Session 8 Disease and Pest Resistance II
Ear Diseases, Fusarium Head Blight, Viroses

Oral Presentation

Off-Season Testing of Barley in China for Fusarium Head Blight

J. D. Franckowiak1, B. X. Zhang2, R. D. Horsley1, B. J. Steffenson3, K. P. Smith4
and S. M. Neate5

1Plant Sciences, North Dakota State University, Fargo, ND 58105, USA,
E-mail: j.franckowiak@ndsu.nodak.edu;
2Plant Protection, Zhejiang University, Hangzhou, 310029, P. R. China; 3Plant Pathology
and 4Agronomy and Plant Genetics, University of Minnesota, St. Paul, MN 55108, USA;
5Plant Pathology, North Dakota State University, Fargo, ND 58105, USA

Accessions and breeding lines of barley must be evaluated multiple times to adequately assess levels of resistance to Fusarium head blight (FHB), incited by Fusarium graminearum. Inoculated field nurseries grown in Minnesota and North Dakota are used to assess FHB reactions; however, spring breeding lines can be tested only once during a year and winter and wild barley accessions cannot be tested at all. Off-season FHB nurseries were established in 1995 at Zhejiang University, Hangzhou in eastern China because FHB is endemic in the region and other confounding head diseases are not present. Under these short-day conditions, most accessions flowered within a two-week window of time. The China nursery has been particularly useful for evaluating of winter barleys, wild barleys, and East Asian spring cultivars; finding new sources of FHB resistance; identifying Chinese cultivars as an important source of FHB resistance; mapping of QTLs for FHB resistance to chromosome 2H; confirming associations between spike type, maturity, and plant height; gaining knowledge about maturity genes; and providing another site for FHB testing of breeding materials.

Variation for Resistance to Fusarium Head Blight in Spring Barley

H. Buerstmayr1, L. Legzdina2, B. Steiner1 and M. Lemmens1

1Division of Biotechnology in Plant Production, Department for Agrobiotechnology Tulln,
University of Natural Resources and Applied Life Sciences Vienna, A-3430 Tulln, Austria,
E-mail: herman.buerstmarvr@boku.ac.at;
2Priekuli Plant Breeding Station, Priekuli LV-4126, Latvia

Fusarium head blight (FHB) is a fungal disease of barley and other cereals, causing substantial yield and quality losses, mainly due to the contamination of the harvest with mycotoxins. We evaluated genetic variation for resistance to FHB and its association with other plant characters in diverse barley germplasm in order to identify useful lines for resistance breeding. The 143 barley lines consisted of 88 current European spring barley lines and cultivars, 33 accessions from the genebank at IPK Gatersleben, and 22 lines obtained from North American institutions. We conducted artificially inoculated field experiments with Fusarium graminearum Schwabe during two seasons. FHB severity was evaluated by repeated assessment of visual symptoms. On a set of 49 lines several trichothecene mycotoxins were analyzed. Variation for FHB severity was quantitative. The lines with lowest FHB severity were CIho 4196 and PI 566203. Also within the European spring barley collection variation for FHB severity was highly significant. There was a significant negative correlation between plant height and FHB severity (r = 0.55). FHB severity assessed in the field and the amount of deoxynivalenol in the harvested grains were positively correlated (r = 0.87). Several lines with a useful level of FHB resistance were found or confirmed and are recommended as crossing partners.

Evaluation of Barley Resistance to Fusarium Head Blight Infection and Mycotoxin Contamination of Grain

V. Sip1, L. Tvaruzek2, J. Chrpova1, S. Sykorova1, L. Leisova1, L. Kucera1 and J. Ovesna1

1Research Institute of Crop Production, 161 06 Prague-Ruzyne, Czech Republic, E-mail: sip@vurv.cz; 2Agricultural Research Institute Kromeriz, Ltd., 767 01 Kromeriz, Czech Republic

The results are based on field infection trials with spring barley cultivars registered in the Czech Republic and resistance sources obtained from the USA and CIMMYT (Chevron, CI 4196, MP 7). In experiments lasting for three (two) years at two locations (Ruzyně, Kroměříž) cultivar resistance to pathogen invasion (after spraying of inoculum) and pathogen spread within a spike (using brushing method Prague-Ruzyně) was examined. Four isolates of F. culmorum and F. graminearum were used for inoculations. Deoxynivalenol (DON) content was determined by both the ELISA and GC method. The examined traits measuring severity of infection and DON content were highly influenced by cultivar, experimental year, inoculation technique and fungus isolate. Determinations of DON by ELISA and GC were closely related (r = 0.94; P = 0.000). It came from GC analysis that the isolate F.g. 608 was predominant nivalenol producer, while the other isolates were DON producers. It is, therefore, stressed the importance to examine fungus isolate for its chemotype. Using pathogenic fungus isolate and under conditions suitable for disease development, DON content was found significantly correlated with percentage of infected grains (% of sprouting seeds, % of Fusarium colonies) and reduction of thousand grain weight.

Breeding for Virus Resistance of Barley: Amalgamation of Classical and Biotechnological Approaches

W. Friedt1 and F. Ordon2

1Institute of Crop Science and Plant Breeding I, Justus-Liebig-University Giessen, D-35392 Giessen, Germany, E-mail: wolfgang.friedt@agrar.uni-giessen.de; 2Institute of Epidemiology and Resistance,
Federal Centre of Breeding Research for Cultivated Plants, D-06449 Aschersleben, Germany

The barley crop in Europe and other parts of the world is threatened by two virus diseases, caused by soil-borne yellow mosaic viruses and aphid-transmitted Barley yellow dwarf virus (BYDV). The Barley yellow mosaic virus complex is caused by at least three viruses, i.e. Barley mild mosaic virus (BaMMV), Barley yellow mosaic virus (BaYMV), and BaYMV-2. Resistance of current German cultivars is based on two genes, rym4 providing complete resistance (immunity) against BaMMV and BaYMV-1 but not effective against BaYMV-2, and rym5 functional against all three viruses. The goal of breeding high-yielding resistant cultivars has been achieved in two decades only, initially starting from the identification of resistant sources amongst adapted cultivars and exotic germplasm. Additional resistance genes are available in the barley gene pool to diversify genetic resistance and to prevent the potential breakdown of virus resistance. Regarding BYDV no full resistance but genetic tolerance has been reported. In order to achieve more information on the mode of inheritance of tolerance, different doubled haploid (DH) populations were tested for variation in grain yield and other agronomic traits after BYDV-PAV infection. By QTL analysis of relative grain yield affected by BYDV two chromosomal regions have been identified which explain nearly 50% of the phenotypic variance. Such genetic variation can now be rapidly exploited by methods of biotechnology like anther and microspore culture to produce homozygous DH cultivars. In addition, molecular markers potentially allow breeders selection on the genotypic rather than the phenotypic level, offering new opportunities for resistance breeding. The present state of breeding for virus resistance or tolerance and variety development as well as future perspectives will be discussed.

Status of Russian Wheat Aphid Resistance in U.S. Barley

D. W. Mornhinweg1, P. P. Bregitzer2, D. A. Obert 2, R. W. Hammon3, F. B. Peairs 3
and D. R. Porter1

1USDA-ARS, Stillwater, OK 74075, USA,
E-mail: dmornhinweg@pswcrl.ars.usda.gov;
2National Small Grains Germplasm Research Facility, USDAARS, Aberdeen, ID 83210, USA;
3Colorado State University, Fort Collins, CO 80523, USA

Russian wheat aphid, Diuraphis noxia, Mordvilko, was first detected in the Southern U.S. in the spring of 1986. It quickly spread throughout the intermountain regions of the western U.S. and into Canada by the summer of 1988. Losses in wheat and barley were severe those 2 years. Aphid populations have since declined and losses have become sporadic and less severe throughout the intermountain regions with the exception of a corridor of persistent damaging infestation in E Colorado and Wyoming and W Nebraska, where barley is no longer grown due to the presence of RWA. Although several unadapted RWA-resistant barley germplasm lines have been released to breeders by the USDA-ARS, a RWA-resistant barley cultivar has yet to be released. The release of a large set of nearly 50 barley germplasm lines with resistance from multiple sources and in many different adapted backgrounds is imminent. Several feed barley cultivars are also close to release in both spring and winter types. The status of these barleys will be discussed. RWAs in Colorado in the spring of 2003 were observed to damage wheat cultivars previously resistant to RWA. The resistant status of barley germplasm lines to this new RWA will also be discussed.

Poster Presentation

Cloning of AFLP Fragments Linked to Resistance Genes for Fusarium Head Blight in Barley

H. A. Agrama1, L. S. Dahleen2 and R. I. McArthur3

1Department of Plant Pathology, North Dakota State University, and Northen Crop Science Laboratory, USDA-ARS, Fargo, ND 58105, USA,
E-mail: agramah@fargo.ars.usda.gov;
2 Northen Crop Science Laboratory, USDA-ARS, SU Station, Fargo, ND 58105-5677, USA;
3Department of Plant Sciences, North Dakota State University, Fargo, ND 58105, USA

Genetic mapping of resistance genes to Fusarium head blight (FHB) in barley, caused by Fusarium graminearum, revealed multiple locus inheritance. A segregating population of seventy-five double haploid lines, developed from the three-way cross Zhedar 2/ND9712//Foster, was used for genome mapping and FHB evaluation. Four amplified fragment length polymorphism (AFLP) markers were identified and mapped to chromosomes 2H and 5H at regions closely associated with FHB resistance and low deoxynivalenol (DON) concentration. An additional AFLP marker was associated with disease susceptibility. These five AFLP fragments were purified, cloned and sequenced. Sequence-characterized amplified regions (SCARs) will be produced and used for assessing the presence of FHB resistance genes for comparisons in other barley genotypes. These markers may therefore be useful in marker-assisted selection of resistance genes in barley breeding programs and will facilitate map-based cloning.

Mapping QTL for Resistance to Pathotypes of Cochliobolus sativus in Barley

H. Bilgic1, B. J. Steffenson1 and P. M. Hayes2

1Department of Plant Pathology, University of Minnesota, St. Paul, MN 55108 USA,
E-mail: bilgi001@umn.edu;
2Department of Crop and Soil Sciences, Oregon State University, Corvallis, OR 97331, USA

Spot blotch, caused by Cochliobolus sativus, is an important disease of barley in many production areas. The parents of the doubled haploid mapping population Calicuchima-sib/Bowman (C/B) exhibited differential reactions to pathotypes 1 and 2 of C. sativus. To elucidate the genetics of resistance, the C/B progeny were evaluated to both pathotypes at the seedling stage in the greenhouse and at the adult plant stage in the field. Seedling resistance to pathotype 1 was conferred by a single QTL on chromosome 4H, and adult plant resistance was conferred a single QTL on chromosome 3H. Bowman contributed both resistance alleles. Seedling and adult plant resistance to pathotype 2 were conferred by a single gene on chromosome 1H (contributed by Calicuchima-sib). Progeny with resistance to both pathotypes were identified and may be useful in programs breeding for spot blotch resistance.

Fusarium Head Blight of Barley in Eastern Canada

T. M. Choo1, B. Vigier1, R. A. Martin2 and K. M. Ho1

1Eastern Cereal and Oilseed Research Centre, Agriculture and Agri-Food Canada,
Ottawa, K1A 0C6, Canada,
E-mail: chootm@agr.gc.ca;
2Crops and Livestock Research Centre, Agriculture and Agri-Food Canada,
Charlottetown, C1A 4N6, Canada

Fusarium head blight (FHB) of barley is a devastating disease in Eastern Canada. It is caused by four major Fusarium species (F. graminearum, F. poae, F. sporotrichiodes, and F. avenaceum). These fungi can produce seven kinds of mycotoxin in barley (deoxynivalenol (DON), nivalenol, zearalenone, 3-acetyl DON, 15-acetyl DON, T-2, and HT-2). In a recent survey, 72% of the 116 barley samples were contaminated with DON and 5% of the samples contained more than 5 mg/kg of DON. The recommended tolerance level of DON for swine in Canada is only 1 mg/kg. Therefore, measures to control FHB are needed. At present, none of the six-row cultivars are resistant to FHB. Some two-row cultivars such as Island and AC Alberte contain low DON. Two-row barley generally contains less DON than six-row barley. Purple barley is more resistant to FHB than yellow barley under severe infection. The length of glume awns appears to be associated with FHB resistance. Under artificial inoculation, DON content is likely to be high if plants are short and susceptible to lodging. FHB infection reduces seed weight. The rate of reduction of two-row barley is similar to that of six-row barley. The reduction could be partly due to genetic linkages.

Population Structure of Bipolaris sorokiniana in Western Canada

H. Ghazvini and A. Tekauz

Cereal Research Centre, Agriculture and Agri-Food Canada, Winnipeg, Manitoba, R3T 2M9, Canada, E-mail: atekauz@agr.gc.ca

Spot blotch, caused by Bipolaris sorokiniana, has become one of the most damaging diseases of barley (Hordeum vulgare) in the eastern prairie region of Canada. In addition, the pathogen is also a causal agent of black point and common root rot on this host. Depending on barley genotype, pathogen virulence and local conditions, spot blotch can cause yield reductions of up to 20%. Determination of a pathogens population structure can provide useful information regarding pathogenic variability, and the usefulness of resistance genes available for deployment in the host. Three pathotypes of B. sorokiniana were identified in North Dakota, the main spring barley growing state of the USA, based on infection responses on three barley differential lines. In this study, the infection responses of 87 isolates of B. sorokiniana from western Canada were evaluated on 12 barley differentials to determine the pathogens variability in the region. The results indicate additional pathogenic variation compared to that reported previously for North Dakota, and two of the three previously designated pathotypes, 0 and 1, may each have two sub-groups. Barley differential line TR251 had the best overall seedling resistance to all isolates tested.

Additional studies on this subject and electronmicroscopic investigations of leaf morphology are in progress. Furthermore recently interspecific hybrids have been developed and will be tested for their BYDV-reaction.

Yield Loss in Barley Inoculated with High and Low Virulence Isolates of Bipolaris sorokiniana

H. Ghazvini and A. Tekauz

Cereal Research Centre, Agriculture and Agri-Food Canada, Winnipeg, Manitoba, R3T 2M9, Canada, E-mail: atekauz@agr.gc.ca

In 2001 spot blotch, caused by Bipolaris sorokiniana, was the predominant leaf spot of barley in Manitoba and occurred in every field surveyed. Despite this general disease incidence, isolates of B. sorokiniana sampled varied in virulence and could be classified into two main groups of high (HV) or low virulence (LV) on barley seedlings. To determine if these isolate groups cause differential levels of damage and grain yield loss, 6 barley lines, including resistant (TR 251, Newdale), moderately resistant (Stander, Robust) and B. sorokiniana-susceptible(CDC Bold, Conlon) genotypes were tested in field trials in 2003. Barleys were inoculated three times using single HV (WRS 1986) or LV (WRS 1949) pathogen isolates. Average grain yield loss caused by the HV and LV isolates was 11% and 6%, respectively. Resistant barley TR 251 had lower reductions in yield (3% and 5%) compared to susceptible Conlon (23%) and CDC Bold (13%) inoculated with HV and LV isolates, respectively. However, Stander barley had the lowest overall yield loss, suggesting it may possess a superior and usable level of adult plant resistance. The results indicate that both LV and HV isolates of B. sorokiniana can cause significant losses, and should be tested, and in addition, that adult plant resistance should be examined, as components of an effective strategy in breeding for resistance to B. sorokiniana.

Greenhouse Screening and NIRS for Fusarium Head Blight
in Barley

T. S. Grewal1, B. G. Rossnagel1, G. Arganosa1, M. Savard2 and G. J. Scoles1

1Department of Plant Sciences/Crop Development Centre, University of Saskatchewan,
Saskatoon, SK S7N 5A8, Canada, E-mail: brian.rossnagel@usask.ca;
2Eastern Cereal and Oilseeds Research Centre, AAFC, Ottawa, ON K1A 0C6, Canada

Fusarium head blight (FHB) is the most significant disease of barley in Canada and some other countries. Main losses are grain quality reduction due to the presence of deoxynivalenol (DON) making barley unsuitable for malting or feed. Effective and efficient indoor screening techniques are critical for breeding resistant barley cultivars as field screening methods are not efficient because of environmental effects on disease development. Plant architecture, heading date and other diseases can confound FHB development in field experiments. Indoor screening techniques are also important for breeding programs where FHB is not present but has the potential to be a devastating disease. Inoculation methods (infected canaryseed, injection and spray methods) and humidity chamber conditions have been evaluated to standardize effective screening technique under controlled conditions. NIRS methodology also shows promise for simplified estimation of barley DON levels. Coleoptile segments from susceptible and resistant barley lines were tested for their sensitivity to Fusarium spp. mycotoxins (Eudes et al. 2000). Preliminary results show greater inhibition of coleoptile segment elongation in susceptible lines. This technique may be useful for mass screening in early generations with only putative resistant genotypes needing to be screened at heading.

Hordeum bulbosum a Source for BYDV Resistance

A. Habekuss, E. Schliephake and F. Ehrig

Institute of Epidemiology and Resistance, Federal Centre for Breeding Research on Cultivated Plants (BAZ), D-06449 Aschersleben, Germany,
E-mail: a.habekuss@bafz.de

Hordeum bulbosum is known to be valuable as source to improve resistance of Hordeum vulgare to different fungi and also to the barley mosaic virus complex. In a pre-screening of 15 Hordeum bulbosum genotypes maintained for several years under natural Barley yellow dwarf virus (BYDV) infection in the field and in the greenhouse one accession free of BYDV was identified by ELISA. In numerous inoculation tests with high infection pressure using BYDV-PAV and BYDV-MAV and 10 to 20 individuals of Rhopalosiphum padi or Sitobion avenae per plant the accession remained symptomless and no virus could be detected. First results of penetration behaviour of R. padi females on the accession, studied with the EPG method, show that the number and the duration time of phloem penetration are much lower on H. bulbosum than on H. vulgare cv. Erfa.

QTL Analysis of Resistance to Fusarium Head Blight
in Barley RI Populations

K. Hori, T. Kobayashi, K. Sato and K. Takeda

Research Institute for Bioresources, Okayama University, Kurashiki 710-0046, Japan,
E-mail: khori@rib.okayama-u.ac.jp

We evaluated Fusarium head blight (FHB) resistance using cut-spike test in two recombinant inbred populations derived from Russia 6 × H.E.S. 4 (RI1) and Harbin two-row × Turkey 6 (RI2). We developed high-density linkage maps including AFLP and SSR markers on two populations. At the same time, the resistant gene analog (RGA) markers mapped on the high-density linkage maps developed from degenerated primers based on highly conserved domain of R-genes, or from sequence specific primers based on barley ESTs that showed high homology to R-gene sequences. In the RI1 population, the results of simple interval mapping (SIM) and composite interval mapping (CIM) for FHB resistance detected two QTLs located on the long arm of chromosome 2H and one QTL located on the short arm of chromosome 5H. One of the QTLs located on chromosome 2H was coincident with vrs1 locus that determines inflorescence row type. In the RI2 population, CIM detected one QTL on chromosome 2H, which seemed identical with the one detected in RI1 since one RGA marker was mapped on 1.1 cM (RI1) and 2.1 cM (RI2) distances from this QTL. The combination of high-density map and closely linked markers to QTLs for FHB resistance is useful for the marker-assisted selection or the identification of candidate genes.

Identification and Mapping of Resistance Genes against the Barley Yellow Mosaic Virus Complex in Barley

K. Humbroich1, H. Jaiser2, A. Schiemann2, P. Devaux3, A. Jacobi4, W. Friedt1 and F. Ordon5

1Institute of Crop Science and Plant Breeding I, Justus-Liebig-University Giessen, D-35392 Giessen, Germany, E-mail: katrin.humbroich@agrar.uni-giessen.de; 2Pajbjergfonden, Dyngby, DK-8300 Odder, Denmark; 3SA Florimond Desprez Veuve and Fils, 59242 Cappelle-en-Pevele, France; 4W. von Borries-Eckendorf GmbH and Co, D-33818 Leopoldshöhe, Germany; 5Institute of Epidemiology and Resistance,
Federal Centre for Breeding Research on Cultivated Plants (BAZ), D-06449 Aschersleben, Germany

Barley yellow mosaic virus disease which is caused by a complex of three viruses, i.e. BaMMV, BaYMV and BaYMV-2, is one of the most important diseases of winter barley in Europe as well as in large parts of Asia due to a permanent spread and high yield losses frequently observed in susceptible barley cultivars. Due to the soil-borne nature of the disease, the availability of resistant cultivars is the only way to ensure cultivation of these crops in the increasing area of infested fields. In order to identify donors of resistance carrying genes different from rym4 and rym5 which are at present widely used in European barley breeding 120 gene bank accessions, resistant against BaYMV in Japan, were analysed by the SSR marker Bmac29 being to some extend diagnostic for this locus. Out of the 120 accessions analysed 43 genotypes were detected carrying rym5 and 12 carrying rym4. Those which are not carrying rym5 or rym4 are potential candidates for detecting new resistance genes after carrying out extensive tests for allelism. Besides this, DH populations derived from crosses to different exotic germplasms are analysed by bulked segregant analysis using genome covering SSRs. In this respect the BaMMV resistance of cv. Chikurin Ibaraki 1 was assigned to chromosome 6H by the analysis of 163 F1-doubled haploid lines of a cross to the susceptible cultivar Igri. Analysis of additional DH-populations is currently in progress.

Spike Morphology and Flowering Behavior Affecting the Resistance to Fusarium Head Blight in Barley

N. Kawada1, M. Yoshida2 and T. Tohno-oka3

1National Agricultural Research Center for Kyushu Okinawa Region, Chikugo, Fukuoka, 833-0041, Japan,
E-mail: kawada@affrc.go.jp;
2National Agricultural Research Center for Kyushu Okinawa Region, Nishigoshi, Kumamoto, 861-1192, Japan; 3National Institute of Crop Science, Tsukuba, Ibaraki 305-8518, Japan

Barley varieties show a wide range of resistance to Fusarium head blight (FHB) and two-rowed barley have been recognized to be more resistant than six-rowed one. And also, from our test for the resistance, two-rowed and closed-flowering, cleistogamy, barley cultivars form Japan belonged to the highest resistant group. In order to define the effect of spike morphology and flowering behavior to the resistance, we investigated the degree of the resistance for near-isogenic lines (NILs) of barley differing for these traits, using two testing methods, pot-plant and cut-spike, in which spikes exactly at anthesis were inoculated with macroconidia suspension of F. graminearum. The largest difference of FHB severity was observed between chasmogamy and cleistogamy NIL pair and obviously greater than that of two-rowed and six-rowed spike NIL pair. Significant differences were not observed between lax/dense spike, normal/uzu-type and wax/wax-less spike NIL pair. The results indicate that cleistogamy and genetic background of Japanese two-rowed varieties make a great contribution to the FHB resistance and are useful germplasm for the resistance breeding.

Mapping of Resistance to BaYMV/BaYMV-2 in the Japanese Winter Barley Accession HHOR 4224

I. Kraemer1, A. Habekuss1, F. Ordon1, R. Pickering2 and G. Proeseler1

1Institute of Epidemiology and Resistance, Federal Centre for Breeding Research on Cultivated Plants (BAZ), D-06449 Aschersleben, Germany, E-mail: a.habekuss@bafz.de;
2Crop and Food Research, Christchurch, New Zealand

A comprehensive screening program on winter barley accessions of the Gatersleben World Collection was carried out to select genotypes with resistance/tolerance to Barley mild mosaic virus (BaMMV), Barley yellow mosaic virus 1 and 2 (BaYMV-1, -2) and Barley yellow dwarf viruses (BYDV). Doubled haploid (DH) lines from crosses between resistant/tolerant accessions and susceptible varieties were developed to perform classical and molecular genetic studies. The inheritance of complete mosaic resistance in the barley accession HHOR 4224 was studied on DH progeny from a cross with the susceptible genotype HHOR 10714. Reactions to the viruses were assessed in natural contaminated fields and confirmed by DAS-ELISA. The DH lines could be grouped in four phenotypic classes suggesting that resistance to BaMMV and to BaYMV-1/BaYMV-2 in HHOR 4224 is conferred by two independent genes. One of the genes determining resistance to BaYMV-1/BaYMV-2 was localised on chromosome 5H using SSRs and AFLPs. A linkage map was constructed using the mapping programme JoinMap 3.0. The resistance locus is flanked by the SSRs Bmac 096 and Bmac 303 at map distances of 1 cM. Current experiments are focused on mapping the BaMMV resistance of HHOR 4224.

Genetic Associations of FHB Reaction and Morphological Traits
in Barley

N. N. Krasheninnik and J. D. Franckowiak

Department of Plant Sciences, North Dakota State University, Fargo, ND 58102, USA,
E-mail: j.franckowiak@ndsu.nodak.edu

Partial resistance of barley, Hordeum vulgare, accessions to Fusarium head blight (FHB), incited by Fusarium graminearum, is often associated with morphological traits such as spike type, plant height, and maturity. Disease occurrence and severity is often lower in tall, late, two-rowed lines, which are agronomically undesirable. Also, expression of traits such as late heading, tall culms, and two-rowed spikes can interfere with accurate measurement of FHB resistance and may increase escape from infection. Several QTLs for FHB resistance have been mapped in chromosome 2H, which contains genes for the six-rowed spike type (vrs1), three long-day sensitive maturity factors (Eam1, Eam6, and Eam11), and at least two short culm traits (hcm1 and lin1). Near-isogenic lines, mapping populations, and segregating progenies were evaluated to study the linkage vs. pleiotropy hypothesis and to estimate linkage distances among morphological traits in chromosome 2H. The results suggest that QTLs for FHB reaction and several morphological traits are tightly linked. A significant effort will be necessary to break several unfavorable linkages or to replace the maturity and plant height genes currently present in barley cultivars adapted to the Upper Midwest of the USA.

Yield Reduction in Naked and Hulled Barley Doubled Haploids Inoculated with Fusarium culmorum (W.G.Sm.) Sac.

K. Krystkowiak1, T. Warzecha2, A. Kuczynska1, M. Rebarz1, T. Adamski1, Z. Kaczmarek1
and M. Surma1

1Institute of Plant Genetics, Polish Academy of Sciences, 60-479 Poznań, Poland,
E-mail: kkry@igr.poznan.pl;
2Agricultural University of Cracow, 31-140 Cracow, Poland

The influence of inoculation with Fusarium culmorum on performance of yield-related traits in naked and hulled barley doubled haploids was studied in experiment carried out in 3 years and 2 localities. Doubled haploids were derived by bulbosum method from F1 hybrids between naked (1N86) and hulled (RK63/1) breeding lines. In each year and locality the experiment was established in a randomised complete block design with three replications. At the stage of full anthesis in each replication 50 ears of each line were inoculated with a single isolate of Fusarium culmorum (IPO 348-01). The following traits were analysed in inoculated and control lines: kernel number and kernel yield per ear, 1000-ker-
nel weight and percentage ofplump kernels (> 2.5 mm). The inoculated lines were compared withnon-inoculatedusing univariate and multivariate statistical methods. Analysis of variance revealed a significant effect of inoculation, environment and genotype on the variation of studied traits. Effect of inoculation appeared to be many times higher than that of the other sources of variation. Generally, reduction in yield-related traits was higher in naked than hulled lines.

Comparison of Hulless and Covered Barley for Infection
with Fusarium Head Blight and Accumulation of -Trichothecene Mycotoxins in Grain

L. Legzdina1 and H. Buerstmayr2

1Priekuli Plant Breeding Station, Priekuli LV-4126, Latvia, E-mail: lindaleg@navigator.lv;
2Division of Biotechnology in Plant Production, Department for Agrobiotechnology Tulln, University of Natural Resources and Applied Life Sciences Vienna, A-3430 Tulln, Austria, E-mail: buerst@ifa-tulln.ac.at

We have evaluated 145 covered and 29 hulless barley genotypes under artificial infection with Fusarium graminearum Schw. in a field experiment in Tulln in 2002. The mean area under disease progress curve (AUDPC) of covered and hulless barley did not significantly differ. We chose from the tested barley genotypes 29 hulless-covered barley pairs which had approximately the same disease severity based on visual symptoms on the heads for analysis of mycotoxin (-trichothecene) content in harvested grain. The average content of deoxynivalenol of the 29 covered and hulless barley samples was 15,520 g/kg and 12,964 g/kg. The results showed that the mean deoxynivalenol, 3-acetyl-deoxynivalenol and 15-acetyl-deoxynivalenol content of covered barley was significantly higher than that of hulless barley (P < 0.01), whereas for nivalenol the difference between the mean values of covered and hulless barley was not significant. The results support the hypothesis that hulless barley appears to be less prone to accumulation of mycotoxins in the harvested crop than covered barley. Explanation of this finding may be that a considerable proportion of the mycotoxin resides in the barley hulls.

Barley Seed Health as Affected by Seed-Borne Bipolaris sorokiniana

G. D. Martens and A. Tekauz

Cereal Research Centre, Agriculture and Agri-Food Canada, Winnipeg, Manitoba, R3T 2M9, Canada, E-mail: atekauz@agr.gc.ca

Spot blotch, caused by Bipolaris sorokiniana (teleomorph: Cochliobolus sativus), in barley (Hordeum vulgare) has increased significantly in Manitoba, Canada. Based on isolations from infected leaf tissue, spot blotch was the most prevalent disease in barley fields surveyed in the province in 2001 and 2002. B. sorokiniana also can infect developing kernels to cause discoloration and shriveling of the grain. Sampling of barley kernels from two-rowed barley fields severely affected by spot blotch has indicated a 90100% incidence of B. sorokiniana. The effects of high levels of B. sorokiniana seed-borne infection on subsequent germination and plant emergence have not been studied in barley. Also, the effects of management options (new seed treatments, seed placement, and varietal selection) to enhance germination and emergence need to be determined. Initial results indicated that germination and emergence of highly infected, untreated barley seed were significantly reduced, but that these negative effects could be mitigated by certain seed treatments. Seed treatment also significantly increased yields, indicating that this could be an effective management tool. However, yields were highest with seed lots having low levels of B. sorokiniana infection, suggesting that prevention of infection should be a component of a holistic management strategy.

Proteinases Secreted by Fusarium and Their Inhibitors
in Barley Grains

S. Marttila1, A. Pekkarinen2, E. Liljeroth1, B. L. Jones3 and M.-L. Niku-Paavola4

1Department of Crop Science, Swedish University of Agricultural Sciences, 230 53 Alnarp, Sweden,
E-mail: salla.marttila@vv.slu.se; 2Miller Brewing Company, Milwaukee, WI 53201-0482, USA;
3Route 1, Kooskia, ID, USA; 4VTT Biotechnology, FIN-02044 Espoo, Finland

Recently, a fungal disease, Fusarium head blight (FHB), has become a serious problem for cultivated barley around the world. Infected grains have poor quality: they are small, shrivelled, discoloured, and often contain mycotoxins. The problem is especially difficult for malting barley. More knowledge about the defense mechanisms that barley uses against Fusarium infection is needed in order to breed for more tolerant malting barley cultivars. When grown on barley grains, F. culmorum secretes trypsin-like and subtilisin-like alkaline proteinases which can hydrolyze barley storage proteins. Barley grains contain inhibitor proteins, such as the Bowman-Birk trypsin inhibitor, BASI and CI-2A, which can decrease the activity of these fungal proteinases. We have studied the spatial and temporal localization of the two fungal proteinases and their barley inhibitors in infected and non-infected barley grains by immunomicroscopy. All three proteinase inhibitors were present both in the non-infected and infected grains. The fungal proteinases were detected only in the non-infected grains. Both proteinases were located in the outer layers of the grain, in collapsed starchy endosperm cells and especially in the deformed aleurone cells. The inhibitors were localized at the same tissues. The intensity of the inhibitor labelling decreased at the later stage of infection, possibly due to formation of proteinase/inhibitor-complex.

Improvement of Malting Barley Resistance to Fusarium Head Blight Using Molecular Markers

Z. Nesvadba1, T. Vyhnanek2, M. Spunarova1, J. Ovesna3, L. Tvaruzek1, I. Jeziskova2
and J. Spunar1

1Agricultural Research Institute Kromeriz, Ltd., 767 01 Kromeriz, Czech Republic,
E-mail: nesvadba@vukrom.cz; 2Department of Botany and Plant Physiology, Mendel University
of Agriculture and Forestry Brno, 613 00 Brno, Czech Republic, E-mail: vyhnanek@mendelu.cz; 3Department of Molecular Biology, Research Institute of Crop Production, 161 06 Prague-Ruzyne,
Czech Republic, E-mail: ovesna@vurv.cz

The worldwide-distributed fungal disease called Fusarium Head Blight (FHB) and successive grain contamination by mycotoxins cause a considerable decrease in yield, quality and economic use of cereal crops. The infection of cereals by Fusarium fungi affects not only hygienic safety, but as well as grain technological quality. For instance, deoxynivalenol passes from malting barley grain infected by FHB up to a final product, beer, and is one of factors inducing beer gushing. Therefore, a great attention has been paid to diseases caused by fusaria in the Czech Republic over the recent years. A set of spring and winter barley varieties artificially infected with head fusaria were subjected to a test analysis. Based on visual and laboratory assessments and determination of the deoxynivalenol concentration, initial types displaying different resistance/susceptibility to FHB were selected. Hybridization of resistant and susceptible resources was carried out. Doubled haploid lines were derived from the hybrid progenies using in vitro induced androgenesis and used for further testing. RAPD markers and AFLP analyses were employed to detect differences in FHB resistance of barley lines.

Supported by the Grant Agency of the Czech Republic, Project No. 521/03/0938.

Characterization of Barley Yellow Mosaic Disease Resistant Gene rym1 and Breeding of a Novel BaYMV and BaMMV Resistant Malting Barley

Y. Okada1, R. Kanatani2, S. AraI1 and K. Ito1

1Biosources Research and Development Laboratories and 2Malting Department, Gunma Brewery, Sapporo Breweries Ltd., Kizaki, Nitta, Gunma 370-0393, Japan, E-mail: yoshihiro.okada@sapporobeer.co.jp

Cv. Mokusekko 3, a Chinese barley landrace is completely resistant to all strains of Barley yellow mosaic virus (BaYMV) and Barley mild mosaic virus (BaMMV) prevailed at present. It is also well known that cv. Mokusekko 3 has at least two resistant genes rym1 and rym5, only rym5 has been utilized for BaYMV resistant barley breeding in Japan. In order to clarify the effect of rym1 on BaYMV and BaMMV and to utilize the gene for resistant barley breeding, the susceptibilities of lines carrying only rym1 against BaYMV and BaMMV were investigated. Our results showed that rym1 was completely resistant to BaYMV-I, -II, BaMMV-Ka1 and -Na1, and had an acceptable level of resistance to BaYMV-III. Therefore, to introduce rym1, a novel BaYMV resistant gene, to malting barley, the cv. Mokkei 01530 was slected from a cross between cv. Mokusekko 3, a donor for rym1, and cv. Haruna Nijo, an excellent malting variety. Mokkei 01530 was completely resistant to BaYMV-I and had an acceptable level of resistance to BaYMV-III. In comparison with its high quality parent cv. Haruna Nijo, malting quality of cv. Mokkei 01530 was almost identical with cv. Haruna Nijo. Our results clearly indicated that rym1 is another promising gene for the breeding of BaYMV and BaMMV resistance malting barley.

Molecular Mapping of Virus Resistance in Barley (H. vulgare L.)

F. Ordon1, W. Friedt2, K. Scheurer2, B. Pellio2, K. Werner2, C. Weiskorn2, G. Neuhaus2,
F. Nissan-Azzouz2, W. Huth3, A. Habekuss1, J. Le Gouis4, P. Devaux5 and A. Graner6

1Institute of Epidemiology and Resistance, Federal Centre for Breeding Research on Cultivated Plants (BAZ), D-06449 Aschersleben, Germany, E-mail: f.ordon@bafz.de; 2Institute of Crop Science and Plant Breeding I, Justus-Liebig-University, D-35392 Giessen, Germany; 3Federal Biological Research Centre,
D-38104 Braunschweig, Germany; 4INRA URGAP, 80200 Estrées-Mons, France; 5SA Florimond Desprez Veuve and Fils, 59242 Cappelle-en-Pevele, France; 6Institute of Plant Genetics and Crop Plant Research (IPK), D-06466 Gatersleben, Germany

Virus diseases, i.e. soil-borne Barley yellow mosaic virus disease (BaMMV, BaYMV BaYMV-2) and the aphid transmitted Barley yellow dwarf virus (BYDV) have gained evident importance in European barley breeding. Resistance to Barley yellow mosaic virus disease has been identified in exotic germplasms and several recessive resistance genes, e.g. rym4 (3H), rym5 (3H), rym9 (4H), rym11 (4H), rym13 (4H) have been mapped. Recently the BaYMV/BaYMV-2 resistance of cv. Chikurin Ibaraki 1 has been assigned to chromosome 5H in the region of rym3 while the BaMMV resistance (rym14) of this accession has been located on chromosome 6H. Genetic analyses of DH-populations to map additional genes is in progress. For the above mentioned genes easy to handle PCR-based markers are available (RAPDs, AFLPs, SSRs, STSs, SNPs), which besides their use in marker assisted breeding procedures have been the basis for genotyping of germplasm collections, pyramiding of resistance genes using different strategies, and the starting point for a map based cloning approach. In contrast to Barley yellow mosaic virus disease, no complete resistance to BYDV is known in the barley gene pool, but tolerant accessions have been identified and QTL for BYDV-tolerance have been detected on chromosomes 2HL and 3HL in different crosses and different trials explaining about 50% of the phenotypic variance. Future work aims at the identification of genes involved in tolerance against BYDV by cDNA-AFLPs. Respective markers will facilitate a more efficient use of genetic diversity with regard to virus resistance in barley and are suited to combine tolerance to BYDV with resistance to soil-borne viruses.

Pathogenic Variability of Fusarium Head Blight Pathogens on Spring Barley

U. Scholz

Division of Biotechnology in Plant Production, Department for Agrobiotechnology Tulln, University of Natural Resources and Applied Life Sciences Vienna, A-3430 Tulln, Austria, E-mail: uwe.scholz@boku.ac.at

Fusarium Head Blight (FHB) threatens the barley (Hordeum vulgare) production in Austria in humid and warm weather and has the potential of reduced food and feed safety for barley products. Sources of resistance to F. graminearum have been profoundly identified in North American Spring Barley Collections. However, very little is known about the reaction of barley to other Fusarium species to effectively manage FHB resistance. Two F. graminearum susceptible and resistant six- and two-rowed spring barley cultivars were investigated for their susceptibility towards Austrian isolates of Fusarium graminearum, F. culmorum, F. poae, F. avenaceum and F. sporotrichioides in pot and field experiments under moderate and severe disease pressure, respectively. In both experiments, the six-row spring barley variety Chevron was resistant against all Fusarium species, while both susceptible lines, the two-rowed barley line ICB 111809 and six-rowed barley variety Stander were highly susceptible for all Austrian Fusarium species. Overall, F. graminearum presented high aggressiveness at moderate and high disease pressure, while F. poae and F. avenaceum exhibited higher aggressiveness at lower humidity and F. sporotrichioides and F. culmorum were more adapted to more humid screening conditions. The ranking of Fusarium species severity on Stander and Chevron 14 and 21 days after inoculation was very similar between moderate and severe disease pressure. Based on observations for both two-rowed barley lines, a potential species × genotype interaction requires further investigation.

Unlocking the Secondary Gene Pool of Barley as a Genetic Resource for Resistance to Barley Yellow Dwarf Virus (BYDV)

M. Scholz1, B. Ruge1, A. Habekuss2 and P. Wehling1

1Institute of Agricultural Crops, Federal Centre for Breeding Research on Cultivated Plants (BAZ),
D-18190 Gross Lüsewitz, Germany; 2 Institute of Epidemiology and Resistance, Federal Centre for Breeding Research on Cultivated Plants (BAZ), D-06449 Aschersleben, Germany,
E-mail: m.scholz@bafz.de

Barley yellow dwarf virus (BYDV) causes high yield losses in most of the major cereal crops worldwide. In contrast to the widely used resistance sources to BYDV based on the Ryd2 gene and conferring tolerance to BYDV, a new type of resistance has been identified within the tetraploid wild species Hordeum bulbosum (2n = 4x = 28), (Habekuss et al. this volume). Interspecific reciprocal crosses between the tetraploid H. bulbosum accession and diploid (2n = 2x = 14) and tetraploid (2n = 4x = 28) cultivars, respectively, of H. vulgare were used to transfer resistance to BYDV into cultivated barley. Triploid (2n = 3x = 21) and tetraploid (2n = 4x = 28) hybrids were obtained with the aid of embryo culture. The number of embryos produced per seed and regeneration rate of plants was strongly influenced by the genotype of the H. vulgare parents and the direction of the cross. Barley anchor markers were used to verify the hybrid character of the interspecific F1 offspring. Fifty hybrids were tested for their resistance to BYDV via inoculation with viruliferous aphids (BYDV-PAV, Rhopalosiphum padi). Thirteen of these 50 F1 plants did not develop symptoms and stayed virusfree according to ELISA. Twenty-five diploid plants have been produced by backcrossing one triploid BYDV resistant hybrid with partial fertility to H. vulgare. Their resistance to BYDV remains to be tested.

Development of Tissue-Specific Gene Promoters for Targeting Anti-Fusarium Gene Expression in Barley

R. W. Skadsen1, M. L. Federico2, T. Abebe2 and H. F. Kaeppler2

1U.S. Department of Agriculture, ARS, Cereal Crops Research Unit, Madison, WI 53711, USA,
E-mail: rskadsen@wisc.edu;
2University of Wisconsin, Agronomy Department, Madison, WI 53706, USA

We identified lemma and pericarp epithelium tissues as rapidly infected by Fusarium graminearum. Genes specifically expressed in these tissues were cloned so that their promoters could be used to express antifungal protein genes. These included a lipid transfer protein homologue (LTP6) highly expressed in the pericarp epithelium but not in vegetative leaves and a jacaline-like gene, Lem2, preferentially expressed in the lemma/palea, compared with the flag leaf. Ltp6 is also expressed in coleoptiles and embryos; mRNA levels increase in response to salt, cold, abscisic acid and salicylic acid in a pattern distinct from other barley Ltps. Transient expression analysis of the promoter showed that 246 bp of upstream sequence confers tissue-specific expression and retains most promoter activity. Substitution of a novel MYC binding site abolishes most of the activity. All 4 Lem2 genes are located in a single BAC and map to chromosome 5(1H). Lem2 is specifically expressed in the lemma/palea and coleoptile. The two LEM2 jacalin-like domains may be involved in pathogen recognition. SA and MeJA induce Lem2 within 2 h, suggesting that it is a defensive gene. Particle bombardment with the cloned Lem2 promoter showed strong expression of sgfp in the lemma/palea and very weak expression in leaves.

QTL Mapping Fusarium Head Blight Resistance in Barley

K. P. Smith, L. Nduulu, C. Gustus, A. Sallam and K. Beaubien

Department of Agronomy and Plant Genetics, University of Minnesota, St. Paul, MN 55108, USA,
E-mail: smith376@umn.edu

Enhancing genetic resistance to Fusarium Head Blight (FHB) is complex because resistance is quantitative, resistance QTLs are coincident with plant morphology QTLs that can influence disease severity, and allelic effects are inconsistent across environments. We have investigated four sources of FHB resistance to identify molecular markers that will enable pyramiding resistance genes through marker assisted selection. Two of eleven QTL identified in a mapping study with the cultivar Chevron were validated on chromosomes 2 and 6 and are associated heading date (HD), which is conditioned by the Eam6 locus, and grain protein, respectively. Two of three QTLs identified in a mapping study with the cultivar Frederickson have been validated. One of these is coincident with the QTL on chromosome 2 identified in the Chevron study. The other is associated with the Vrs1 locus. We have created fine mapping populations to determine whether these plant morphology genes (Eam6 and Vrs1) condition resistance to FHB or are tightly linked to resistance loci. We have developed mapping populations using new FHB resistant cultivars Atahualpa and Hor211. Preliminary data indicate that these sources contain new genes that could complement genes discovered in Frederickson and Chevron. We will present current data on the positions and effects of QTLs for FHB resistance derived from these four sources of resistance.

Correlations among Components of Fusarium Head Blight in Barley

A. Tekauz

Cereal Research Centre, Agriculture and Agri-Food Canada, Winnipeg, Manitoba, R3T 2M9, Canada, E-mail: atekauz@agr.gc.ca

The reactions to Fusarium Head Blight (FHB) of a group of feed barley cultivars from western Canada were assessed at four field locations in southern Manitoba in 2000. Disease components measured included the FHB Index, total Fusarium spp., F. graminearum, F. poae, Fusarium damaged kernels (FDK) and deoxynivalenol (DON). Significant (P 0.05) positive correlations were found among all components, except for F. poae; correlations were highest (0.86, P < 0.001) between F. graminearum and DON. Several of the cultivars tested were as susceptible (S) to FHB as the S check AC Lacombe, while others were moderately resistant (MR), similar to the MR check AC Metcalfe. Six-rowed barleys alone were in the S group, but the MR group included both two-and six-rowed types. Both groups included hulled and hulless cultivars. The MR level of FHB resistance identified in the feed cvs. AC Hawkeye (six-rowed, hulless,) and Tukwa (six-rowed, hulled) is noteworthy, as this level is rarely found in six-rowed genotypes. Together with the two-rowed cvs. Phoenix, Seebe and Tercel, these genotypes may provide alternative resistance(s) to that previously detected in two-rowed malting cultivars such as AC Metcalfe, CDC Kendall and CDC Stratus.

Field Reaction of Selected Spring Barley World Collection Accessions to Fusarium Head Blight Infection

L. Tvaruzek1, J. Spunar2, M. Spunarova2, Z. Nesvadba2, J. Milotova2 and L. Ji3

1Department of Integrated Plant Protection and 2Genetics and Breeding Department,
Agricultural Research Institute Kromeriz, Ltd., 767 01 Kromeriz, Czech Republic,
E-mail: tvaruzek@vukrom.cz;
3College of Plant Protection, Agricultural University of Hebei, Baoding, P. R. China

360 spring barley genotypes were tested in field trial under the artificial infection with conidia suspension of Fusarium culmorum. Inoculation was made in flowering. 23 entries showed highly significant low accumulation of DON (mg/kg) mykotoxin in harvested grains. It was positively correlated with low FHB symptoms development on spikes. Mean level of DON of the trial was 9.2 mg/kg, minimum concentration was 0.36 mg/kg DON and maximum reached 35.4 mg/kg DON. The lowest DON accumulation combined with zero incidence of powdery mildew (Blumeria graminis f.sp. hordei) and low and medium net blotch (Drechslera teres) infection showed cv. Princesse and Union firlbecks. Plant height was for both of these cultivars 80 cm in full heading. Other cultivars with low DON grain content (under the level 4.0 mg/kg) were: Selecta Hanak 1, Spartan, Jersey, KM 1192, Digger, Diplom, Domen, Hodoninský kvas, Opal, Diamant, Branisovický C, Nolc-Dreg.-Velerany, Viva, Deternický Kargyn, Ratborsky, Aramir, Krajova St. Hrozenkov, Grosso, Ackermann donaria, Kompakt and Ingrid.

Supported by the Ministry of Education, Youth and Sports of the Czech Republic, Project No. 7528 and by the Grant Agency of the Czech Republic, Project No. 521/03/0938.