grown in the artificially infested commercial soil with Ggt

Ave

rage

Dis

ease

Sev

erit

y In

dex 6,0

5,0

4,0

3,0

2,0

1,0

0,0C-Dacke-1

Dacke-0.063

Dacke-0.25

Dacke-1

C-Apogee-1

Apogee-0,063

Apogee-0,25

Apogee-1

2-weeks3-weeks5-weeks

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and an isolate collection has been deposited in international culture collections. Isolation protocols and bioassay procedures for patho-genicity 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 develop-ment. 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 pre-requisite for both accurate screening of re-sistance with equal infection pressure in every pot, and realistic soil inoculation ex-periments, 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. eu-teiches has been completed (Figure 3). Sig-nificant 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 distri-bution in Skåne and Hallands län during th-ree 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.

Table­2.­Design­of­field­trials­where­effects­of­P.­pisi­and­A.­euteiches­are­studied­(4­blocks­x­3­fields).

reports and knowledge disseminationStandard 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.Typical symtoms of club root caused by

P. Brassicae on white mustard. Photo: Ann-Charlotte Wallenhammar

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Detection of clubroot in field experimentsSoils from 31 field experimental sites of va-riety trials in winter oilseed rape and sum-mer 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 indica-tion of the inoculum level in this field.

Detection of clubroot in infested fieldsA field with a high population density of P. brassicae was identified in 2010, and the presence of P. brassicae DNA has been de-termined by qPCR at 24 individual sampling points chosen by GPS. The sampling was re-peated 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. brassicaeThe knowledge of different pathotypes pre-valent in Swedish soils has been improved. The term ‘pathotype’ is used in place of ‘race’. Two sets of differential hosts, inclu-ding Williams and the European Clubroot Differentials (ECD), were used for pathoty-pe differentiation. A root dip method accor-ding 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 repea-ted 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 symp-toms on three selected isolates. The results confirmed the results according to the bioas-say. A set of bio-assays were also performed by Lantmännen SW Seed.

Further, seven different cultivars of Rap-hanus sativus ssp. oleifera were tested for susceptibility at the request of Agortus AB.

airborne inoculum of S. sclerotiorumThe real-time PCR method developed in pre-ceding 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 samp-ling of airborne spores. This was done in or-der to better understand the correlation bet-ween sclerotia in the soil, airborne spores and actual infection.

For analysis of DNA in airborne samples, a new type of DNA extraction was develo-ped, 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 underta-ken in 10 fields in 2011.

Soils were also sampled. A disease support

foReCASt bASeD on bIoASSAy AnD fIelD tRIAlS

9

system will be developed based on predictive tests, field data and local climate.

rapid diagnosis of patogens of red clover in soil and rootsRed 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 Ther-moseed treatment of clover seeds was also investigated in green house, agar plates and field experiments.

The methods developed will facilitate ana-lysis 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. 2013. Root rot of pea and faba bean in southern Sweden caused by Phy-tophthora pisi, sp. nov. Plant Disease, http://dx.doi.org/10.1094/PDIS-09-12-0823-Re. (only published online at the time of writing).heyman, f., Almquist, C., Jonsson, A., Wallenhammar, A-C., lindahl, b., Stenlid J. 2008. evaluation of a quantitative PCR method for detection and quantification of Aphanomyces euteiches in soil samples. Developed and altered manuscript from dis-sertation by f. heyman 2008, SlU.hosseini, S., Karlsson, M., funk Jensen, D., heyman, f. 2012. Quantification of Phytopht-hora pisi DnA and RnA transcripts during in planta infection of pea. eur J Plant Pathol. 132:455-468Wallenhammar, A-C., Almquist, C., Söder-ström, K., Jonsson, A. 2012. In-field distribu-tion of Plasmodiophora brassicae measured using quantitative real-time PCR. Plant Pathology 61: 16-28.

reports and knowledge disseminationStudier av infektionsprocessen av Sclero-tinia sclerotiorum i vårraps och vidareut-veckling av modell för riskbedömning 2009. Slutrapport av projekt h0860030 Stiftelsen lantbruksforskning.

oral and poster presentationsheyman, f. 2010. A new Phytophthora

species causing root rot in pea and other legumes. 9th conference of the european foundation for Plant Pathology; evora, Portugal, nov 15-18 2010. (poster)Jonsson .A, Wallenhammar A-C and Dixelius C. 2012. long-term soil data sets reveals shifts in population of Plasmodiop-hora brassica. 6th International Symposium on brassica and 18th Crucifer Genetics Workshop,; 12th-16th november 2012, Catania, Italy. (oral)Wallenhammar. A-C, Almquist C., Söder-ström M., Jonsson A. 2012.In-field distribu-tion 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., Söder-ström, M., Jonsson, A. 2011. In-field distri-bution 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. 459.Wallenhammar, A-C., Almqvist, C., Redner, A. 2011. 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. 460.

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PAthoGen bIoloGy – ReStInG StRUCtUReS AnD APPlIeD GenoMICS

gene sequencing of P. brassicaeA method to obtain pure P. brassicae in suf-ficient quality and quantity for de novo who-le 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 combi-ned, and a draft assembly of the first P. bras-sicae genome has been established. The as-sembled 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 obtai-ned show very low similarity to DNA se-quences in the public databases, suggesting that P. brassicae genes might be quite uni-que 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 cab-bage 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 ex-ploited 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 pro-tist species, and it’s life cycle which is not yet completely described. The data will also substantially improve our knowledge associ-ated to phylogeny and evolution of Rhizaria organisms, a species-rich supergroup of uni-cellular eukaryotes with only very limited information on genome data to date.

Initial testing of Swedish isolates as well as international isolates from Canada, Ger-many 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 publis-hed genome data for Verticillium dahliae with sclerotia forming and non-sclerotia for-ming 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 similari-ties 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 geno-me data. To improve the genome data of Ver-ticillium species and to further understand V. longisporum species, the major Brassica

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pathogen in Sweden, DNA and RNA materi-al for sequence analyses are in the pipeline, financed outside of BioSoM. This will pro-vide 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 con-tent detected in V. longisporum isolates and help to understand the preference of V. long-isporum for Brassica hosts. High-quality se-quence data will also support advances on qPCR for detection of Verticillium in soil.

Beta vulgaris – Rhizoctonia solani interactionIn 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 Biele-feld University, Germany.

In order to shed light on R. solani taxo-nomy and anastomosis groupings, collection of isolates from various plant hosts and soils was initiated 2012. In next step their DNA sequences will be amplified to generate in-formation for extensive phylogenetic ana-lysis including mating type associated ge-

nes. This workpackage will after phase I be transferred to a specific VR industry grant outside BioSoM.

Genome and transcriptome data sets ge-nerated 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. 2011. Genome-wide comparative analysis reveals insight into maize fungal pathogens. (Manus-cript in t. Martin’s PhD thesis 2011:66). Roos, J., hopkins, R., Kvarnheden, A., Dixelius, C. 2011. the impact of global warming on plant diseases and insect vectors in Sweden. eur J Plant Pathol 129:9-19. web version http://pub.epsilon.slu.se/5568/1/roos_j_etal_110111.pdf

reports and knowledge disseminationPCR primer sequences related to Verticillium and qPCR protocols delivered to Scanbi Ab 2011. PM bioSoM.

oral and poster presentationsRoos, J., Schwelm, A., Dixelius, C. 2011. Plant defence to Verticillium wilt and fungal sclerotia biology. In: Proc. 13th International Rapeseed Congress, June 5-9, Prague, Czech Republic, p. 235.Schwelm, A fogelqvist. J and Dixelius C. 2012 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. 2012 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. 2012 first glance at the genome of the club root pathogen Plasmodiophora brassiae.at the MPMI Conference, Kyoto, Japan 29 July-2 August 2012. (poster)

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SoIl ChARACteRIStICS AnD InfeStAtIon of PAthoGenS

Boron enhances P. brassicae toleranceThe effects of selected nutrients on the de-velopment of club root on Chinese cabbage were tested in pot experiments in three loca-tions, the greenhouse at Uddetorp Agricul-tural College (Skara), the growth chamber at University in Skövde and in growth chamber at SLU, Skara. There were no significant ef-fects of different doses of N, S and Mn on di-sease 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 (5x106 spores/g soil), DSI de-creased from 64 in non-treated control to 35 in plants treated with 11.5 kg B/ha. This re-sult was confirmed by a second experiment (2.5 to 23 kg B/ha).

field experimentsDuring 2012 two field trials using boron (Bortrac 150, Yara) were conducted. Field ex-periments 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 tre-atment and despite a moderate inoculum load prior to growing season (2.3x105 spores/g soil). On the other hand, almost no infected plants (2 out of 300) were found during symptom as-sessment in the winter oilseed rape trial, even though initial infestation of this field was hig-her 4x106 spores/g soil. The field trials will be

followed during phase II, also in cooperation with Yara AB.

Micro-bioassayTraditional 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-cham-ber. 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 mi-croscope observations of roots, real-time PCR technique for assessing the amount and develop-ment stage of pathogen in the roots, as well as the background of boron-plant-pathogen.

nutrient treatments against A. euteiches pea root rotThe influence of chosen nutrients on pea root rot was studied in pot experiments in the greenhouse at Findus R&D, Bjuv. No well-defined 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.

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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. sclerotiorumThe possibilities of using cotyledon assay as a down-scaled bioassay for S. sclerotiorum

PapersStoltz, e., Wallenhammar, A-C. 2012. Micron-utrients reduces root rot development in red clover (Trifolium pratense). Journal of Plant Diseases and Protection, 119 (3), 92-99..Wallenhammar, A-C., Almquist, C., Söder-ström, K., Jonsson, A. 2012. In-field distribu-tion of Plasmodiophora brassicae measured using quantitative real-time PCR. Plant Pathology 61: 16-28.

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.

Figure­3.­A­field­with­probe­points­positioned­and­pathogen­count.

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The focuS of IMPleMenTaTIon during the first years was on presenting the BioSoM programme to farmers, advisors and resear-chers through posters and presentations at diffe-rent meetings and exhibitions, to start using the available qPCR methods in some field trials and to screen some fields for the presence of soil-borne pathogens.

Sampling for biological soil mapping has been performed at Lanna Research Station SLU, Bjertorp, a farm managed by Lantmän-nen SWSeed, at Uddetorp Agricultural College, Skara, and on individual fields at Åkerby, Öre-bro, 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 po-pulation of clubroot has varied over time at ex-perimental sites in Skåne as well as in western Sweden (Figure 4). The crop rotation includes oilseed rape every fourth year in Skåne and eve-ry 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 conti-nue in phase II.

Since the dissemination of the results from BioSoM is a very important task, a communica-tion plan for the programme was developed by the Steering Committee during 2011. A collabo-ration has also been initiated with researchers at the University of Skövde working with cogni-tion and machine/man interaction. The aim is to identify possibilities to use knowledge from de-cision-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 (Deci-

sion 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 compe-tence in many specialized areas. Farmers’ deci-sion-making is characterized by a very complex situation in which biological, technical, econo-mic, 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 far-mers’ decision-making is studied in their natu-ral environment, so-called naturalistic decision making (NDM). The cognitive theoretical fram-ework is the so called distributed cognition. This highlights cognition as a complex dynamic sys-tem between people and technology, where deci-sion-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 post-doctoral researchers from the University of Skövde and a project leader from precision agri-culture 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 sup-port. 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 far-mers during an entire growing season. The vi-sits are made both when the adviser and the far-mer meet and when the farmer is alone.

The experiences so far have been that the far-mers has great confidence in the advisor and that the farmer’s situation is characterised by a great mix of levels of decision-making – strate-

IMPleMentAtIon AnD USe In R&D AnD PRACtICe

15

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 programmeThis programme covers aspects from gene/ge-nome levels to field and soil management and on to advisory services to farmers. The wide competencies of the partners in the program-me and the stakeholders are of the utmost im-portance 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 2014.

The possibility to quantify the status of soil-borne pathogens would enable farmers to im-prove management, choose more appropriate cultivars (resistant if available), reduce unneces-sary use of plant protection (fungicides) and use some of the precision agriculture tools now av-ailable for crop production.

In order to meet new climate-related chal-lenges and regulatory frameworks, there is an urgent need to strengthen cooperation and to develop methods for improved implementation of new knowledge between academia and agri-cultural-associated stakeholders Furthermore, it is essential to improve communication to far-mers and advisors and to find new possibilities for communication using all the available infor-mation technologies through collaboration with other universities.

Transfer of knowledgeTransfer of the knowledge generated in the programme is secured through a ‘working net-work’, i.e. a network with many ways of inte-

raction and communication between stakehol-ders, 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 stakehol-

der companies such as Syngenta and Euro-fins 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 dis-cussion of activities and problems in the WP projects etc.

Popular science articlesWallenhammar, A-C. & Jonsson, A. 2008. biologisk markkartering ger koll på sjukdomar. Arvensis 5, 11.Wallenhammar, A-C. 2008. DnA på kronbladen avslöjar bekämpningsbehovet. Arvensis 2, 13.Wallenhammar, A-C. 2009. Rapsens fotboja. Arvensis 4, 18-19.netterlund, h. & Wallenhammar, A-C. 2010. Rätt Rät-tika. Arvensis 3, 14-15.Jonsson A 2012. biologiska markanalys – kan minska sjukdomsrisker. Idetidskriften C. Årgång 24, nr 2. nilsson S-Å, engdahl Axelsson C. 2012. Kontrollera risken för klumprotsjuka. Svensk frötidning 3, 16-17.Wallenhammar A-C. 2012. Konsten att hantera klum-prosjuka. Svensk frötidning 3, 11-14

Figure­4.­Change­in­level­of­infestation­of­Plasmo-diophora­brassicae­in­soil­(ie­DNA­fg/g­soil)­from­long-term­fertility­experiment­at­Ekebo,­1971-2011.

DN

A (f

g/g

soil)

30 000

25 000

20 000

15 000

10 000

5 000

0

Ekebo

1971 1975 1979 1983 1987 1991 1995 1999 2003 2007 20115 5 5 5 7 1110 5210 23900 25800 1432 37

DNA (fg/g soil

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