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Presented at the 18th North American Barley Researchers Workshop, July 17-20, 2005, Red Deer, Alberta, Canada
PCR detection and quantification of Fusarium species
Fusarium head blight (FHB) is presently the most significant disease of barley in Canada and many areas of the world. FHB is caused by many Fusarium species and it is very difficult to correlate visual symptoms with deoxynivalenol (DON) concentration as symptomless kernels may carry the pathogen. Conversely many blighted kernels may not be contaminated with DON. Symptoms may be caused by non-trichothecene producing Fusarium species. Identification of Fusarium species based on morphological characteristics by growing on selective media is cumbersome and requires considerable expertise and experience. In addition DON estimation is slow, labour intensive and expensive and demand for this service outstrips supply. PCR-based assays to quantify fungal DNA in infected plant tissue could indirectly estimate DON levels. PCR assays have been standardized to detect Fusarium species with species–specific primers (reported by Parry and Nicholson 1996, Schilling et al. 1996, Nicholson et al. 1998, Yoder and Christianson 1998, Aoki and O'Donnell 1999, Williams et al. 2002) using 22 isolates of seven different Fusarium species. The protocol to detect F. graminearum associated with FHB symptoms produced under artificial and natural infections has been standardized. Primer-pair Fg16N amplified the desired band (280 bp) in all infected barley samples from a greenhouse experiment and no band was amplified from uninoculated and uninfected samples. The vast majority of samples from artificially and naturally infected samples from barley fields showed the desired band. Protocol to quantify F. graminearum DNA using a competitor DNA template (obtained from Dr. P. Nicholson, John Innes Centre, Norwich, UK) has been standardized. Fungal concentration ranging from 1 pg to 100 ng with a constant competitor DNA template concentration (1fg or 3fg/µl) was evaluated and fungal DNA concentration as low as 100 pg (0.1ng) was detected. Work is in progress to quantify fungal DNA from FHB infected barley.
References
Aoki, T. and O'Donnell. 1999. Mycologia. 91:597-609.
Nicholson P. et al. 1998. Physiological and Molecular Plant Pathology. 53:17-37.
Parry D. and Nicholson P. 1996. Plant Pathology. 45:383-391.
Schilling A.G. et al. 1996. Phytopathology. 86:515-522.
Williams K.J. et al. 2002. Australian Plant Pathology. 31: 119-127.
Yoder, W. and Christianson, L. 1998. Fungal Genetics and Biology. 23: 68-80.
Tajinder S. Grewal, Brian G. Rossnagel, Graham J. Scoles
Crop Development Centre/Department of Plant Sciences, University of Saskatchewan, 51 Campus Drive, Saskatoon, SK, S7N 5A8 Canada
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Can we use Australian identified molecular markers for barley net blotch resistance in western Canadian barley breeding programs?
Net blotch of barley caused by Pyrenophora teres Drechs. is an important disease in western Canada (Tekauz 1990) and elsewhere (Steffenson 1997). Two types of leaf symptoms are associated with the net blotch disease: the net form (NFNB), caused by P. teres f. teres, which causes a dark brown crisscross venation pattern that sometimes turn chlorotic; and the spot form (SFNB), caused by P. teres f. maculata, which causes dark brown circular or elliptical spots accompanied by chlorosis of the surrounding leaf tissue (Khan and Tekauz 1982). Yield losses of 20 to 30 % in susceptible cultivars have been reported in western Canada (van den Berg 1988) and up to 40 % in other parts of the world (Khan 1987). More important than yield losses, the pathogen reduces thousand kernel weight, plumpness and test weight, negatively affecting malting and feed quality. The most effective and economical method to control this disease is the use of resistant cultivars, however most commonly grown barley cultivars are susceptible to most isolates of P. teres (Tekauz 1990, 2000). The variability observed in P. teres and failure to find lines resistant to all isolates suggests breeding for resistance should emphasize pyramiding resistance genes to develop broad-based durable resistance. Molecular markers allow breeders to rapidly introgress resistant genes into elite lines and to pyramid more than one resistant gene into a cultivar. Molecular markers linked to net blotch resistance in barley have been recently reported from Australia (Cakir et al. 2003, Raman et al. 2003, Williams et al. 2003). There is need to determine whether we can use Australian developed molecular markers in western Canadian barley breeding programs. Thirty-nine barley lines were screened with 6 NFNB (WRS102, WRS858, WRS1607, WRS1906, LO256, LO246) and 4 SFNB (WRS857, WRS1881, LO233, LO231) isolates at the seedling stage in the U of S, College of Agriculture Phytotron. Parents of Australian barley mapping populations used to identify/validate net blotch markers in Australia, western Canadian lines, US lines and one Ethiopian accession and lines from International collections were included. The majority of the Australian parent lines were susceptible to western Canadian isolates. Australian 'R' lines Franklin, Alexis, Kaputar, Baudin, Hamelin were susceptible to the majority of the isolates tested. However, Pompadour, Halcyon, Tilga and Chebec were resistant to some isolates. Accession CI9214 showed the best overall resistance to all isolates and several western Canadian lines/cultivars viz. TR253, TR251, TR244, CDC Helgason and CDC McGwire were resistant to the majority of the isolates. We will evaluate Pompadour/Stirling, WPG8412/Stirling and Sloop/Halcyon populations to validate Australian NFNB markers using western Canadian NFNB isolates. For SFNB marker validation, potential populations are CI9214/Stirling, Keel/Gairdner, Chebec/Harrington and Tilga/Tantangara.
Selected references
Cakir, M. et al. 2003. Australian J. Agric. Res. 54:1369-1377.
Khan, T.N.1987. Australian J. Agric. Res. 38:671-689.
Raman, H. et al. 2003. Australian J. Agric. Res. 54:1359-1367.
Tekauz, A. 1990. Canadian J. Plant Pathol. 12:141-148.
Williams, K.J. et al. 2003. Australian J. Agric. Res. 54:1387-1394.
Tajinder S. Grewal, Brian G. Rossnagel, Graham J. Scoles
Crop Development Centre/Department of Plant Sciences, University of Saskatchewan, 51 Campus Drive, Saskatoon, SK, S7N 5A8 Canada
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Selection for improved scald resistance in the Crop Development Centre barley improvement program
Scald, caused by Rhynchosporium secalis, is a significant barley disease in western Canada in most years, especially in the moist regions of Alberta and north west Saskatchewan. Control depends primarily on genetic resistance in varieties planted and cultural practices including crop rotation and tillage to bury crop residue. Sources of resistance have been identified; however, many resistance sources break down rather quickly. Success in using the strategy of genetic resistance to control scald has been hampered by evolution and pathogenic variability within scald populations. Improved scald resistance is a part of the Crop Development Centre (CDC) barley improvement program and involves repeated testing of selected lines in a search for putative sources of resistance as well as to monitor scald resistance inherent in the program. Annual testing is conducted in collaboration with T.K. Turkington et al, AAFC, Lacombe at screening nurseries at Lacombe and Edmonton, Alberta. Scald reaction data has been collected at both locations for more than 10 years. Data from 2000 through 2004 is presented for a number of resistant CDC selections and six check varieties. Intermediate resistance derived from CDC Dolly is holding but appears to be less effective than it was initially. Resistance derived from PC11 continues to be effective. Resistance tracing to Arizona hulless waxy (AzHull) remains effective with a large number of selections from crosses with that resistance currently being tested at advanced levels in the CDC program. Lines derived from Senor demonstrate moderately resistant reactions. Resistance from Hordeum spontaneum or H. bulbosum, introduced to the CDC program via New Zealand accessions, has endured well. Several CIMMYT lines, notably Calicuchima, 18 IBON-128 and 18 IBON-75 exhibit highly resistant reactions. Progeny of BT474, a six row CDC breeding line with no apparent resistance source(s) in its pedigree and now serving as a resistant nursery check, will be screened for scald reaction in 2005 Alberta nurseries. Several selections from crosses with BT474 have now reached advanced yield test stages of the CDC program.
B.G. Rossnagel (1), D. Voth (1), T. Zatorski (1), D.D. Orr (2) and T.K. Turkington (2)
(1) Crop Development Centre, University of Saskatchewan, Saskatoon, SK, S7N 5A8
(2) Agriculture and Agri-Food Canada, Lacombe Research Station, Lacombe, AB, T4L 1W1
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Selection for improved FHB tolerance in the Crop Development Centre barley improvement program
Continued financial support by the WGRF, CWB and AAFC MII fund to operate a collaborative Fusarium Head Blight (FHB) screening project, initiated in 2000, at AAFC Brandon, attests to the serious impact of FHB on both barley growers and users. As complete resistance has not been observed in barley, genetic resistance from additive action of multiple minor genes offers the greatest potential against FHB. The Crop Development Centre (CDC) barley improvement program’s participation in this collaborative project was initially limited to searching for putative sources of resistance for the breeding program and screening existing breeding lines to select for improved reaction to FHB. Repeated testing, 2001 to 2004, identified 9 hulled genotypes that consistently demonstrate DON concentrations comparable to the resistant check CI4196. Two of these lines, 2ND16092 (from NDSU) and HDE84194-622-1 (a Chinese accession from Shanghai Academy) have been used as parents in the CDC program on the basis of their FHB reaction. 114 selections from 7 populations derived from 2ND16092 and 65 selections from 2 populations derived from HDE84194-622-1 will be tested in 2005 for FHB response as Preliminary yield test entries. The remaining two-rowed hulled CDC lines all share TR251 as a common parent. SB00106, the most promising in an agronomic sense, also showed promise as an entry in the 2003 and 2004 North American Barley Scab Evaluation Nursery, where its DON level across all sites was among the lowest. SB00106 was entered in the Western Two Row Coop Test as TR04378 in 2004 and chosen to replace CI4196 as the resistant check in the 2005 Brandon FHB nursery. The larger proportion of hulless, 30 versus 9 hulled lines, repeatedly selected and tested for low DON concentration, emphasize the relationship between hull removal and DON accumulation. DON concentrations for all selected hulless lines were lower than the checks. The predominance of several parents (i.e. CI4196, CDC Freedom and TR251) in the pedigrees of these selections indicate the heritable nature of the low DON accumulation trait. SH00749, selected from an early cross between CDC Freedom and CI4196 (population 99T511-03) was in turn used as a parent to improve FHB tolerance. 115 selections from 4 populations derived from SH00749 will be tested in the 2005 FHB nursery as pre-yield test entries. The FHB response of several of the best CDC selections for lower DON accumulation, with few exceptions, seem to hold up well in other FHB screening nurseries.
B. Rossnagel (1)*, D. Voth (1), T. Zatorski (1), J. Tucker (2), W. Legge (2), and M. Savard (3)
*Corresponding author: brian.rossnagel@usask.ca
(1) Crop Development Centre, University of Saskatchewan, Saskatoon, SK, S7N 5A8
(2) Agriculture and Agri-Food Canada, Brandon Research Station, Brandon, MB, R7A 5Y3
(3) Agriculture and Agri-Food Canada, Eastern Cereal and Oilseed Research Centre, Ottawa, ON K1A 0C6
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An AFLP derived tightly linked marker for true loose smut resistance (Un8)
True loose smut resistance in barley is conferred by the single dominant gene Un8, and is the major source of resistance in cultivars bred for western Canada. Since smut is seed borne, conventional disease screening involves the manual (hand held syringe) inoculation of 10 to 12 florets per spike (line). Mature seed from inoculated plants is harvested and grown to anthesis before plants can be evaluated for the presence of smut, a process which needs to be repeated for putative resistant lines because of potential escapes. Cost estimates for conventional screening are near $5 per line. This expense and the simple genetics of the resistance make smut resistance an excellent candidate for marker-assisted selection (MAS). PCR based markers for Un8 (Eckstein et al., 2002) have been in routine use in the Crop Development Centre barley breeding program for more than five years. While the markers are robust and simple to use, the 6cM of genetic distance (recombination) renders approximately 50% of crosses as monomorphic (unscreenable). AFLP on bulked-segregant DNA samples has generated a polymorphism that is more tightly linked with the resistance in 149 DH lines from the cross Harrington x TR306. AFLP primers E32-M58 amplify a short fragment of DNA from the resistant DNA bulk and resistant parent only. This fragment is consistently amplified from constituent lines of the resistant DNA bulk, is absent from the susceptible lines, and co-segregates appropriately with the disease reaction in all lines that showed recombination with our previous marker. Efforts to convert the AFLP polymorphism to a simple PCR-based marker are in progress to simplify linkage analysis on an additional 367 lines from the same population. The polymorphic fragment has been isolated, cloned and sequenced, and consists of 40 bases of genomic sequence from which allele specific PCR-based markers cannot be designed. A 1052bp fragment containing the original 40 bases has been identified through anchored PCR. This locus has been sequenced from resistant and susceptible genotypes in order to design allele specific primers. Analysis of sequence to date indicates that the locus likely has numerous locations in the barley genome and efforts to identify nucleotide variation between resistant and susceptible genotypes at the Un8 linked locus continue. This tightly linked marker will be useful in reducing the error percentage in MAS, and will increase the amount of breeding material that can be evaluated through MAS. Linkage estimates obtained from the larger population will form the basis for closer examination of the locus and perhaps the isolation of the gene itself. The eventual isolation of the Un8 gene will allow for the characterization of previously un-phenotyped materials such as can be done with the Rpg1 resistance gene (Brueggeman et al., 2002) and markers (Eckstein et al., 2003).
References
Brueggeman, R., Rostoks, N., Kudrna, D., Kilian, A., Han, J., Druka, A., Steffenson, B., and A. Kleinhofs. 2002. Proc. Natl. Acad. Sci. USA 99:9328-9333.
Eckstein, P.E., Krasichynska, N., Voth, D., Duncan, S., Rossnagel, B.G., and G.J. Scoles. 2002. Can. J. Plant Pathol. 24:46-53.
Eckstein, P., Rossnagel, B., and G. Scoles. 2003. Barley Genetics Newsletter 33:7-11.
Peter Eckstein, Donna Hay, Brian Rossnagel, and Graham Scoles
Department of Plant Sciences/Crop Development Centre, University of Saskatchewan, Saskatoon, SK, CANADA S7N 5A8
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Cytological karyotyping of Pyrenophora teres
Net blotch, caused by the fungal pathogen Pyrenophora teres, is a common disease of barley that adversely affects seed quality characteristics like plumpness and test weight. Despite its importance, there is limited information available about the P. teres genome. Karyotype analysis of this pathogen was initiated to address this and to provide a foundation for further genetic work including the creation of a molecular linkage map.
One technique available for karyotyping fungal pathogens is the germ tube burst method (GTBM) which allows cytogenetic analysis by releasing mitotic metaphase chromosomes from the actively growing tip of conidial germ tubes. Previous cytological work with fungi focused on meiotic stage chromosomes present during sexual reproduction, however, the sexual stage of many important phytopathogenic fungi is either unknown or difficult to induce in the lab, restricting the use of this method. Pulsed field gel electrophoresis (PFGE) is another popular method of karyotyping fungi which is useful for estimating the molecular weights of chromosomes, but tends to underestimate chromosome numbers due to the inability to resolve chromosomes of similar size. The combination of the GTBM and PFGE make a powerful tool for karyotype analysis.
The GTBM was applied to four isolates of P. teres. A time course analysis was conducted to determine the conidial germination rate and the point at which the maximum number of germ tube nuclei were in metaphase. Germination began after 30 min and was near 100% by 120 min. The proportion of metaphase stage nuclei in germlings reached a maximum of 15% at regular intervals of 60-70 min. Culture media was amended with hydroxyurea to attempt to synchronize mitosis and increase the proportion of metaphase nuclei, but only a marginal increase was observed. Cells within the germling contained an average of seven nuclei and the nuclei within each cell appeared to be at the same mitotic stage. These observations were not significantly different between the four isolates studied. Nine chromosomes were observed for each isolate after making a minimum of 20 chromosome counts. Observations on chromosome size were recorded. This karyotype analysis solidifies the only previous estimation of chromosome number in P. teres, made using PFGE. In that study, only six bands could be resolved, but densitometric analysis of the larger, unresolved bands led to a chromosome number estimate of nine. This study shows the power of the GTBM to accurately determine karyotypes in fungi.
Beattie, A.D.*, G.J. Scoles, B.G. Rossnagel
*Corresponding author: adb164@duke.usask.ca
University of Saskatchewan/Crop Development Center, 51 Campus Drive, Saskatoon, SK S7N 5A8
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A molecular linkage map of Pyrenophora teres
Genetic linkage maps provide basic information about a species’ genome organisation and are important tools required for positional cloning of genes. The rice blast pathogen, Magnaportha grisea, provides a good example of how a linkage map of this fungus has allowed map-based cloning of several avirulence genes. The goal of this project was to construct a linkage map of the barley net blotch pathogen, Pyrenophora teres, that will support future mapping studies.
A mapping population of 80 single ascospore progeny were isolated from a cross between parental isolates WRS 1906 x WRS 1607. The parents were screened with 144 AFLP primer combinations from which 23 primer pairs were selected to screen across the entire population. Because WRS 1906 is avirulent on the barley variety 'Heartland' while WRS 1607 is highly virulent, the population was also evaluated for virulence on Heartland and segregated 38 avirulent: 42 virulent (X2= .2, P= .70). This suggested a single gene controlled the avirulent phenotype and this locus was placed on the linkage map. Finally, the mating type (MAT1/MAT2) locus was mapped on the population using a set of PCR primers specific to this locus. The map consists of 110 unique loci distributed over 18 linkage groups. Only eight (7.3%) markers displayed a Mendelian segregation ratio different from 1:1. The total map length is approximately 650 cM.
The present chromosome number estimate for P. teres is nine based on pulsed field gel electrophoresis (PFGE) and cytological observations. This clearly indicates that many of the linkage groups represent common chromosomes. Currently markers from each linkage group are being hybridized to PFGE-separated chromosomes in order to assign linkage groups to specific chromosomes. Mapping of the chromosome telomeres has also been initiated to determine how fully the current map represents the genome and to better define the region around the avr locus near the terminus of linkage group 6.
Beattie, A.D.*, G.J. Scoles, B.G. Rossnagel
*Corresponding author: adb164@duke.usask.ca
University of Saskatchewan/Crop Development Center, 51 Campus Drive, Saskatoon, SK S7N 5A8
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The barley stem rust resistance gene Rpg5 encodes NBS-LRR and protein kinase domains in a single gene
The biotrophic fungus Puccinia graminis causes stem rust of barley. Several major genes for resistance (i.e. Rpg1-Rpg5) have been described in barley. To understand the molecular basis of stem rust resistance, we have focused on the isolation and characterization of the genes involved in the incompatible interactions between barley and Puccinia graminis.
The Rpg1 gene was recently cloned and predicted to code for a receptor-like kinase with dual kinase domains (Brueggeman et. al., PNAS 99:9328, ’02), representing a novel class of plant disease resistance genes. A genetic locus believed to contain the barley stem rust resistance genes Rpg5 and rpg4 was delimited genetically to two BAC clones and completely sequenced. The Rpg5 locus confers resistance to the rye stem rust pathogen Puccinia graminis f. sp. secalis, isolate 92-MN-90 and the rpg4 locus confers resistance to P. graminis f. sp. tritici pathotype, Pgt-QCC. Annotation of the BAC sequences revealed several candidate resistance genes. Allele sequencing from resistant and susceptible cultivars and recombinant lines resulted in a single candidate Rpg5 gene. The Rpg5 gene was confirmed by allele sequencing and it also appears to be required for rpg4-mediated resistance. This was indicated by the presence of recombinants resistant to isolate 92-MN-90 (Rpg5), but not to QCC (rpg4). Recombinants resistant to QCC, but susceptible to 92-MN-90 were never isolated among over 5,000 gametes examined. In-silico translation of the Rpg5 sequence from the resistant line Q21861 revealed a protein containing NBS-LRR and protein kinase domains, all in one gene. Several cases are known in the literature where an NBS-LRR gene and a protein kinase gene are required for resistance to a pathogen, but this is the first case where all three domains are encoded by a single gene. Further validation of the gene is underway using a viral induced gene silencing approach as well as complementation by Agrobacterium mediated transformation. Characterization and validation of this gene will be presented and possible mechanism of resistance discussed.
Brueggeman, R. (1)*, T. Drader (1), A. Druka (2), T. Cavileer (3), B. Steffenson (4), J. Nirmala (1), H. Bennypaul (1), K. Gill (1) and A. Kleinhofs (1,5)
*Corresponding author Robert Brueggeman: bigbass@wsu.edu
(1) Washington State University, Crop and Soil Science, Pullman, WA 99163, USA
(2) Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, Scotland, UK
(3) University of Idaho, Dept. of Biological Sciences, Moscow, ID 83844, USA
(4) University of Minnesota, Department of Plant Pathology, St. Paul, MN 55108-6030, USA
(5) Washington State University, School of Molecular Biosciences, Pullman, WA 99163, USA
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In vitro selection for pre-screening barley for resistance to Fusarium head blight
Cultivation of susceptible wheat and barley cultivars has resulted in FHB epidemics in the Midwest USA and Manitoba. The use of barley cultivars with genetic resistance to FHB is the most cost effective and environmentally sound means to manage this disease. However, the limited occurrence of FHB caused by Fusarium graminearum Schwable in Alberta, especially in the central and northern regions, requires developing methodology to screen resistance under controlled conditions. Barley genotypes (Hordeum vulgare L.) with known field reactions to fusarium head blight (FHB) were used for in vitro ground grain and leaf detached assays. In the ground gain assay, the FHB reaction of the genotypes was measured based on the extent of mycelial growth of F. graminearum (PW027) on a mixture of agar and ground grain at room temperature (23±1oC). In addition, the deoxynivalenol (DON) level in a mixture of F. graminearum biomass and ground grain of each line was determined using an ELISA-based assay. Fusarium graminearum showed larger colony diameters (mm) for susceptible ‘AC Lacombe’ and ‘Stander’ compared with resistant genotypes, ‘Chevron’ H94051001, I92130, and H93120, except for resistant CI4196. Fusarium graminearum produced more DON on susceptible ‘AC Lacombe’ compared to resistant ‘Chevron’, I92130, and H93120, except for resistant CI4196 and H94051001. Larger colony diameters and higher DON content were also observed for susceptible genotypes compared with resistant ones in a repeated test with another 16 barley genotypes. Detached leaves of six barley genotypes were inoculated with single isolates of F. graminearum (PW027) and F. culmorum (PW0T6) for evaluation of partial disease resistant (PDR) components at 10oC and 23±1oC, and DON content at 23±1oC. Both isolates were pathogenic at 23±1oC with shorter incubation period and greater lesion sizes on detached leaves compared to 10oC. PW027 was more virulent than PW0T6 at both temperatures resulting in shorter incubation and larger lesions for all barley genotypes but not for spore production. Fusarium graminearum appeared to better differentiate between resistant and susceptible barley genotypes at 23±1oC compared with F. culmorum. However, there was no clear pattern in DON content between resistant/tolerant and susceptible genotype. The results of in vitro evaluations tended to agree with previous field reactions in terms of FHB reaction and DON level of the genotypes. Thus, both in vitro assays may be alternate selection methodologies for FHB resistance. The detached leaf assay has the advantage of measuring specific disease components, allowing elucidation of the potential nature and genetic components of resistance to FHB operating for this assay. The potential of screening barley embryos of different genotypes against DON as a method of identifying resistant genotypes was also evaluated. In a preliminary test, 20 days old embryos of susceptible ‘AC Lacombe’ and resistant H94051001 were evaluated in Murashige Skoog media amended with different concentrations of DON. DON was found to considerably reduce the embryo weight of ‘AC Lacombe’ compared with that of H94051001. Further embryo experiments are being carried out, with screening of more barley genotypes against different DON concentrations.
Kumar, K. (1), Xi, K. (1), Helm, J.H. (2), Turkington, T.K. (3), and Jennifer Zantinge (1)
(1) Field Crop Development Centre, Alberta Agriculture, Food and Rural Development (AAFRD), 6000 C & E Trail, Lacombe, AB T4L 1W1
(2) Field Crop Development Centre, AAFRD, 5030-50 Street, Lacombe, AB T4L 1W8
(3) Lacombe Research Centre, Agriculture and Agri-Food Canada, 6000 C & E Trail, Lacombe, AB T4L 1W1
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Diversification strategies for barley disease management in Alberta
Barley is an important feed grain crop in Alberta that accounts for 40-50% of Canadian production. To ensure a constant supply of feed many farmers often grow barley continuously for several years; however, continuous barley production leads to a build-up of plant diseases and a general reduction in yield potential over the long-term. A three-year experiment was established at Lacombe in 1998 to determine if barley cultivar rotation could be used to reduce the impact of leaf diseases, while maintaining crop productivity. Treatments consisted of various sequences of two cultivars with varying degrees of scald and net blotch resistance, ‘Kasota’, and ‘AC Lacombe’; a previously scald-resistant cultivar ‘CDC Earl’; a susceptible check, ‘Harrington’; and a non-host, triticale cultivar ‘Wapiti’. Rotations were established in 1998 with comparisons being made in 1999 and 2000. In 1999, significant rotation differences occurred for scald and net blotch severity, with disease severity usually highest and yield lowest when a barley cultivar was grown on its own residue, especially for cultivars other than ‘Kasota’. Statistical analysis using contrasts indicated that yield and kernel weight were lower, while scald and net blotch levels were higher for barley cultivar monoculture compared with barley cultivar rotation. In 2000, when a barley cultivar was grown on its own residue, scald severity was usually highest compared to barley cultivar rotation. A similar trend was also observed for net blotch, especially for ‘Harrington’ and ‘AC Lacombe’. Poor stand establishment in some plots precluded the detection of yield differences among some treatments in 2000. Contrasts for 2000 indicated that higher levels of scald and net blotch, and decreased kernel and test weights occurred for barley cultivar monoculture compared to barley cultivar rotation. In both 1999 and 2000, barley planted on triticale residue generally had the highest yield, kernel weight and test weight, while having the lowest disease levels compared to planting barley on barley residue. Barley cultivar rotation can be a potential short-term strategy to help reduce leaf disease levels and maintain crop productivity for Alberta barley producers where crop rotation options are limited due to feed requirements or market factors. However, continuous production of barley, even utilizing cultivar rotation, will not provide long-term sustainable leaf disease management, especially for scald. The summer of 2004 was the third year of a separate trial that was established to look the interactive effects of agronomic factors such as rotational diversity, seeding rate, and time of silage removal on crop health, competitiveness, disease levels, productivity and quality in a cereal silage production system. In 2004, all plots except for the continuous triticale were seeded to the barley cultivar ‘Seebe’. The rotation treatments of ‘CDC Helgason’ barley/’Pronghorn’ triticale/’Seebe’ and ‘Pronghorn’/’AC Mustang’ oats/’Seebe’ had significantly greater emergence compared to the continuous ‘Seebe’ treatment, which had the lowest emergence. The remaining rotation treatments had intermediate emergence and were not significantly different from the continuous ‘Seebe’ rotation. Overall, silage yield on a dry weight basis was highest for the ‘Pronghorn’/’AC Mustang’/’Seebe’ rotation (8060 kg/ha), intermediate for the ‘CDC Helgason’/’AC Mustang’/’Seebe’ rotation (7822 kg/ha) and lowest for the remaining rotations (7005-7279 kg/ha, LSD = 400 kg/ha). The spot-form of net blotch was the main leaf disease present and it was highest for the continuous ‘Seebe’ rotation (13.8% leaf area diseased on the flag -2 leaf) and lowest for the ‘Pronghorn’/’AC Mustang’/’Seebe’ rotation (5.9%) with the other rotations having intermediate disease levels (8.2-11.7%, LSD = 2.1). Rotation had a significant influence on root mass assessed in the fall. Root mass was highest for the ‘Pronghorn’/’AC Mustang’/’Seebe’ rotation (42.4 g/2 m length of row), lowest for the continuous ‘Seebe’ rotation (29.1 g), and intermediate for the remaining rotation treatments (34.4-37.0 g, LSD=8.2 g). Rotation treatments had an impact on silage yield and this appeared to be related to crop health as indicated by leaf disease levels and root mass measurements. A second 3-year cycle of this trial is being repeated starting in 2005.
Turkington, T.K.(1)*, K. Xi (2), G.W. Clayton (1), K.N. Harker (1), J.G. O’Donovan (1), and N. Lupwayi (1)
*Corresponding author: turkingtonk@agr.gc.ca
(1) Lacombe Research Centre/Beaverlodge Research Farm, Agriculture and Agri-Food Canada, Lacombe, AB, T4L 1W1;
(2) Field Crop Development Centre, Alberta Agriculture, Food and Rural Development, c/o Lacombe Research Centre, Agriculture and Agri-Food Canada, Lacombe, AB, T4L 1W1
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Resistance of western Canadian barley genotypes to scald in Alberta
Barley (Hordeum vulgare L.) production in Alberta has averaged around 6 million metric tonnes from 2 million hectares annually from 1995 to 1999, and accounted for close to 50% of the total barley production in western Canada. Scald caused by Rhynchosporium secalis J. J. Davis is one of the major foliar diseases causing significant yield loss in Alberta as a result of intensive barley production, and cool and wet environmental conditions that favor disease development. In the current study, nine barley differentials were grown up in hill plots at multiple sites to monitor scald development during 1997 – 1999 (Period 1), twelve during 2000 – 2001 (Period 2), and twelve during 2003 – 2004 (Period 3). Thirty-eight genotypes and commercial cultivars with different levels of resistance were also evaluated for scald reaction from 2003 to 2004 at these sites. Differentials Abyssinian, Atlas, Atlas 46, Atlas 68, Hudson, Osiris, Kitchin and Turk were resistant to scald at all sites, other differentials including Brier, La Mestita, Modoc and Trebi showed intermediate levels of scald, indicating that the majority of major genes for scald resistance are effective against R. secalis pathotypes in Alberta. The differentials showed highly significant correlations in scald severity among all three periods. Period 1 and Period 2 (R2=0.86**) were, however, more closely correlated, compared with Period 1 and Period 3 (R2=0.63**), suggesting the development of considerable variability in R. secalis in response to major genes for resistance during 1997 to 2004 in Alberta. Analysis from ten station-years of data classified thirty-eight genotypes and cultivars into three major clusters corresponding to scald reaction: resistant, intermediate and susceptible. Those in Cluster I that were resistant included AC Stacey, Kasota, Mahigan, Manny, Niobe, Seebe and five other genotypes from the Field Crop Development Centre. Eighteen commercial cultivars in Cluster II were intermediate in scald reaction and these included AC Lacombe, CDC Dolly, CDC Earl, CDC Guardian, CDC Kendall, Falcon, Nisku and Peregrine. The three susceptible barley cultivars including Harrington were classified into Cluster III with several malting cultivars including AC Metcalfe, CDC Sisler and Excel, and three feed barleys, AC Bacon, AC Rosser and CDC Candle. The number of cultivars that were classified to be resistant was up from previous studies. A substantial number of feed cultivars were classified to be intermediate in resistance in the present study due partly to the overall moderate level of scald severity during 2003 – 2004 in Alberta. Changes in R. secalis pathotypes and conducive weather conditions may increase severity, resulting in breaking down of resistance, as demonstrated in previous study. Given the fact that no malting cultivar was resistant and only one was classified as intermediate in scald reaction, there is a need to incorporate scald resistance genes into malting cultivars for production in Alberta.
K. Xi (1), T.K. Turkington (2) and C. Bos (1)
(1) Field Crop Development Centre, Alberta Agriculture, Food and Rural Development, c/o Lacombe Research Centre, 6000 C&E Trail, Lacombe, AB Canada T4L 1W1
(2) Agriculture and Agri-Food Canada, Lacombe Research Centre, 6000 C & E Trail, Lacombe, AB Canada T4L 1W1
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Mapping and molecular marker development of scald resistance in ‘Seebe’ barley
Scald (Rhynchosporium secalis) of barley is prevalent in central Alberta, Canada and causes considerable yield and quality losses. Scald can rapidly change in pathotype composition and frequency, thereby making it difficult to develop durable scald resistance in barley. Previous studies have shown that the cultivar ‘Seebe’ carries durable genetic resistance, however, barley breeders have found this trait difficult to transfer into new barley lines. Therefore, we are trying to develop molecular markers for scald resistance from ‘Seebe’. Recombinant inbred lines were developed from the cross of ‘Harrington’ (scald susceptible) and ‘Seebe’ (scald resistant). Progeny of about 175 individual F2 seedlings were advanced by single-seed descent to the F8 generation. Disease resistance to a major scald race was phenotyped at the seedling stage in a greenhouse. By utilizing bulked segregant analysis (BSA), resistant and susceptible pooled populations were compared by AFLP analysis. A total of 255 AFLP primer combinations were used to analyze the genetic population and several EcoRI-MseI and PstI-MseI fragments were found linked to scald disease resistance. We are also analyzing this population with SSR markers. Our goal is to map and identify molecular markers flanking the genes contributing to scald resistance in ‘Seebe’.
Key words: scald resistance, marker development, Hordeum vulgare, barley
Zantinge, J.L.*, J.H. Helm, Z. Hartman, J.B. Russell, and K. Xi
*Corresponding author: jennifer.zantinge@gov.ab.ca
Field Crop Development Centre, Alberta Agriculture, Food and Rural Development, 5030 – 50th Street, Lacombe, AB T4L 1W8, Canada
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Breeding for multiple disease and multiple gene resistance in barley
Combining genes for disease resistance is very difficult, as most breeding programs can only test for the diseases present at their breeding sites. In addition, pyramiding genes or developing multiple gene resistance is difficult to detect when testing at only one location. Multiple disease and gene resistance involves breeding against more than one pathogen and more than one gene per pathogen. Each pathogen may have several races that are able to attack varieties and render a resistant variety ineffective in a short period of time, presenting a significant challenge to plant breeders.
Over the years the ICARDA/CIMMYT barley program in Mexico has given us an excellent opportunity to screen for multiple gene resistance for Scald in barley and at the same time look at multiple disease resistance to Stripe Rust, Barley Yellow Dwarf Virus, Leaf Rust, and Fusarium Head Blight (FHB). In Canada not only have we screened for Scald, but have also screened for Loose Smut and Covered Smuts as well as Net Blotch (net and spot forms), Spot Blotch, and FHB. Over the last 5 years we have screened over 2000 breeding lines at 4 locations in Canada and 3 locations in Mexico. New combinations of resistance genes have been found with some lines containing genes for resistance to 5 and 6 diseases. We found multiple gene combinations for scald resistance that have 3 or more genes and should give durable resistance to this disease in both countries. In order to classify breeding lines according to resistance gene combinations, we are currently analyzing overall similarity computed from multivariate disease resistance data and matching it to the pedigree.
The best lines will be used in the breeding program in order to rapidly incorporate even greater disease resistance into new varieties for Alberta producers. We will also develop several populations to begin the process of mapping on as many of these genes as possible. Continuation of this research is necessary to anticipate and cope with the changes in disease problems likely to occur in the future. Up to this point in time, stripe rust has not been a problem in Alberta on barley; however, in 2004 this disease was found on barley at Olds, Trochu, Calmar and Lacombe. If this disease continues its move north it will be devastating to Alberta’s barley crop. FHB also is not presently a problem in Alberta but seems to be moving west. FHB has cost the barley industry millions of dollars in the Midwest in the United States and in Manitoba in Canada.
Helm, J.H. (1), H. Vivar (2), F. Capettini (2), K. Xi (1), P. Juskiw (1), and J. Zantinge (1)
(1) Alberta Agriculture, Food and Rural Development; Field Crop Development Centre, Lacombe, Alberta
(2) ICARDA/ CIMMYT, Texcoco, Mexico
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Variation in virulence among net blotch isolates infecting barley
Pyrenophera teres, causal agent of net blotch of barley, is widely distributed throughout North American production areas. Previously, researchers have shown variation in virulence of isolates within and between locations. The purpose of this research was to continue monitoring diversity of isolates in locations of interest to the BARI barley breeding program. Isolates were collected randomly from symptomatic plants from 2000 to 2004. A set of 25 differential varieties was developed based on work of other researchers. Differentials were planted in Rootrainers containing Metromix 200 and grown in the greenhouse. Isolates were grown on V8 Juice agar at 18C with 12 hr light per day for 7 days. Plants were inoculated at the 2-leaf stage with a suspension of a single isolate, incubated in the dark at 18C and 100% RH for 24 hr, and returned to the greenhouse. Plants were evaluated for disease on a 0 (R) to 9 (S) scale 7-10 days later. To date, 16 isolates have been characterized on 25 differential varieties. Isolates ranged from the least virulent (PT01/6 from Bottineau, ND), infecting 5 differentials, to the most virulent (PT04/14 from Minot, ND), infecting 20. Of these 25 differentials, only 2 (9839 and CIHO 5822) were resistant to all isolates and 3 (Alexis, Bonanza and Klages) were susceptible to all isolates. There was no correlation of virulence to year or location. This study will continue in order to assist breeding programs developing resistance to net blotch.
Linnea G Skoglund
Corresponding author: linnea.skoglund@anheuser-busch.com
Busch Agricultural Resources, Inc. 3515 Richards Lake Road, Fort Collins, CO 80525
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Assessment of artificial inoculation methods and deoxynivalenol levels in barley lines representing various candidate sources of resistance to Fusarium head blight
Fusarium Head Blight (FHB) is one of the most devastating diseases affecting the production of barley and other cereal grains throughout Canada and the world. FHB infection results in drastic decreases in crop yield and severe reduction in grain quality. The most common species causing FHB in North America is Fusarium graminearum (Schwab). The fungus produces mycelical extensions that rapidly spread among the florets, leaving shrivelled kernels and floral pieces covered with a pink or white film of mycelium displaying elevated deoxynivalenol (DON) levels. 19 barley lines representing FHB candidate resistance and susceptible sources were point inoculated or spray inoculated in the greenhouse with 40,000M/ml of F. graminearum macroconidia. Following inoculation barley plants were kept at 24°C and 95% humidity for 3 days to favour disease establishment and were then returned to normal growing conditions at 21°C without humidity control (45%). 18 days post-inoculation heads were collected and spikelets displaying symptoms of FHB infection were rated for progression of the disease. These lines were also evaluated in the Brandon, MB nursery from 2000-2004. The number of discoloured spikelets produced by point inoculation, indoor spray inoculation, and disease and DON assessment were compared for each of the 19 lines. Barley lines representing candidate sources of FHB resistance or susceptible FHB sources showed varying degrees of symptoms due to fungal infection in the three tests. Barley lines representing more or intermediate FHB resistant sources showed consistent levels of resistance in the three tests. Artificial inoculation methods and DON quantification enable us to rate the level of resistance of the barley lines, with confidence. A higher level of FHB resistance will guarantee lower risks for the farmer associated with crop losses due to reduced grain yield, low quality grain, and DON contamination.
Geddes, J. (1), Eudes, F. (1), Legge, B. (2), Tucker, J. (2)
(1) Agriculture and Agri-Food Canada, Lethbridge, AB T1J 4B1
(2) Agriculture and Agri-Food Canada, Brandon, MB R7A 5Y3
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Reactions of barley lines to leaf rust, caused by Puccinia hordei
Resistant barley cultivars are the most economical and efficient means of controlling leaf rust caused by Puccinia hordei G. Otth. However, changing virulence in P. hordei has rendered ineffective many of the known resistance genes. In this study, isolates ‘Race 8’, ‘90-3’, ’90-15’, ‘89-3’, and ‘Neth 202’ of P. hordei were used in the greenhouse to differentiate resistance genes in a collection of 82 selected barley lines. These lines exhibited resistance in the previous tests against isolate ‘Race 8’ at the seedling stage. Barley line ‘C2-02-134-2-2’ exhibited low reaction types to all the tested leaf rust isolates, suggesting that in addition to an already introduced resistance gene, Rph15, it possesses one or more new resistance genes. The F2 population of a cross made between ‘C2-02-134-2-2’ and a susceptible line ‘ZA47’ which was challenged with isolate ‘Race 8’, segregated into a 15:1 resistant to susceptible ratio (X2=0.853) based on disease reaction. The 15:1 segregation ratio indicates that ‘C2-02-134-2-2’ possesses two genes, Rph15 and a new single dominant resistance gene. In the F2, the Rph15 phenotype (00;) was separated from the new resistance gene phenotype (0;12-). To isolate the new gene from Rph15, the 10 F2 plants bearing the new phenotype were transplanted and selfed and the F3 will be screened for homogeneity of disease reaction. The identification of the new resistance gene(s) and incorporation of them into barley cultivars will add in reducing yield losses due to leaf rust.
Y. Sun (1), J. D. Franckowiak (2), and, S. M. Neate (1)
(1) Department of Plant Pathology, North Dakota State University, Fargo, ND 58105
(2) Department of Plant Sciences, North Dakota State University, Fargo, ND 58105 USA
The above posters were presented at the 18th North American Barley Researchers Workshop, July 17-20, 2005, Red Deer, Alberta |
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