Annual Progress Report on Malting Barley Research March, 2007

<strong>Annual</strong> <strong>Progress</strong> <strong>Report</strong>
on
Malting Barley Research
March, 2007
Information presented in this report is
confidential and not for publication,
citation or reproduction in any form.
American Malting Barley Association, Inc.
740 N. Plankinton Ave., #830; Milwaukee, Wisconsin 53203; (414) 272-4640; http://www. AMBAinc.org

OFFICERS AND STAFF
Chairman of the Board ............... DAVID S. RYDER, Miller Brewing Co.
Vice Chairman ........................... DOUGLAS E. EDEN, Cargill Malt
Secretary/Treasurer .................... DALE WEST, International Malting Co. - US
President ..................................... MICHAEL P. DAVIS, AMBA
VP & Technical Director ........... SCOTT E. HEISEL, AMBA
Administrative Assistant ............ JOANN M. WALLDREN, AMBA
BOARD OF DIRECTORS
DAVID S. RYDER, Miller Brewing Co. (Chairman)
LARRY BELL, Bell’s Brewery, Inc.
DOUGLAS E. EDEN, Cargill Malt
KEN R. GROSSMAN, Sierra Nevada Brewing Co.
ROBERT S. MICHELETTI, Rahr Malting Co.
GORDON LANE, Briess Malt & Ingredients Co.
JOHN SERBIA, Anheuser-Busch, Inc.
DALE WEST, International Malting Co. - United States
TECHNICAL COMMITTEE
MICHAEL P. DAVIS (Chairman), American Malting Barley Association, Inc.
ALLEN BUDDE, USDA/ARS Cereal Crops Research Unit, WI
SUSAN B. KAY, Miller Brewing Co.
PAUL KRAMER, Rahr Malting Co.
DAVID R. KUSKE, Briess Malt & Ingredients Co.
JOHN MALLETT, Bell’s Brewery, Inc.
MARY-JANE MAURICE, International Malting Co. - United States
GIL W. SANCHEZ, Sierra Nevada Brewing Co.
JOHN RENZ, Cargill Malt
LESLIE J. WRIGHT, Anheuser-Busch, Inc.

-i-
INTRODUCTION
The March 2007 <strong>Annual</strong> <strong>Progress</strong> <strong>Report</strong> on Malting Barley Research was compiled from
reports submitted by barley researchers. The reports include research supported in whole or in
part by the American Malting Barley Association and other barley research conducted during the
2006 crop season and the winter of 2006/2007.
The American Malting Barley Association made grants totaling $399,900 to state and
federal research institutions for the support of malting barley research programs in eight states
during the 2006/2007 fiscal year. This substantial industry support augments state and federal
funds allocated for barley research.
The overall objective of the American Malting Barley Association research program is
the development of barley varieties that combine superior malting and brewing qualities with
superior agronomic characteristics. Attainment of this goal involves both basic and applied
research in breeding, cytogenetics, genetics, biochemistry, molecular biology, plant physiology,
plant pathology, and production. We gratefully acknowledge the cooperation and contributions
of both state and federal researchers and administrators in helping to achieve this objective.

ANNUAL PROGRESS REPORT ON MALTING BARLEY RESEARCH
March, 2007
TABLE OF CONTENTS
Page
INTRODUCTION ............................................................................................................... i
CALIFORNIA
UNIVERSITY OF CALIFORNIA – DAVIS
Two-rowed and Six-rowed Malting Barley Germplasm Development in California
L.W. Gallagher........................................................................................................... 6
IDAHO
USDA-ARS NATIONAL SMALL GRAINS GERMPLASM RESEARCH FACILITY
Development of Two and Six-row Spring and Winter Malting Barleys for the Intermountain
West: Variety and Germplasm Development.
D.E. Obert ................................................................................................................ 10
MINNESOTA
UNIVERSITY OF MINNESOTA
Minnesota Barley Improvement Project
K.P. Smith ................................................................................................................ 17
Management and Epidemiology of Barley Diseases
R. Dill-Macky, A. Elankkad and K. Wennberg ....................................................... 28
Investigations on Barley Diseases and Their Control
B.J. Steffenson ........................................................................................................... 33
MONTANA
MONTANA STATE UNIVERSITY
Developing Improved Malt Barley Varieties for Montana and the Western US
T.K. Blake ................................................................................................................ 43
Epidemiology and Control of Barley Leaf Diseases Caused by Fungal Pathogens
M.R. Johnston .......................................................................................................... 48
NORTH DAKOTA
NORTH DAKOTA STATE UNIVERSITY
Breeding and Genetics of Six-rowed Malting Barley
R.D. Horsley ............................................................................................................ 52
Studies on Barley Diseases and Their Control
S. Neate .................................................................................................................... 61
Malting and Brewing Quality of Barley
P.B. Schwarz ............................................................................................................ 74

USDA-ARS NORTHERN CROP SCIENCE LABORATORY
Net Blotch of Barley: Survey of Pathogen Virulence
T. L. Friesen............................................................................................................... 92
OKLAHOMA
USDA-ARS PLANT SCIENCE RESEARCH LABORATORY
Germplasm Enhancement for RWA Resistance
D.W. Mornhinweg, D.R. Porter, and G.J. Puterka .............................................. 97
OREGON
OREGON STATE UNIVERSITY
The Oregon Barley Improvement Program
P.M. Hayes, J. Kling, P. Szucs, A. Corey and T. Filichkin ....................................... 102
WISCONSIN
USDA-ARS CEREAL CROPS RESEARCH UNIT
Malting Quality Analysis of New Barley Selections
A.D. Budde, C. Martens and M.R. Schmitt ............................................................ 111
Fermentable Sugar Production by Barley Malts
S.H. Duke and C.A. Henson ..................................................................................... 115

Two-rowed and six-rowed malting barley germplasm
development in California.
Researcher: Lynn Gallagher, Department of Plant Sciences,
University of California, Davis, CA
Executive Summary
This project will help meet the goals of AMBA when breeding objectives are met
wherein a developmental pipeline is filled with six-rowed and two-rowed germplasm
having favorable alleles (genes) conferring resistances to several diseases combined
with superior malting characteristics in barley lines highly adapted to the Western
growing region. The foliar disease resistances being incorporated are primarily those
against Barley Yellow Dwarf Virus (BYDV), barley stripe rust, scald, net blotch and
Cereal Yellow Dwarf Virus (CYDV). Resistance to leaf rust and powdery mildew are
additional possibilities. Promising initial results for two-rowed barley are indicated for
advanced lines selected from crosses of an ICARDA/CIMMYT line (developed in
California) to Oregon St. material. These new lines will be available for distribution after
harvest in late May ‘07. Very few six-rowed malting barleys are sufficiently adapted to
growing conditions found in the Central Valley of California to be of breeding value. The
first six-rowed crosses for malting quality are in the F4 generation. Additionally sixrowed
Oregon St. winter barleys were crossed to a small number of spring barelys
from northern states to form base populations for improved advanced line creation.
Objectives, Methodology, and Results
The main objective is to combine multiple disease resistances, superior malting
characteristics, and high grain yield potential into barley lines adapted to the Western
growing region such that they may be used as a resource by other breeders. This
objective will be achieved by traditional breeding methods using a modified bulkpedigree
breeding scheme. Results are indicated in the paragraphs below.
Objectives met in one-year funding period
Promising advanced lines have been identified from the cross of the parents BCD 47,
BU, and SAL to Triumph/Tyra//Arupo “S” *2/Abyssinian (TTA), selection 119. BCD 47
was derived from the cross Orca/3/Harrington/ /CI10587/Galena. SAL was derived from
the cross BCD 47/3/CI10587/Galena/ /Baronesse. BU was derived from BCD 47
/3/CI10587/Galena// Baronesse ‘S’. These three lines were developed by Pat Hayes at
Oregon St. Univ. Hundreds of advanced lines from BCD 47 X TTA population were
selected in May and seeds planted at Davis in Nov. ’06 along with more lines from the
BU/TTA and SAL/TTA crosses. BCD 47 was the highest grain yielder in the Western
6

Spring Regional over two years at Davis and had near-malting quality. All of the parents
in these three crosses have short stature. The two-rowed effort is more advanced than
the six-rowed effort and the developmental pipeline is full of two-rowed material.
The Oregon St. Univ. six-rowed winter barley (STAB47/KAB51-20) was crossed into
the winter by spring gene pool to create new populations. In July ‘06 winter by spring
segregating populations in the F2 and F3 generations were sent to Pat Hayes for
selection in a second environment. STUC 6 continues to be the best six-rowed source
of BYDV and stripe rust resistance combined with near-malting quality. Segregating
populations of STUC 6 crossed to 6B96-9339, Drummond, and 94AB13449 are among
the F3 bulks. The project has continued to identify suitable malting barley parents and to
create new populations. New crosses to be made in April ’07 will use Tradition, Robust
‘RWA’ (Russian Wheat Aphid resistant) lines developed by the USDA, and 01NZ706,
which is ant643/WA9138-87//6B95-8253 supplied by Ditter vonWetstein and the highest
grain yielder in the Western Spring Regional barley trial grown at Davis in ‘06.
Segregating populations of Conrad crossed to ICARDA/CIMMYT lines were planted
as F3 bulks. Subsamples were sent previously to BARI for selection in a second
environment . Orca and ND22202 crosses to Mexican materials are in the F2
generation. Madre Selva barley was used as a source of resistance to CYDV. Materials
planted at Davis from other programs include the following: advanced lines from the
cross BCD 47 to Baronesse, BARI elite lines, and Tango X Lacey F3 lines. Twenty new
crosses for malting quality were made in April ’06. Subsequently the F1’s were grown
out at Ft. Collins by BuschAg. The harvested seeds were planted at Davis in Nov. ’06.
Reserve F1 seeds were planted at Davis in Nov. ’06. New two-rowed parents likely to
be used in crossing are Craft and 98AB11993, the latter of which was highly productive
in the Western Spring Regional barley trial grown at Davis. The most time consuming
part of the project is given to selection, harvesting, seed processing and preparation for
planting subsequent generations in the germplasm pipeline.
Most significant accomplishment
The project has created its first malting advanced line germplasm worthy of distribution
to other interested breeders (Table 1). Entries in bold type were created at UC Davis.
Crosses involving Oregon St. parents (described above) developed by Pat Hayes were
made to a selection from the ICARDA/CIMMYT cross of TTA, which was originally
planted at Davis in 1995 as a F2 population supplied by Hugo Vivar. The TTA
population was selected for its absence of foliar diseases, short stature, and
productivity. The TTA population and subsequent selections were resistant to all
diseases noted at Davis over several years during the selection process. BCD 47 like
TTA appeared resistant to stripe rust, scald and net blotch but BCD 47 was susceptible
to barley yellow dwarf virus and powdery mildew at Davis. Several lines from the TTA
population were evaluated by CCRU and one line was selected for crossing to BCD 47,
BU, and SAL. Three lines (BCD/TTA-MQ29, BCD/TTA-N, and BCD/TTA-V) had CCRU
quality scores above 60 compared to the highest CCRU Harrington check quality value
of 50. The lines having the MQ 28 and MQ29 designation have two years of favorable
7

data. Many additional lines had quality scores in the 50’s. Harrington is not grown at
Davis because its susceptibility to several diseases results in very poor malting quality
and low grain yields. Checks include the following: Orca, which has a good grain yield
at Davis, ND 22202 from No. Dakota, three lines from BARI designated by the 2B99
prefix, and two ICARDA/CIMMYT lines which are among the most recent donors of
multiple disease resistances. Grain yield data will be available by Sept. ‘07.
The project continues to identify six-rowed parents useful in creating new
populations. Agronomically these lines have several major defects that result in their
being poorly adapted to the Central Valley. Generally they have inadequate grain yield,
shatter badly, and are susceptible to several California barley diseases. Drummond,
94AB13449, and B98-9339 have already been crossed to STUC 6,derived from Stander
X UC 960, to create new segregating populations which are now in the F3 generation.
Additionally five crosses were made to Oregon St. winter lines using STUC 6 as a
parent.
Other barley research and future direction of program
The barley breeding program has been traditionally a feed barley program. The feed
barley portion of the program has decreased in size. The hulless barely “Tamalpais” ,
which was tested as UC 1134, will be released and commercialized in the Central
Valley. UC 1135, a sib of UC 1134, is exceptionally high in grain Beta-glucan (>7%).
Components of the barley program include the goals of feed, forage, human
consumption, and malting barley, the latter of which is becoming the dominant portion of
the breeding effort. Six-rowed parents which are likely to be used in the ’07 crossing
cycle include: Tradition from BARI, 94Ab13449 from Idaho, 01NZ706 from Washington,
STAB47/KAB51-20 from Oregon, and Robust-RWA (Russian Wheat Aphid) from
Minnesota via USDA, OK. New two-rowed parents will include the following: Craft and
98Ab11993 depending upon agronomic appearance in the field in April ’07. These six-
and two-rowed parents will be crossed to material having a broader range of
resistances to important diseases found in the Central Valley.
Project personnel None
Publication Gallagher, L.W., L.F. Jackson, and H.E. Vogt, Registration of ‘Ishi’
barley, Crop Sci. 46:1396 (2006).
8

Table 1. 2006 UCD Two-Rowed Malting Barley Selections - Davis, CA
Kernel on Barley Malt Wort Barley Wort DP Alpha- Beta- Qual- Over-
Weight 6/64" Color Extract Wort Clar- Protein Protein S/T (deg amylase glucan ity all
ENTRY (mg) (%) (Agtron) (%) Color ity (%) (%) (%) ASBC) (20°DU) (ppm) Score Rank
BCD/TTA-N 44.4 97.5 66 81.5 1.7 1 11.3 4.59 42.5 177 80.3 50 65 1
BCD/TTA-MQ29 41.9 91.0 66 81.2 1.6 1 12.5 4.86 41.4 183 111.1 52 64 2
BCD/TTA-V 45.3 96.6 57 79.5 1.5 1 11.6 4.66 41.5 144 80.5 77 62 3
SAL/TTA-MQ28 39.8 90.4 66 80.3 1.6 1 11.4 4.48 40.1 151 88.6 46 59 4
SAL/TTA-E 43.9 96.3 52 79.5 2.3 1 12.3 5.34 44.3 117 85.4 52 59 4
SAL/TTA-I 43.6 96.5 65 79.3 1.9 1 12.9 5.45 43.0 159 91.0 38 59 4
SAL/TTA-K 44.3 91.1 60 78.7 1.5 1 12.6 4.98 40.6 182 96.3 28 59 4
BCD/6/A… 44.9 94.5 60 78.3 1.7 1 12.5 4.92 40.1 126 84.1 58 59 4
BCD/TTA-W 40.9 88.6 72 80.9 1.6 1 11.6 4.86 42.9 143 106.2 59 59 4
BCD/TTA-C 40.7 92.7 61 80.2 1.8 1 11.3 4.70 43.0 119 78.2 32 58 10
BCD/TTA-J 38.4 89.4 63 79.9 1.5 1 11.6 4.52 41.3 143 94.5 82 57 11
SAL/TTA-L 41.3 88.8 54 78.5 1.9 1 12.7 5.07 40.3 178 101.0 66 56 12
BU/TTA-A 42.2 92.8 71 79.2 1.5 1 12.9 5.25 42.6 171 76.0 120 55 13
SAL/TTA-F 44.0 97.3 63 81.7 2.2 1 10.8 4.93 47.3 119 95.1 18 52 14
BCD/TTA-Q 41.5 92.4 71 80.4 1.5 1 10.3 4.24 42.4 131 84.8 83 52 14
BCD/TTA-R 42.5 93.6 72 82.4 2.2 1 9.7 4.44 47.2 118 106.2 42 52 14
BCD/TTA-I 38.6 81.6 64 79.3 1.6 1 11.1 4.49 41.9 134 74.6 42 51 17
BCD/TTA-U 41.3 94.2 70 79.2 1.6 1 11.6 4.51 39.3 150 86.5 122 49 18
BCD/TTA-D 42.0 82.4 65 79.3 2.0 1 11.2 5.06 46.6 103 67.3 111 47 19
BCD/TTA-T 44.9 95.5 69 81.0 1.4 1 9.9 3.97 43.5 117 81.5 17 47 19
2B99-2039 34.5 77.8 70 81.3 1.4 1 11.8 4.88 41.9 126 95.1 131 47 19
2B99-2763-10 34.8 76.1 70 79.3 2.3 1 12.8 4.80 38.9 144 77.4 173 47 19
ND22202 39.5 93.7 49 79.4 1.7 1 12.7 5.03 41.3 111 73.5 266 46 23
SAL/TTA-A 43.7 94.1 52 77.6 1.5 1 13.0 4.64 36.0 163 58.8 101 46 23
BCD/TTA-G 41.3 91.7 63 78.0 1.4 1 12.9 4.87 39.1 153 78.8 163 46 23
ORCA 49.8 98.6 47 77.9 1.6 1 13.1 4.66 36.4 149 67.5 41 45 26
SAL/TTA-D 43.9 95.0 55 78.0 1.7 1 13.0 4.83 38.4 129 70.5 83 45 26
BU/TTA-C 39.5 80.3 66 80.1 1.6 1 9.9 4.48 48.4 123 77.0 38 44 28
BCD/TTA-B 46.9 98.2 46 78.1 1.4 1 11.8 3.82 32.7 107 49.0 48 44 28
BCD/TTA-L 41.5 88.4 66 78.5 1.6 1 12.5 4.74 39.2 173 94.9 163 44 28
SAL/TTA-B 44.6 97.3 65 79.8 1.2 1 11.5 3.91 34.6 151 67.6 173 43 31
2B99-2771-1 34.1 84.2 75 80.7 2.2 1 11.9 4.64 40.6 122 82.8 260 43 31
BCD/TTA-H 37.5 89.5 68 81.1 1.5 1 9.3 4.11 47.8 105 75.5 36 41 33
30IB299 47.2 95.4 44 77.7 2.1 1 13.1 4.01 31.6 143 40.4 121 34 34
29IB20 49.9 97.2 44 77.8 1.8 1 12.2 3.83 32.7 79 49.4 321 29 35
Malt Check
HARRINGTON 37.5 86.1 78 81.1 2.5 1 12.5 5.77 47.0 91 73.6 189 40
HARRINGTON 38.6 86.2 78 81.5 1.6 1 12.6 5.49 45.0 115 86.1 170 50
Minima 34.1 76.1 44 77.6 1.2 9.3 3.82 31.6 79 40.4 17 29
Maxima 49.9 98.6 75 82.4 2.3 13.1 5.45 48.4 183 111.1 321 65
Means 42.1 91.4 62 79.6 1.7 11.8 4.65 40.9 138 80.8 95 50
Std Dev 3.7 6.0 9 1.3 0.3 1.1 0.42 4.1 25 16.1 74 8
Coeff Var 8.8 6.5 14 1.6 17.0 9.0 8.96 10.1 18 20.0 78 16
Malt Check Data are Excluded from Rank Sorting and Statistics
For Wort Clarity - 1 = clear, 2 = slightly hazy, 3 = hazy
9

Development of 2 and 6-row Spring and Winter Malting Barleys for the
Intermountain West: Variety and Germplasm Development.
Executive Summary
Major objectives and expected benefits:
DE Obert
USDA-ARS
National Small Grains Research Facility
Aberdeen, ID 83210
Our project mission is two-fold: 1) develop and provide to industry and producers
superior widely adapted malt barley cultivars, and 2) develop improved barley
germplasm which can be used by other public and private barley breeding programs. In
addition to variety development our work also provides valuable information to private
industry, other breeders, and barley producers via multiple location testing of lines from
other breeding programs. Therefore we assist in meeting the mission of AMBA by the
development of improved varieties, assisting other breeding programs in their effort to
release improved cultivars, and providing management advice to barley producers.
Our major objective is to develop malting barleys that can be grown over large and
diverse production areas to provide a uniform supply of barley for the malting industry.
This goal is being accomplished by testing all phases of our breeding lines at all
locations in Idaho that either currently grow malt barley or that have potential for
expanded malting barley production. In addition, we also test our elite spring lines in
Montana and our advanced and elite winter lines in Kansas, Oregon, and Washington.
Expected benefits include 1) more widely adapted cultivars to irrigated and rain-fed
areas of current production and 2) lines that expand the potential for malting barley
production into under utilized cropping systems, such as winter production in
environments currently used only for feed barley.
Objectives and Accomplishments in 2006:
A significant accomplishment was the production of 160 A of Charles, a winter tworowed
malt variety being grown for plant-scale evaluation. Sublette, a two-rowed spring
cultivar was grown on 250 A for plant-scale production but has been eliminated from
additional evaluation. In addition, 95Ab2299, a winter two-rowed type, was approved for
plant-scale evaluation and seed increase has begun for plant-scale evaluation
beginning the fall of 2009.
In addition, objectives from the 2006 grant proposal were met: 1) An additional year of
evaluation was provided for our elite materials and selection for very diverse
10

environments was practiced, 2) elite lines from Busch Agricultural Research (BARI)
were evaluated allowing us to provide valuable information to area producers, and 3)
testing locations were expanded to include a winter testing location in Hutchinson, KS.
This location allows us to gain knowledge concerning the winterhardiness and heat and
drought tolerance.
Before 2003 our winter lines were tested only at Aberdeen, ID. In 2004 we were also
able to evaluate at Parma, ID. In the fall of 2004 our winter lines were planted at
Aberdeen, Filer, and Parma, ID., Pendleton, OR, and Pullman, WA. In the fall of 2006
we also planted these trials at Colby and Hutchinson, KS. These expanded winter
testing locations provide a thorough representation of literally hundreds of thousands of
acres of highly productive winter small grain areas, giving us confidence we can
continue to release improved winter malting barley cultivars which can be grown on
large production areas. The additional locations in Kansas represent an area that is
currently planted exclusively to winter wheat. We, at this point, don’t propose that winter
malting barley will soon become common in these areas, but these experimental plots
should provide us some idea of the amount of heat and drought tolerance that exists in
our germplasm. With continuing water restrictions in Idaho this may become a more
pressing issue in the near future.
We evaluated six elite lines from BARI in exchange for their testing our elite malt test at
two additional locations: Idaho Falls, ID and Fairfield, MT. In the past our program has
focused almost exclusively on Idaho but with the expansion of testing our spring lines in
Montana, and our winter lines in Oregon and Washington, we are now able to test for
broader regional adaptation.
Objectives, Methodology, and Results:
As in previous years our major objective is to develop, advance, and release improved
malting barleys. The developmental phase of this included the development of 48 tworowed
and 181 six-rowed F2 segregating populations that were advanced over the
winter in New Zealand. In addition, 38 F3 populations, with superior malt potential, were
advanced in New Zealand. The remainder of the F3 and F4 populations were advanced
in bulk at Aberdeen in 2006. A single F6 spike from 50 F5 plants per population was
harvested and advanced via single seed descent to the F7 generation over the summer
in the greenhouse. A single plant was then grown over the winter and these will be
planted as a single 2-row yield plot in 2007. This will allow us to save two years in the
developmental process by 1) advancing the generation in the greenhouse and 2) begin
yield testing in 2007 as a two-row plot. Approximately 12,000 of these 2-row plots will be
tested then reduced to approximately selected 500 lines next season. In the summer of
2007 we will derive lines from F3 populations that were advanced in New Zealand this
winter and therefore begin yield trials in 2008 for crosses that were made in 2005-06.
11

The results from 2006 were somewhat variable. Our trials mirrored the mixed results
obtained by barley growers. Yields were not particularly good and test weight and plump
values were also poor at several locations. We had a favorable spring and plots were
planted in a timely manner. A very dramatic increase in temperatures resulted in the
loss of optimum yield, test weight, and plump kernels. Our elite trials included
Aberdeen, Ashton, Fenn (new site), Filer, Potlatch, Soda Springs, and Tetonia, ID. In
addition, in cooperation with BARI, at Idaho Falls, ID and Fairfield, MT. Preliminary and
advanced lines were evaluated at the majority of these locations except Ashton and
Idaho Falls, ID and Fairfield, MT, where only the elite malt was evaluated. The
Advanced Yield Malt trial was planted as Aberdeen, Fenn, File, Soda Springs, and
Tetonia. Aberdeen and Filer were irrigated. Results for selected two-rowed lines are
shown in Table 1 and six-rowed selections in Table 3. Please note that we had not yet
received any malt data from 2006 and therefore quality data for 2006 is not included in
the summaries presented in the following tables.
Table 1. Agronomic data for 2006 Advanced Yield Malt two-rowed selections.
Entry Yield (Bu/A) Test WT (#/Bu) Plump (%)
02Ab17060 97 51.7 85.7
02Ab17271 94 51.9 80.3
01Ab7163 90 51.6 82.7
02Ab17464 88 53.8 93.0
02Ab17344 86 51.3 91.3
02Ab17373 85 51.0 79.0
Merit 85 50.2 78.3
Harrington 83 52.6 90.0
Table 2. Selected two-rowed lines compared to Harrington
Entry Yld GrnPro Extrct WortPro S/T DP AA BG
Loc Years 11 4 4 4 4 4 4 4
02Ab17271 83 12.3 81.1 5.3 46.2 126 71 131
02Ab17373 78 12.7 80.7 5.5 45.4 139 86 170
02Ab17344 82 12.2 80.6 5.3 46.6 121 72 99
Harrington 75 12.4 80.5 5.1 44.0 134 76 196
Table 3. Agronomic data for 2006 Advanced Yield Malt six-rowed selections.
Entry Yield (Bu/A) Test WT (#/Bu) Plump (%)
01Ab9671 95 49.2 84.3
01Ab9730 92 48.0 77.0
01Ab7844 87 51.6 89.7
01Ab9573 85 48.8 91.7
01Ab9663 84 51.0 89.0
Legacy 84 51.3 82.3
Morex 70 50.7 84.0
12

Table 4. Selected six-rowed lines compared to Morex
Entry Yld GrnPro Extrct WortPro S/T DP AA BG
Loc
Years
16 9 9 9 9 9 9 9
01Ab7844 99 11.9 79.1 5.2 46.2 127 62.8 195
01Ab9573 96 10.9 80.1 5.2 51.3 127 61.7 224
01Ab9663 98 10.8 81.9 5.3 52.4 137 73.8 129
01Ab9671 106 10.9 81.0 5.2 50.8 124 81.6 122
01Ab9739 102 11.3 80.3 5.2 48.9 128 66.6 177
Morex 82 13.5 78.7 5.2 41.8 164 65.9 237
Results of the elite malt trials are presented in Tables 5-6.
Table 5. Performance of selected elite malt two-rowed lines compared to
Harrington.
Entry Yld Plmps GrnPro Extrct WortPro S/T DP AA BG
Loc Years 37 22 24 24 24 24 24 24 24
93Ab835 91 83.5 14.1 78.7 5.7 42.4 148 77.8 90
97Ab6643 91 84.9 13.8 79.4 5.3 40.3 147 60.5 190
Harrington 91 79.7 13.9 78.6 5.4 40.7 147 69.3 202
Loc Years 31 18 18 18 18 18 18 18 18
01Ab10055 96 79.1 14.0 79.0 5.81 44.3 153 71.9 138
01Ab10062 97 82.7 13.5 79.5 5.44 42.9 148 69.8 107
Harrington 95 82.1 13.5 79.1 5.46 42.7 145 69.5 198
Table 6. Performance of selected elite six-rowed lines compared to Morex.
Entry Yld Plmps GrnPro Extrct WortPro S/T DP AA BG
Loc Years 31 15 15 15 15 15 15 15 15
01Ab7841 103 77.0 11.3 78.4 4.8 46.0 114 59.1 176
92AB1368 98 80.7 11.6 79.4 5.1 46.8 121 67.1 262
Morex 82 70.4 13.1 78.4 4.9 39.4 165 55.6 204
Loc Years 38 14 14 14 14 14 14 14 14
98Ab12905 94 81.2 11.0 80.9 5.22 50.5 118 72.0 220
Morex 79 68.9 13.1 78.2 4.87 39.3 167 55.2 208
13

Our winter lines were evaluated at Aberdeen and Parma, ID, and Pendleton, OR, and
Pullman, WA. A severe hail storm at Aberdeen destroyed all plots and therefore we
collected no agronomic data in 2006. The quality data presented is from 2005 and
previous years.
Table 7. Performance of selected two-rowed lines in the Winter Advanced and
Elite Trials.
Entry Yld Plmps GrnPro Extrct WortPro S/T DP AA BG
Loc-Years 7 5 5 5 5 5 5 5 5
02Ab339 177 81.5 10.3 81.8 4.4 46.1 110 86.6 92
03Ab669 173 76.1 10.9 81.8 5.0 48.4 118 91.8 55
02Ab671 171 81.0 10.9 81.6 5.1 50.5 132 90.2 53
Charles 150 87.9 10.9 81.2 4.8 47.4 112 81.6 176
Loc-Years 6 5 5 5 5 5 5 5 5
95Ab2299 174 67.4 11.1 80.6 4.9 46.6 138 88.8 135
94Ab1219 173 82.1 11.3 78.6 4.5 41.7 133 63.2 300
00Ab14 172 77.9 11.7 79.6 4.6 40.7 156 73.0 280
94Ab1386 169 91.8 11.9 80.6 5.2 47.4 158 80.2 170
Charles 168 87.0 11.2 80.0 4.7 44.5 123 78.2 175
Table 8. Performance of selected six-rowed lines in the Winter Advanced and Elite
Trials.
Entry Yld Plmps GrnPro Extrct WortPro S/T DP AA BG
Loc-Years 8 5 5 5 5 5 5 5 5
00Ab280 173 46.4 11.3 80.2 4.8 44.5 127 59.2 134
00Ab363 157 85.6 10.3 79.8 3.9 40.1 118 50.8 319
88Ab536B 157 65.2 11.3 78.8 4.4 42.3 136 60.0 217
Loc-Years 9 6 6 6 5 6 6 5 6
93AB669 187 56.7 10.2 78.5 4.5 41.5 131 60.0 259
94Ab1777 181 71.4 9.7 79.7 4.6 46.5 108 50.4 449
88Ab536B 158 69.3 10.4 78.6 4.4 41.9 143 60.0 221
Loc-Years 5 5 5 5 5 5 5 5 5
01Ab273 166 65.8 10.5 80.4 4.5 46.0 127 68.0 118
02Ab201 179 69.2 10.9 79.0 4.4 42.7 133 62.6 344
02Ab2320 183 66.0 11.3 78.4 5.0 47.2 138 71.4 320
02Ab2472 182 74.2 10.9 80.0 4.9 47.1 117 80.0 238
88Ab536B 162 68.1 11.5 78.8 4.8 43.4 145 64.4 211
14

Other Barley Research:
Feed Program: The total number of crosses made for the barley breeding program in
2006 was approximately 500. Of these, 350 were malt types. Thus the remaining
crosses were feed and specialty types for high beta-glucan and low phytic acid. The
non-malt part of the breeding program is tested in early generations in conjunction with
the malt types, and only after line derivation are they separated. Feed types are tested
at three fewer locations than are malt types and feed populations are typically not
advanced via winter nurseries.
Mapping studies:
We are advancing several mapping populations: one for high beta-glucan and four for
high diastatic power. The high beta-glucan population (F7:9) will be evaluated for QTL in
2008 at three locations. This is a collaborative effort with Dr Eric Jackson at Aberdeen.
The high diastatic power populations are currently at the F3 stage and are being
advanced via single seed descent in the greenhouse. The high DP work is a
collaborative effort with Eric Jackson, Al Budde, Mark Schmidt, and Cynthia Henson.
Stripe Rust effort:
We are collaborating with Dr Pat Hayes (and others) to introgress QTL for stripe rust
resistance from the i-Bison lines into our breeding program using marker assisted
selection. This research is in the beginning phase as we have recently developed F1
populations between the stripe rust donor and our elite breeding lines. In addition to the
mapping effort per se, we plan to incorporate these sources of resistance into our best
winter (Charles) and spring (Sublette) malting lines.
CAP grant:
We evaluated our 96 CAP breeding lines (spring types) for phenotypic characteristics
and malt quality parameters at three locations in 2006 (Aberdeen, Filer, and Fenn for
six-rowed types, and Aberdeen, Filer, and Soda Springs for two-rowed types). In
addition, our program also has 96 winter lines being evaluated as Pat Hayes has
chosen to allot his portion of the breeding lines to examine our winter breeding lines.
Future Program Direction:
We envision that our program will continue to expand as far as the number of
populations and derived lines evaluated with a very large expansion first beginning in
2008 as we go from 12,000 to over 20,000 lines being evaluated per year. We
anticipate that malting barley production will continue to grow in Idaho and Montana and
we plan to release cultivars to meet the market demand. A major source of new barley
could be winter types and we will have a full program in 2008-09. Our future allotment
of resources will continue to provide more resources for our winter program as it
continues to grow. Our breeding program will continue to focus primarily on malt types
versus feed types because malting barley is critical to Idaho and Montana growers.
15

Project Personnel: Don Obert, Dave Burrup, Chris Evans, Karla Reynolds, Kathy
Satterfield
Recent Publications: (2005-06):
Obert, DE, DM Wesenberg, DE Burrup, BL Jones, and CA Erickson. 2006. Registration
of ‘Charles’ Barley. Crop Sci. 46:468-469.
Obert, DE, DM Wesenberg, DE Burrup, CA Erickson, JC Whitmore, and BL Jones.
2006. Registration of ‘Sublette’ Barley. Crop Sci. 46:989-991.
J. Michael Bonman, Harold E. Bockleman, Blair J. Goates, Don E. Obert, Patrick E.
McGuire, Calvin O. Qualset, and Robert J. Hijmans. 2006. Geographic distribution of
common and dwarf bunt resistance in landraces of Triticum aestivum subsp. aestivum.
Crop Sci. 46:1622-1629.
CA Erickson, D Obert, DE Burrup, JC Whitmore, and DM Wesenberg. 2006.
Registration of ‘Creel’ Barley. Crop Sci. 46:1812-1813.
DW Mornhinweg, DE Obert, DM Wesenberg, CA Erickson, and DR Porter. 2006.
Registration of seven winter feed barley germplasm lines resistant to Russian wheat
aphid. Crop Sci 46:1826-1827.
Hoffman, DL, J Chong, EW Jackson, and DE Obert. 2006. Characterization and
mapping of a crown rust gene complex (Pc58) in TAM O-301. Crop Sci. 46:2630-2635.
EW Jackson, DE Obert, M Menz, G Hu, JB Avant, J Chong, and JM Bonman.
Characterization and Mapping Oat Crown Rust Resistance Using Three Assessment
Methods. Phytopathology (Accepted).
P Bregitzer, V Raboy, DE. Obert, J Windes, and JC Whitmore. Registration of ‘Herald’
barley. Crop Sci. (Accepted).
An Hang, Don Obert, Ann Inez N. Gironella and Charlotte S. Burton. Evaluation of
Amylose Starch and β-glucan in Barley: Their Relationships to Protein Content,
Agronomic traits, and Environmental Factors. Crop Sci. (Accepted).
P Bregitzer and DE Obert. Registration of 95SR316A barley germplasm. 2006. Crop
Sci: Submitted.
16

Executive Summary
AMBA <strong>Progress</strong> <strong>Report</strong> – 3/09/07
MINNESOTA BARLEY IMPROVEMENT PROJECT
Kevin P. Smith
Department of Agronomy and Plant Genetics
University of Minnesota, St. Paul, MN 55108, U.S.A.
The overall aim of this research is to develop new six-rowed barley varieties with
acceptable malt quality, improved disease resistance, and high yield potential. This
research will directly assist in AMBA’s mission to provide an adequate supply of high quality
malting barley. Barley improvement at the University of Minnesota is a cooperative effort of
the Department of Agronomy and Plant Genetics, the Department of Plant Pathology, and
the Research and Outreach Centers of the University of Minnesota. Specific breeding
goals include high yield, enhanced lodging resistance, resistance to Fusarium head blight
(FHB), net blotch, spot blotch, stem rust, and Septoria speckled leaf blotch (SSLB), and
favorable malting and brewing characteristics. To meet these objectives, we are
conducting a comprehensive breeding and genetics research effort funded by state and
federal grants. This AMBA project supports breeding activities (making crosses, population
development, trait evaluation, breeding line selection) directed toward the development of
new varieties. This past year one breeding line, M109, was entered into AMBA plant-scale
brewing evaluation with the 2006 crop. Breeding line M115 was rated satisfactory for its
second year in AMBA pilot-scale malting and will advance to plant-scale in 2008. Our first
breeding line with enhanced FHB resistance was rated satisfactory in AMBA pilot malt
testing (2005 crop) and entered into a second year of testing with the 2006 crop.
During the previous year, we made crosses, among elite parents, designed to combine
desirable traits. We developed populations through single seed decent and successfully
conducted our standard set of yield and disease trials in Minnesota to evaluate breeding
lines. Growing conditions were generally dry during most of the season, however, we
obtained good quality data from all of our planned trials and experiments at five locations in
Minnesota. Specific expected outputs on a yearly basis are: 1) development of breeding
populations segregating for useful genes; 2) barley germplasm with specific desirable
traits; 3) a steady flow of variety candidates into the AMBA pilot malting and plant-scale
brewing programs.
The following are some of the most significant accomplishments from the previous year:
• Variety candidate M109 was grown for AMBA plant scale brewing evaluation with the
2006 crop. Sufficient production was obtained from 3 locations in Minnesota and
North Dakota to permit plant-scale brewing.
• Three of the four variety candidates (M115, M122, M124) we submitted to AMBA pilot
malting evaluation were rated satisfactory with the 2005 crop. This is the second year
M115 was rated satisfactory. We should have sufficient seed of M115 in 2008 to grow
17

for plant-scale brewing evaluation. M122 is our first line with enhanced FHB
resistance.
• We continue to make progress in developing breeding lines with resistance to FHB, net
blotch and Septoria speckled leaf blotch and have crosses made in which we will be
using SSR markers to select for resistance alleles.
• Graduate student, Federico Condon, successfully defended his PhD thesis in April,
2006. Using a historical set of varieties and breeding lines, he has estimated genetic
gain for important traits in the breeding program, characterized genetic diversity in
response to breeding using molecular markers, and conducted association mapping to
identify desirable genes for agronomic, disease, and quality traits.
Objectives, Methodology and Results – AMBA Funded Project
The overall objective of this project is to develop new six-rowed malting varieties that are
attractive to producers and the malting and brewing industry. To accomplish this objective,
we conduct numerous field and greenhouse experiments to measure a wide range of
important traits. Varieties and breeding lines were evaluated in yield trials in five locations in
Minnesota (St. Paul, Morris, Crookston, Stephen, and Roseau). Advanced yield trials were
conducted at all five locations, intermediate trials at three locations (St. Paul, Morris, and
Crookston) and preliminary yield trials (PYT) at three locations (St. Paul, Morris, and
Crookston). The Mississippi Valley Nursery (MVN) was grown at Morris and Crookston.
Both locations had favorable growing conditions and grain samples were reasonably plump
and had acceptable levels of protein. Thus, both locations were submitted for malt quality
analysis at the USDA-ARS Cereal Crops Research Unit. The Canadian Six-Row Coop
Test was grown at Crookston. We grew the AMBA pilot trial in Morris and Crookston and
submitted grain from Crookston for testing of the 2005 crop.
EVALUATING CURRENT VARIETIES
Yield averages for barley in Minnesota were 60 bu/A compared to 43 bu/A last year
resulting in production of about 5.4 million bushels. Given the drought that occurred in
most of the upper Midwest, these yields were quite high. The dry weather conditions
resulted in a nearly complete absence of FHB and DON in harvested grain. Robust was
displaced as the leading variety at 40.7 % of the planted acreage by Lacey at 44.9%.
Tradition, Royal, and Legacy followed at 4.2%, 3.4%, and 1.3 %, respectively. Grain yield
data for the current varieties is reported in Table 1 in bu/A for five locations planted in
Minnesota. Tradition, Legacy, and Lacey were the highest yielding varieties in the state
based on the multi-location three-year average.
18

Table 1. Agronomic performance of M109 and M115 and check cultivars using data from
nurseries grown in Minnesota from 2004 to 2006 (mean of 10 trials).
Variety / Line Hdng (DAP) Height (cm) Yield (Bu/A) Lodging
M109 57.8 79 104.5 1.9
M115 57.8 77 104.2 1.9
Tradition 59.7 84 103.3 1.6
Legacy 59.4 86 102.5 2.4
Lacey 59.3 82 101.6 2.6
Stander 59.0 81 97.2 2.3
Drummond 58.5 83 97.0 1.9
Conlon 55.7 80 96.1 3.1
Stellar-ND 58.1 84 95.7 2.6
Robust 58.4 86 94.5 2.5
MNBrite 58.8 87 93.3 4.8
ADVANCED LINE EVALUATION
Varieties currently grown in Minnesota were evaluated with 20 advanced breeding lines in
trials at St. Paul, Morris, Crookston, Stephen and Roseau. Variety candidate, M109, was
grown for carlot testing in the AMBA plant-scale brewing evaluation with the 2006 crop.
M109 has been consistently higher yielding (Table 1 ) than current varieties grown in the
region which should make it attractive to growers. In trials conducted in North Dakota and
Montana over three years, M109 yielded two to nine percent higher than the other newer
varieties and 14% higher than Robust (Table 2). Thus, M109 appears to be widely adapted
to the six-rowed barley production area.
Table 2. Performance of M109 in eleven locations* in North Dakota and Montana in 2004 -
2006 compared with six current malting varieties.
Days to Plant Stem Test
Entry heading height Lodging breakage Yield weight
(Days after May 31) (cm) (1-10†) (1-5‡) (bu/ac) (lb/bu)
Sta. Yrs. 22 20 7 6 22 10
M109 26.9 73.5 4.1 3.1 86.1 51.9
Lacey 27.5 75.7 3.4 2.9 84.0 52.3
Tradition 27.8 78.4 4.3 3.0 83.6 51.8
Stellar-ND 27.8 76.8 2.9 2.6 83.5 51.5
Drummond 27.0 78.4 2.6 2.5 83.0 51.4
Legacy 29.4 78.8 5.0 3.3 78.9 51.1
Robust 27.6 80.6 4.4 3.2 75.2 51.6
* Data courtesy of Rich Horsley, NDSU. Locations include: Fargo, Carrington, Osnabrock,
Minot, Sydney MT (overhead and flood irrigated), Mott, Halliday, Williston (recrop and
fallow),
†Lodging 1=no lodging and 10=severe lodging.
‡Stem breakage 1=no stem breakage at harvest and 5=severe stem breakage at harvest
19

Three of the four lines submitted for 2005 crop AMBA pilot malt testing (M115, M122, M124)
were rated satisfactory. M115 was rated satisfactory for its second year and is eligible for
plant-scale brewing evaluation. M115 is shorter with better lodging resistance than Lacey
(Table 1). In AMBA pilot malt quality evaluations, M115 has higher malt extract, slightly
higher S/T, slightly lower diastatic power, and lower protein compared to Robust (Table 3).
Table 3. Evaluation of M115 in AMBA Pilot Malting Evaluations for 2004 and 2005 crop
years mean of Morris and Crookston, MN locations.
2004 2005
CHARACTER M115 Robust M115 Robust
BARLEY
Skinned & Broken Kernels (%) 2.0 2.0 0.6 2.5
3-Day Germination (%) 85.8 86.8 98.1 98.5
On 7/64 (%) 38.3 32.4 28.6 24.5
Plump (On 6/64 + 7/64) (%) 92.4 92.8 91.7 90.2
Moisture (%) 11.1 11.4 11.4 10.9
Total Protein (% d.b.) 12.3 13.0 11.8 ** 12.6
DON (ppm) 1.7 1.3
MALT
Moisture at Steep-out (%) 44.6 * 43.8 45.1 43.8
On 7/64 (%) 74.9 ** 66.8 69.9 59.8
Extract, Fine Grind (% d.b.) 80.5 ** 78.9 80.5 * 78.6
Carbohydrate Extract (% d.b.) 74.9 * 73.6 nd nd
Extract Constant 85.6 ** 84.3 nd nd
F-C Difference 1.1 1.0 1.0 1.0
Wort Viscosity 1.44 1.45 1.38 1.39
Wort Color (Deg. Lov.) 2.29 ** 1.93 2.60 * 2.11
Wort Turbidity (Hach NTU) 6.7 5.6 5.3 4.6
Diastatic Power (Deg. L) 150 ** 167 161 * 182
DP/Total Protein 13.0 13.5 nd nd
Alpha Amylase (D.U.) 71.2 ** 52.3 76.3 * 61.0
Soluble Protein (% d.b.) 5.66 * 5.27 5.98 * 5.67
Total Protein (% d.b.) 11.6 ** 12.3 11.1 * 12.1
Soluble/Total Protein (% d.b.) 49.9 ** 43.6 53.6 * 46.8
Moisture (%) 4.0 3.9 3.7 3.8
Beta-Glucan (ppm) 159 138 62 82
Friability (%) 85.8 ** 81.8 92.6 92.8
Free Amino Nitrogen
(FAN) 226 209 261 * 240
*, ** significantly different from Robust at p=0.05, p=0.01, respectively
20

Lines M122 and M124 were rated satisfactory in their first year of AMBA pilot-scale malt
evaluations. M122 is superior to Robust in yield and agronomically competitive with other
current varieties. M122 not as resistant to lodging as many of the current varieties and
similar to Robust in both height and lodging. M122 continues to demonstrate resistance to
FHB with about 40% of the disease severity and 50% the level of DON. This level of
resistance has been consistently observed in trials over the last four years. Grain samples
from 2006 trials have been sent to the CCRU Malt Quality Lab at Madison, however no
data is available yet. Please refer to last years report for quality data from 2005.
Table 4. Agronomic and FHB performance of M122 compared to check varieties 2003-
2006.
Days to Plant FHB
Variety/Line Yield Lodging Heading Height Severity DON
(Bu/A) (%) (Days) (Inches) (% of Robust) (% of Robust)
Sta. Yrs. 16 9 16 15 23 24
M122 105 41 58 34 42 51
Robust 96 39 58 34 100 100
Stander 99 29 58 31 135 155
Lacey 100 28 58 32 -- --
MNBrite -- -- -- -- 65 70
M124 is superior to Lacey in yield, but similar to Robust in lodging (Table 5). It is also
similar to Lacey in response to the current diseases in the region including FHB. This is the
last line that we have advanced to AMBA pilot malt evaluation from our breeding program
that does not have enhanced disease resistance over the currently available varieties.
2006 crop year quality data is not yet available. From previous years data, M115 is higher
in malt extract and lower in grain protein compared to Robust. Both M122 and M124 were
submitted for 2 nd year evaluation in AMBA pilot-scale malt evaluation with the 2006 crop.
Table 5. Agronomic performance of M124 compared to check varieties 2003-2006.
Days to Plant
Variety/Line Yield Lodging Heading Height
(Bu/A) (%) (Days) (Inches)
Sta. Yrs. 16 8 16 16
M124 101 35 59 32
Robust 93 34 58 35
Stander 94 26 59 32
Lacey 97 26 59 32
21

Two first year entries, M128 and M129, were submitted to AMBA pilot malt evaluation with
the 2006 crop. Both of these lines are from our FHB breeding program and have levels of
resistance equal to or greater than MNBrite. M128 has the resistance sources Atahualpa,
MNBrite and Zheddar in its pedigree. M128 is slightly higher in yield and similar in lodging
to Robust. It is intermediate in height to Robust and Lacey with about a 30% reduction in
FHB severity and DON compared to Robust (Table 6).
Table 6. Agronomic and FHB performance of M128 compared to check varieties 2003-
2006.
Days to Plant FHB
Variety/Line Yield Lodging Heading Height Severity DON
(Bu/A) (%) (Days) (Inches) (% of Robust) (% of Robust)
Sta. Yrs. 14 3 14 14 10 11
M128 101 34 57 33 65 73
Robust 100 38 57 35 100 100
Stander 103 36 57 31 135 157
Lacey 102 35 57 31 -- --
MNBrite -- -- -- -- 84 75
M129 has the resistance source AC Oxbow in its pedigree. M129 is equal in yield, slightly
shorter, and slightly better in lodging resistance compared to Robust. M129 exhibits about
a 50% reduction in disease severity and 23% reduction in DON (Table 7).
Table 7. Agronomic and FHB performance of M129 compared to check varieties 2003-
2006.
Days to Plant FHB
Yield Lodging
Variety/Line Heading Height Severity DON
(Bu/A) (%) (Days) (Inches) (% of Robust) (% of Robust)
Sta. Yrs. 14 3 14 14 10 10
M129 100 30 57 34 52 77
Robust 100 38 57 35 100 100
Stander 103 36 57 31 156 153
Lacey 102 35 57 31 -- --
MNBrite -- -- -- -- 83 83
INTERMEDIATE AND PRELIMINARY YIELD TRIALS
Fourty-five lines were grown and evaluated in our intermediate yield trials at 3 locations (St.
Paul, Morris, and Crookston). Nineteen of these lines have partial resistance to FHB and
another three have enhanced resistance to Septoria and one to net blotch. Our preliminary
yield trials grown at St. Paul, Morris, and Crookston included 183 lines. Of these lines, 94
came from our FHB screening program and have partial resistance to FHB.
22

EARLY GENERATION LINES
Forty F5 populations were grown at Crookston and St. Paul. The populations were largely
of two types, i.e., traditional elite by elite and elite by FHB resistant. Nine of the F5
populations (elite by elite) along with eight populations segregating for resistance to net
blotch or septoria were advanced through the F3 and F4 generations via single seed
descent (SSD) in greenhouses in fall and winter 2005-2006. These populations were
planted as F5 head rows in Crookston or Stephen (net blotch) in the summer of 2006.
Selection in these crosses was based on heading date, height, lodging, kernel plumpness,
and spike characteristics. The Septoria and net blotch populations were planted in
inoculated nurseries. We were unable to get good Septoria disease expression and
therefore unable to select for resistance. These lines were screened for resistance to
Septoria in the Fall greenhouse and resistant lines will be advanced to 2007 trials. Disease
pressure at our net blotch nursery was light, so we also conducted greenhouse disease
screening in the Fall. Twenty-four of the F5 populations (elite by FHB) were advanced to
F3 in a fall 2005 greenhouse in Minnesota and then to the F4 as single plants in
Christchurch, New Zealand. The New Zealand nursery allowed us to harvest enough seed
from individual plants for replicated FHB nurseries in a timely manner for planting.
Selection on FHB populations in the 2006 field season was based on FHB resistance,
heading date, height, and lodging. Grain harvested from selected rows was analyzed for
DON. In addition, we plant a single row outside of the disease nursery for malting quality
analysis in Madison, WI. Heads from selected F5’s in 2006 were planted in single rows in
a winter nursery (2006/2007) in Yuma, AZ to produce seed for 2006 PYTs. Based on 2006
FHB, DON, and malting quality data and also phenotype in the Yuma nursery, about 200
lines will be selected for entry to preliminary yield trials in 2007.
A total of 51 F2 populations were grown in the field at St. Paul and Crookston in 2005. In
addition, we grew eight F2 populations in the summer greenhouse in cones arranged in a
96-well format to facilitate marker assisted selection (MAS). In these populations, we
selected for markers linked to a QTL identified on chromosome 6(6) (Wingbermuehle et al.,
2004) and markers linked to a QTL for resistance to Septoria speckled leaf blotch. Marker
genotyping was done in collaboration with Dr. Shiaoman Chao at the USDA Genotyping
Lab in Fargo, ND.
SCREENING FOR DISEASE RESISTANCE
FHB
Steady progress has been made in breeding for resistance to FHB. Funding through the
US Wheat and Barley Scab Initiative and the Minnesota Small Grains Initiative have
allowed us to sustain a major effort in disease resistance breeding and studies aimed to
understand and exploit the genetics of disease resistance. Our FHB screening effort is
conducted in collaboration with Dr. Ruth Dill-Macky and Dr. Charla Hollingsworth. We are
advancing resistant lines in our program tracing to different sources of resistance and
continue to make crosses with new sources of resistance as they are identified. We have
been working with Dr. Brian Steffenson in selecting new sources of FHB resistance to
introduce into the breeding program. Each year we have increased the number of entries
23

in our advanced yield trials that have improved levels of resistance to FHB. Last year we
entered three variety candidates with enhanced FHB resistance, M122, M128 and M129
into AMBA pilot testing. This year we will enter three new lines with enhanced FHB
resistance into AMBA pilot testing.
SPOT BLOTCH
Spot blotch (SB) has been controlled relatively well in six-rowed barley in the Midwest
utilizing resistance tracing back to NDB112. It has been assumed that this source of
resistance has been fixed in the Minnesota elite breeding material for some time due to the
generally narrow genetic base of this germplasm. However, since the extensive effort to
breed for resistance to FHB, many wide crosses have been made to FHB resistant sources
that are susceptible to SB. Because of the increased probability of losing resistance to SB
in many of the FHB populations, we have resumed screening for SB in collaboration with
Dr. Brian Steffenson.
STEM RUST
Until very recently, the situation with stem rust (SR) was similar to SB. A single gene,
Rpg1, has provided durable resistance to SR in Midwestern six-rowed varieties. However,
with the FHB breeding effort, we could possibly loose this gene in our breeding lines in the
absence of selection. We have established collaborative greenhouse screening with Dr.
Brian Steffenson to screen early generation segregating populations and all PYT entries for
resistance to SR. In addition, we are using a PCR marker, designed from the Rpg1 gene
sequence, to screen breeding material.
Recently, a new race of the stem rust pathogen, Ug99, has emerged in Africa. None of the
currently grown varieties have resistance to this race. We are working with Drs. Yue Jin
and Brian Steffenson to assess the level of resistance in our breeding program and deveop
a strategy to introduce resistance to Ug99 into our germplasm. So far this race has not
been detected in the United States, but if and when it does the disease could be
devastating without adequate resistance.
NET BLOTCH
Net Blotch (NB) is probably the second most important disease in Minnesota behind FHB.
In collaboration with Dr. Ruth Dill-Macky and Dr. Charla Hollingsworth, we have established
both greenhouse and field screening programs to select for resistance to NB. We
evaluated elite lines, resistance sources, and six populations for net blotch reaction in the
field in a nursery located at Stephen, MN in 2006. Unfortunately, dry conditions resulted in
very light disease pressure. We have been able to continue to screen for NB reaction on
seedlings in a greenhouse assay in collaboration with Dr. Dill-Macky. An undergraduate in
my lab has also recently mapped a QTL for net blotch resistance. We are working with the
Genotyping Lab in Fargo to develop markers that could be used for MAS.
SEPTORIA SPECKLED LEAF BLOTCH
Septoria Speckled leaf blotch (SSLB) is an important pathogen in the Midwest. We
have a breeding effort, in collaboration with Dr. Brian Steffenson, to develop resistance
to this disease. We are utilizing two sources of resistance. For the source, CI4780, we
24

have evaluated a PCR based marker, developed in Dr. Steffenson’s lab, for use in
selection. We usedave been using this marker for selection in F2 populations. We
initiated a field screening program in 2003 at Crookson, MN which was overseen by Dr.
Steffenson and Dr. Char Hollingsworth. In 2005 and again in 2006, we were unable to
establish sufficient levels of disease in the field for evaluation of resistance. We were
able to screen lines in the greenhouse in collaboration with Dr. Steffenson. One of our
new breeding lines entered into the AMBA Cooperative Pilot Trial has resistance to
Septoria. Pending analysis of 2006 quality data, this line may be entered into AMBA
pilot malt evaluations,
Genetic Analysis Of Elite Minnesota Barley Germplasm
A Ph.D. graduate student, Federico Condon, has defended his thesis etitled “ Genetic
Gain, Diversity, And Marker-Trait Associations In Minnesota Barley Germplasm”.
This was one of the pilot projects that help to shape the proposal for the Barley
Coordinated Agricultural Project (CAP).
Barley CAP Project
One aspect of out research funded through the USDA Barley Coordinated Project (CAP) is
to map malting quality QTL using breeding germplasm. Carol Powers is a PhD student on
this project who was initially funded through AMBA and is now funded through the Barley
CAP. Her project involves mapping, validating, and conducting marker assisted selection
for malting quality traits.
Recent Publications
Peer-Reviewed
Wenzl, Peter, Haobing Li, Jason Carling, Meixue Zhou, Harsh Raman, Edie Edie, Phillippa
Hearnden, Christina Maier, Ling Xia, Vanessa Caig, Jaroslava Ovesna, Mehmet Cakir,
David Poulsen, Junping Wang, Rosy Raman, Kevin P Smith, Gary J Muehlbauer, Ken J
Chalmers, Andris Kleinhofs, Eric Huttner and Andrzej Kilian. 2006. A high-density
consensus map of barley linking DArT markers to SSR, RFLP and STS loci and agricultural
traits. BMC Genomics 2006, 7:206
S. J. Yun, L. Gyenis, E. Bossolini, P. M. Hayes, I. Matus, K. P. Smith, B. J. Steffenson, R.
Tuberosa, and G. J. Muehlbauer. 2006. Validation of Quantitative Trait Loci for Multiple
Disease Resistance in Barley Using Advanced Backcross Lines Developed with a Wild
Barley. Crop Sci. 2006 46: 1179-1186.
Zhong, S., H. Toubia-Rahme, B. J. Steffenson, and K. P. Smith. Molecular Mapping and
Marker-Assisted Selection of Genes for Septoria Speckled Leaf Blotch Resistance in
Barley. Phytopathology 96:993-999
25

Steffenson, B. J and Smith, K. P. 2006. Breeding Barley for Multiple Disease Resistance
in the Upper Midwest Region of the USA. Czech J. Genet. Plant Breed. 41: 79–85.
Brakke, M., K. P. Smith, P. Baepler, and J.D. Walker. 2006. Using Problem-Based
Learning to Enhance Students’ Motivation to Learn. Creative College Teaching
Journal "Problem-Based Learning : Successful Examples from Across the
Disciplines"
Abstracts and Proceedings
Wenzl, Peter, Haobing Li, Jason Carling, Meixue Zhou, Harsh Raman, Edie Paul, Phillippa
Hearnden, Christina Maier, Ling Xia1, Vanessa Caig, Jaroslava Ovesná, Mehmet
Cakir, David Poulsen, Junping Wang, Rosy Raman, Kevin P. Smith, Gary J.
Muehlbauer, Ken J. Chalmers, Andris Kleinhofs, Eric Huttner, Andrzej Kilian. 2006.
A high-density consensus map of barley linking DArT markers to SSR, RFLP and
STS loci and agricultural traits. In: Proceedings of the International Triticeae
Mapping Initiative 2006 Workshop. Victor Harbor, Australia, 9-1-06.
Roy, J.K., G. Muehlbauer, K. P. Smith, J. Carling, A. Kilian, V. Jan, B. Steffenson. 2006.
Association Mapping Of Disease Resistance Genes In Wild Barley. In: Plant and
Animal Genome XIV Abstracts, San Diego, CA. Jan 14 – 18, 2005, P299
Beaubien, K. A. and K. P. Smith. 2006. Identifying Marker-Trait Associations In
Contemporary Midwest Breeding Germplasm. In: Plant and Animal Genome XIV
Abstracts, San Diego, CA. Jan 14 – 18, 2005, P317
Roy, J.K., G. Muehlbauer, K. P. Smith, J. Carling, A. Kilian, V. Jan, B. Steffenson. 2006.
Genetic Diversity And Linkage Disequilibrium-Based Association Mapping Of
Disease Resistance In Wild Barley. In: Plant and Animal Genome XIV Abstracts,
San Diego, CA. Jan 14 – 18, 2005, W257
Shiaoman Chao, S., J. Anderson, K. Glover, K.P. Smith. 2006. Use Of High Throughput
Marker Technologies For Marker-Assisted Breeding In Wheat And Barley. In: Plant
and Animal Genome XIV Abstracts, San Diego, CA. Jan 14 – 18, 2005, W43
Bilgic, H., S. Cho, L. Nduulu, K. P. Smith, and G. J. Muehlbauer. 2006. Microarray
Analysis of Gene Expression in Barley During Fusarium graminearum
Infection. In: Proceedings of the In Vitro Biology Meeting, Minneapolis, MN, June 3-7,
2006
Smith, K.P. 2006. A community research effort to apply genomics approaches to
understand and exploit the genetics of malting quality in barley. In: Proceedings of
the American Society of Brewing Chemists 2006 <strong>Annual</strong> Meeting. Palm Springs,
California, June 17-21, 2006. P-52.
Project Personnel
Barley Project
Kevin P. Smith, Associate Professor, Project Leader
26

Edward Schiefelbein, Research Scientist
Guillermo Velasquez, Plot Technician
Charles Gustus, Research Scientist
Karen Beaubien, Junior Research Scientist
Lex Nduulu, Post Doc
Federico Condon, Research Assistant
Carol Powers, Research Assistant
Jon Massman, Research Assistant
Collaborators
Ruth Dill-Macky, Department of Plant Pathology, UM
Gary J. Muehlbauer, Department of Agronomy and Plant Genetics, UM
Brian J. Steffenson, Department of Plant Pathology, UM
Char Hollingsworth, Department of Plant Pathology, UM
John V. Wiersma, Northwest Research and Outreach Center, UM
George A. Nelson, West Central Research and Outreach Center, UM
Shiaoman Chao, USDA-ARS Fargo, ND
Yue Jin, USDA-ARS, St. Paul, MN
27

MANAGEMENT AND EPIDEMIOLOGY OF BARLEY DISEASES
Ruth Dill-Macky, Amar Elakkad and Karen Wennberg
Department of Plant Pathology
University of Minnesota
St. Paul, MN 55108, USA
Executive Summary: This is an applied research program directed to the control of
the plant disease issues of importance to the barley industry in Minnesota and the
Upper Midwest. The emphasis of the research is directed to research on the foliar
pathogens of barley. Fusarium head blight (FHB or scab) presents the greatest threat
to the production of malt quality barley in Minnesota and research on FHB has been the
principle focus of the program. The foliar diseases of importance in Minnesota include
net blotch, spot blotch, Septoria leaf blotch, and the rusts (leaf and stem rust). Barley
Pathology Research at the University of Minnesota is a cooperative effort of the
Department of Plant Pathology, the Department of Agronomy and Plant Genetics and
the experiment stations of the University of Minnesota. The joint efforts of these
research programs have results directly in the development of new malting barley
varieties suitable for production in the upper Midwest of the United States.
In 2006 this project was instrumental in the screening of over 14,350 field rows and 250
pots in the greenhouse for resistance to FHB. The program also conducted germplasm
screening in the field and greenhouse to the foliar pathogens of barley. In 2006 the
project was involved in the screening of 600 rows in the field and 1,300 pots in the
greenhouse for response to Pyrenophora teres (net blotch).
Detailed <strong>Report</strong>:
Objectives: The project incorporates two interrelated objectives:
1. To improve the management and control of diseases in barley. Emphasis is placed
on the control of the foliar diseases with the greatest economic impact on
commercial barley production. Improving our understanding of the diseases of
barley and developing effective management options facilitates disease
management: primarily through the use of host resistance and cultural control
practices.
2. The program provides support to the barley breeding program as part of ongoing
collaborative efforts to develop and maintain germplasm with improved
resistance to plant pathogens.
Fusarium Head Blight Research – Methodology and Results
Field Screening Program: A large effort is maintained each field season assessing
Fusarium head blight reactions within breeding populations for the University of
Minnesota’s barley breeding program at the three Minnesota screening locations. The
28

small grains pathology input to this project involves the oversight of inoculum
preparation during the winter and spring, assisting with summer plot maintenance
including the operation of the mist-irrigation system, and spray applications of inoculum
during the summer field season.
In the 2006 field season a total of 14,350 rows were screened in breeding nurseries.
The number of field rows evaluated was much the same as in the previous two years.
Field nurseries were conducted at three locations; St Paul with 6,800 rows, Morris with
800 rows and Crookston with 6,750 rows. Nurseries at all three locations were primarily
inoculated and mist-irrigated to promote disease development, although we also
established dryland (without irrigation) nurseries at both St Paul and Crookston to
provide additional screening under weather conditions which more closely resemble
those of commercial crops. In 2006 approximately 4000 liters of macroconidial
inoculum (800,000 macroconidia ml -1 ) was produced in our lab and used by five
separate research programs (for barley, wheat and oat research).
Greenhouse Screening Program: During the fall, winter, and spring seasons
greenhouse tests for resistance to Fusarium head blight are conducted in the
greenhouse. Testing of approximately 250 pots of barley was completed in 2006. The
inoculation of barley plants in the greenhouse is conducted using an airbrush sprayer to
deliver macroconidial inoculum to one face of the barley spike. Point inoculations, as
used widely in screening wheat for resistance to FHB, do not differentiate between
resistance and susceptible barley varieties. This spray inoculation technique has
proven useful to detect QTL associated with reaction to deoxynivalenol accumulation in
barley.
Research on Foliar Diseases – Methodology and Results
This program provides support to the barley breeding program as part of ongoing
breeding efforts to develop improved germplasm with improved disease resistance.
Screening breeding germplasm for resistance to kernel discoloration, leaf rust, loose
smut, net blotch, powdery mildew, and spot blotch are conducted on an ongoing basis
with the barley breeding program.
In 2006 evaluations were conducted to identify resistant germplasm in breeding
populations following natural infections of leaf spot diseases at the Northwest Research
and Outreach Center, Crookston and the West Central Research and Outreach Center,
Morris.
In 2006, we established a net blotch nursery of approximately 600 rows at Stephen.
However, due to the extremely dry conditions in 2006, little disease development was
observed. Nevertheless, straw infected with the net blotch pathogen was collected by
for inoculation of the nursery in 2007. Despite two disappointing years (the 2005 trial
was lost to flooding) we remain confident that future screening nurseries in Stephen will
be successful. The only two nurseries we have lost in over ten years have been these
29

last two. In years when field screening is not successful additional greenhouse
screening has been conducted to ensure that the breeding programs have adequate
data for germplasm selection. In 2006 over 1300 barley entries were tested as
seedlings for their reaction to the net blotch pathogen Pyrenophora teres in the
greenhouse at St. Paul. Approximately 40% of the lines tested exhibited resistance and
were advanced in the breeding program.
Other Barley Research
With financial support from the USWBSI we are undertaking a project to examine our
ability to reduce the inoculum potential of Fusarium-infested residues by aiding residue
decomposition and targeting biocontrol agents to the pathogen within these residues.
The field experiments we will conduct will utilize a relatively new piece of equipment (a
mower/shredder attachment for a combine) for finely chopping residue. This new
equipment is being rapidly adopted by corn growers in areas where no-till and/or Btcorn
are produced and may assist in our control of this residue borne pathogen by
identifying ways to reduce the inoculum potential of Fusarium-infested residues without
removing the residues from the soil surface where they play a vital role in conserving
soil from wind and water erosion.
Three field experiments, one examining barley residues, and the others corn and wheat,
will be conducted. Each experiment will examine both the fine chopping of Fusariuminfested
crop residues and biological control agents (BCAs: Trichoderma,
Streptomyces, or Bacillis), fungicides (prothioconazole) and bentonite clay applications
to the residues. Applications of BCAs and fungicides will be made in either the fall or
spring. We anticipate that the finely chopping will accelerate the rate of residue
decomposition and that in combination with BCAs that the survival/inoculum production
capacity of Fusarium in the residues will be diminished. Negatively impacting the
survival of F. graminearum (G. zeae) will reduce inoculum production and thus the
potential of disease development and DON production in subsequent wheat or barley
crops.
Future Direction of Program
A new race of stem rust detected in Africa has raised concern in wheat and barley
workers across the world. This new race is highly virulent and poses a serious threat to
the security of wheat and barley. In the Upper Midwest the majority of spring wheat
cultivars and all the barley cultivars are susceptible to this race. As rust spores may be
disseminated over large distances this rust is likely spread to other cereal production
regions of the world. A proactive research effort is needed to identify and incorporate
effective resistance into adapted barley germplasm. Research at the USDA-ARS
Cereal Disease Laboratory and University of Minnesota has identified potential sources
of resistance to this race in barley. We (Dill-Macky, Jin and Steffenson) are seeking
funding to facilitate a collaborative research effort in barley to address this new threat to
barley production.
30

Project Personnel
Small Grains Pathology Laboratory Personnel
Ruth Dill-Macky, Associate Professor, Project Leader
Amar M. Elakkad, Research Plot Technician
Karen J. Wennberg, Assistant Scientist
Beheshteh Zagaran, Junior Scientist
Cooperators
Yue Jin – USDA-ARS, Cereal Disease Laboratory, St Paul, MN 55108
Gary J. Muehlbauer - Department of Agronomy and Plant Genetics, University of
Minnesota, St Paul, MN 55108
George A. Nelson - West Central Research and Outreach Center, University of
Minnesota, Morris, MN 56267
Kevin P. Smith - Department of Agronomy and Plant Genetics, University of Minnesota,
St Paul, MN 55108
Brian J. Steffenson - Department of Plant Pathology, University of Minnesota, St Paul
MN 55108
Jochum J. Wiersma - Northwest Research and Outreach Center, University of
Minnesota, Crookston, MN 56716
John V. Wiersma - Northwest Research and Outreach Center, University of Minnesota,
Crookston, MN 56716
Recent Publications (2006 publications)
Fuentes, R.G., Mickelson, H.R., Busch, R.H., Dill-Macky, R., Evans, C.K. Thompson,
W.G., Wiersma, J.V., Xie, W., Dong, Y., and Anderson, J.A.. (2005). Resource
allocation and cultivar stability in breeding for Fusarium head blight resistance in
spring wheat. Crop Science, 45:1965-1972.
Anderson, J.A., Busch, R.H., McVey, D.V., Kolmer, J.A. Jin, Y., Linkert, G.L., Wiersma,
J.V., Dill-Macky, R., Wiersma, J.J. and Harland G.A. (2006). Registration of
“Ulen” wheat. Crop Science, 46: 979-980.
Anderson, J.A., Liu, S., Zhang, X., Jin, Y., Dill-Macky, R. and Chao, S. (2006). Markerassisted
selection for FHB resistance in wheat. p. 3. In: Proceedings of the
CIMMYT Fusarium Head Blight Workshop on Global Fusarium Initiative for
International Collaboration, 14-17 March, 2006. El Batan, Mexico, 137 p.
Chakraborty, S., Scott, J.B., Akinsanmi, O.A., Liu, C.J., Mitter, V. and Dill-Macky, R.
(2006). Fusarium pathogens of wheat in Australia. p. 42-45. In: Proceedings of
the CIMMYT Fusarium Head Blight Workshop on Global Fusarium Initiative for
International Collaboration, 14-17 March, 2006. El Batan, Mexico, 137 p.
Dill-Macky, R. (2006). Implications of population variability on the management of
Fusarium head blight. p. 72. In: Proceedings of the CIMMYT Fusarium Head
Blight Workshop on Global Fusarium Initiative for International Collaboration, 14-
17 March, 2006. El Batan, Mexico, 137 p.
31

Dill-Macky, R., Evans, C.K., Culler, M.D., Elakkad, A.M. and Wennberg, K.J. (2006).
Considerations in designing nurseries for screening FHB response in wheat and
barley. p.109. In: Proceedings of the CIMMYT Fusarium Head Blight Workshop
on Global Fusarium Initiative for International Collaboration, 14-17 March, 2006.
El Batan, Mexico, 137 p.
Dill-Macky, R., Mudge, A.M., Dong, Y. and Manners J.M. (2006). Systemic
colonization and production of deoxynivalenol throughout wheat plants following
inoculation of crown tissues with Fusarium graminearum. p. 66. In: Book of
Abstracts, 9 th European Fusarium Seminar, 19-22 September, 2006,
Wageningen, The Netherlands, p. 194.
Hill, N.S., Neate, S.M., Cooper B., Horsley, R.D., Schwarz, P.B., Dahleen, L.S., Smith
K.P, and Dill-Macky, R. (2006). ELISA Analysis for Fusarium in Barley:
Application in Field Nurseries. Abstract 1827b, In American Society of Agronomy
- Crop Science Society of America - Soil Science Society of America, 2006
International Meetings, 12-16 November, 2006, Indianapolis IN.
Dill-Macky, R., Mudge, A.M., Dong, Y. and Manners J.M. (2006). Systemic
colonization and production of deoxynivalenol throughout wheat plants following
inoculation of crown tissues with Fusarium graminearum. In: Proceedings of the
2006 National Fusarium Head Blight Forum, Research Triangle Park, North
Carolina, USA, December 10-12, 2006, p. 45.
Di, R., Blechl, A., Dill-Macky, R., Tortora, A. and Tumer N.E. (2006). Expression of a
truncated form of ribosomal protein L3 in transgenic wheat confers resistance to
deoxynivalenol and Fusarium head blight. In: Proceedings of the 2006 National
Fusarium Head Blight Forum, Research Triangle Park, North Carolina, USA,
December 10-12, 2006, p. 64.
Makander, R., Nalam, V., Essig, J.S., Schapaugh, M.A., Trick, H., Bockus, W., Dill-
Macky, R. and Shah, J. (2006). Enhancing resistance to Fusarium graminearum
by expression of Arabidopsis thaliana NPR1 in wheat. In: Proceedings of the
2006 National Fusarium Head Blight Forum, Research Triangle Park, North
Carolina, USA, December 10-12, 2006, p. 66.
Shin, S.H., Lewis, J.M., Mackintosh, C.A., Elakkad, A.M., Wennberg, K.J., Heinen, S.J.,
Dill-Macky, R. and Muehlbauer, G.J. (2006). Transgenic wheat with enhanced
resistance to Fusarium head blight. In: Proceedings of the 2006 National
Fusarium Head Blight Forum, Research Triangle Park, North Carolina, USA,
December 10-12, 2006, p. 67.
Hill, N.S., Neate, S., Cooper, B., Horsley, R., Schwarz, P., Dahleen, L.S., Smith, K.P.,
Dill-Macky, R., O'Donnell, K. and Reeves, J. (2006). Is there value in quantifying
Fusarium mycelium for breeding FHB resistance? In: Proceedings of the 2006
National Fusarium Head Blight Forum, Research Triangle Park, North Carolina,
USA, December 10-12, 2006, p. 98.
Chakraborty, S., Liu, C.J., Mitter, V., Scott, J.B., Akinsanmi, O.A., Ali, S., Dill-Macky, R.,
Nicol, J., Backhouse, D. and Simpfendorfer S. (2006). Pathogen population
structure and epidemiology are keys to wheat crown rot and Fusarium head
blight management. Australasian Plant Path., 35: 643-655.
32

Investigations on Barley Diseases and Their Control
Brian J. Steffenson
Department of Plant Pathology
University of Minnesota
ANNUAL PROGRESS REPORT
Executive Summary
Plant diseases are one of the most important constraints to barley (Hordeum
vulgare) production and quality in the United States. Our Cereal Disease Resistance
Project is part of the Minnesota Barley Improvement Program team that develops sixrowed
malting barley cultivars for the Midwest. The primary mission of the Cereal
Disease Resistance Project at the University of Minnesota is the control of economically
important barley diseases. For many diseases, this goal is best achieved through the
development of cultivars with genetic resistance. Thus, the long-term goal of this
project is to develop the knowledge base, resources, and germplasm for achieving
durable disease resistance in malting barley cultivars. In addition to these goals, it is
also essential to conduct disease surveys and monitor pathogen populations for new
virulence types. In 2006, breeding lines were sown for evaluation for resistance to spot
blotch, Septoria speckled leaf blotch (SSLB), and net blotch. Durable resistance has
been achieved for spot blotch. Our evaluations ensure that this resistance is not lost
when exotic material is introgressed into the breeding program. Indeed, we identified
several susceptible lines from Busch Agricultural Resources, Inc. (BARI) breeding
program in this tests. These lines have now been discarded. We are also working on
increasing the level of resistance to SSLB and net blotch in the Minnesota program.
Toward this end, we have identified a number of agronomically advanced lines with high
levels of resistance to both diseases. Our annual disease survey was conducted on
July 12-14 in 2006. Fusarium head blight, net blotch, and SSLB continue to be common
diseases on barley, but their severity was reduced due to dry weather in 2006.
Bacterial blight also was found in a few fields in 2006. Additional pathogen isolates
were collected from this survey and were stored in our pathogen collection. Pathogen
isolates are an essential resource for resistance breeding efforts and the identification of
novel sources of disease resistance. They are also useful as a historical record of
virulence shifts in pathogen populations. Our research goals all directly address
AMBA’s primary objective of developing malting barley cultivars with improved
agronomic and quality characters. The deployment of superior malting cultivars with
disease resistance will help ensure that an adequate supply of high quality malting
barley is available to the malting and brewing industry.
Objectives, Methodology, and Results
(AMBA Funded Project)
Plant diseases are one of the most important constraints to barley (Hordeum
vulgare) production and quality in the United States. The primary mission of the Cereal
33

Disease Resistance Project at the University of Minnesota is the control of economically
important barley diseases. For many diseases, this goal is best achieved through the
development of cultivars with genetic resistance. In addition to the development and
deployment of disease resistant cultivars, it is also essential to conduct disease surveys
and monitor pathogen populations for new virulence types. Disease surveys are useful
for identifying economically important pathogens of barley and determining their impact
on yield and quality.
The long-term goal of this project is to develop the knowledge base, resources,
and germplasm for achieving durable disease resistance in malting barley cultivars.
This is done in cooperation with the Minnesota Barley Improvement Program led by Dr.
Kevin Smith and the Small Grains Pathology Project led by Dr. Ruth Dill-Macky.
The specific objectives of this research for FY06-07 are to:
1) evaluate Minnesota breeding lines for resistance to Septoria speckled leaf
blotch, spot blotch, and stem rust;
2) survey commercial barley fields for diseases in Minnesota;
3) collect pathogen isolates from infected barley cultivars and determine their
virulence phenotype;
4) increase, maintain, and distribute pathogen stocks for testing barley
germplasm for resistance and also barley stocks for testing pathogens for
virulence;
5) cooperate with other investigators conducting research on malting barley and
barley diseases.
Evaluation of barley breeding germplasm for disease resistance. Entries
from the University of Minnesota (44 lines) and Busch Agricultural Resources Inc.
(BARI) (203 lines) barley improvement programs were evaluated for resistance to the
spot blotch pathogen in the field at St. Paul. This screening is done every year to
ensure that the durable spot blotch resistance in six-rowed germplasm is maintained in
populations derived from non-elite parents, especially those used in breeding for
Fusarium head blight (FHB) resistance. Seventeen lines from BARI were susceptible to
spot blotch, indicating that the durable resistance genes had been lost. These lines
were discarded. All of the Minnesota lines were resistant to spot blotch. Over the past
several years, we have been working on increasing the level of resistance to net blotch
and SSLB. Toward this end, we planted about 500 accessions for evaluation to the two
diseases at Crookston and Stephen, respectively, in cooperation with Dr. Charla
Hollingsworth at the University of Minnesota Northwest Research and Outreach Center.
Unfortunately, due to the extremely dry weather conditions in the Stephen area, very
low disease levels were observed in the nursery. At Crookston, disease development in
the SSLB nursery was insufficient to distinguish between resistant and susceptible lines.
The breeding lines were therefore evaluated in the greenhouse as seedlings.
Significant progress in breeding has been made as a number of the most resistant lines
identified also possessed good yield and quality characteristics. Several of these lines
have been advanced into late generation evaluation tests.
34

Minnesota barley disease survey and collection of pathogen isolates.
Disease surveys are useful for identifying economically important pathogens of barley
and determining their impact on yield and quality. A survey was made in the
northwestern part of Minnesota (the primary barley production area in the state) to
ascertain the prevalence and importance of barley diseases in 2006. Nine fields were
visited during the survey conducted on July 12-14. FHB was the most common disease
and detected in 67% of surveyed fields, with observed severities ranging from 0% to
0.2% (Table 1). Net blotch was the next most common disease and was observed in
56% of fields. Net blotch severity ranged from trace to 30%. Other diseases identified
in the survey included bacterial blight, SSLB and crown rust. FHB continues to be a
major disease problem on six-rowed barley in the region as it has for the past 14 years.
However, the severity of FHB was among the lowest observed for this survey in many
years. This was likely due to the very dry weather in northwestern Minnesota. Net
blotch has and continues to be a common disease in barley. In 2005, bacterial blight
was widespread in Minnesota. In 2006, this disease was found again in a number of
fields. Crown rust can be a common disease in Upper Midwest barley fields—its
severity in any one year can vary dramatically.
Determine the virulence phenotype of pathogen isolates and maintain a
pathogen isolate collection. Pathogen isolates are an essential resource for
resistance breeding efforts and the identification of novel sources of disease resistance.
We have over 1000 isolates of various barley pathogens stored at the University of
Minnesota. Over the past year, we tested the viability of all isolates and are in the
process of compiling a database (place of collection, cultivar of origin, virulence pattern,
etc.) on them. We collected more pathogen isolates from the 2006 barley disease
survey. These isolates will be purified and stored for future virulence evaluations. We
conduct the leaf rust virulence surveys for the United States. In 2006, seven isolates of
P. hordei (leaf rust) were received from around the country. Our lab continues to serve
the barley community by testing germplasm for resistance to various diseases and
supplying cooperators with pathogen cultures and germplasm for their studies. As
coordinator for disease and pest resistance genes for the International Barley Genetics
Committee, I also supply information to cooperators on the genetics and mapping of
resistance genes.
Other barley research projects funded from other sources. We conduct a
number of other barley research projects in addition to those funded by AMBA. These
are listed below.
Other Barley Research Projects
Sub-cellular localization and functions of the barley stem rust resistance
receptor-like serine/threonine-specific protein kinase Rpg1. The Rpg1 gene
confers resistance to many pathotypes of the stem rust fungus Puccinia graminis f. sp.
tritici and has protected barley from serious disease losses for over 60 years. Rpg1
encodes a constitutively expressed protein with two tandem kinase domains.
35

Fractionation by differential centrifugation and aqueous two-phase separation of the
microsome proteins located Rpg1 mainly in the cytosol, but also in the plasma
membrane and intracellular membranes. Recombinant Rpg1 autophosphorylates in
vitro intramolecularly only serine and threonine amino acids with a preference for Mn 2+
cations and a Km of 0.15 and Vmax of 0.47 nmol·min -1 ·mg -1 protein. The inability of wild
type Rpg1 to transphosphorylate a recombinant Rpg1 inactivated by site directed
mutation confirmed that Rpg1 autophosphorylation proceeds exclusively via an
intramolecular mechanism. Site-directed mutagenesis of the two adjacent lysine
residues in the ATP anchor of the two-kinase domains established that the first of the
two tandem kinase domains is non-functional and that lysine 461 of the 2 nd domain is
the catalytically active residue. Transgenic barley, expressing Rpg1 mutated in either
the kinase 1 or 2 domains, were fully susceptible to P. graminis f. sp. tritici revealing
requirement of both kinase domains for resistance. In planta expressed Rpg1 mutant
protein confirmed that mutation in domain 2, but not 1, rendered the protein incapable of
autophosphorylation.
Molecular mapping and marker-assisted selection of genes for Septoria
speckled leaf blotch resistance in barley. Septoria speckled leaf blotch (SSLB),
caused by Septoria passerinii, has emerged as one of the most important foliar
diseases of barley in the Upper Midwest region of the USA. To map and tag genes for
SSLB resistance, we developed two populations derived from the resistant accessions
CIho 4780 and CIho 10644 and the susceptible malting cultivar Foster. Segregation
analysis of F2 plants and/or F2:3 families from the Foster/CIho 4780 and Foster/CIho
10644 populations revealed that a single dominant gene conferred resistance at the
seedling stage. Bulked segregant analysis identified an AFLP marker, E-ACT/M-CAA-
170, that cosegregated with the SSLB resistance gene Rsp2 in the Foster/CIho 4780 F2
population. Southern hybridization analysis with DNA from the wheat/barley addition
lines localized E-ACT/M-CAA-170 on the short arm of the barley chromosome 5(1H).
RFLP analysis with DNA clones previously mapped to the short arm of chromosome
5(1H) placed Rsp2 at a position flanked by the markers Act8 and ksuD14. A Sequence
Characterized Amplified Region (SCAR) marker (E-ACT/M-CAA-170a) was developed
that not only cosegregated with Rsp2 in the Foster/CIho 4780 population, but also
resistance gene Rsp3 in the Foster/CIho 10644 population. This result indicates that
Rsp3 is closely linked to Rsp2 on the short arm of chromosome 5(1H). The utility of
SCAR marker E-ACT/M-CAA-170a for selecting Rsp2 in two different breeding
populations was validated.
Molecular mapping of loci conferring resistance to different pathotypes of
the spot blotch pathogen in barley. Spot blotch, caused by Cochliobolus sativus, is
an important disease of barley in many production areas and is best controlled through
the deployment of resistant cultivars. Information on the genetics of resistance in
various sources can be useful in developing effective breeding strategies. Parents of
the doubled haploid mapping population Calicuchima-sib/Bowman-BC (C/B) exhibit a
differential reaction to pathotypes 1 and 2 of C. sativus. To elucidate the genetics of
spot blotch resistance in this population, C/B progeny were evaluated to both
36

pathotypes at the seedling stage in the greenhouse and at the adult plant stage in the
field. At the seedling stage, progeny segregated 84 resistant : 26 susceptible based on
the qualitative analysis of infection response (IR) data to pathotype 1. This fit best to a
3:1 ratio, indicating that two genes were involved in conferring resistance. Quantitative
analysis of the raw IR data to pathotype 1 revealed a single quantitative trait locus
(QTL) on chromosome 4(4H) explaining 14% of the phenotypic variance. Adult plant
resistance to pathotype 1 was conferred by QTL on chromosome 2(2H) and
chromosome 3(3H), explaining 21% and 32% of the phenotypic variation, respectively.
Bowman contributed the resistance alleles on chromosome 3(3H) and chromosome
4(4H), whereas Calicuchima-sib contributed the resistance allele on chromosome
2(2H). Resistance to pathotype 2 was conferred by a single gene (designated Rcs6) on
chromosome 5(1H) based on qualitative analysis of data. Rcs6 was effective at both
the seedling and adult plant stages and was contributed by Calicuchima-sib. This result
was corroborated in the quantitative analysis of raw IR (seedling stage) and disease
severity (adult plant stage) data as a single major effect (r 2 =0.93 and 0.88, respectively)
QTL was identified on chromosome 5(1H). Progeny with resistance to both pathotypes
were identified in the C/B population and may be useful in programs breeding for spot
blotch resistance.
Rpr1, a gene required for Rpg1-dependent resistance to stem rust in barley.
Rpg1 is a stem rust resistance gene that has protected barley from severe losses for
over 60 years in the United States and Canada. It confers resistance to many, but not
all pathotypes of the stem rust fungus Puccinia graminis f. sp. tritici. A fast neutron
induced mutant showing susceptibility to stem rust pathotype Pgt-MCC was identified in
barley cv. Morex, which carries Rpg1. Genetic and Rpg1 expression level analysis
showed that the mutation was a suppressor of Rpg1 and was designated Rpr1
(Required for Puccinia graminis resistance). Genome-wide expression profiling, using
the Affymetrix Barley1 microarray containing approximately 22,840 probesets, was
conducted between Morex and the rpr1 mutant. Southern analysis confirmed that three
genes (Contig4901_s_at, HU03D17U_s_at, and Contig7061_s_at) are deleted in the
rpr1 mutant. These three genes mapped to chromosome 4(4H) bin 5 and cosegregated
with rpr1-mediated susceptible phenotype. The loss of resistance was
presumed to be due to mutation in one or more of these genes. However, the
possibility exists that there are other genes within the deletions, which are not
represented on the Barley1 microarray. The Rpr1 gene was not required for Rpg5- and
rpg4-mediated stem rust resistance, indicating that it is specific to the Rpg1-mediated
resistance pathway.
Analysis of ergosterol in single kernel and ground grain by gas
chromatography-mass spectrometry. A method for analyzing ergosterol in a single
kernel and ground barley and wheat was developed using gas chromatography-mass
spectrometry (GC-MS). Samples were saponified in methanolic KOH. Ergosterol was
extracted by “one step” hexane extraction and subsequently silylated by Ntrimethylsilylimidazole/trimethylchlorosilane
(TMSI/TMCS) reagent at room temperature.
The recoveries of ergosterol from ground barley were 96.6, 97.1, 97.1, 88.5, and 90.3%
37

at the levels of 0.2, 1, 5, 10, and 20 µmg/g (ppm), respectively. The recoveries from a
single kernel were between 93.0 ~ 95.9%. The precision (coefficient of variance) of the
method was in the range of 0.79 to 12.30%. The method detection limit (MDL) and the
method quantification limits (MQL) were 18.5 and 55.6 ng/g (ppb), respectively. The
ergosterol analysis method developed can be used to handle 80 samples daily by one
person, making it suitable for screening cereal cultivars for resistance to fungal
infection. The ability for detecting low levels of ergosterol in a single kernel provides a
tool to investigate early fungal invasion and to study mechanisms of resistance to fungal
diseases.
Identification and chromosomal location of major genes for resistance to
Pyrenophora teres in a doubled-haploid barley population. Net blotch, caused by
Pyrenopora teres, is one of the most economically important diseases of barley
worldwide. We used a barley doubled-haploid population derived from the lines
SM89010 and Q21861 to identify major quantitative trait loci (QTLs) associated with
seedling resistance to P. teres f. teres (net-type net blotch (NTNB)) and P. teres f.
maculata (spot-type net blotch (STNB)). A map consisting of simple sequence repeat
(SSR) and amplified fragment length polymorphism (AFLP) markers was used to
identify chromosome locations of resistance loci. Major QTLs for NTNB and STNB
resistance were located on chromosomes 6H and 4H, respectively. The 6H locus
(NTNB) accounted for as much as 89% of the disease variation, whereas the 4H locus
(STNB resistance) accounted for 64%. The markers closely linked to the resistance
gene loci will be useful for marker-assisted selection.
Quality risks associated with the utilization of Fusarium head blight
infected malting barley. Fusarium head blight (FHB) has adversely affected the
quality of barley grown in the northern Great Plains of the United States and the eastern
Prairie Provinces of Canada since 1993. Objectives of this study were to document the
occurrence of deoxynivalenol (DON) on barley within North Dakota and Minnesota,
investigate relationships among FHB, DON, and malt quality, and to determine at what
level FHB/DON-contaminated barley can be safely utilized for the production of quality
malt. Since 1993, mean DON levels have ranged from 10.3 to 0.4 µg/g with a
corresponding 81 to 32% of the regional barley crop in excess of 0.5µg/g. Strong
relationships were not observed between either kernel size or kernel weight and DON.
As a consequence, cleaning is unlikely to achieve insignificant reductions in DON levels
in most cases. In terms of barley and malt quality, the strongest relationships were
observed between barley DON and malt DON and malt DON and wort color. However,
malt DON levels could not be reliably predicted from barley at

germplasm with the highest level of FHB resistance possible. Our specific activities
involve the systematic evaluation unique Hordeum germplasm from the USDA and
foreign genebanks for resistance to FHB. The screening of the entire six-rowed spring
barley collection (8,100 accessions) from the USDA National Small Grains Collection
(NSGC) is complete. Additionally, we have now completed the evaluation of nearly one
half of the six-rowed winter barley and wild barley (Hordeum vulgare subsp.
spontaneum) collections of the NSGC.
Future Direction of the Program
Given that the mission of my position at the University of Minnesota is to improve
and enhance small grain cereal crops with genes derived from wild species, I will
continue to work on exploiting wild cereal species. However, I remain deeply committed
to improving malting barley varieties for enhanced disease resistance at the University
of Minnesota and at other institutions. If I were to receive an AMBA grant in the future,
my activities for these funds will be strictly focused on the improvement of malting
barley varieties.
Personnel:
Brian J. Steffenson, Professor/Project Leader
Stephanie Dahl, Junior Scientist and Lab Manager
Tamas Szinyei, Junior Scientist
Claudia Miller-Castell, Post-doctoral Research Associate
Joy Roy, Post-doctoral Research Associate
Pablo Olivera, Graduate Student
Ben Alsop, Graduate Student
Cooperators:
Michael C. Edwards, USDA-ARS, Fargo, ND
Paul B. Schwarz, NDSU, Fargo, ND
Richard D. Horsley, NDSU, Fargo, ND
Kevin Smith, UM, St. Paul, MN
Gary Muehlbauer, UM, St. Paul, MN
Ruth Dill-Macky, UM, St. Paul, MN
Harold Bockelman, USDA-ARS, Aberdeen, ID
Andy Kleinhofs, WSU, Pullman, WA
Richard Pickering, Crop & Food Research, Christchurch, New Zealand
Stephen Neate, NDSU, Fargo, ND
Patrick M. Hayes, OSU, Corvallis, OR
Publications:
Nirmala, J., Brueggeman, R., Maier, C., Clay, C., Rostoks, N., Gamini Kannangara, C.,
von Wettstein, D., Steffenson, B., and Kleinhofs, A. 2006. Sub-cellular localization and
functions of the barley stem rust resistance receptor-like serine/threonine-specific
protein kinase Rpg1. Proc. Nat. Acad. Sci (USA) 103:7518-7523.
39

Zhong, S., Toubia-Rahme, H., Steffenson, B. J., and Smith, K. P. 2006. Molecular
mapping and marker-assisted selection of genes for Septoria speckled leaf blotch
resistance in barley. Phytopathology 96:993-999.
Bilgic, H., Steffenson, B.J., and Hayes, P.M. 2006. Molecular mapping of loci conferring
resistance to different pathotypes of the spot blotch pathogen in barley. Phytopathology
96:699-708.
Zhang, L., Fetch, T., Nirmala, J., Schmierer, D., Brueggeman, R., Steffenson, B. J., and
Kleinhofs, A. 2006. Rpr1, a gene required for Rpg1-dependent resistance to stem rust in
barley. Theor. Appl. Genet. 113:847-885.
Steffenson, B. J., and Smith, K. P. 2006. Breeding barley for multiple disease resistance
in the Upper Midwest region of the USA. Czech J. Genet. Plant Breed. 42:79-85.
Dong, Y., Steffenson, B. J., and Mirocha, C. J. 2006. Analysis of ergosterol in single
kernel and ground grain by gas chromatography-mass spectrometry. J. Agric. Food
Chem. 54:4121-4125.
Friesen, T. L., Faris, J. D., Lai, Z., and Steffenson, B. J. 2006. Identification and
chromosomal location of major genes for resistance to Pyrenophora teres in a doubledhaploid
barley population. Genome 49:855-859.
Schwarz, P. B., Horsley, R. D., Steffenson, B. J., and Barr, J. M. 2006. Quality risks
associated with the utilization of Fusarium head blight infected malting barley. J. Am.
Soc. Brew. Chem. 64:1-7.
Mammadov, J. A., Steffenson, B., and Saghai Maroof. M. A. 2006. Physical mapping of
the Rph5 region in barley. Abstract P330. Plant & Animal Genomes XIV Conference,
January 14-18, 2006, Town & Country Convention Center, San Diego, CA.
http://www.intl-pag.org/14/abstracts/PAG14_P330.html
Roy, J. K. Muehlbauer, G., Smith, K., Carling, J., Kilian, A., Valkoun, J., and Steffenson,
B. 2006. Association mapping of disease resistance genes in wild barley. Abstract
P299. Plant & Animal Genomes XIV Conference, January 14-18, 2006, Town & Country
Convention Center, San Diego, CA. http://www.intlpag.org/14/abstracts/PAG14_P299.html
Drader, T. B., Johnson, K. A., Brueggeman, R. S., Kim, H. R., Kudrna, D., Wing, R.,
Steffenson, B., and Kleinhofs, A. 2006. Candidates for the barley spot blotch resistance
gene, Rcs5. Abstract P318. Plant & Animal Genomes XIV Conference, January 14-18,
2006, Town & Country Convention Center, San Diego, CA. http://www.intlpag.org/14/abstracts/PAG14_P318.html
40

Mirlohi, A., Brueggeman, R., Steffenson, B., and Kleinhofs, A. 2006. Identification of
new stem rust resistance gene Rpg1 alleles in Hordeum. Abstract P331. Plant & Animal
Genomes XIV Conference, January 14-18, 2006, Town & Country Convention Center,
San Diego, CA.
http://www.intl-pag.org/14/abstracts/PAG14_P331.html
Zhang, L., Castell-Miller, C., Steffenson, B., and Kleinhofs, A. 2006. Parallel expression
profiling of barley-stem rust interactions. Abstract P721. Plant & Animal Genomes XIV
Conference, January 14-18, 2006, Town & Country Convention Center, San Diego, CA.
http://www.intl-pag.org/14/abstracts/PAG14_P721.html
Brueggeman, R., Nirmala, J., Maier, C., Clay, C., Rostoks, N., Steffenson, B., Gamini
Kannangara. C., Von Wettstein, D., and Kleinhofs, A. 2006. In vivo and in vitro
characterization of the barley stem rust resistance gene Rpg1 kinase domains. Abstract
P792. Plant & Animal Genomes XIV Conference, January 14-18, 2006, Town & Country
Convention Center, San Diego, CA. http://www.intlpag.org/14/abstracts/PAG14_P792.html
Nirmala, J., Maier, C., Steffenson, B., and Kleinhofs, A. 2006. Towards identification
and characterization of signaling components interacting with the barley stem rust
resistance gene product Rpg1. Abstract P794. Plant & Animal Genomes XIV
Conference, January 14-18, 2006, Town & Country Convention Center, San Diego, CA.
http://www.intl-pag.org/14/abstracts/PAG14_P794.html
Roy, J. K. Muehlbauer, G., Smith, K., Carling, J., Kilian, A., Valkoun, J., and Steffenson,
B. 2006. Genetic diversity and linkage disequilibrium-based association mapping of
disease resistance in wild barley. Abstract W257. Plant & Animal Genomes XIV
Conference, January 14-18, 2006, Town & Country Convention Center, San Diego, CA.
http://www.intl-pag.org/14/abstracts/PAG14_W257.html
Bilgic, H., Castell-Miller, C. V., and Steffenson, B. J. 2006. Sequence polymorphisms in
the stem rust resistance gene Rpg1 in barley. Phytopathology 96:S12.
Steffenson, B. J. and Jin, Y. 2006. Resistance to race TTKS of Puccinia graminis f. sp.
tritici in barley. Phytopathology 96:S110.
Castell-Miller, C. V., Bilgic, H., and Steffenson, B. J. 2006. Toward the high-resolution
mapping of a quantitative trait locus conferring durable spot blotch resistance in barley
Phytopathology 96:S20.
41

Table 1. Results of the 2006 Minnesota barley disease survey (9 fields visited).
Disease/Pathogen Number of fields Range in Disease
with disease Severity or Incidence
Fusarium head blight 6 0.2%
(Fusarium spp.)
Net blotch 5 Trace to 30%
(Pyrenophora teres f. teres)
Bacterial Blight 3 Trace to 5%
(Xanthomonas translucens pv. translucens)
Septoria speckled leaf blotch 2 Trace to 25%
(Septoria and Stagonospora spp.)
Crown Rust 3 Trace to 25%
(Puccinia coronata)
42

Developing Improved Malt Barley Varieties for Montana and the Western US
Dr. Tom Blake, Professor, Montana State University
MISSION:
Our mission is to develop the next generation of malting barley varieties for barley
growers in Western North Dakota, Montana, Idaho and the Pacific Northwest. The MSU
barley improvement program has aggressively utilized agronomically superior parents for
the past two breeding cycles, crossing them with excellent malt quality parents and
selecting desirable recombinants. This use of high yield potential, exceptionally durable
lines like Baronesse, Stark, MT851195 and MT860756 as parents has enabled us to select
malt barley lines with agronomic performance nearly equal to the best feed barley
varieties currently available (table 1,2). The continuing diversion of corn to ethanol
production is likely to result in high feedgrain prices for the foreseeable future, making it
even more important to demonstrate that our malt barley varieties are agronomically
competitive with feed barley varieties.
The MSU barley improvement program is devoted to improving the farmgate value of the
Montana and US barley crops. We seek to reverse the slide in barley acreage from 1986
(more than 12 million acres nationally) to 2006 (less than 3 million acres nationally). We
will need to make barley economically more competitive with wheat to reverse this
unfortunate trend. While increasing yield will help, increasing quality and the reliability
of our dryland malt barley crop will be critical.
In the past year, we successfully won AMBA recommendation for ‘Craft’ (PI646158),
our first malt barley release with obviously improved agronomic performance. Our
objectives for the next two years are to guide Geraldine and Hockett successfully through
the AMBA plant scale evaluation and recommendation process (tables 1 and 2).
Our project has taken the lead in moving qGPC6H, the ‘Karl’ low grain protein gene on
chromosome 6, into all of our regionally adapted varieties. The primary action of this
gene appears to be to extend grainfill duration, enabling greater starch storage and
perhaps increasing grain yield and possibly malt extract (table 3). We recently purchased
a used Phoenix micromalting unit, and will explore how qGPC6H impacts quality and
agronomic performance in a wide variety of genetic backgrounds and environments.
Maximizing US barley’s potential as a starch production and storage system will improve
our competitive position relative to corn, while also improving barley’s malting quality.
Over the next five years we need to optimize barley straw as a substrate for cellulosic
ethanol production, an objective the MSU program began working on two years ago.
PRIMARY OBJECTIVE:
The primary objective of the MSU barley improvement program is to improve the
farmgate value and competitiveness of barley for Montana growers. Malt barley provides
growers with added value, and Montana’s position as a malt barley provider has
improved over the past decade. Enabling Montana’s dryland barley growers to reliably
produce high quality malt barley is our project’s primary goal.
43

Detailed <strong>Report</strong> on Objectives, Methodology and Results – AMBA Funded Project
The MSU barley improvement program utilizes single seed descent to speed our
pedigree-based breeding program. Montana’s cool, dry summers provide poor
environments for most pathogens, making it possible for our program to focus largely on
quality and adaptation. Our use of exotic sources of specific genes for which we develop
tightly linked molecular markers to utilize in backcrossing has resulted in several
advances in our germplasm pool, with the most recent being the deployment of qGPC6H
throughout our malting germplasm base.
We evaluate approximately 20,000 F5 families each year from 100-150 unique crosses.
From these we select about 2000 plants that provide seed for the following years’
unreplicated 2-row evaluation. Approximately 250 of these are selected for advancement
to our preliminary yield trial, planted on dryland at Huntley, MT and under irrigation at
Bozeman’s A.H. Post research farm. Sixty of these lines are advanced into our 64-entry
5 location Early Yield Trial, and the twenty best of these advance to our 11 location
statewide yield trial. Lines with malting potential are evaluated by the USDA malt barley
lab at Madison, WI. Lines demonstrating superior agronomic and malting performance
are advanced into the AMBA Pilot Testing System, and successful lines are proposed for
plant scale evaluation. The results of this system can be seen in tables 1 and 2.
Table 1: Montana’s Statewide Trials, 1997-present: Dryland Environments
ID Pedigree
#
yrs
Yield
(bu/ac)
#
yrs
Protein
(%)
#
yrs
Test
Wt
(lbs/bu)
#
yrs
Plump
(%)
#
yrs
Plant
Ht
(in)
#
yrs
Heading
Date
MT950186 Haxby 57 76.6 45 13.7 57 51.4 54 67.1 55 29.6 54 176.9
MT910189 Hockett 57 72.7 45 13.7 57 50.1 54 73.1 55 28.7 54 176.0
PI568246 Baronesse 56 76.0 45 14.0 56 48.8 54 64.4 55 28.4 54 178.9
BZ594-19 WPB Xena 51 74.5 39 14.1 51 48.9 48 62.4 49 30.1 49 178.4
MT960228 Eslick 50 75.9 40 14.2 50 49.0 49 61.2 49 28.1 48 179.2
SK 76333 Harrington 50 70.2 39 14.1 50 47.9 49 65.7 50 29.3 48 178.1
MT960101 Geraldine 46 72.4 36 14.5 46 48.5 45 54.5 46 27.4 44 180.3
MT970229 MT890021/Stark 46 76.4 39 14.1 46 50.5 45 71.8 45 29.0 44 178.6
MT970116 Craft 45 72.5 38 14.3 45 50.3 44 70.1 44 30.8 43 177.4
6B952482 Tradition 29 71.1 29 14.3 29 47.7 28 65.5 28 31.2 28 175.3
BZ596117 Boulder 29 78.4 23 13.9 29 50.4 29 73.4 28 29.3 27 179.2
YU501385 Baronesse/Camas 18 80.0 16 14.4 18 50.5 18 67.7 18 28.9 17 177.3
2B965057 Conrad 17 72.3 17 15.4 17 48.0 17 68.3 16 27.5 17 180.5
TR232 Metcalfe 16 73.0 14 14.2 16 49.7 16 67.5 22 31.4 15 175.6
Mean 28 74.4 25 14.6 28 49.3 27 68.5 28 29.3 27 177.7
In Table 1, we see the results from Montana’s dryland statewide trial, grown at 6
locations each year. Haxby (PI646160) is our highest yielding, highest testweight feed
barley variety for dryland producers. Hockett, Geraldine and Craft approach Haxby’s
superb local adaptation, and surpass Harrington in yield and test weight.
44

Table 2. Montana’s Statewide Trials, 1997-present: Irrigated Environments
ID PEDIGREE
#
yrs
Yield
(bu/ac)
#
yrs
Protein
%
#
yrs
Test Wt
(lbs/bu)
#
yrs
Plump
(%)
#
yrs
Plant Ht
(inches)
#
yrs
Heading
Date
2B965057 Conrad 15 119.79 14 13.24 15 51.44 15 75.95 15 42.71 12 175.65
6B952482 Tradition 25 116.63 22 12.43 25 49.98 25 78.66 25 46.12 20 172.73
BZ594-19 WPB Xena 33 125.47 27 12.22 33 51.13 33 75.36 33 43.86 25 175.51
BZ596117 Boulder 25 122.93 19 12.47 25 52.70 25 78.33 25 44.75 20 175.02
MT910189 Hockett 48 116.80 36 12.42 48 51.85 48 77.98 48 42.39 36 172.86
MT950186 Haxby (check) 47 121.18 35 12.18 47 53.08 47 76.27 47 43.12 35 176.00
MT960101 Geraldine 40 124.20 30 12.28 40 51.32 40 70.78 40 40.20 31 176.93
MT960228 Eslick 44 122.57 33 12.32 44 51.73 44 75.44 44 41.46 34 175.40
MT970116 Craft 40 122.69 32 12.17 40 52.93 40 80.23 40 44.64 31 174.40
SK 76333 Harrington 29 110.86 22 12.41 29 50.62 29 75.33 29 43.70 25 175.08
TR232 Metcalfe 10 114.59 10 12.67 10 50.59 10 74.83 10 43.51 8 176.28
MT970229 MT890021/Stark 40 124.34 32 12.41 40 52.48 40 80.41 40 43.71 31 176.23
Means 23 120.5 20 12.643 23 52.022 23 76.81 23 43.405 18 174.949
Table 2 further demonstrates the advantages provided by Geraldine, Hockett and Craft
relative to Harrington and Metcalfe when grown in high yield environments. In today’s
economic environment, the 10-13 bu/ac advantage that Geraldine provides relative to
Harrington and Metcalfe will result in a roughly $40/acre advantage to producers.
Should Geraldine be recommended by AMBA, it will find favor with Montana’s high
yield environment producers because of its lack of a yield potential penalty relative to the
best feed barley varieties in production.
Table 3. 2005 Madison quality reports for new and continuing entries into 2007
AMBA Pilot Scale Evaluation
Kernel on Barley Malt Barley Wort Alpha- Beta-
Weight 6/64" Color Extract Protein Protein S/T DP amylase glucan Overall
Entry Rowed (mg) (%) (Agtron) (%) (%) (%) (%) (°ASBC) (20°DU) (ppm) Rank
Harrington 2 40.9 88.0 83 80.2 11.9 4.73 40.4 121 69.3 133 8
2B914947 2 38.4 *83.6 81 79.9 11.8 4.86 44.4 132 74.6 88 4
2B965057 2 43.1 96.2 75 79.7 12.8 4.87 39.5 134 73.8 33 2
6B932978 6 *34.0 *79.2 81 77.7 13.1 4.93 38.7 171 69.1 107 13
6B952482 6 37.9 94.7 81 79.8 12.0 4.43 38.3 170 62.2 60 7
Morex 6 35.4 *83.9 83 78.2 12.5 4.33 37.0 169 55.7 154 11
MT010158 1 2 45.5 96.7 71 80.4 12.4 4.82 41.2 167 50.2 96 2
MT010160 1 2 44.0 91.3 81 80.5 12.5 4.89 41.9 173 68.7 210 8
MT020155 2 2 46.0 94.4 78 78.6 12.8 4.56 36.3 124 57.3 280 10
MT020205 2 2 43.4 92.0 78 77.7 14.2 4.88 36.5 185 81.6 269 15
MT030042 2 2 44.8 96.0 80 81.7 12.2 4.80 42.3 122 82.1 143 1
1 Continuing entries 2 New entries
45

The MSU barley improvement program remains a consistent contributor to the AMBA
Pilot Scale Evaluation Program. We select our best-performing lines that malt well in the
Madison Micromalt Evaluation Program and advance these to pilot scale evaluation. In
2007, we have two lines that passed first year evaluation, MT010158 and MT010160, and
have added three new lines, MT020155, MT020205 and MT030042 (Table 3).
Other Barley Research and Future Direction of Program
We extensively and effectively utilize molecular markers. While perhaps not speeding
the process of line development, they provide a level of certainty in selection. The ‘Karl’
low grain protein allele provides a perfect example. We initially mapped the gene (See et
al., 2002), and since then fine-mapped the gene with markers derived from an analogous
system found in wheat (Uauy et al., 2006). While tracking the genetics underlying this
trait, we identified lines that carry a double recombination event surrounding the Karl
allele, making it possible to backcross this gene into well-adapted, high quality lines
without dragging a large part of Karl’s chromosome 6 along. In a 2-replication yield trial
we grew last season, utilizing BC4 derivatives (the Lewis allele backcrossed into Karl,
and the Karl allele backcrossed into Lewis), we found that the Karl allele had a profound
impact on Lewis yield and quality (see Table 4). Yield and starch percentage were
improved, and grain protein percentage was reduced.
In wheat, this appears to be the result of a defect in a transcription factor (Uauy et al.,
2006), while in barley the molecular basis for this effect remains unclear. The
phenotypic effect of the allelic difference seems to be to increase the duration of grainfill
in the low grain protein lines. If there are no negative pleiotropic effects on malting
quality, this should enable the development of low protein percent, high starch percent
barley lines with increased yield potential.
Table 3. 2006 Replicated qGPC6H Yield Trial
Karl, Karl High Grain Protein BC4 lines; Lewis, Lewis Low Grain Protein BC4 lines
When superscripts are different, means differ at p

As a Barley CAP collaborator, our CAP effort is focused on the genetics underlying
response to drought. Last year we grew the spring barley lines in the cap, providing grain
to the WSU barley food research component of the CAP program, reserving seed for a 2location
replicated trial this spring. Following the 2007 harvest, we will determine the
degree of drought adaptation among lines in our 868 entry 2 location trial.
We are also working diligently to uncover the basis for variation in straw digestibility, as
an inducement for companies like Iogen to utilize barley straw as a feedstock for
cellulosic ethanol production. We demonstrates substantial variation in in rumen forage
digestibility in a sample of the lines from the USDA barley core collection. We will
complete our analyses of these lines this spring, and will regrow the core collection at the
Post Farm in the 2007 crop year for a second years’ analysis.
Project Personnel
Dr. Tom Blake, Professor Barley breeding and genetics, Montana State University
Dr. Victoria Carollo, Research Assistant Professor, Montana State University
Stan Bates, Research Associate
Mackenzie Ellison, Research Associate
Jeremy Jewell, Graduate Research Assistant
Jessica Patrick, Kyle Patrick, Duke Pauli, Chris Shafer, MSU undergraduate students
Recent Publications
1. Blake, T. Bowman, J. Hensleigh P., Boss D., Carlson G., Kushnak G., Eckhoff J.
Submitted. Release of ‘Haxby’ Barley. Crop Science.
2. Blake, T. Bowman, J. Hensleigh P., Boss D., Carlson G., Kushnak G., Eckhoff J.
Submitted. Release of ‘Hays’ Barley. Crop Science.
3. Blake, T. Bowman, J. Hensleigh P., Boss D., Carlson G., Kushnak G., Eckhoff J.
Submitted. Release of ‘Eslick’ Barley.
4. Blake, T. Bowman, J. Hensleigh P., Boss D., Carlson G., Kushnak G., Eckhoff J.
Submitted. Release of ‘Craft’ Barley. Crop Science.
5. Singh M, Chabane K, Valkoun J, Blake T. 2006. Optimum sample size for
estimating gene diversity in wild wheat using AFLP markers. Genetic Resources
and Crop Evolution 53:23-33.
Literature Cited
1. Uauy C, Distelfeld A, Fahima T, Blachl A, Dubcovsky J. 2006. A NAC Gene
Regulating Senescence Improves Grain Protein, Zinc and Iron Content in Wheat.
Science 314, 1298-1301.
2. See D, Kanazin V, Kephart K, Blake T., 2002. Mapping the genes controlling
variation in barley grain protein content. Crop Science 42: 680-685.
3. Wesenberg D.M., Hayes R.M., Standridge N.N., Burger W.C., Goplin E.D., and
Petr F.C. 1976. Registration of Karl barley. Crop Sci. 16: 737.
47

ANNUAL PROGRESS REPORT
Epidemiology and Control of Barley Leaf Diseases Caused by Fungal Pathogens
1. EXECUTIVE SUMMARY
Dr. M. R. Johnston
Department of Plant Sciences
Montana State University
Bozeman, MT 59717
Stripe rust Puccinia striiformis f. sp. hordei remains an important fungal disease of barley
in some areas of North America. Resistant cultivars are considered an economical means
of disease control.
Genes conferring broad based resistance to stripe rust have been mapped in a
“Baronesse” background. The next logical step would be the transfer of these genes into
lines/cultivars acceptable to the malting industry as well as to the grower. The (ongoing)
project develops quantitative, more durable types of resistance in an agronomically
desirable background. At the same time more information on the degree of dominance
and possible background effects will be gathered.
2. OBJECTIVES
2.1. Transfer QTLs for stripe rust resistance from several of the best two “Baronesse”
derivatives (based on disease resistance and genome architecture) into the malting
cultivars, “Legacy” and “Metcalfe” which were recommended by AMBA.
2.2. Epidemiological studies on virulence and spread of Puccinia striiformis hordei in
Montana.
2.3. Maintain inoculum of several fungal pathogens. Analyze virulence patterns of
different strains to be used in further genetic analysis of resistance.
3. MATERIALS AND METHODS:
3.1. Spring/summer 06 continue to produce missing F1 combinations.
Fall/Winter06: Cross each F1s back to malting parent to produce BC1 populations of
about 300 plants each for a total of 1200 plants.
Subsequently begin screening of BC1 with markers (would be part of a future
project).
48

3.2. A barley nursery composed of differential cultivars will again be evaluated in the
Gallatin Valley and possibly the Flathead Valley of Montana where stripe rust of
barley appeared for the first time in 1996. Commercial cultivars/lines will be
evaluated for stripe rust reaction in areas were the fungus appears naturally.
Virulence types will be compared to stripe rust of barley from other locations. Wild
grasses will be surveyed for the presence of the fungus as soon as the snow cover has
melted and green tissue is visible.
3.3. We maintain several of isolates of Puccinia striiformis hordei, stripe rust of barley,
which have been isolated from the Gallatin Valley over the past few years. These
isolates are periodically increased and purified by single uredosorus isolation. The
virulence will be determined by the reaction of a set of differential cultivars, which
are also maintained at MSU. Inoculum as well as differential cultivars are available
to other research projects at MSU and other locations worldwide.
Isolates of numerous other barley pathogens are being maintained at MSU. They are
being utilized for class room demonstration, disease screenings and are also
available to other researchers.
4. Results
4.1. F1 populations from all but a few combinations of the malting cultivars Metcalfe and
Legacy are now available. Where the seed supply was sufficient an F1 reaction to
stripe rust has been determined on 10 seedlings (Tab.1). Most F1 populations gave, as
expected, a susceptible reaction. Quantitative resistance is usually based on a number
of recessive genes. We have long suspected that Bison 2-22 with high resistance to
barley stripe rust located on chromosome 7H is based on a mayor gene. F1 results
confirm this hypothesis. Since rust fungi are capable to mutate and overcome
monogenic resistance rather easily, this gene should not be used by itself, but
combined with other sources. Resistant reactions could also be observed on QTLs
located on chromosome 5H indicating some dominance in these loci.
The screened plant have been saved and transplanted. If time allows the first
backcrosses could be made in late spring of 2007. There is also some left over F1 seed
from all combination of Metcalfe and Legacy with the resistance donors.
49

Table 1. Barley stripe rust reactions of QTL lines, parents and F1 plants from
crosses of malting barley cultivars Metcalfe(M) and Legacy(L).
# Bison Line QTL BSTR Reaction 1 F1 BSTR Seedling
Reaction
Sdlg 2
Tllrg 2
Hdg 2
M L
4 69 5 3 R 3
7 7
5
6
128
191
1H
7
4
6
1
R
R
7
-
8
-
7
8
104
129
4H
3
4
1
1
R
R
-
-
7
-
9 87 8 7 S 7 -
10
11
111
136
5H
8
7
7
8
I
I
7
3
7
7
12 217
4 2 I - 3
13 2-22 7H 0 1 R 0
14 216-4 4 1 R - 7
15 243-4 1Hx4H 5 3 I - 7
16 136-2
4 1 R - -
17
18
218-1
110-3
1Hx5H
5
6
1
5
R
I
-
-
-
7
19
20
217-2
22-4
4Hx5H
4
4
1
2
I
R
-
-
7
7
21 95-2 1Hx4Hx5H 4 1 R - 7
22 Metcalfe Malting Parent 8 - - - -
23 Legacy Malting Parent 8 - - - -
24 Baronesse 7 - - - -
25 BCD12 4 - - - -
26 BCD47 4 - - - -
1 Scale 0 – 9; 0-3 = resistant, 4-6 = intermediate, 7-9 = susceptible
2 Sdlg = seedling; Tllrg = tillering; Hdg = heading
3 R = resistant, I = intermediate, S = susceptible
4.2. Barley stripe rust was not observed in any nursery in 2006, although we experienced
a severe outbreak of wheat stripe rust on susceptible cultivars in western and central
Montana. Stripe rust of wheat can over-winter on winter wheat under snow cover in
Montana, whereas winter barley is not grown in the state. Any over-wintering would
Thus have to occur on susceptible wild grasses.
4.3. We continue to maintain collections of numerous barley pathogen; Puccinia hordei,
leaf rust; Puccinia graminis ,stem rust; Puccinia striiformis hordei, stripe rust;
Rhynchosporium secalis, scald; Pyrenophora teres, net blotch and Erysiphe graminis,
powdery mildew. All cultures with the exception of powdery mildew are preserved
by various means. Powdery mildew is maintained on living plants in isolation in a
cold room at 40F.
50

5. Related Barley Projects
Barley Portion of USDA/ARS stripe rust initiative: Precise phenotyping of barley stripe
rust resistance gene introgression.
Barley elite lines or cultivars from areas at greatest risk for stripe rust development
(Montana, California, Idaho and Washington) are being utilized for gene introgression.
Numerous crosses have been made by cooperators. First F1 populations are currently
being screened at MSU.
6. Publications
Richardson, K., Chen, X., Johnston, M., Capettini, F Vales, I., Kling, J., Mundt, C.,
and Hayes, P. Barley stripe rust resistance QTL alleles are effective across growth stages,
races, and environments. Submitted to Phytopathology.
51

Breeding and Genetics of Six-rowed Malting Barley
Richard D. Horsley
Department of Plant Sciences
North Dakota State University
Executive Summary
The objective of the project is to develop and release improved six-rowed malting barley
varieties acceptable to barley producers in North Dakota and adjacent areas in the
United States, and to those who use or process this barley. This objective is being
accomplished using traditional breeding methodologies. Traits receiving top priorities
are improved malt quality, resistance to Fusarium head blight (FHB) and foliar diseases,
reduced deoxynivalenol (DON) accumulation, and improved agronomic performance. In
the short-term, varieties with acceptable malt quality will be developed that accumulate
25% less DON than Robust. Long-term goals are to develop varieties that accumulate
75% less DON than Robust. Today’s growers have many choices of crops to produce.
All new varieties with acceptable malting and brewing quality also must have sufficient
agronomic performance to make them competitive with other barley varieties and other
crops. Our improved varieties must consistently meet the quality needs of the malting
and brewing industries and the demands of the growers.
For the first time, a line (ND20448) from our breeding project with improved FHB
resistance and acceptable malt quality was found satisfactory in its first year of Pilot
Scale Evaluation. ND20448 accumulates about 30% less DON than Robust, yields
intermediate to Robust and Drummond, and appears to have acceptable malt quality.
In 2006, 391 of the 729 experimental lines we evaluated in replicated yield trials came
from our FHB-resistance breeding project.
The North Dakota Agricultural Experiment Station released Stellar-ND, tested as
ND16301, in February 2005. Stellar-ND has a high yield potential across a wide range
of growing conditions in the northern Great Plains, excellent straw strength during the
growing season and at harvest, and excellent malt quality. Miller Brewing found Stellar-
ND to be satisfactory in two years of Plant Scale evaluation and Anheuser-Busch found
Stellar-ND to be satisfactory in one-year of testing. The second-year of Plant Scale
testing by Anheuser-Busch will be done using grain produced in 2006.
Objectives, Methodology and Results – AMBA Funded Project
The six-rowed barley improvement research program at the North Dakota Agricultural
Experiment Station (NDAES), North Dakota State University, Fargo, is a cooperative
effort among the Departments of Plant Sciences and Plant Pathology. The fundamental
objective of the program is to develop and release improved barley varieties acceptable
to barley producers in North Dakota and adjacent areas in the United States, and to
those who use or process this barley. Basic and applied research is conducted at NDSU
on barley to provide information that will facilitate achievement of the barley
improvement goals, improve cultural practices, and enhance our understanding of
barley.
52

Barley Breeding - Advanced Testing Program - General
Important commercial barley varieties, new varieties, and promising advanced
selections were evaluated in seven trials at six locations in North Dakota and two trials
in Sidney, MT in during 2006. In addition, "off-station" trials with new and check
varieties were conducted by the Carrington, Dickinson, Hettinger, Langdon, North
Central (Minot), and Williston Research Extension Centers. Pure seed increase plots of
experimental lines (30) in Varietal and Advanced Yield Trials were grown at Fargo and
Casselton. Pure seed increase plots of experimental lines (112) in the Intermediate
Malting Barley Yield Trial and the Intermediate Low-protein Malting Barley Yield Trial
were grown at Fargo. Regional trials grown were the Mississippi Valley Barley Nursery,
the Western Regional Spring Barley Nursery, the Western Regional Dryland Spring
Barley Nursery, the Canadian Western Cooperative Six-row Trial, and the Canadian
Western Cooperative two-row Trial. We evaluated lines in the USDA-CSREES Barley
CAP for resistance to FHB and DON accumulation in our Osnabrock FHB Nursery and
for resistance to preharvest sprouting (PHS) in the greenhouse.
Plant emergence in yield trials was uniform at all locations. Moisture at planting and up
to heading was adequate. Following heading precipitation was below average and
temperature was above average across North Dakota. The below average precipitation
during this period prevented development of FHB and foliar diseases. Yields and kernel
plumpness were reduced due to the hot, dry conditions. Rain during harvest at our
Osnabrock, ND research site caused damage due to PHS in some entries.
The NDAES released Stellar-ND, tested as ND16301, in February 2005. Stellar-ND
yields greater than Robust and similar to Lacey and Tradition (Table 1). Stellar-ND has
a heading date similar to Lacey, and lodging resistance intermediate to that of
Drummond and Tradition. Straw strength of Stellar-ND at harvest maturity is similar to
that of Drummond and Tradition, making it a candidate for straight combining. Kernel
plumpness and malt extract of Stellar-ND are greater than that of Robust, Lacey,
Legacy, Drummond, and Tradition (Table 2). Miller Brewing found Stellar-ND to be
satisfactory in two years of Plant Scale Evaluation and Anheuser-Busch found Stellar-
ND to be satisfactory in one-year of testing. The second-year of Plant Scale Evaluation
by Anheuser-Busch will be done using grain produced in 2006.
Twenty-one experimental lines were grown at six North Dakota locations (Fargo,
Carrington, Minot, Osnabrock, Nesson Valley, and Williston) and at Sidney, Montana in
their third or fourth year of yield trials. The USDA-ARS Cereal Crops Research Unit,
Madison, WI, evaluated selected lines for malt quality and Dr. Stephen Neate evaluated
lines for resistance to spot and net blotch in greenhouse tests.
53

Table 1. Agronomic comparisons of six-rowed varieties grown in North Dakota yield
trials, 1999-2006.
Days to Plant
Stem Test
Yield heading height Lodging breakage weight
Variety (bu/ac) (days after 5/31) (inches) (1-9)† (1-5)‡ (lb/bu)
Station years 42 41 40 14 15 21
Stellar-ND 86.6 26.2 30.7 2.6 2.6 49.6
Robust 79.8 26.6 32.6 4.0 3.3 50.5
Lacey 86.6 26.3 30.3 3.2 2.7 50.7
Legacy 84.4 28.2 31.6 4.0 3.2 49.6
Drummond 84.5 26.0 31.6 2.3 2.3 50.1
Tradition 86.3 26.3 31.3 3.4 2.5 50.1
†Lodging score of 1 = no lodging, 9 = severe lodging.
‡Stem breakage score of 1 = no stem breakage at harvest, 5 = severe breakage at
harvest.
Table 2. Malt quality comparisons of Stellar-ND and other six-rowed barley varieties
grown in North Dakota yield trials, 1999-2005†.
Barley Plump Malt Wort Diastatic Alpha-
protein kernels extract protein S/T power Amylase
Entry (%) (%) (%) (%) (%) ( o L) (20 o DU)
Station years 16 16 16 16 16 16 16
Stellar-ND 13.3 84.2 79.8 5.97 47.3 187 71.6
Robust 14.0 74.4 78.7 5.86 43.5 177 56.7
Lacey 13.6 77.8 79.1 5.90 45.3 173 66.7
Legacy 13.3 70.0 79.2 6.29 49.3 175 83.4
Drummond 13.6 78.2 79.0 5.79 44.4 186 67.1
Tradition 13.5 80.6 78.9 5.50 42.3 193 65.9
† Data courtesy of the USDA-ARS Cereal Crops Research Unit, Madison, WI.
Four lines currently are being evaluated in the AMBA Pilot Scale Evaluation Program.
The lines are ND20299 (ND16924/ND17082), ND20448 (ND16918/C98-10-155-3),
ND20508 (ND16918*2/CIho 6610), and ND21306 (Drummond*2/FEG4-66L). ND20448
and ND20508 are in their second year of Pilot Scale Evaluation and both were rated
satisfactory last year by AMBA. ND20299 and ND21306 are in their first year of
evaluation. Based on unacceptable agronomic and DON data from 2006 (data not
presented), ND20508 will not be evaluated in yield trials in 2007. ND20299, ND20448,
and ND21306 will be evaluated in 2007 and comparisons of these lines to currently
grown varieties are presented in Tables 3-8.
54

Table 3. Agronomic comparisons of ND20299, Robust, and Drummond grown in North
Dakota yield trials, 2002-2006.
Grain Days to Plant Stem Test
Variety yield heading height Lodging breakage weight
(bu/ac) (days after 5/31) (inches) (1-9)† (1-5)‡ (lb/bu)
Station years 30 29 27 9 7 9
ND20299 87.1 27.0 29.2 3.5 2.9 50.1
Robust 76.7 27.9 31.2 4.1 3.3 50.6
Drummond 83.5 27.0 30.3 2.5 2.6 50.6
†Lodging of 1 = no lodging and 9 = severe lodging.
‡ Stem breakage of 1 = no breakage at harvest and 5 = severe breakage at harvest.
Table 4. Malt quality comparisons of ND20299, Robust, and Drummond grown in North
Dakota yield trials, 2002-2005†.
Barley Plump Malt Wort Diastatic Alpha-
protein kernels extract protein S/T power Amylase
Entry (%) (%) (%) (%) (%) ( o L) (20 o DU)
Station years 10 10 10 10 10 10 10
ND20299 12.7 90.6 79.4 5.59 46.1 172 63.4
Robust 13.9 81.3 78.8 5.85 43.6 171 55.2
Drummond 13.4 85.3 79.0 5.71 44.3 180 65.2
†Data courtesy of the USDA-ARS Cereal Crops Research Unit, Madison, WI.
Table 5. Agronomic comparisons of ND20448, Robust, and Drummond grown in North
Dakota yield trials, 2002-2006.
Grain Days to Plant Stem
Variety yield heading height Lodging breakage DON
(bu/ac) (days after 5/31) (inches) (1-9)† (1-5)‡ (ppm)§
Station years 30 29 27 9 7 7
ND20448 79.0 27.3 31.3 3.1 2.6 5.5
Robust 77.1 27.9 31.3 4.1 3.2 7.8
Drummond 83.0 27.1 30.3 2.5 2.6 --
†Lodging of 1 = no lodging and 9 = severe lodging.
‡ Stem breakage of 1 = no breakage at harvest and 5 = severe breakage at harvest.
§DON = deoxynivalenol. Data provided by Dr. Paul Schwarz, Dept. of Plant Sciences,
NDSU.
55

Table 6. Malt quality comparisons of ND20448, Robust, and Drummond grown in North
Dakota yield trials, 2003-2005†.
Barley Plump Malt Wort Diastatic Alpha-
protein kernels extract protein S/T power Amylase
Entry (%) (%) (%) (%) (%) ( o L) (20 o DU)
Station years 9 9 9 9 9 9 9
ND20448 12.9 90.8 79.5 5.89 48.5 153 71.0
Robust 13.7 81.3 79.0 5.80 43.9 171 56.1
Drummond 13.2 85.3 79.2 5.68 44.7 181 65.8
†Data courtesy of the USDA-ARS Cereal Crops Research Unit, Madison, WI.
Table 7. Agronomic comparisons of ND21306, Robust, and Drummond grown in North
Dakota yield trials, 2003-2006.
Grain Days to Plant Stem Test
Variety yield heading height Lodging breakage weight
(bu/ac) (days after 5/31) (inches) (0-9)† (1-5)‡ (lb/bu)
Station years 19 18 17 6 6 6
ND21306 83.3 28.6 29.8 2.5 2.5 51.2
Robust 74.7 28.7 31.7 3.9 3.4 50.6
Drummond 80.3 27.8 30.5 2.1 2.6 51.2
†Lodging of 1 = no lodging and 9 = severe lodging.
‡ Stem breakage of 1 = no breakage at harvest and 5 = severe breakage at harvest.
Table 8. Malt quality comparisons of ND21306, Robust, and Drummond grown in North
Dakota yield trials, 2003-2005†.
Barley Plump Malt Wort Diastatic Alpha-
protein kernels extract protein S/T ‡ power Amylase
Entry (%) (%) (%) (%) (%) ( o L) (20 o DU)
Station years 6 6 6 6 6 6 6
ND21306 13.5 87.3 78.5 5.32 40.2 169 66.5
Robust 14.8 81.8 77.8 5.84 41.0 182 55.5
Drummond 14.2 85.8 78.7 5.82 41.9 193 67.8
†Data courtesy of the USDA-ARS Cereal Crops Research Unit, Madison, WI.
Intermediate and Preliminary Yield Trials
Seventy-four lines were grown in the Intermediate Malting Barley Yield Trial at six
locations in North Dakota (Fargo, Carrington, Osnabrock, Minot, Nesson Valley, and
Williston) and Sidney, Montana for their second year of evaluation. Thirty-eight
experimental low-protein barley lines were grown in the Intermediate Low-protein
Malting Barley Yield Trial grown at three locations in North Dakota (Minot, Nesson
Valley, and Richardton) and Sidney, Montana for their second year of evaluation.
Selected lines from both trials were evaluated for malt quality by the USDA-ARS Cereal
Crops Research Unit at Madison, WI and for spot blotch and net blotch reactions in
greenhouse tests conducted by Dr. Neate. About 625 experimental lines were grown in
56

Preliminary Malting Barley Yield Trials at the Osnabrock and Nesson Valley research
sites.
Early Generation Selections
Single spikes from the F3 and F4 generations were sown at the Osnabrock and Nesson
Valley research locations. About 8,600 head rows representing material from 74
crosses were sown. Selection of head rows in the field was based on maturity, plant
height, straw strength, kernel plumpness, kernel color, FHB resistance, awn type, spike
length, spike erectness, and spike density. About 2,100 selections were made and
submitted to Dr. Paul Schwarz’s laboratory (Dept. of Plant Sciences) for malt quality
prediction tests. Selections were evaluated for barley kernel assortment, barley grain
protein, test weight, and kernel color. Spikes from the selected rows were sown in
Yuma, AZ to increase seed for 2007 yield trials. Rows from the selections with the best
malt quality estimates, and acceptable straw strength, maturity, and uniformity will be
harvested and advanced to the Preliminary Malting Barley Yield Trials.
One-hundred three F2 populations were grown at the Osnabrock and Nesson Valley
research locations. Selection among and within populations was based on the same
criteria used in the F3 and F4 progeny rows. Thirty-seven F1 populations from crosses
made in the 2006-spring greenhouse were grown in the field at Nesson Valley. Onehundred
thirty-two crosses were made during fall of 2006 in the greenhouse to combine
favorable agronomic characteristics, disease resistance, and malt quality traits. Over
50% of the crosses made used parents that showed resistance to FHB and/or septoria
speckled leaf blotch.
Breeding for Fusarium Head Blight Resistance
Since the inception of our work on breeding for FHB resistance, a main objective has
been to transfer FHB-resistance from unadapted resistant accessions to our elite
Midwest six-rowed malting barley germplasm. In 2006, 391 of the 729 lines we
evaluated in replicated yield trials came from our FHB-resistance breeding project. In
2007 the percentage of lines from this breeding project will continue to increase. For
example, 15 of the 20 experimental lines in our 2007 Advanced Yield Trial (AYT) will
come from our FHB-resistance breeding project. This AYT is very important since it is
the source of parents that will be used for the 2007 fall crossing block and the source of
lines for entry in the 2008 AMBA Pilot Scale Evaluation Program.
A limitation in our effort to develop FHB-resistant varieties is the well documented
linkage block near the centromeric region of chromosome 2H that is found in most of
the resistant accessions. The linkage block includes loci controlling FHB resistance,
DON accumulation, heading date, plant height, and maturity. This linkage block helps
to explain our inability to identify progeny from crosses to FHB-resistant lines that have
acceptable plant height, even in F2 populations with > 15,000 plants. A strategy we are
using to overcome the negative linkages in chromosome 2H is to control plant height
and maturity using genes from outside the critical region. For example, parents
containing the sdw1 gene in chromosome 3H were crossed to tall FHB resistant lines as
a means for reducing plant height in FHB resistant progeny. About 36% of the
57

selections made in the 2006 head row nurseries were crosses that combined the
semidwarf and low DON accumulation characters. Lines from these selections will be
entered in the 2007 Preliminary Yield Trials.
For the first time, a line (ND20448) from our breeding project with improved FHB
resistance and acceptable malt quality may be a candidate for Plant Scale Evaluation in
2008. Seed increases of this line will be done this summer in North Dakota and next
winter in Arizona to produce sufficient seed for Plant Scale Testing in 2008.
Other Barley Research and Future Direction of Program
Western North Dakota Malting Barley Program
The Western Malting Barley Project was initiated by the North Dakota Legislature in
2001 because of the increased interest of western North Dakota growers in producing
malting barley and the industry sourcing grain from this area. Because of the dryer
growing conditions of western North Dakota, FHB may be less problematic; however,
these same dry conditions also can result in barley with unacceptably high grain protein
and low kernel plumpness. One of the goals of the Western Malting Barley Project is to
develop malting barley varieties specifically adapted to dryland production in western
North Dakota. To overcome the problems of high protein, we have been crossing into
our lines genes for low-protein using conventional breeding methods. These genes for
low-protein can reduce grain protein by as much as 2.5 percentage units.
Another goal has been to develop varieties specifically adapted for production under
irrigation. To maximize production of irrigated malting barley, we are breeding lines that
incorporate the low-protein character and reduced plant height. We believe that these
two characters together in a variety will allow a grower to increase yield by increasing
the amount of nitrogen fertilizer they can add, while still keeping grain protein ≤ 13.0%
and the crop standing. Research is described below on a soil fertility study related to
this effort.
Special Projects
1. Rongshuang Lin, a Ph.D. candidate from China, is conducting several studies to
gain a better understanding of pre-harvest sprouting (PHS) in barley. In 2006,
Rongshuang determined that QTL for PHS-resistance map to the distal end of the
long arm of chromosome 5H using three mapping populations (Chevron/Stander,
Harrington/Morex, and Robust/Stander). Preliminary results indicate that the QTL
identified in Harrington/Morex may be different from those identified in the other
populations and that QTL for PHS in chromosome 5H are linked to a QTL controlling
malt alpha-amylase activity.
2. Martin Hochhalter, a research specialist on the project, is working toward an M.S
degree. His research is an agronomic study on the fertilization requirements of lowprotein
barley produced on dryland or irrigated conditions. He is evaluating 24
barley genotypes, including Robust, Drummond, Lacy, Legacy, Tradition, and
Conlon; six conventional height genotypes with the low-protein character; six semi-
58

dwarf genotypes with the conventional-protein character; and six semi-dwarf
genotypes with the low-protein character. Each of these genotypes is being
subjected to four different fertility regimens and all pertinent agronomic and malt
quality data are collected. In 2005, the research was conducted under dryland
conditions in Minot and Williston, ND. In 2006, research under dryland conditions
was conducted at Richardton and Williston, and research under irrigated conditions
was conducted at the Nesson Valley research site near Tioga, ND. In 2007 the
research will be conducted again at the Nesson Valley irrigated research site.
3. Fabio Pedraza, a Ph.D. student from Columbia, is conducting research that will
focus on determining the genetic basis for the dissimilarities in the malt quality of
Robust and Stander, and to use this information to design a marker assisted
selection (MAS) breeding strategy for developing varieties that meet the needs of
different brewers. Fabio is using SNP data derived from the USDA-CSREES-
NRI/Barley CAP to generate primers that may be useful for MAS.
4. Adisu Negeri, a Ph.D. student from Ethiopia, is conducting research to identify QTL
controlling FHB resistance and heading date in the Chinese variety Shenmai 3.
Unlike most other Chinese accessions with FHB-resistance, Shenmai 3 has plant
height and maturity similar to Midwest barley. As part of his project, Adisu will
develop a genetic map comprised of diversity array technology (DArT), simple
sequence repeat (SSR), and sequence tagged sites (STS) markers. This map will
be used to identify QTL associated with FHB and DON accumulation, days to
heading, and plant height, and to determine the linkage among agronomic and
morphological traits previously reported to be associated with FHB severity and
DON accumulation.
Future Direction of the Program
The direction of the program will continue as it has the past several years, with
development of improved six-rowed malting barley varieties with FHB resistance being
our top priority. The biggest change to our program for 2007 will be incorporating the
two-rowed program into our program due to the retirement of Dr. Jerome Franckowiak
in July 2007. The transitioning of the two-rowed project with the six-rowed project will
take about two years. There is excellent malting barley germplasm in the two-rowed
program as evidenced by the release of the variety Pinnacle in January 2007. Pinnacle
was tested as 2ND21863. The two-rowed project is being continued using funds from
the USDA-ARS National Wheat and Barley Scab Initiative. However, varieties from this
component of the program will be best adapted to the areas of North Dakota where FHB
is a problem. Development of two-rowed varieties for the dryland areas of western
North Dakota will be slowed because of reduced funding.
Project Personnel
R.D. Horsley, Professor and barley breeder
Martin Hochhalter, Research Specialist II and M.S. Candidate
Jason Faller, Ag. Research Technician III
59

Rongshuang Lin, Ph.D. Candidate
Fabio Pedraza, Ph.D. Candidate
Adisu Negeri, Ph.D. Candidate
Recent Publications
Peer-reviewed
Horsley, R.D., J.D. Franckowiak, P.B. Schwarz, and S.M. Neate. 2006. Registration of
Stellar-ND barley. Crop Sci. 46:980.
Horsley, R.D., J.D. Pederson, P.B. Schwarz, K. McKay, M.R. Hochhalter, and M.P.
McMullen. 2006. Integrated use of tebuconazole and Fusarium head blight
resistant genotypes in controlling Fusarium head blight and deoxynivalenol
accumulation. Agron. J. 98:194-197.
Horsley, R.D., D. Schmierer, C. Maier, D. Kudrna, C.A. Urrea, B.J. Steffenson, P.B.
Schwarz, J.D. Franckowiak, M.J. Green, B. Zhang, and A. Kleinhofs. 2006.
Identification of QTL associated with Fusarium head blight resistance in barley
accession CIho 4196. Crop Sci. 46:145-156.
Schwarz, P.B., R.D. Horsley, B.J. Steffenson, B. Salas, and J.M. Barr. 2006. Quality
risks associated with the utilization of Fusarium Head Blight infected malting barley.
Accepted: J. Am. Soc. Brew. Chem. 64:1-7.
Abstracts
C. Boyd, C. Maier, S. Sushailo, R. Horsley, and A. Kleinhofs. 2006. Genetic and
physical mapping of the balrey chromosome 2 (2H) Vrs1 region Fusarium head
blight resistance QTLs. P. 87-90. In S.M. Canty, A. Clark, and D. Van Sanford
(eds.) Proc of the 2006 National Fusarium Head Blight Forum, Research Triangle
Park, NC, 10-12 Dec 2006. Michigan State University, East Lansing, MI.
60

STUDIES ON BARLEY DISEASES AND THEIR CONTROL
Stephen Neate
Department of Plant Pathology
North Dakota State University
Fargo, ND 58105
ANNUAL PROGRESS REPORT
Fiscal year 2006/2007
EXECUTIVE SUMMARY
Diseases are the most important factor in limiting the yield and quality of malting barley
production in the upper mid-west of the US. The objectives of the Barley Pathology Project at
North Dakota State University are to maintain and enhance resistance to barley diseases and
develop timely, practical methods for disease control to ensure that the quantity and quality of
malting barley in the upper Midwest are not limited by disease. We will achieve this goal through
the support of the development of cultivars with genetic resistance to current and emerging
diseases, as well as through the development of cultural and chemical management strategies.
To accomplish this goal we have an ongoing program of surveying and research as well as
working closely with state and industry breeders throughout the region.
In 2006/07 we,
• Conducted field trials and or screened in China or several locations in North Dakota
breeding material and elite selections for breeders and geneticists at NDSU, USDA and
several mid-west breeding programs.
• Screened 872 lines in two separate tests in the greenhouse or in the field for resistance to
spot blotch. Of the two-rowed lines tested in the winter of 2006/07, 31 % were resistant
and of the six-rowed lines 49% were resistant.
• Screened 494 lines in the greenhouse for resistance to net blotch, but due to low disease
development we are currently repeating those tests.
• Surveyed 108 fields for 12 barley diseases and posted weekly graphics summarizing the
disease progress with accompanying interpretation on the NDSU web site, which allowed
farmers and industry to make more informed disease control decisions. Diseases were
lower in 2006 than for the last 5 years, both for FHB, leaf spots and rust. Insect levels were
moderate especially later in the season.
• Identified the chromosomal location of the leaf rust resistance gene Rph13 and identified a
new leaf rust resistance gene. Leaf rust is a common late season disease of barley in the
upper mid-west. The new gene is particularly interesting as it has a slow-rusting phenotype
which is highly desirable. We have identified one microsatellite (SSR) marker closely linked
to Rph13 which if combined with other markers may be useful for marker assisted
selection.
• Identified that the septoria speckled leaf blotch pathogen is genetically diverse throughout
ND and western MN and that although a sexual stage has not been identified it is likely to
exist. This information is critical for making decisions about how to deploy resistance
genes for this important disease.
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• Our continued collaboration on leaf and head disease screening contributed to the
development of a good disease resistance package in line ND20448, a line with less FHB
than Robust and good malting characteristics. ND20448 was found acceptable in its first
year of AMBA Pilot Scale Evaluation.
• Our continued collaboration on leaf and head disease screening contributed to the
development of a good disease resistance package in Stellar-ND, which has been found
satisfactory in one year of Anheuser-Busch plant scale testing and two years of Miller
Brewing plant scale testing.
DETAILED REPORT
1. Evaluate elite barley germplasm and segregating breeding populations for
resistance to Fusarium Head blight (FHB) and the accumulation of deoxynivalenol.
Working together with the breeders, Prof R. Horsley and Prof. J. Franckowiak, NDSU (until
retirement in July 06), the barley pathology project has led the establishment, inoculum
production, inoculation, maintenance and screening for FHB resistance in irrigated and
dryland plots of barley in North Dakota. In 2006, a total of 162 NABSEN rows were
screened for Fusarium Head Blight in multiple field locations including irrigated experiments
at Fargo, China and Langdon.
The barley pathology group also screened under both irrigated and non-irrigated
environmental conditions about 300 rows of unique lines being tested for Fusarium
resistance from the combined program of Dr Lynn Dahleen at Cereal Crops Research Unit
USDA Fargo and Dr Phil Bregitzer at Small Grains Research Unit USDA, Aberdeen Idaho.
The North American Barely Scab Evaluation Nursery (NABSEN) trials were co-ordinated
and ND trials were established at Langdon ND, Fargo ND and Hangzhou China with part
funding from the US Wheat and Barley Scab Initiative. The 48 experimental entries and the
6 resistant or susceptible controls include lines from NDSU 6-rowed breeding program,
NDSU 2-rowed breeding program, Minnesota breeding program, Busch-Ag breeding
program, CIMMYT breeding program and the Agriculture & Agri-Food Canada Research
breeding program as well as the USDA Cereal Crops Research Unit Barley Genetics
program. This collaboration facilitates exchange of germplasm between the participating
breeding programs and allows comparisons of performance across a range of
environments.
Approximately 1,000 rows of lines from the diverse germplasm screening projects at NDSU
Barley Pathology, Minnesota Barley Pathology, Agriculture Canada Brandon and
ICARDA/CIMMYT Barley Breeding Program were evaluated in off-season trials at Zheijiang
University in Hangzhou China. A total of 157 rows of the same material were evaluated in
Fargo ND and Langdon ND. Assessments were made when entries were at the soft dough
stage of development. Characters recorded included, infected kernels per head, number of
infected heads and total kernels per head.
2. Screen barley cultivars for resistance to spot blotch.
Field: A total of 189 plots of advanced 2-rowed or 6-rowed breeder’s lines were screened
for spot blotch (Cochliobolus sativus) resistance in the field at Fargo in irrigated and
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inoculated plots. The inoculum used was isolate SB85 of C. sativus. Due to the extremely
dry conditions, even with irrigation and inoculation, good disease severity did not develop
on the susceptible controls and the screening was abandoned.
To compensate for the loss of the field screening, the 189 lines were sown in the
greenhouse in the fall of 2006 and tested for seedling spot blotch resistance by inoculation
with isolate ‘SB85’ of C. sativus. One hundred and forty lines showed good resistance, 41
lines showed intermediate resistance and the remainder were segregating ie showing a
range of symptoms in the one pot.
Greenhouse, 2-rowed: 182 lines were tested for seedling spot blotch resistance in the
greenhouse by inoculation with isolate ‘SB85’ of C. sativus. The resistant check NDB112
exhibited the expected ‘2 to 3’ type of resistant reaction and the susceptible check ND 5883
exhibited the expected ‘7 to 8’ type of susceptible reaction. Fifty seven tested lines were
resistant to spot blotch 22 lines showed an intermediate reaction and 101 lines were found
to be susceptible. One line was found to be segregating ie showing a range of symptoms in
the one pot; further selection is needed for this line.
Greenhouse, 6-rowed: 312 lines were tested for seedling spot blotch resistance in the
greenhouse by inoculation with isolate ‘SB85’ of C. sativus. The resistant check NDB112
exhibited the expected ‘3 to 4’ type of resistant reaction and the susceptible check ‘ND
5883’ exhibited the expected ‘7 to 8’ type of susceptible reaction. One hundred and fifty
three lines were resistant to spot blotch, 80 lines showed an intermediate reaction, 77 lines
showed a susceptible reaction and 2 lines were segregating. This is significantly greater
resistance than has been seen in 6 rowed breeding materials in the last 5 years.
Screen breeding lines for resistance to net blotch. In our first screening in the
greenhouse of 182 two-rowed and 312-six-rowed breeding lines for net blotch resistance in
December 2006 we got poor disease symptoms on “NDB112” the resistant check. The 494
lines were re-sown in February 2007 and inoculated in March 2007. They are currently
being assessed for resistance.
3. Leaf disease resistance screening of unique barley germplasm
To study the genetic diversity in P. hordei, 45 isolates with diverse virulence patterns and
geographical origins were analyzed using AFLP markers. Six AFLP primer-pair
combinations were used to generate a total of 782 polymorphic markers. The P. hordei
isolates clustered into five groups: Group I contained a single rare isolate that is virulent on
all resistance genes except for Rph13 and Rph15; group II contained a single isolate
virulent on the resistance gene Rph15; group III contained two isolates; group IV contained
24 isolates, 11 from the US and 13 from diverse locations around the world; and group V
contained 17 isolates, 8 from California, 6 from other US states and 3 from Europe. The
study revealed that molecular diversity in P. hordei can be associated with virulence, but
not geographic origin.
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A second aim of this research was to study the leaf rust resistance gene Rph13 in the
barley line “PI531849”, to identify putative resistance related markers, and to map the gene
using molecular markers. Crosses were made between the resistant parent “PI 531849”
containing resistance gene Rph13 and “Bowman”, a cultivar susceptible to leaf rust.
Inheritance studies were performed by screening F2 progeny and F2:3 families against
pathotype “Race 8” of P. hordei to determine the leaf rust resistance genes in “PI 531849”.
Data showed a single resistance gene, Rph13, acted in a dominant manner. Using
diversity array technology (DArT) and simple sequence repeat markers (SSRs), Rph13 was
mapped to chromosome 3H. The SSR marker EBmac0541 was mapped 0.5 cM distal to
Rph13 and may be useful for MAS if further flanking markers can be identified.
A selection of races of P. hordei were used in the greenhouse to screen for resistance in a
collection of 82 barley lines from the breeding program at NDSU. Resistance was identified
in barley line “C2-02-134-2-2” which had a different phenotype from the resistance gene,
Rph15 which had already been shown to exist in that line. In the F2 generation of a cross
between C2-02-134-2-2 and ZA47, the Rph15 phenotype (00;) was separated from the
second resistance gene phenotype (0;12-). A cross was then made between barley line
“4A” which contained only the second gene and “Bowman”, and the progeny were use in an
inheritance study and for gene mapping by DArT. The new resistance was shown to be due
to a single dominant gene. The gene was mapped onto barley chromosome 7H. When
inoculated with a set of P. hordei isolates the gene showed different phenotypic reactions
from that of Rph3 and Rph19 already known to be on chromosome 7H, which is some
evidence that it is a new gene.
We are very happy with the progress in this project as we achieved significantly more in
than was proposed in the AMBA project proposal.
4. Conduct detailed field surveys to determine the incidence and severity of barley
diseases in Western North Dakota. After 4 years of funding the SBARE Malting Barley
for Western North Dakota project has been incorporated into the ND Ag Experiment Station
Budget. I now apply annually to the Experiment Station for the $12,000 to employ crop
scouts and their travel to continue the survey. The money allocated does not fully cover
the cost and a small amount has come from AMBA funds to support this effort. In 2006 I
again lobbied successfully for ND Ag Experiment Station funds to continue the service, but
the intention is to redirect the SBARE Malting Barley for Western North Dakota funds
toward agronomic research and it is likely that this service will be discontinued in future
years.
To achieve this aim I have developed collaborations with Dr Marcia McMullen (small grains
pathology extension Fargo), Dr Janet Knodel (plant protection/entomology extension
Fargo) and Mr. Roger Ashley (small grains agronomy Dickinson) to sample barley fields
across the state for leaf, stem head and root diseases with an emphasis on Western North
Dakota. Prior to the initiation of this survey only about 60 barley fields were sampled
annually in North Dakota with patchy distributions in the West of the state. Disease survey
staff was employed between June and August 2006 to systematically sample barley fields
for seedling and mature plant diseases as well as for insects. Due to the severe drought in
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much of North Dakota in 2006, few fields were detected with diseases. A total of 105 fields
were sampled. This was less than half of the number sampled in 2005 because the area of
barley sown in 2006 was significantly less than in previous years and disease was low so
that finding crops to survey took much more of the scout’s time.
Scouts examined 10 plants in five locations per field, sampling in a W pattern. Information
collected at each site included district, county, field location, GPS location, growth stage,
previous crop, and incidence and severity of major diseases and insects. Data were
recorded on hand held iPAQ computers equipped with an Excel spreadsheet. Information
was forwarded at the end of each week to NDSU at Fargo. Data were mapped using ARC-
INFO GIS software, and on Monday mornings reports were uploaded to the web at
http://www.ag.ndsu.nodak.edu/aginfo/ndipm/. Extension staff, consultants, farmers and the
general public are able to view the site immediately. On an accompanying web page Dr
McMullen summarizes and interprets the information contained in the disease maps. An
example of part of the June 19-23 pest report by Dr McMullen follows,
“WHEAT AND BARLEY DISEASE OBSERVATIONS, June 19-23
……. NDSU IPM’s summer field scouts surveyed ….. 11 barley fields during the week of
June 19-23. The average growth stage of these crops during that week was Zadoks 40 =
the boot stage, with a wide range of growth stages, from 2 leaf to soft dough.……
Barley: Leaf spots were commonly found by field scouts, but were not severe. The barley
growth stage averaged Zadoks 40 = boot stage at the time. Two of the four headed barley
fields had loose smut, with a 4 and 10% incidence of infected tillers.
Fusarium head blight: For Fusarium head blight, some NDAWN stations indicated that
weather had been favorable for moderate risk of Fusarium head blight…. This area of risk
is small and is similar to last week’s areas of risk, primarily in the northcentral tier of
counties. ”
In 2006 diseases were less common and less severe than in the last three years, probably
because of the well below average rainfall during the tillering and heading stages of crop
growth. Early in the season almost no disease was found on barley except for some minor
spot blotch symptoms and an occasional BYDV affected plant. By late June, leaf spots
were common but not severe and some fields that had not been seed treated were showing
symptoms of loose smut. By the beginning of July spot blotch was being detected in all
fields and aphid and thrip numbers had increased to damaging levels although no FHB was
detected in fields that had emerged heads. As July progressed aphids and thrips became
more common and patches of BYDV affected plants were frequently found, presumably as
the aphids are a vector for this disease. FHB at low severity was detected in only a very
few fields. By the end of July most crops were ripe, but those that were planted later and
were still green had little leaf disease detectable and no FHB was detected.
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Due to the overall low incidence and severity of leaf and head diseases in 2006 carry-over
inoculum will be low in 2007 which should reduce disease levels particularly if conditions
are not suitable for disease development.
Figure 1. Typical output on the web site which is available for each pest and
disease. Animated gifs (simple movies) are also available that allow farmers to see
changes occurring as the season progresses.
The disease data are being used by the barley pathology program, together with the tworowed
and six-rowed barley breeding programs to set priorities for research in disease
resistance for the development of Western region barley varieties. In addition farmers can
access the data on the web and use it and the accompanying interpretation as a decision
aid for fungicide spraying or changes to management in following seasons.
Tissue samples from all diseased fields were returned to NDSU at Fargo, isolations of the
causal organism was made and isolates were stored for future and current work on
pathogen race distribution and evolution.
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5. Collect isolates of barley pathogens and determine their virulence phenotype.
In 2006 we had difficulty in making isolations from leaf lesions probably due to the fact that
there were few well developed infections on the material collected.
A survey of fields was undertaken in July 2006 for common root rot (CRR) caused by
Bipolaris sorokiniana which finds dry soil conditions favorable for development.
Cochliobolus sativus was isolated from approximately 80% of the more than 200 sub-crown
internodes showing symptoms. After identification we subcultured the isolates using a
single conidium in order to ensure that we did not have a mixed culture. The single
conidium isolates were dried down and are in stage at -80 C. We were able to add these
collections to the large collection we amassed in 2002-2006 and they will be used by the
grad student Sanjay Gyawali to study diversity of the pathogen.
6. Molecular Markers and Resistance Genes to Septoria in Barley. Dr Seonghee Lee a
PhD student (August 2002-July 2006) completed his research on identifying molecular
markers for resistance genes to Septoria leaf blotch in barley. Septoria speckled leaf
blotch (SSLB) caused by the pathogen Septoria passerinii is a common leaf disease in
cultivated barley in North Dakota. The disease causes economic losses in yield and malting
quality, and genetic resistance is the preferred method of control. Identification of molecular
markers for SSLB resistance genes will allow marker-assisted selection (MAS) in breeding.
To develop molecular markers for resistance, susceptible cultivars were crossed with three
resistant lines, each containing one the three genes. The F2 generation and F2.3 families
were evaluated for resistance in the green house and F2.3 families in the field. Sequence
tagged site (STS) markers SUBC285, SOPC2, SOPAH5, and SOPBA12 each closely
linked to one of the three genes, were produced from RAPD markers. Another STS marker,
MWG938 linked to Rsp2, was also identified. The STS markers were screened on breeding
material with know phenotype as well as diverse genetic material. The STS markers linked
to Rsp genes will be very be useful for maker-assisted selection and for gene pyramiding
with other genes in barley breeding programs trying to incorporate SSLB resistance in their
cultivars.
In order to utilize the three single dominant genes it is important to know the diversity in the
pathogen and its capacity to develop virulence to the resistance genes. The genetic
structure of Septoria passerinii from nine field populations was examined at several scales;
within lesions, among lesions in a leaf, among leaves in a field, and among fields in ND and
western MN, using amplified fragment length polymorphism (AFLP) markers. A total of 390
isolates were sampled from seven barley fields located in ND and MN. AFLP DNA
fingerprints identified 176 different genotypes among 390 (non-clone corrected) isolates in
the nine different fields. In two intensively sampled sites, ND16 (Williston ND) and ND17
(Langdon ND), one to four different genotypes only were found within a lesion. A higher
level of genetic and genotypic diversity was found within a leaf where six to nine different
genotypes were found on each leaf. The genetic diversity within a leaf was similar to the
genetic diversity within a field. The pattern of genetic variation within and among lesions on
a leaf was similar to that observed in the closely related Mycosphaerella graminicola which
is known to have regular cycles of sexual reproduction in the field. A lack of correlation
between geographical distance and genetic distance was found, and this suggests the
potential for a high level of gene flow between different geographical regions. The
population genetic structure described in this study for S. passerinii in North Dakota and
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western Minnesota is consistent with that of a sexually reproducing fungus. This diversity
and likelihood of a sexual stage in S passerinii indicate that single dominant resistance
genes may have a limited life if employed widespread throughout the region. Consideration
should be given to pyramiding more than one of the genes into breeding material.
7. Control of FHB in Barley by reducing inoculum load of the pathogen. PhD Student
Scott Halley tested in the field at Langdon in 2006, 15 chemical treatments plus an
untreated control on Fusarium infested crop stubble for their ability to inhibit pathogen
sporulation and head blight disease. Sporulation was measured by bagging and exposing
barley heads for predetermined times and then washing and culturing to determine the
number of spores on the heads. Sporulation was also measured by placing volumetric
spore collectors and open Petri plates with selective isolation media under the barley
canopy. Due to the dry season little infection was seen and few spores were measured.
Experiments will have to be repeated in 2006 to obtain useful data.
In a second study undertaken with Mr. Halley, we investigated the effect of fungicide
application at different stages of tiller development in an attempt to explain the poor control
of FHB fungicides sprayed on barley compared to the control obtained on wheat. Tillers
that were spayed with fungicide at maturities earlier or later than that recommended
suggests that two-rowed and six-rowed barleys as well as hard red spring wheat may
benefit from additional fungicide applications to protect these tillers in some environments.
An application of tebuconazole fungicide only reduced FHB incidence at head fully
emerged growth stage but did not affect tillers that had not reached this growth stage. As
barley does not develop synchronously there is always a proportion of tillers at spraying
that are younger and older than the recommended age Deoxynivalenol concentrations
were reduced in one environment when the growth stages were head partially emerged
and head fully emerged but were not reduced by other growth stages.
Experiments are also continuing with Prof Nick Hill at the University of Georgia. We have
collected, purified and shipped several shipments of spores to Prof Hill to determine the
specificity of his ELISA method of Fusarium graminearum quantifications, the minimum
detection limits and the impact of method of shipment on sensitivity. All experiments are
ongoing.
OTHER FUNDS AND FUTURE DIRECTIONS
1. Common Root Rot Resistance Gene Mapping for Barley CAP
Research in 2006 was the first year of a four year project funded by the CSREES CAP
Barley project. Common root rot (CRR) caused by Bipolaris sorokiniana has been
identified as a disease of importance in barley in North Dakota and in adjacent Canadian
Provinces. Losses in yield average 5 to 15% and are more consistent than leaf diseases
that occur in epidemics. Canadian researchers have been breeding for resistance to CRR
and Canadian Pathologists have been supplying information to growers for more than 40
years so that they can manage the disease and use resistant cultivars. No information or
resistant lines are available in North Dakota.
We organized and took receipt of the subset of OPAH2 lines available in sufficient
quantities (about 500g) for large scale field trials for testing of disease reaction to common
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oot rot. In collaboration with Scott Halley at the Langdon Research Extension Centre we
chose sites expected to have moderate levels of the CRR pathogen due to their history of
consecutive cereal crops. We planted these lines in 4’x10’ plots in Langdon on unirrigated
and uninoculated plots with 8 replications. At hard dough stage plants were randomly
pulled from each plot until we had at least 20 sub-crown internodes 1-2cm long from each
plot. Sub-crown internodes are being stripped of their papery covering prior to disease
assessment. Approximately 9000 plants were pulled. At maturity plots were harvested and
seed retained for quality assessment. When information about resistance genes for CRR is
available it will be made available to breeders to incorporate resistance into current
breeding programs.
2. FHB Resistance Gene Mapping for Barley CAP
In 2006 we collaborated on the first year of a four year project on mapping FHB using
association genetics. This project is funded by the CSREES CAP and led by Kevin Smith,
University of Minnesota. The collaborative trial included 96 entries from each of the four
Midwest breeding programs. We planted two misted and inoculated FHB nurseries (Fargo
and Langdon ND) in single row plots, two replicates per location in an augmented block
design including the checks Robust, Stander, MNBrite, Chevron. Due to extremely hot dry
conditions at maturity causing premature ripening only one replicate was able to be
assessed for disease at each site however data on DON toxin levels, heading date and
harvest was able to be undertaken on both replicates at both sites. The information about
resistance genes for FHB which is generated in this project will be made available to
breeders to speed up the incorporation of resistance into current breeding programs.
3. Pre-harvest crop management to reduce FHB
The aim of this project is to investigate FHB and DON in barley after use of pre-harvest
desiccant herbicides and swathing to speed-up the maturity of the crop and reduce losses
due to lodging and head shattering.
As Fusarium is both a pathogen and saprophyte and is favored by warm temperatures and
high humidity, the act of swathing a crop could result in an increase in Fusarium levels
during the latter stages of crop maturity. As rainfall during grain ripening and maturity are
variable between years, simulated rainfall is achieved by irrigation of the swath. The
amounts of simulated rainfall quantity and frequency are proportional to historical rainfall
patterns at each trial site. DON, visual infection and microbial analyses are done on the
treatments.
Unexpected effects from combinations of disease and herbicides have been reported for
decades so that the reports on herbicide-disease interactions with FHB and glyphosate
which originated in Canada require critical experimentation to verify their validity. A range of
herbicides registered for use as pre-harvest desiccants were tested over the range of
recommended growth stages. Susceptible and moderately resistant cultivars were tested
under a range of environmental conditions. The crop was assessed for DON, visual
infection, microbial analysis and important; yield and quality components.
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The outcome of this project will be a series of management recommendations for industry
on the use of pre-harvest desiccant herbicides and swathing and their impact on FHB and
DON in barley.
4. Development of farmer friendly barley disease publications and websites
The first publication in the series, the Barley Disease Handbook was completed with
funding from the ND Barley Council and distributed at field days in 2005. Multiple copies
have been distributed to several AMBA members across the US as well as at the AMBA
field day. The initial print run of 2,500 was exhausted within 12 months and the ND Barley
Council commissioned a second print run in 2006. Plans are underway to update NDSU’s
single glossy sheet barley disease identification handouts many of which were designed in
the 1980’s and 1990’s.
The Barley Disease Handbook and the NDSU Barley pathology web site are now online for
industry, farmers and the public to access information about barley diseases.
Neate, S.M. Barley Disease Handbook [Web Page].
http://www.ag.ndsu.nodak.edu/aginfo/barleypath/barleydiseases/index.htm
Neate, S.M. NDSU Plant Pathology Barley Disease Research website [Web Page].
http://www.ag.ndsu.nodak.edu/aginfo/barleypath
Presentations at National and International Professional Meetings
1. 2006 Joint Meeting of NCERA-184 and WCERA-97 Small Grain Pathologists: Fargo, June
2006. Septoria speckled leaf blotch resistance in barley. Invited Speaker.
2. New strategies for management of plant pathogenic soil microorganisms - genetically
modified plants. Session 2.3P World Congress of Soil Science, Philadelphia July 2006.
3. Incidence and Severity of Spot Blotch on Barley in North Dakota 2002-2006. Spot Blotch
special topics session, 3 rd International workshop on barley leaf blights, Edmonton July 23-
27 2006.
Invited Chairman, National and International Meetings
1. 18th World Congress of Soil Science, Philadelphia, USA New Strategies for Management of
Plant Pathogenic Soil Microorganisms Symposium, Commission 2.3P. July 9-15, 2006
2. American Phytopathological Society North Central Division 2006 <strong>Annual</strong> Meeting, Oral
Presentations. June 13-15, 2006
Committees or Organization Involvement Related to Barley Pathology
1. Symposium on "Management of pathogenic microorganisms - control through natural
suppression or genetic modification." 18th World Congress of Soil Science, Philadelphia,
July 2006. (Chair of Organizing committee).
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2. United States Wheat and Barley Scab Initiative Cultural, Chemical and Biological Control
Research Area Committee, 2005-present. (Vice-Chair of Committee).
3. United States Wheat and Barley Scab Initiative Steering Committee, 2003-present. (Barley
representative).
4. International Society of Plant Pathology, Rhizoctonia working group, 1998-present.
(Committee Chairman).
Farmer/Industry Talks
1. ND State Barley Show, Osnabrock ND, March 23 th 2006.
Personnel:
Stephen Neate, Associate Professor / Project Leader – State Funded
Open Position, Research Specialist I – State Funded
Pat Gross, Research Specialist I – 100% AMBA Funded
Sanjaya Gyawali, PhD Student – 100% Barley CAP Funded
Scott Halley, Extension Pathologist, Langdon and PhD Student – State Funded
Undergraduate Student – 10% USWBSI funded
Statement of Context: The Barley Pathology project does not have a funded field technician
and the North Dakota State funded laboratory/greenhouse technician resigned in February to
take up a higher paid position in industry. At present the state funded laboratory technician
position is frozen, but we hope to be allowed to hire at the end of summer. For the last 4 years,
funds from AMBA have allowed the employment of a field technician with 30 years experience
in small plot experimental work. The AMBA funded field technician position is indispensable to
the operation of the Barley Pathology program and without both a field and
laboratory/greenhouse technician, much of the support to the 6-rowed breeding program and
most of the practical and industry relevant research will not take place.
Graduate student Seonghee Lee finished in June 2006 and Sanjaya Gyawali started in January
2006. The most valuable work to the barley industry has come from my graduate students.
Their work has resulted in a series of molecular markers that can be used in marker assisted
selection for control of septoria speckled leaf blotch the most important barley disease in the
Midwest. In addition the new stem rust race recently found in Africa was a wake-up-call to the
industry and work by my students has kept my group current in mapping and identifying new
rust resistance genes. Students working with existing staff are the cheapest and most flexible
way to address new issues faced by the barley industry.
Cooperators:
Disease Survey
Mr. Roger Ashley, Dickinson Research Extension Center, ND
Ms. Janet Knodel, NDSU Fargo, ND
Prof. Marcia McMullen, NDSU, Fargo, ND
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FHB, Spot Blotch, Net Blotch and CRR Germplasm screening
Dr Phil Bregitzer, USDA-ARS, Aberdeen ID
Dr. Flavio Capettini, ICARDA/CIMMYT, Mexico.
Dr. Lynn Dahleen, USDA-ARS, Fargo, ND
Mr Scott Halley Langdon Research Extension Centre, ND
Prof. Richard Horsley, NDSU, Fargo, ND
Prof Brian Steffenson, University of Minnesota, MN
Leaf Rust Genetics
Dr. T Friesen, USDA-ARS, Fargo, ND
Prof. Shaobin Zhong, University of Hawaii, Honolulu, HI
Septoria Genetics
Dr. Stephen Goodwin, UDSA-ARS, Purdue University, IN
Prof Bruce McDonald, Federal Institute of Technology, Zurich, Switzerland
FHB Disease Management
Prof Nick Hill, University of Georgia, GA
Mr. Mark Halvorsen, North Central Research and Extension Centre, Minot ND
FHB Barley Disease Modeling
Prof Jeff Stein, Small Grains Pathology, South Dakota State University
Publications:
1. Hill, N.S., Neate, S.M., Cooper, B., Horsley, R.D., Schwarz, P.B., Dahleen, L.S., Smith,
K.P. and Dill-Macky, R. (2006) ELISA Analysis for Fusarium in Barley: Application in
Field Nurseries. ASA-CSSA-SSSA International <strong>Annual</strong> Meetings, Indianapolis, November
12-16, 2006.
2. Hill, N.S., Schwarz, P., Dahleen, L.S., Neate, S.M., Horsley, R., Glenn, A.E., O’Donnell, K.
(2006) ELISA Analysis for Fusarium in Barley: Development of Methodology and Field
Assessment. Crop Science 2006 46: 2636-2642.
3. Horsley, R.D, Franckowiak, J.D., Schwarz, P.B. and Neate, S.M. (2006) Registration of
'Stellar-ND' Barley. Crop Science 46:980-981.
4. Lee, S. and Neate, S.M. (2006) Distribution and frequency of mating types in Septoria
passerinii in North Dakota Barley. p135. Proceedings 3 rd International Workshop on Barley
Leaf Blights, Edmonton Canada July 2006. Agriculture and Agri-Food Canada, Lacombe,
Alberta.
5. Lee, S. and Neate, S.M. (2006) Molecular mapping of Rsp1 and Rsp3 resistance genes
against Septoria passerinii in barley using DArT technology. Phytopathology 96:S66.
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6. Lee, S. and Neate, S.M. (2006) Population genetic structure of Septoria paserinii in North
Dakota Barley. p136. Proceedings 3 rd International Workshop on Barley Leaf Blights,
Edmonton Canada July 2006. Agriculture and Agri-Food Canada, Lacombe, Alberta.
7. Lee, S-H. and Neate, S.M. (2006) Molecular Mapping of Rsp1, Rsp2, and Rsp3 Genes
Conferring Resistance to Septoria Speckled Leaf Blotch in Barley. Phytopathology 97:155-
161.
8. Lee, S-H. and Neate, S.M. (2006) Population genetic structure of Septoria passerinii in
North Dakota barley. Phytopathology (Accepted October 24 th 2006)
9. Lee, S-H. and Neate, S.M. (2006) Sequence tagged site (STS) markers to Rsp1, Rsp2, and
Rsp3 genes for resistance to septoria speckled leaf blotch in barley. Phytopathology 97:162-
169.
10. Manoharan, M., Dahleen, L.S., Hohn, T., Neate, S.M., Yu, , X.-H., Alexander, N.J.,
McCormick, S., Schwarz, P., and Horsley, R. (2006) Expression of 3-OH trichothecene
acetyltransferase in barley (Hordeum vulgare L.) and effects on Fusarium head blight. Plant
Science 161:699-706.
11. Schwarz, P.B., Neate, S.M. and Rottinghaus, G.E. (2006) Widespread Occurrence of Ergot
in Upper Midwestern U.S. Barley, 2005. Plant Disease 90:527.
12. Sun, Y., Zhong, S., Steffenson, B.J., Friesen, T.L. and Neate S.M., Relationship between
amplified fragment length polymorphism (AFLP) and virulence variation in Puccinia hordei
Can. J. Plant Pathol. (accepted September 2006)
73

MALTING AND BREWING QUALITY OF BARLEY
Dr Paul Schwarz
Department of Plant Sciences
North Dakota State University
Fargo, ND 58105
EXECUTIVE SUMMARY
The primary goals of the Barley Quality Program at NDSU are to provide individuals in
the barley improvement program with barley and malt quality analytical services, 2.) to
conduct applied research that addresses issues of immediate concern and improves the
understanding of malting quality, 3.) to conduct the annual survey of regional barley
crop quality, and 4.) to provide training in cereal chemistry with particular emphasis on
the science of malting and brewing.
Major Objectives and Expected Benefits (goals 1 and 2: AMBA funded projects):
Barley and Malt Quality Analyses: Malt quality analyses provided to individuals in the
NDSU barley variety development program (and others) directly assists AMBA in
meeting its mission of providing the malting and brewing industries with an abundant
supply of high quality malting barley. Early generation screening for malt quality is
important in streamlining the overall malting barley variety development process.
Elimination of undesirable materials at an early stage increases the overall quality of
materials submitted for laboratory malting. Barley and malt quality analyses conducted
for breeding research projects contribute to a more complete understanding of the
genetics of malting quality. Likewise, quality analyses conducted for barley production
studies help maintain barley as a competitive crop within the region.
Research Studies - Applied quality research directly addresses the AMBA mission to
increase the understanding of malting barley. Projects which have addressed immediate
issues have had major impact. Our past research work on pre-harvest sprouting and
deoxynivalenol are important examples of immediate issues.
74

Objectives Met in the One-Year Funding period
Barley and Malt Quality Analyses
• Screened 3350 early generation samples for barley quality, and 130 variety plot
samples for malt quality.
o Data for the NDSU Variety Development Program was used to make
decisions on lines submitted for advanced testing.
• Micro-malted and provided malt quality analyses (n=181) for a study on
transgenic barley showing partial resistance to FHB.
• Micro-malted and provided malt quality analyses (n=98) for a study on adaptation
of European germplasm in western ND and eastern MT.
o These lines may prove of value under irrigation conditions, and may
provide growers with a competitive crop (barley) until adapted domestic
cultivars are released.
Research
• Identified significant interference from beta-amylase in the standard ASBC
method for the determination of alpha-amylase in malt (method Malt-7)
Most Significant Accomplishment
• Provided value services to barley grower, industry and researcher stakeholders,
while maintaining a good balance of applied barley research.
75

MISSION: The primary purpose of AMBA is to encourage and support an adequate
supply of high quality malting barley for the malting and brewing industry and increase
our understanding of malting barley.
PRIMARY OBJECTIVE: Develop malting barley varieties with improved agronomic and
quality characters to keep malting barley competitive with other crops so that growers
continue to plant and produce an adequate supply of suitable quality for improved
utilization by the industry.
OBJECTIVES, METHODOLOGY AND RESULTS –
AMBA FUNDED PROJECT(S)
1.) Early Generation Quality Testing Program and Cooperative Malt Quality
Research. Approximately 3350 early generation lines and cultivars (NDSU Malting
Barley Variety Development Program), grown in 2006, were submitted for preliminary
screening. This was up by approximately 160 samples from 2005. These samples were
screened by near infrared reflectance (NIR) for protein and color. Kernel assortment
and test weight were additionally determined on 3200 of the samples, which is an
increase of approximately 1000 from 2005. Lines and varieties (130) from the 2006
NDSU variety plot trials were micro-malted and analyzed for standard quality
parameters.
Micro-malting and malt quality testing services are provided to research cooperators.
This work supports research. These samples are not part of the normal barley varietal
development program.
• 181 barley samples were malted analyzed for Dr Lynn Dahleen and Dr Phil
Bregitzer. These samples were for a study on the expression of anti-fungal
genes in barley, and their impact on both Fusarium Head Blight resistance and
malt quality.
• 98 samples were malted and analyzed for Dr Rich Horsley, as part of study on
the adaptation of European lines and cultivars in North Dakota and Montana.
2.) Applied Malt Quality Research. Much of the current methodology used in malt
analysis was developed from the late 19 th to mid 20 th century. Changing technology as
well increased analytical demands and expectations have identified some methods as
problematic. As example, we recently reassessed methodology for Congress mashing
(Schwarz et al 2007). The current project, which was directly funded by AMBA in 2006
involves the determination of alpha-amylase in malt.
Measurement of Malt Alpha-Amylase. The determination of alpha-amylase in malt by
automated flow analysis has been challenging since its introduction in the 1970’s.
76

These automated methods have been the subject of numerous ASBC technical
subcommittees, but a rugged, reproducible method has not been accepted. Principle
problems with the methods have included alternative calibration methods and
chemistries (starch-iodine and ferricyanide). The iodine reaction with beta-limit dextrin is
the foundation of the original 1939 Sandstedt-Kneen-Blish (SKB) publication and
subsequently, the ASBC manual method (ASBC Methods 1992). The method is based
upon indirect measurement of a substrate property, and is perhaps of questionable
kinetic soundness. On the other hand, reducing sugar methods, such as the ferricyanide
method are based upon the direct measurement of reducing sugars formed as a result
of hydrolysis of alpha-1, 4 glucosidic linkages in starch by alpha-amylase. We contend
that some of the difficulties in inter-lab measurement of malt alpha-amylase can be
resolved by improving the understanding of the kinetics involved in various methods.
The objective of the project is to compare various methods on a kinetic basis.
Background and Hypothesis. The standard ASBC method for alpha-amylase in malt
is based upon the reaction of beta-limit dextrin (starch) with iodine (figure 1). Iodine
binds within helical sections of the starch or beta-limit dextrin chain and yields a blue
color. As amylases degrade starch, this helical structure and iodine binding are lost, and
the color gradually changes to red. The measurement of alpha-amylase is based on this
color change.
Iodine
Figure 1. Iodine binding within a cyclodextrin. This is analogous to binding within helical
sections of the starch chain.
77

With starch as a substrate, iodine binding is impacted by both alpha-and beta-amylases.
In order to make the method specific for alpha-amylase, beta-limit dextrin is used as
substrate. Beta-limit dextrin is prepared by exhaustively reacting starch with a purified
beta-amylase. Beta-amylase hydrolyzes starch from the non-reducing end to yield
maltose. As it can not pass alpha-1,6 linkages in the starch, a beta-limit dextrin product
results in addition to the maltose (figure 2).
Beta-Amylase
Alpha- 1, 6 branch
+ Maltose
starch
Beta-Limit Dextrin
Figure 2. Preparation of beta-limit dextrin from starch using purified beta-amylase.
Arrows show points at which beta-amylase can hydrolyze alpha-1,4 linkages in starch.
In theory, the beta-limit dextrin can no longer be attacked by beta-amylase, and its use
as a substrate makes the method specific for alpha-amylase. Alpha-amylase is an
endo-enzyme, meaning it attacks the beta-limit dextrin internally and forms small
oligosaccharides (figure 3). Loss of iodine binding is thus believed only to be associated
only with the alpha-amylase in the malt sample.
78

Beta-limit dextrin
Alpha-Amylase
Alpha- 1, 6 branch
Small dextrin + sugars
Figure 3. Endo-activity of alpha-amylase on beta-limit dextrin substrate. Products are
small dextrins and sugars. As the size of the substrate decreases, the ability to bind
iodine is lost.
However, the situation is not as simple as presented in figure 3. Starch (amylopectin) is
described as being composed of A, B and C-Chains. The C-chain is the central chain
carrying the reducing group, while the outermost A-chains are unbranched. While the Bchains
are branched, they may have very long unbranched segments. Figure 4 shows a
representative diagram of a portion of the starch molecule (the complete molecule may
contain > 1 x 10 6 glucose residues). The shaded circles show glucose residues that
would be initially removed by beta-amylase (as maltose) in the preparation of beta-limit
dextrin. However, when the beta-limit dextrin is used in the assay of alpha-amylase, its
action opens up these unbranched B-chain residues, and they then become susceptible
to beta-amylase. The glucose residues that would become susceptible to beta-, only
after attack by alpha-amylase are shown with striped circles. The length of these
susceptible unbranched B-regions can be considerable (>50 glucose) and they are
associated with iodine binding. As such, it seems possible that the use of beta-limit
dextrin in the assay of malt alpha-amylase may not completely eliminate interference
from beta-amylase.
79

A-Chain
A-Chain
A-Chain
B-Chain
C-Chain
Figure 4. Portion of an idealized starch (amylopectin) molecule. Gray circles show
glucose residues that would be removed by beta-amylase in the preparation of betalimit
dextrin substrate. Striped circles show residues within the beta-limit dextrin that
would be susceptible to beta-amylase only after initial treatment with alpha-amylase.
Methods and Results. In the first portion of this study high performance size exclusion
chromatography (HPSEC) was used to compare the change in the molecular weight of
the beta-limit dextrin (HPSEC) substrate to the loss in iodine binding when incubated
with alpha-amylase. As shown in figure 5, there was more than a 7-fold reduction in
molecular weight within the first min of reaction, while iodine binding was only lost
slowly. The absorbance at 12 min corresponds approximately to the end-point of the
manual method. These results suggest that fragments of the beta-limit dextrin well
below 30 kDa still show considerable iodine binding. As this extensive degradation is
required to achieve the color end-point, the presence of substrate that is susceptible to
beta-amylase is likely to influence the final results.
80

M w kDa
800
600
400
200
0
0.4
0 2 4 6 8 10 12
Reaction Time (min)
molecular weight
iodine binding
Figure 5. Change in the weight average molecular weight (Mw) and iodine binding of
beta-limit dextrin when incubated with purified malt alpha-amylase under conditions of
the ASBC manual method for alpha-amylase.
The second portion of the study focused on study of the reaction of beta-limit dextrin
with purified malt alpha- and beta-amylase. Malt alpha-amylase was purified from a
crude Sigma (Sigma-Aldrich, St Louis) enzyme preparation by heat shock and
ultrafiltration. The freeze-dried enzyme preparation was standardized to yield
approximately 30, 60, and 90 DU in the manual method. Beta-amylase was from
Megazyme (Ireland). In the first series of experiments the loss of iodine binding with
alpha-amylase was monitored both in the presence and absence of beta-amylase.
2.2
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
OD 650
81

Figure 6. Degradation of beta-limit dextrin with alpha-amylase (30 DU) and alpha- and
beta-amylase. End-point line indicates the normal color endpoint of the manual assay.
As shown in figure 6, supplementation with beta-amylase did change in the initial rate of
color loss. However, the impact on the final endpoint was only about 1 min. The would
translate to about 4 DU. The value for alpha-amylase in the absence of added beta-
was 40, and in the presence of beta was 44. These results are significant but perhaps
the magnitude is not of great concern in routine analytical situations.
Beta-limit dextrin is prepared by exhaustive treatment of soluble starch with betaamylase.
In the next series of experiments we tested the beta-limit dextrin substrate for
the presence of residual enzyme activity. The beta-limit dextrin substrate was assayed
both as normal, and following heat treatment. As shown in figure 7, the heat treated
beta-limit dextrin substrate took over 20 min to reach the color endpoint when digested
with only alpha-amylase (■-■-■). However, when assayed as normal, with no heat
treatment conversion was in approximately 12 min (▲-▲-▲). This translates to 24 DU
in the heat treated substrate and 40 in the normal assay. Residual beta-amylase in the
beta-limit dextrin substrate is clearly making a very large contribution to the value for
malt alpha-amylase.
82

Figure 7. Time course of I2-binding with heated and unheated beta-limit dextrin in the
presence of alpha-amylase (30 DU) and alpha- and beta-amylase.
Addition of beta-amylase to the heat treated beta-limit dextrin substrate returns
conversion time to what is normally observed (12 min) (Figure 7, ▼-▼-▼ or ●-●-●). At
30 DU (figure 7) the amount of beta-amylase added (10 or 100 units/ml) did not seem to
greatly influence the final result. As such it might be assumed that at saturating levels of
beta-amylase there is little to no influence on the final determination of malt (alphaamylase)
DU. However, as the amount of alpha-amylase was increased from 30 to 90
DU, supplementation with beta-amylase did appear to impact the final estimate of DU
(data not shown). Ongoing research is addressing the significance of the beta-amylase
interference in actual malt samples with varying levels of alpha-amylase and diastatic
power.
83

OTHER BARLEY SERVICE AND RESEARCH WORK
(supported outside AMBA funding)
1.) Quality Testing Services
(A) Regional Barley Crop Quality. The regional survey of North Dakota and Minnesota
barley has been conducted by this laboratory for over 25 years, and represents an
excellent example of industry-grower cooperation. The survey provides timely
information on the quality of regional barley as harvest progresses, and has been an
important means of documenting crop quality issues such as pre-harvest sprouting and
DON. The NDBC utilizes the survey in marketing efforts. Samples and data from the
crop survey have been widely utilized in research and economic studies. With all
impacts considered, the annual crop survey is an important tool in maintaining barley as
a competitive crop within the region.
The 2006 survey of the regional barley crop was prepared in cooperation with the North
Dakota Barley Council and the USDA North Dakota Agricultural Statistics Service.
AMBA. A total of 233 six-rowed and 35 two-rowed samples were collected throughout
North Dakota and the primary barley producing regions of Minnesota. Samples (n=9)
were also collected in eastern Montana. Data was provided to AMBA. A comparative
summary of data is presented in Table I
Table I.
North Dakota and Minnesota Six-rowed Barley Crop Quality, 2003–2006
Kernel Plumpness (%) Test Weight (lbs/bu) Protein (%) Kernel Color
2006 58.7 45.9 12.8 3
2005 75.7 46.0 12.9 5
2004 80.4 47.5 12.6 7
2003 76.3 47.8 12.8 6
Samples (n=282) were tested for DON. The average level of DON observed in the 2006
North Dakota and Minnesota Regional six-rowed barley crop was

10
9
8
7
6
5
4
3
2
1
0
1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006
Figure 8. Average DON levels present in the North Dakota and Minnesota Barley Crop,
1993-2005.
(B) Deoxynivalenol Testing Program. The overall goal of DON testing in our
laboratory is to expedite the development of scab resistant malting barley cultivars for
the upper Midwest. This portion of the program is largely funded through USDA and ND
state appropriated funds. DON analysis services are provided to barley breeders,
geneticists, and pathologists at NDSU, University of Minnesota, Virginia Polytech., and
Busch Agricultural Resources, Inc. Allotments are provided to all cooperators at no
charge. Samples beyond individual allotments are analyzed at a minimal charge, which
includes only the cost of expendable supplies, chemicals, and sample grinding labor.
Approximately 15,000 barley samples from the 2006 crop were analyzed (or are in
process) for DON by GC-ECD (Table II). This number compares to 10,000 in 2005.
Samples included breeder’s lines, crop survey samples, and samples from research
studies. The 2005 crop samples were analyzed beginning August 2006 and will be
complete in April 2006.
A barley DON check sample service is operated as a service to 15 academic and
industry labs. Many of these are AMBA member companies, or are affiliated with
member companies. Two samples are shipped to each participant on a monthly basis.
85

Table II
2006 Crop Samples Analyzed for DON in the Department of Plant Sciences
Sample Type Sample
Number
Barley Breeding
4,668
(BARI-Cooper, Dr Griffey,
Dr. Horsley, Dr Smith)
2006 Regional Crop Survey 282
Barley Pathology (Dr Neate) 4652
Barley Genetics (Dr Dahleen) 899
Research (Dr Schwarz) 2993
AMBA 20
Standards 1282
Other 503
Tot 15,299
(C) IBMS Grower Survey. A barley production and information survey was sent to
5000 barley producers in Idaho, Montana, and North Dakota by the Institute of Barley
and Malt Sciences (IBMS) in late November 2006. The questionnaire was produced
through the cooperative efforts of the IBMS, AMBA, the Idaho Barley Commission, the
North Dakota Barley Council, the Montana Wheat and Barley Committee, the
USDA/CSREES funded barley Coordinated Agricultural Project (CAP) The survey was
mailed by the ND Ag Statistics Service, who also conducted telephone follow-up.
Questions on the survey include such topics as yield, acreage, transportation, factors
influencing barley growers decisions to produce barley, information sources, farming
practices, producer support system effectiveness and satisfaction levels for growers.
Approximately 1200 surveys had been received by December 15, 2006. Results are
currently being analyzed. Survey incentive prizes were donated by AMBA, Anheuser-
Busch, Cargill Malt, International Malting Co, Molson-Coors Brewing, and Rahr Malting
Co.
86

2. Other Research
(A) Predicting Seed Storage Viability with Accelerated Aging: Widespread
occurrence of pre-harvest sprouting in 2002 raised concern over the storage viability of
sprouted grain. We found that while severely sprouted samples rapidly lost viability in
storage, common measures of sprouting were not good overall predictors (r≤0.50) of
germination following storage for 4 and 7 months (Schwarz et al. J. ASBC 62(4):147,
2004). Germination following accelerated seed aging was found to be a more reliable
predictor (r=0.73) of germination following 7 months of storage. Accelerated aging is
performed by heating seed at 41 o C for 48 hr at 80% RH prior to germination testing.
This treatment resulted in a 0 to 50% reduction in the observed germinative energy.
The objective of this study is to evaluate the utility accelerated seed aging to predict the
long term storage viability of barley. The study was initiated with the 2004 crop, and
sample collection will be continued for through 2006. Where possible, samples will be
selected to include some sprouting. Storage is at room temperature (24 o C), 7 o C, and
under outdoor ambient conditions. Germinative will be determined 3 times each year,
and for a total of 3 years storage. Accelerated aging is performed, as described above,
following harvest. Data for the 2004 -2006 samples is shown in Table III.
Table III
Germination and Sprouting Parameters of the 2004 - 2006Barley Samples
Selected for Study of Storage Viability (data is 4 months post-harvest)
Germinative
Energy
(4 ml, %)
2004 2005
2006 2004
Accelerated Aging
Germinative Energy
(%)
2005 2006 2004
Water Sensitivity
(4-8 ml, %)
2005 2006 2004
RVA
Stirring Number
2005 2006
N 15 15 15 15 15 15 15 15 15 15 15 15
Mean 92 95 98 72 86 93 33 24 7 101 127 135
Minimum 85 77 84 8 77 40 9 4 0 5 65 4
Maximum 100 100 100 93 100 100 75 48 40 216 191 242
(B) Barley CAP Project (Dr Yin Li). We are screening 960 breeder’s lines for betaglucanase
activity as part of the Barley CAP Project (Barley Coordinated Agricultural
Project). Lines will be screened at low and elevated temperatures to detect possible
differences in thermostability. A subset of the 960 lines will also be analyzed for
lipoxygenase activity.
The barley Coordinated Agricultural Project (CAP) is a community effort of 30 scientists
from 19 institutions (http://barleycap.coafes.umn.edu/ ). The University of Minnesota is
87

the lead institution for this project. The overall theme of the barley CAP is to integrate
and utilize state-of-the-art genomic tools and approaches in plant breeding programs,
thereby facilitating the development of superior barley cultivars and access to
agronomic and economically important genes.
(C) Determination of Factors Controlling Malt Extract (Dr Yin Li). Using a
chemometrics approach, we hope to define analytical and process components which
have the greatest influence on the development of malt extract. This is valuable, given
the greater demands for consistency and uniformity in malting, advances in malting
technology (process control), and the potential to improve our understanding of quality
through genomics. Common malt analytical procedures have origins in the late 1800’s
to 1940’s and have not significantly changed since adopted by ASBC. Extract is a
complex trait, and quantitative trait loci (QTL) for malt extract have been identified on
each of the 7 barley chromosomes. A statistical approach to factors contributing to
extract may also help to identify the type of genes that are of primary importance. The
overall objectives will be to statistically evaluate chemical and physical factors that
contribute to laboratory extract and its composition. Three separate studies have been
conducted to date.
• Part I was a study of how operational parameters of grind, grist:water ratio and
temperature profile impact the determination of laboratory extract and associated
wort quality parameters (Schwarz et al 2007).
• Part II was a study of factors influencing extract in a historical population of 16
six-rowed cultivars dating to 1910. Extract values in these samples range from 75
too 80%. A range of chemical and physical factors was evaluated across
modification levels. Research is approximately 80% complete.
• Part III was a study of factoring influencing extract in a single cultivar (Tradition).
Extract (CG) in the original unfractionated samples ranged from 77 to 80%.The
second study looked at a population of historical varieties dating back to 1910
(D) Bound Mycotoxins (Ms Zhou Bing). We have recently identified that barley can
contain significant amounts of bound DON that is not extractable under normal assay
conditions (room temperature extraction with acetonitrile:water). This “bound” DON can
represent a very small amount to almost 100% of the DON extracted under normal
conditions. The bound nature of the DON does not appear due to simple matrix effects,
and finer milling and heating during extraction have a negligible effect. Small but
significant amounts of bound DON can be released following treatment with amylolytic
enzymes. However, the largest amounts of bound DON are found following
hydrolysis/solvolysis of the sample in trichoro acetic acid.
The bound nature of the DON has a number of interesting implications. First, there is a
possibility that it might be released during digestion of grain based foods/feeds, or
during food processing. As an example the occasional increase in DON that is seen
during the malting of FHB contaminated grain, might be explained by release of bound
88

forms (through enzymolysis during malting) rather than by synthesis of new DON.
Second the binding of DON may represent a defense mechanism of the plant. We are
currently optimizing methods for bound DON, and intend to explore the above
hypothesises.
(E) Cell Wall Degrading Enzymes of Fusarium graminearum (Ms Xinrong Dong).
Cell wall degrading enzymes such as cellulases and xylanases might be important in
the pathogenicity of Fusarium graminearum. As such we are isolating and
characterizing xylanase enzymes from Fusarium. Two xylanases have been identified in
Fusarium cultures grown on wheat bran. One of the enzymes has been purified
approximately 40-fold by a combination of ultrafiltration, ion-exchange and gel filtration
chromatography.
(F) Use of Ozone for Controlling the Growth of Fusarium during Malting (Dr Dennis
Tobias). Treatment of Fusarium (liquid) cultures and barley grain with gaseous ozone at
11-26 mg O3/ml for up to 2 hr was found to be effective in dramatically reducing
Fusarium survival. This method will be optimized and scaled-up to evaluate
effectiveness in pilot-steeping.
(G) Multiplex real-time PCR for Detection, Identification and Quantification of
Fusarium species (Dr Dennis Tobias). There has been considerable concern over a
possible shift in Fusarium chemotypes, and thus associated toxin profiles and amounts.
The Fusarium species associated with FHB in Midwestern barley was last extensively
determined in the mid-1990’s. A major impediment to this work is the difficult and time
consuming nature of traditional methods for species determination (plating and
microscopic evaluation). The goal of this project is to develop and real-time PCR
method that can be used for relatively quick and high throughput analysis of grain
samples. This tool is needed both for breeding/pathology and toxicology research. The
limited work to date has focused on primer selection.
(H) Fusarium mycotoxins in Potato Tubers Infected with Fusarium Dry Rot (Mr.
Javier Delgado). We are studying the mycotoxin profiles associated with Fusarium
graminearum induced dry-rot of potato. These results have epidemiological implications
to FHB in cereals, as potato is often used in rotation with wheat, barley, and corn.
FUTURE DIRECTION OF PROGRAM
Future Direction. Malting technology is undergoing perhaps the most significant
change in the past 100 years, largely through the introduction of new process control(s)
methods. This technology change will demand a greater understanding of malt quality,
and greater level of consistency in the malting barley. Methods of assessing malt
89

quality, on the other hand, remain largely unchanged since the 1940’s, and do not
necessarily address the needs of brewers. Research directed at re-evaluation of some
test methods, and their foundations may be particularly pertinent. We are proposing to
continue with evaluation of factors that control laboratory malt extract, and their
relationship to extract factors that are not routinely determined, such as fermentability. A
chemometrics approach will be utilized.
Nevertheless, Fusarium Head Blight remains the most critical problem facing barley
producers and the malting and brewing industries. We will continue to work in
conjunction with breeders and pathologists on the development of FHB resistant
cultivars.
LABORATORY PERSONNEL
Dr Paul Schwarz Professor
Dr Yin Li Post-Doctoral Scientist
Dr Dennis Tobias Post-Doctoral Scientist
Mr. John Barr Chemist (Malt Quality)
Ms Karen Hertsgaard IBMS Barley Information Specialist
Mr. James Gillespie Chemist (DON)
Ms Debra Hatzenbeller Chemist (DON)
Mr Javier Delgado Graduate Research Assistant (Plant Pathology)
Ms Xinrong Dong Graduate Research Assistant
Ms Zhou Bing Graduate Research Assistant
Ms Jun Ying Yan Laboratory Assistant (DON)
Mr. Evan Cummings Laboratory Assistant (DON)
COOPERATORS
Dr Phil Bregitzer USDA-ARS, Aberdeen, ID
Dr Lynn Dahleen USDA-ARS NCSL, Fargo, ND
Dr Leif-Alexander Gabe Technical University Berlin, Germany
Dr Neil Gudmestad Dept. of Plant Pathology, NDSU
Dr Guo-Qing He Zhejiang University, PRC
Dr Charlene Wolf-Hall Dept. of Veterinary Sciences, NDSU
Dr Nick Hill Dept. of Crop and Soil Sci., University of Georgia
Dr Richard Horsley Dept. of Plant Sciences, NDSU
Dr Stephen Meinhardt Dept. of Plant Pathology, NDSU
Dr Stephen Neate Dept. of Plant Pathology, NDSU
Dr Gary Secor Dept. of Plant Pathology, NDSU
90

PUBLICATIONS (2006-07)
Schwarz, P.B., Li, Y., Barr, J.M., and Horlsey, R.D. Effect of operational parameters on
the determination of laboratory extract and associated wort quality factors. Accepted: J.
Am. Soc. Brew. Chem. February 2007.
Li, Y., Xu, Y., Schwarz. P.B., and Gu, G. Organic acids of commercial beers in China: A
chemometric study. Accepted: J. Am. Soc. Brew. Chem. September 2006.
Manoharana, M., Dahleen, L.S., Hohnc, T.M., Neate, S.M., Yue, X.-H., Alexander, N.J.,
McCormick, S.P. Bregitzer, P., Schwarz, P.B., and Horsley, R.D. Expression of 3-OH
trichothecene acetyltransferase in barley (Hordeum vulgare L.) and effects on
deoxynivalenol. Plant Science (6): 699-706, 2006.
Schwarz, P.B., Hill, N., and Rottinghaus, G.E. Fate of ergot (Claviceps purpurea)
alkaloids during malting and brewing. J. Am. Soc. Brew. Chem. 65(1):1-8, 2007.
Hill, N.S., Schwarz, P.B., Dahleen, L. S. Neate, S.M., Horsley, R., Glenn, A.E. and
O'Donnell , K. ELISA analysis for Fusarium in barley: development of methodology and
field assessment. Crop Sci. 46: 2636-2642, 2006.
Schwarz, P.B., Neate, S.M., and Rottinghaus, G.E. Widespread occurrence of ergot
(Claviceps pupurea) in upper Midwestern U.S. barley. Plant Disease 90:527, 2006.
Horsley, R. D., Franckowiak, J.D. P.B. Schwarz, P.B. and S.M. Neate, S.M.
Registration of ‘Stellar-ND’ Barley. Crop Sci 46:980-981, 2006
Horsley, R.D. Schmierer, D. Maier, C., Kudrna, D. Urrea, C., Steffenson, B.J., Schwarz,
P.B., Franckowiak, J.D., Green, M., Zhang, B., Kleinhofs, A. Identification of QTLs
associated with Fusarium Head Blight resistance in barley accession CIho 4196. Crop
Sci. 46: 145-156, 2006.
He, G.-Q., Wang, Z.-Y., Liu, Z.-S., Chen, Q.H., Ruan, H., and Schwarz, P.B.
Relationship of proteinase activity, foam proteins and head retention in unpasteurized
beer. J. Am. Soc. Brew. Chem. 64(1):33-38, 2006.
Schwarz, P.B., Horsley, R.D., Steffenson, B.J., Salas, B., and Barr, J.M. Quality risks
associated with the utilization of Fusarium Head Blight infected malting barley. J. Am.
Soc. Brew. Chem. 64(1):1-7, 2006
91

Net Blotch of Barley: Survey of Pathogen Virulence
Timothy L. Friesen
USDA-ARS
Northern Crop Science Laboratory
Cereal Crops Research Unit
Fargo, ND 58105
Executive Summary: Because net blotch on barley has the potential to cause
severe yield loss as well as a reduction in quality and that most barley lines are
moderately to highly susceptible, it is important that we have an understanding of
host resistance and pathogen virulence in this important host-pathogen system.
This proposal was aimed at identifying pathogen virulence present in the
Pyrenophora teres field populations at Langdon and Fargo, ND in 2004 and 2005 in
an attempt to characterize the variability of virulence of P. teres the causal agent of
net blotch on barley. Successful progress in this research will help our
understanding of the virulence as it relates to specific resistance genes in the host
with the ultimate goal of being able to effectively breed for durable net blotch
resistance. Several good resistance sources have been identified that have not
been overcome by any of the P. teres isolates collected to date. This research will
be conducted each year to monitor changes in the pathogen population in order to
provide important information on the effectiveness of various sources of resistance.
What major objectives or issues are being resolved short and long term, how are
you resolving them, and what are the expected benefits?
In the present study we have identified what resistance genes are effective in the
field population and at the same time identifying which genes are ineffective in
combating net blotch of barley in the ND/MN region in the previous two growing
seasons. We have also identified changes in virulence within the pathogen
population over the two year period tested, showing that the pathogen population
has the ability to change if selection pressures such as the introduction of single
resistance genes were applied. Long term, it is our objective to monitor the change
in the P. teres population to identify what resistance genes have been overcome or
have the potential to be overcome. This monitoring will aid in choosing resistance
genes for developing durably resistant barley cultivars.
What objectives were met and/or outputs produced in the one-year funding period?
Using a varied barley differential set with published differences we have identified
which lines contain the most effective resistance. The most effective resistance
sources in the year 2005 study are barley genotypes CI1179 (Algerian), CI5791,
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CI9214, PI552963 (Heartland), and NDB112. All other resistance sources were
susceptible to at least one isolate collected in 2005.
What were the most significant accomplishments?
The most significant accomplishment in this work was the identification of which
resistant sources are overcome and which ones contain potentially durable net
blotch resistance that could be used in combating net blotch in this region as well as
the identification of virulence changes within the pathogen population from year to
year.
Objective:
Net blotch caused by the fungus Pyrenophora teres Drechs. f. teres Smedeg.
(anamorph: Drechslera teres) is one of the most economically important diseases in
barley growing regions of the United States and the world. This disease is a
perennial problem especially in cool, wet barley growing regions although it has
been seen in dry regions as well (Mathre 1982). The majority of barley cultivars
grown in the United States are moderately to highly susceptible to net blotch of
barley. Therefore, it is critical that we have an understanding of the pathogen
population to properly develop barley cultivars with durable resistance to net blotch.
The objective of this project was to evaluate the North Dakota field population at two
location with two significantly different environments. This study focused on the
level of variability present in the P. teres population as it relates to identified host
resistance in a differential barley set.
Methodologies:
Host differential set
A host differential set of 20 barley lines (Table 1) was chosen based on
published differential reaction of these lines. (Jalli 2004, Wu et al. 2003, Cromey
and Parks 2003, Gupta and Loughman 2001, Jonsson et al. 1997, Steffenson and
Webster 1992, Tekauz 1990, Kahn 1982, Kahn and Boyd 1969). This was done by
evaluating the most thorough publications on differential sets. All lines used in this
differential set have been shown to have different resistance patterns when
inoculated with isolates from around the world indicating that each line harbors at
least one different resistance gene. NDB112 and Heartland (Table 1) have also
been added to this set based on their use as resistance sources in the North Dakota
and Minnesota barley breeding program, respectively.
Field collection and pathotype analysis on the barley differential set
Field plots of the near universal susceptible cultivar Hector were planted on
barley stubble to ensure the best disease possible. Hector plots were established
at Fargo and Langdon, ND in both 2004 and 2005. Diseased leaves were collected
throughout the growing season. Leaves were plated on sterile water agar and
single spore isolates were picked from net blotch lesions. Isolates were grown and
93

induced to sporulate followed by a second round of single sporing to ensure purity.
Phenotypic data was collected by inoculating each field isolate on each of the 22
barley lines. The barley cultivar Heartland was added to the set in 2005 (Table 1)
and therefore no data is available for 2004 collections. For 2005 the differential set
was inoculated with conidia of 25 individual isolates collected at Langdon and 10
isolates collected at Fargo. Inoculations were completed at the two-to-three-leaf
stage as follows. Individual differential lines were planted along with controls using
three conetainers (Stuewe and Sons, Inc., Corvallis, OR) per line and three plants
per conetainer. Conidia were grown and harvested as described by Weiland et al.
(1999). Conidia were diluted to 3000 spores/ml, and 2 drops of Tween 20
(polyoxyethylene sorbitan monolaurate) was added per 100 ml of inoculum. Plants
were inoculated to a heavy mist but not to runoff. Following inoculation, plants were
placed in 100% relative humidity at 21˚C for 24 h, and then placed in a controlled
chamber under a 12 h photoperiod at 21˚C. Disease readings were taken at 7 d
post-inoculation using the 1-10 scale developed by Tekauz (1985) where average
disease reactions of less than 5 were considered resistant and those 5 or greater
were considered susceptible. At least two replicates were completed for each
isolate collected.
Results
The comparison of 2004 and 2005 virulence data showed some striking changes in
virulence patterns found across the isolates tested. The 2004 data showed that
resistance found in Canadian Lake Shore, CI4922, Harbin, Manchuria, Manchurian
and Tifang were not effective against any of the isolates collected at either location
(Table 1). In 2005 this was also the case for isolates collected in Fargo whereas in
Langdon these sources of resistance were much more effective indicating a shift
away from virulence on these
barley lines in the Langdon area
(Table 1). Barley lines Algerian,
CI5791, CI9214, NDB112, and
Prato showed high levels of
resistance in both years whereas
lines Rika and SM89010 had a
reduction in effective resistance
in 2005 especially in the Langdon
collections.
Barley lines NDB112 and
Heartland are used as disease
resistance sources in the North
Dakota and Minnesota barley
breeding programs respectively.
Both of these lines look to be very
effective against the current
pathotypes, with both lines
94
%
%
%
%
virulence virulence virulence virulence
Fargo, ND Langdon, ND Fargo, ND Langdon,
2005
2005
2006
ND
CI
(10 isolates) 2006
Barley line number
(25 isolates)
Algerian 1179 0 0 0 0
Atlas 4118 32 40 90 0
Beecher 6566 24 20 70 0
Can. Lk. Sh. 2750 100 100 100 0
Cape 1026 80 60 90 84
CI11458 11458 92 40 80 0
CI4922 4922 100 100 100 0
CI5791 5791 0 0 0 0
CI9214 4929 0 0 0 0
Harbin 12673 100 100 100 0
Hazera 145351 20 12 90 0
Heartland 552963 0 0
Hector 15514 100 100 100 100
Kombar 15694 32 40 100 68
Manchuria 2330 100 100 100 60
Manchurian 739 100 100 100 56
Ming 4797 100 88 80 0
NDB112 11531 12 4 0 0
Prato 15815 4 0 0 4
Rika 8069 20 20 30 84
SM89010 4 12 0 60
Tifang 4407-1 100 100 100 0

showing good levels of resistance to all isolates tested in 2005 at both locations.
Although there was no apparent shift in the major barley cultivars planted our
result from 2004 and 2005 would show that the P. teres pathogen population has
variability for potential adaptation to new resistance sources. Caution should be
used when relying on a single resistance source for net blotch.
References:
Cromey, M.G. and Parks, R.A. 2003. Pathogenic variation in Dreschlera teres in
New Zealand. New Zealand Plant Protection 56:251-256.
Gupta, S. and Loughman, R. 2001. Current virulence of Pyrenophora teres on
barley in Western Australia. Plant Dis. 85:960-966.
Jalli M.J. 2004. Suitability of a selected barley differential set for Pyrenophora teres
f. teres virulence screening. Proceedings of the 9 th International Barley Genetics
Symposium, Brno Czech Republic, pp. 266-269.
Jonsson R., Bryngelsson, T., and Gustafsson, M. 1997. Virulence studies of
Swedish net blotch isolates (Drechslera teres) and identification of resistant barley
lines. Euphytica 94:209-218.
Kahn, T.N. 1982. Changes in pathogenicity of Drechslera teres relating to changes
in barley cultivars grown in Western Australia. Plant Dis. 66:655-656.
Kahn, T.N. and Boyd, W.J.R. 1969. Physiologic specialization in Drechslera teres.
Aust. J. Biol. Sci. 22:1229-1235.
Mathre, D.E., ed. 1982. Compendium of Barley Diseases. American
Phytopathological Society, St. Paul, MN.
Steffenson, B.J. and Webster, R.K. 1992. Pathotype diversity of Pyrenophora teres
f. teres on barley. Phytopathology 82:170-177.
Tekauz, A. 1990. Characterization and distribution of pathogenic variation in
Pyrenophora teres f. teres and P. teres f. maculata in Western Canada. Can J.
Plant Path. 12:141-148.
Tekauz, A. 1985. A numerical scale to classify reactions of barley to Pyrenophora
teres. Can. J. Plant. Pathol. 7:181-183.
Weiland, J.J., Steffenson, B.J., Cartwright, R.D., and Webster, R.K. 1999.
Identification of molecular genetic markers in Pyrenophora teres f. teres associated
with low virulence on ‘Harbin’ barley. Phytopathology. 89:176-181.
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Wu, H.-L., Steffenson, B.J., and Zhong, S. 2003. Genetic variation for virulence and
RFLP markers in Pyrenophora teres. Can. J. Plant. Pathol. 25:82-90.
Other Barley Research and Future Direction of Program
Additional barley research in our lab is looking at both the host and pathogen sides
of the barley net blotch system. This is being done by developing and evaluating
both pathogen and host populations segregating for virulence and resistance
respectively. Currently we are mapping and identifying closely linked markers to
resistance genes in the Rika X Kombar doubled haploid population. On the
pathogen side we are using P. teres mapping populations that segregate for
virulence/avirulence on several of the barley differential lines in order to identify and
genetically characterize pathogen virulence.
Project Personnel
Tim Friesen – Research Scientist
Philip Meyer – Technician
Danielle Holmes – Technician
Adam Little – Undergraduate research assistant
Recent Publications
Lai Z., Faris J.D., Weiland, J.J., Steffenson, B.J., and Friesen, T.L. 2007 Genetic
mapping of Pyrenophora teres f. teres genes conferring avirulence on barley.
Fungal Genet. Biol. In press (doi:10.1016/j.fgb.2006.11.009)
Friesen, T. L., Faris, J. D., Lai, Z., and Steffenson, B.J. 2006 Identification and
chromosomal location of major genes for resistance to Pyrenophora teres f. teres
and P. teres f. maculata in a barley doubled haploid population. Genome 49:855-
859.
96

Germplasm Enhancement for RWA Resistance
D.W. Mornhinweg, D.R. Porter, and G.J. Puterka
USDA-ARS Plant Science and Water Conservation Research Laboratory
Stillwater, Oklahoma
Executive Summary
Whenever insect pests impact grain yield of barley they affect malting quality. The
major cause of yield loss with RWA feeding is through head trapping which results in
reduced fertility and a severe decrease in plumpness of surviving seed. Not only is less
grain available for malt, but the quality of that grain is greatly decreased. When RWAs
feed on susceptible plants, the new leaves do not unroll and aphids build up in high
numbers inside the unrolled leaves where they are protected from contact insecticides
as well as natural parasites and predators, and wind and rain. In years of severe or
early infestation, chemical control can only be accomplished with repeated applications
of systemic insecticides. These chemicals are not only expensive for barley producers
but could quite possibly end up in malt produced from treated fields. The solution to
these problems is resistant varieties. 109 unadapted germplasm lines have been
developed in previous years as a part of this project after screening the entire NSGC.
Two lines have been officially released to breeders while the others were available upon
request. Inheritance studies, also accomplished in previous years by this project, have
given barley breeders valuable information on how to best utilize these germplasm lines
in their breeding programs.
Negative affects on yield and malting quality are often associated with the use of
unadapted germplasm in a breeding program, so a prebreeding program was instituted
to bring resistant genes into good malting quality backgrounds adapted to all the barley
growing areas of the US. Due to the common occurrence in aphid populations of
biotype change after which the aphid can overcome resistance, all 109 resistant
sources have been utilized in this prebreeding program in hopes of producing
germplasm with genetic diversity for resistance which could protect barley from any
future aphid biotype change. Such a biotype change occurred in the summer of 2003
when wheat varieties resistant to RWA1 were severely damaged and yields greatly
reduced. This new biotype has since been named RWA2. Since that time yet another
3 biotypes (RWA3, RWA4, and RWA5) have been reported and more are suspected.
We have tested potential RWA1 resistant barley germplasm releases against RWA2
and RWA3 and all highly resistant lines have maintained their resistance.
Seven, RWA-resistant, winter, feed barley germplasm lines in a Schuyler background
were released in the fall of 2005. Nineteen, RWA resistant, 6-rowed, spring, barley
germplasm lines in 6-rowed, malting barley backgrounds and 17, RWA resistant, 2-
97

owed, spring, barley germplasm lines in 2-rowed, malting barley backgrounds were
released in 2006. Breeders should be able to utilize these germplasm lines directly in a
breeding program with reduced detrimental affect on malting quality as well as grain
yield. Seven, RWA-resistant, 2-rowed, spring, feed barley germplasm lines were also
released in 2006. There are 34 different sources of resistance involved in these 50
germplasm lines. All 43 spring germplasm releases were tested against 5 biotypes of
RWA in 2006. Resistance appears to be holding up to all biotypes. Analysis is ongoing.
Stoneham and Sidney, drought hardy, spring, 2-rowed, feed barley cultivars were also
released in 2006. These barleys are not only RWA-resistant but also adapted to the
high and dry production areas of eastern Colorado and Wyoming and western Nebraska
and Kansas.
Objectives, Methodology, and results
The prebreeding program is designed to bring resistance genes from unadapted
germplasm lines into adapted malting and feed barley backgrounds for all barley
growing regions in the U.S. It involves repeated backcrossing with intermittent
screening with a time commitment of approximately seven years from the first cross
until homozygous resistant BC3F3 lines can first be evaluated as observation lines in
the field. This is an ongoing process involving many resistant lines and adapted
cultivars currently in all phases of the program. Field testing is ongoing in cooperation
with Phil Bregitzer (spring barley) and Don Obert (winter barley) scientists with the
USDA-ARS in Aberdeen, Idaho; Frank Peairs, Colorado State University; and Gary
Hein, University of Nebraska.
The first RWA-resistant barley cultivar, Burton, was selected and released to breeders
in 2004 cooperatively by ARS in Aberdeen and Stillwater, Colorado State University,
University of Nebraska and New Mexico State University. In the summer of 2005,
foundation seed was grown in Colorado and Nebraska for two new RWA resistant,
drought-hardy, spring feed barley cultivars Stoneham and Sidney. These cultivars were
co-released by USDA-ARS in Stillwater, USDA-ARS in Aberdeen, Colorado State
University and University of Nebraska in 2006. Both are in an Otis background with
resistance from STARS 9301B (Sidney) and STARS 9577B (Stoneham). The need for
genetic diversity is very important and has been demonstrated since the 2005 growing
season with severe yield losses of RWA resistant winter wheat by a new virulent
biotype(s) of RWA in Colorado and Nebraska. A survey of biotypes in Wyoming,
Colorado, Nebraska, Kansas, Oklahoma, Texas, and New Mexico in 2006 by Gary
Puterka, USDA-ARS, Stillwater, has shown RWA2 to be the predominant biotype of
RWA in all these states except New Mexico, where RWA1 still predominates. Both
Sidney and Stoneham are highly resistant to all known biotypes of RWA and out yielded
Otis in 12 large-scale on farm tests conducted in 2006 by cooperators Frank Peairs and
98

Gary Hein in Colorado and Nebraska.
Five malt barleys and ten feed barleys being considered for cultivar release were tested
at 4 locations in Idaho with the cooperation of Phil Bregitzer. Remnant seed from head
selections made in 2005 and evaluated for homozygous resistance to RWA, were
grown as plant rows in Aberdeen Idaho in 2006 by Phil Bregitzer to obtain breeders
seed increase. These rows were tested again in 2006 for homozygosity prior to bulking
seed for release.
Three advanced lines of RWA-resistant feed barleys were entered in elite winter barley
nurseries and grown at several locations in Idaho with the cooperation of Don Obert.
These 3 lines are in a Schuyler background each with a different source of RWA
resistance and are being considered for cultivar release. A large-scale on farm test of
4 winter feed barleys adapted to western Colorado was planted in the fall of 2006.
Heads will be selected for pure seed increase of potential cultivar release.
Ten, winter, feed barley germplasm lines in a Post 90 background with RWA resistance
from 10 sources were screened for homozygosity prior to bulking for pure seed for
germplasm release in 2007. These lines will be resistant to both RWA and greenbug
and are widely adapted growing successfully as far north as Idaho as well as the
southern great plains.
The timely addition of Dr. Gary Puterka to our research unit in Stillwater has enabled
this project to respond to the biotypic changes now occurring in RWA populations in the
barley growing areas of the US. RWA resistance in barley germplasm must be tested
against all biotypes. The need for genetic diversity has never been clearer. Gary
Puterka is currently screening all 43 spring barley germplasm releases (STARS 0601B
– STARS 0643B) to RWA1, RWA2, RWA3, RWA4, and RWA5. Rating is almost
complete and early indications are that resistance has held up to all biotypes. Analysis
is ongoing.
The Barley Core Collection (BCC) was screened for bird cherry-oat aphid (BCOA)
resistance using a new screening technique developed at the USDA-ARS in Stillwater.
The screening is actually and adaptation of flat screening tests established for RWA and
greenbug. Under these testing conditions, normally asymptomatic seedlings become
symptomatic. Morex, used as a susceptible check, died after 3 weeks of infestation
while other lines survived. Seedlings were rated using a rating scale of 1-7 with 1=
resistant and 7=dead. Approximately 10% of the 960 accessions in the BCC survived
the screening and were rescued. All rescued plants were transplanted into pots and are
currently being evaluated for yield and yield components.
Crosses were made and populations developed for inheritance and genetic diversity
studies for aphid resistance (RWA and greenbug) in barley. Crosses were also made
99

towards the development of RWA and Greenbug resistant winter hulless barleys
adapted to Oklahoma with the end use of ethanol production.
Barley accessions recently added to the NSGC were screened for RWA1 resistance.
Other barley research and future direction of the program
A cooperative project with Lynn Dahleen, USDA-ARS, Fargo, ND, entered its second
year to find genetic markers for RWA resistance in STARS 9301B and STARS 9577B to
aid in genetic diversity studies and in marker assisted selection of resistant germplasm
lines. 300 F2:F3 lines from the STARS 9577B population were phenotyped twice for
this project in 2006.
A project has been initiated to develop hulless winter barleys for ethanol production in
the Southern Plains. 80 winter hulless barley accessions and 240 hulled winter barley
lines were grown replicated tests and evaluated at 2 locations in Oklahoma in 2006.
The hulled barleys were in a Post 90 background with RWA resistance from 10 different
sources. Post 90 is also resistant to all currently identified biotypes of Greenbug.
Barley grown in Oklahoma will need resistance to both RWA and Greenbug. All hulless
lines were evaluated for %starch by Patricia Rayas-Duarte at Oklahoma State
University. 19 hulless lines were selected for crossing with 25 RWA-resistant, hulled
germplasm lines.
Project Personnel
Germplasm Enhancement
Dolores W. Mornhinweg, Geneticist
David R. Porter, Research Geneticist
Gary Puterka, Research Entomologist
Cooperators
Don Obert, Research Agronomist, USDA-ARS, Aberdeen, ID
Phil Bregitzer, Research Geneticist, USDA-ARS, Aberdeen, ID
Frank Peairs, Professor, Colorado State University
An, Hang, Research Geneticist, USDA-ARS, Aberdeen, ID
Dave Blatensperger, Professor, University of Nebraska
Lynn Dahleen, USDA-ARS, Fargo, ND
Tim Springer, Research Geneticist, USDA-ARS, Woodward, OK
Brett Carver, Professor, Oklahoma State University
Wynse Brooks, Professor, Virginia Polytechnic Institute and State University
100

Kevin Hicks, Research Leader, USDA-ARS, Wyndmoor, PA
Patricia Rayas-Duarte, Professor, Oklahoma State University
Recent Publications
Bregitzer, P., D.W. Mornhinweg, and B.L. Jones. 2003. Resistance to Russian wheat
aphid damage derived from STARS-9301B protects agronomic performance and
malting quality when transferred to adapted barley germplasm. Crop Sci. 43(6):
2050-2057.
Bregitzer, P., D.W. Mornhinweg, R. Hammon, M. Stack, D.D. Baltensperger, G.L. Hein,
M.K. o’Neill, J.C. Whitmore and D.J. Fiedler. 2005. Registration of ‘Burton’
Barley. Crop Sci. 45: 1166.
Mornhinweg, D.W., D.R. Porter and J.A. Webster. 1999. Registration of Stars-9577B
Russian Wheat Aphid Resistant Barley Germplasm. Crop Sci. 39: 882-883.
Mornhinweg, D.W. 2003. Invasion of Barley Insects – Yield and Quality Snatchers: We
dare not rest. Proceedings 34 th Barley Improvement Conference.
Morhninweg, D.W., L.H. Edwards, E.L. Smith, G.A. Morgan, D.R. Porter, and B.F.
Carver. 2004. Registration of Post 90 Barley. Crop Sci. 44:2263.
Mornhinweg, D.W.,D.E. Obert, D.M. Wesenberg, C.A. Erickson, and D.R. Porter. 2006.
Registration of seven winter feed barley germplasms resistant to Russian wheat
aphid. Crop Sci. 46: 1826-1827.
Mornhinweg, D.W., M.J. Brewer and D.R. Porter. 2006. Effect of RWA on yield and
yield components of field grown susceptible and resistant spring barley. Crop Sci.
46:36-42.
Porter, D.R., D.W. Mornhinweg, and J.A. Webster. 2004. New Sources of Resistance
to Greenbug in Barley. Crop Sci. 44: 1245-1247.
Porter, D.R. and D.W. Mornhinweg. 2004. Characterization of greenbug resistance in
barley. Plant Breeding 123:5 pp. 493-494.
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Project Title: The Oregon Barley Improvement Program
Researchers: Dr. Patrick Hayes, Dr. Jennifer Kling, Dr. Peter Szucs, Ms. Ann
Corey, Ms. Tanya Filichkin
Department: Dept. of Crop and Soil Science
Institution: Oregon State University, Corvallis, OR 97331
Executive Summary
How the OSU program helps AMBA realize its mission and primary objective: The
two principal goals of this project remain (1) to develop six-row winter malting barley
varieties that will assist AMBA in meeting its mission of providing the malting and
brewing industries with an abundant supply of high quality malting barley and (2) to
develop molecular breeding tools that will benefit all barley breeders working to advance
the AMBA cause. We are addressing AMBA’s primary objective – ensuring that barley
is a competitive crop – by incorporating malting quality into high yielding winter habit
varieties that provide growers with a profitable and productive cropping option.
Major issues, solutions, and expected benefits: We have developed a winter barley
germplasm base suitable for making the elite x elite crosses from which most malting
barley varieties derive. It has taken 20 years to reach this point due to necessity of
combining malting quality, disease resistance, and winter hardiness. Our approach to
solving these complex issues has been to develop molecular breeding tools based on
knowledge of gene locations, effects, and interactions. We now have excellent parental
stocks and two years ago embarked on a more extensive crossing program to generate
the segregating generation progeny from which AMBA-approved varieties can be
expected to come. The benefits will be high yielding malting barley varieties that
represent a new, dependable, and alternative source of high quality malting barley.
One-year objectives and outcomes: We developed, tested, characterized, and
selected winter germplasm at multiple locations. We have ceased all spring barely
improvement efforts, except for specialized disease resistance stocks. We have
advanced lines with excellent yields under irrigated and dryland conditions. These high
yielding lines have good disease resistance and potentially acceptable malt profiles. We
have developed perfect markers for target traits and implemented these in our breeding
program. No malting quality data are available for the 2006 crop samples, due to the
construction of a new facility for the CCRU.
Most significant accomplishments: Per the preceding section, we have developed
some very exciting germplasm and we have developed molecular breeding tools to
accelerate future gains. Three lines are in their first year of AMBA Pilot Scale testing. A
new line is in increase for submission to the program in 2007. And the breeding pipeline
is full.
102

Detailed <strong>Report</strong> on Objectives, Methodology and Results – AMBA Funded Project
Objectives:
Our objective is to develop superior varieties that meet AMBA specifications
based on an understanding of the genetic basis of target traits. Our genetics research
is conducted in the context of an ongoing breeding program involving agronomically
relevant germplasm. This effort received a tremendous boost with funding of the Barley
CAP. The idea is that we will make progress towards our goal and when we reach the
goal, we’ll know how we got there. In winter barley, our primary traits of interest are:
malting quality, productivity, winterhardiness and disease resistance.
Methodology:
We work cooperatively on a regional basis. In Idaho we work with Dr. Don Obert
(USDA/ARS, Aberdeen, Idaho) to realize the potential of winter malting barley via
exchange of advanced yield trials and site visits. Dr. Steve Ullrich is our cooperator in
Washington. Our primary winter barley testing site in Oregon is Pendleton, where we
work with Dr. Steve Petrie. Dr. Lee Jackson, UC Davis, provides disease screening. Dr.
Blake Cooper, BARI, provides winter hardiness screening at Fort Collins, CO. We have
initiated a program of controlled freeze testing with the Martonvasar Research Institute
in Hungary. Dr. David Hole, Utah State, now participates in screening of winter barley
head rows at Logan, UT.
In order to focus our energy on winter barley improvement, we have stopped
breeding spring barley, except for targeted development and distribution of genetic
stocks with quantitative resistance to barley stripe rust (see
http://www.barleyworld.org/osubreeding/striperustmapping.php).
Our winter barley field phenotyping efforts are based on regional evaluation of
variety candidates, replicated multi-environment testing of advanced lines, screening of
preliminary yield trials, advance of segregating generations, and crossing to accumulate
favorable alleles. The 2006/2007 winter nurseries are summarized in Table 1. Our
laboratory program directly supports the winter malting barley breeding program. We
have developed diagnostic PCR markers for target traits and used these to genotype
our breeding lines in our own lab or in collaboration with the Regional Genotyping Lab in
Pullman, Washington. Malting quality assessments are conducted by Mr. Al Budde,
USDA/ARS and Dr. Cynthia Henson has collaborated on beta amylase activity and
thermostability assays.
Results:
Breeding program activities and results are detailed in the following tables. These
tables show data only for selections in the AMBA Pilot program or that will be submitted
to the AMBA Pilot program in 2007. The complete 2005/2006 data are too voluminous
for inclusion in this report; they are available on request.
Three lines were submitted to the first year of AMBA Pilot Scale Testing and
passed pre-screening. The pre-screening data are shown in Table 2. These lines are
STAB BC 50-7-3, STAB 47/KAB 51-7 and STAB 113/ KAB 50 – 21. STAB BC 50-7-3 is
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derived from the backcross: Strider/88Ab536//88Ab536. STAB 47 and STAB 113 are
doubled haploid lines from the cross of Strider/88Ab536. KAB 50 and KAB 51 and KAB
lines are doubled haploid lines from the cross of Kold/88Ab536.
Quality: Quality data from the 2006 harvest have not been received, due to the
movement of the CCRU to a new facility. Past data on the three lines in first year AMBA
Pilot, plus a fourth selection that will be submitted in 2007, are summarized in Table 3.
These data show a pattern of lower grain protein (and consequently lower enzyme
activity) but as is apparent from the pre-screening data from Aberdeen, ID, these
selections can achieve high grain protein. It remains to be seen what happens to extract
and enzyme activity at these high levels. An advantage of winter over spring barley will
be nitrogen management options. Fall/Spring split applications of N will allow for more
precise targeting of protein and enzyme levels.
Agronomics: The selections all have high test-weight and a high percentage of
plump seed. The yield potential is truly impressive, as is shown in Table 4. Irrigated
winter barley will save at least one application of scarce and expensive water compared
to spring barley grown at the same location. Disease data, as compared to check
varieties, are shown in Table 5. All selections showed excellent stripe rust resistance.
No BYDV symptoms were reported on any of the four selections at UC Davis nor
observed at Corvallis. The selections show a range of reaction to scald under intense
disease pressure at Corvallis – such intense scald levels are usually not observed in
target production areas.
Winter hardiness: We obtained very useful winter hardiness data from two locations.
The data on selections are shown in Table 6 and perspectives on the entire germplasm
array are shown in Figure 1. Two features stand out. One is that we have a limited
number of winter x spring cross progeny with good winter hardiness profiles. Since both
the high and low lines are part of the CAP set, we will have complete genotype data on
this germplasm, allowing new insights into the genetic control of winter hardiness. This
should further facilitate the conversion of elite spring malting lines to winter habit.
Secondly, a set of elite winter lines from Europe was included in the controlled freeze
tests in Hungary. It is gratifying that our germplasm is competitive with the best
European material.
Advanced, preliminary, and early generation materials
These nurseries, and the numbers of lines therein, are shown in Table 1.
104

Other Barley Research and Future Direction of Program
In addition to winter malting barley development, the Oregon Barley Project is
engaged in a number of other endeavors:
Basic research:
• Winter hardiness genetics
• Barley CAP
Applied research
• Winter barley for human nutrition
• Barley straw for algae control
We plan to continue these areas of endeavor in the future.
Project Personnel
Patrick Hayes, Professor
Jennifer Kling, Senior Research Professor
Peter Szucs, post-doc
Ann Corey, Senior Research Assistant
Tanya Filichkin, Senior Research Assistant
Kale Haggard, Graduate Research Assistant
Juan Rey, Graduate Research Assistant
Phinyarat Kongprakhon, Visiting Scholar
105
Recent Publications (2006 - 2007)
1. Druka, A., G. Muehlbauer, I. Druka,, R..Caldo, U. Baumann, N. Rostoks, A.
Schreiber, R. Wise, T. Close, A. Kleinhofs, A. Graner, A. Schulman, P.
Langridge, K. Sato, P. Hayes, J. McNicol, D. Marshall, and R. Waugh. 2006.
An atlas of gene expression from seed to seed through barley development.
Func. Int. Genomics. 6:202-211.
2. Szűcs, P., I. Karsai, J. von Zitzewitz, K. Mészáros, L.L.D. Cooper, Y.Q. Gu,
T.H.H. Chen, P.M. Hayes, and J.S. Skinner. 2006. Positional relationships
between photoperiod response QTL and photoreceptor and vernalization
genes in barley. Theor. Appl. Genet. 112:1277-1285.
3. Richardson K.L,, M. I. Vales, J. G. Kling, C. C. Mundt, and P. M. Hayes. 2006.
Pyramiding and dissecting disease resistance QTL to barley stripe rust. Theor.
Appl. Genet. 113:485-495.
4. Yun, S.J., L. Gyenis, E. Bossolini, P.M. Hayes, I. Matus, K.P. Smith, B.J.
Steffenson, R. Tuberosa and G. J. Muehlbauer. 2006. Validation of
quantitative trait loci for multiple disease resistances using advanced
backcross lines developed with a wild barley (Hordeum vulgare subsp.
spontaneum). Crop Sci. 46: 1179-1186.

106
5. Bilgic, H., B.J. Steffenson, and P.M. Hayes. 2006. Molecular mapping of loci
conferring resistance to different pathotypes of the spot blotch pathogen in
barley. Phytopathology 699-708.
6. Karsai, I., K. Mészáros, P. Szűcs, P.M. Hayes, L. Láng, and Z. Bedő. 2006.
The Vrn-H2 locus (4H) is influenced by photoperiod and is a major
determinant of plant development and reproductive fitness traits in a
facultative × winter barley (Hordeum vulgare L.) mapping population. Plant
Breeding . 124: 468-472.
7. Kóti, I. Karsai, P. Szűcs, C. Horváth, K. Mészáros, G. Kiss, Z. Bedő, P.M.
Hayes. 2006. Validation of the two-gene epistatic model for vernalization
response in a winter x spring barley cross. Euphytica 152:17-24.
8. Inukai, T., M. I. Vales, K. Hori, K. Sato and P.M. Hayes. RMo1 confers blast
resistance in barley and is located within the complex of resistance genes
containing Mla, a powdery mildew resistance gene. Mol. Plant. Microbe. Inter.
19: 1034-1041.
9. Rossi, C., A. Cuesta-Marcos, I. Vales, L. Gomez-Pando, G. Orjeda, R. Wise,
K. Sato, K. Hori, F. Capettini, H. Vivar, X. Chen, and P. Hayes. Mapping
multiple disease resistance genes using a barley mapping population
evaluated in Peru, Mexico, and the USA. Mol. Breeding. 18:355-366.
10. Singh, J., S. Zhang, C. Chen, L.D. Cooper, P. Bregitzer, A.K. Sturbaum, P.M.
Hayes, and. PG. Lemaux. 2006. Remobilization over multiple generations in
barley facilitates gene tagging in large genome cereals. Plant Molecular
Biology. 62:937-950.
11. Szucs, P., J. Skinner, I. Karsai, A.Cuesta-Marcos, K.G. Haggard, A.E. Corey,
T.H.H. Chen, and P.M. Hayes. 2006. Validation of the VRN-H2/VRN-H1
epistatic model in barley reveals that intron length variation in VRN-H1 may
account for a continuum of vernalization sensitivity. Mol. Genet. Genomics .
(online)
12. Hayes, P.M. and P. Szucs. 2006. Disequilibrium and association in barley:
thinking outside the glass. PNAS (USA) 49:18385-18386.
13. Limin, A., A.Corey, P. Hayes, and D. B. Fowler. 2007. Low-temperature
acclimation of barley cultivars used as parents in mapping populations:
response to photoperiod, vernalization and phenological development. Planta.
In press.

Table 1. 2006/2007 Oregon winter barley nurseries.
_________________________________________________________________________________________________________________________
OSUCAP96; 96 entries; Oregon CAP Lines
• Corvallis 1 rep, purification plot
• Pendleton 1 rep, Augmented Design
• Aberdeen 1 rep, Augmented Design
• BARI observation plots, cold test
• Other locations: Disease screen with Lee Jackson, UC Davis; Fusarium root rot
screen with Richard W. Smiley and Jason Sheedy, CBARC; Food Quality with
Mitch Wise, USDA Madison, WI; LOX, and Beta glucanase with Paul Schwarz,
NDSU; BSR screen with Xianming Chen, USDA, Pullman, WA; Food Quality
with Byung-Kee Baik, WSU, Pullman, WA; Cold Test with Ag Research
Institute, Martonvasar, Hungary
Trial A; 96 entries; ORELT reselections
• Corvallis 1 rep, purification plot
• Pendleton 1 rep, Augmented Design
• Aberdeen 1 rep, Augmented Design
Trial B; 96 entries; ORELT, WBADV, and WBPYT reselections
• Corvallis 1 rep, purification plot
• Pendleton 1 rep, Augmented Design
• Aberdeen 1 rep, Augmented Design
Trial C; 26 entries; ORELT, WBADV, and WBPYT reselections
• Corvallis 1 rep, purification plot
Head Rows (all at Corvallis)
• F5 Head Rows, 16 entries, variable rows per entry
• F4 Head Rows, 29 entries, variable rows per entry
• F3 Head Rows, 117 entries, variable rows per entry
Early generations (all at Corvallis)
• F2 Masa, 54 entries
• TC Masa, 18 entries
• F1 crosses 120 entries
• Top Crosses 18 entries
107

Table 2. Pre-screening data for OSU winter 6-row AMBA Pilot submissions and the
AMBA quality check variety (88Ab536), 2007.
Selection
STAB 47/KAB 51-7
Source plump
(% on 6/64)
STAB 50-7-3
STAB113/KAB 50-21
88AB536
Grain protein
(%)
Aberdeen, ID 89 14.6
Pendleton, OR 91 11.3
Aberdeen, ID 93 14.5
Pendleton, OR 95 11.6
Aberdeen, ID 87 13.7
Pendleton, OR 92 11.1
Aberdeen, ID 84 15.3
Pendleton, OR 82 13.2
Table 3. Malting quality data for OSU winter 6-row malting lines and check varieties.
Eight-twelve and 88Ab536 are the AMBA agronomic and quality checks, respectively.
Average of 2004 and 2005 crop years, CCRU data; 6 station years. Strider and Maja
are OSU feed varieties included as checks to measure progress; Eight-twelve is a feed
variety with no malting quality. Samples were not submitted for analysis.
Selection Malt
extract
(%)
Barley protein
(%)
Wort
protein
(%)
Diastatic
power
( 0 ASBC)
Alpha
amylase
(20 0 DU)
Wort Beta
glucan
(ppm)
88Ab536-B 78.8 12.1 4.5 126 56.6 276
Strider 77.1 12.2 3.5 55 37.1 626
Maja 80.7 10.5 4.2 114 51.5 122
Eight-Twelve NA NA NA NA NA NA
StabBC 50-7-3 79.4 10.5 3.4 91 45.3 467
Stab 47/Kab 51-7 80.5 11.4 4.2 105 53.0 307
S113/K50-21 80.1 11.0 4.1 121 48.2 238
Stab 47/Kab 51-20 80.1 11.1 4.6 142 64.6 182
108

Table 4. Agronomic data for OSU winter 6-row malting lines and check varieties.
Average of 2005, 2006 crop years; 10 station years.
Selection Grain Yield Plump Test weight Height
(lbs/acre) (% on 6/64) (lbs/bu) (inches)
88Ab536-B 6467 75 52 43
Strider 7374 81 53 38
Maja 7045 81 54 40
Eight-Twelve 7099 70 50 38
StabBC 50-7-3 7505 94 54 45
Stab 47/Kab 51-7 7182 87 53 44
S113/K50-21 6955 81 52 44
Stab 47/Kab 51-20 7062 92 53 43
Table 5. Disease resistance data for OSU winter 6-row malting lines and check
varieties.
Selection Barley stripe rust
Corvallis, 2006
(% disease
severity)
Barley stripe rust
UC-Davis, 2005/2006
average
(% disease severity)
Scald rating
Corvallis, 2006
(9 = lesions on flag
leaf)
88Ab536-B 90 60 6
Strider 8 0 3
Maja 6 42 5
Eight-Twelve 93 70 7
StabBC 50-7-3 6 0 2
Stab 47/Kab 51-7 3 0 3
S113/K50-21 11 0 4
Stab 47/Kab 51-20 3 0 7
Table 6. Winter hardiness (% survival) for OSU winter 6-row malting lines and check
varieties under field conditions at Pendleton, Oregon and under controlled environment
conditions (-13 o C) at Martonvasar, Hungary.
Selection Pendleton, Oregon Martonvasar, Hungary Average
AMBA Drill strip CAP trial
88Ab536-B 50 60 60 57
Strider * 48 58 53
Maja * 66 78 72
Eight-Twelve * 75 82 79
StabBC 50-7-3 30 50 84 55
Stab 47/Kab 51-7 50 60 80 63
S113/K50-21 30 55 77 54
Stab 47/Kab 51-20 60 85 93 79
109

Figure 1. Winter survival data for OSU selections, check varieties, and elite European
varieties (in a -13 0 controlled freeze test) and at Pendleton, Oregon (rated 2/2007).
WxW and S X W = selections from winter x winter and spring x winter crosses,
respectively.
# Lines
# lines
14
12
10
14
12
10
8
6
4
2
0
8
6
4
2
0
1-5
1-5
6-10
6-10
11-15
11-15
16-20
16-20
MRI Freeze Test 2006
21-25
21-25
26-30
31-35
36-40
Strider (ave of 13)
(48%)
StabBC 50-7-3
(50%)
26-30
31-35
Strider (58%)
88Ab536 (60%)
41-45
S113/K50-21 (77%)
Maja (78%)
STAB47/KAB51-7 (80%)
46-50
51-55
56-60
Survival %
Pendleton 2006-07
36-40
41-45
S113/K50-21
(55%)
46-50
51-55
56-60
% green foliage
61-65
66-70
71-75
WxW SxW
76-80
88Ab536 (60%)
Stab 47/Kab 51-7
(60%)
Maja (ave of 13)
(66%)
61-65
66-70
71-75
76-80
Eight-Twelve (82%)
STABBC50-7-3 (84%)
81-85
81-85
86-90
86-90
STAB47/
KAB51-20
(93%)
91-95
91-95
WxW SxW
Eight-Twelve
(75%) Stab 47/Kab 51-
20 (85%)
96-100
96-100
110

MALTING QUALITY ANALYSIS OF NEW BARLEY SELECTIONS
2006 CROP YEAR REPORT
Allen D. Budde, Chris Martens and Mark R. Schmitt.
Cereal Crops Research Unit, USDA Agricultural Research Service
MARCH 9, 2007
Executive Summary:
The Malting Quality Analysis project at the Cereal Crops Research Unit (CCRU) in
Madison is an on-going effort to provide information to a number of state and federally
funded barley breeders on the malting quality of new barley selections. Upon receipt of
seed from cooperating breeders, we micro-malt the samples of the various lines, and
then analyze the resulting malt for a number of parameters used to assess the suitability
of the line for development of commercial malting varieties. Our goal is to provide
accurate and timely data on the malting quality of the submitted samples to assist barley
breeders in making their breeding line selections. We also carry out investigations on
the malting barley in order to better understand the biochemical basis for malt
production and malting quality.
The QA lab completed the 2005 crop year analyses in August, 2006, in time to prepare
for the move to a new facility. The 2005 crop year analyses represent the greatest
number of samples that have ever been malted and analyzed in one year at the CCRU.
The QA lab was moved in October of 2006. We encountered a number of expected and
unexpected issues, which have delayed us from initiating malting of breeder’s
submissions. Nearly all issues have been resolved and performance testing is nearly
complete. If performance testing goes well, we hope to begin malting 2006 crop year
submissions the week of March 12. We have plans in place to process an increased
number of samples per week through to initiation of malting the 2007 crop year
samples. Remaining submissions from the 2006 crop year will be malted and analyzed
as time permits.
As a result of discussions at the 2007 Barley Improvement Conference, breeders are
being provided with barley analytical data as the best alternative to our usual malt
analyses. As of March 9, 2007, barley data on 2000 submissions have been reported
back to the breeders.
We have prepared the AMBA pilot and plant scale malts using our Joe White
micromalters and have completed malting 50% of the regional nursery submissions.
Analyses of these malts will be completed prior to the Spring AMBA Technical
Committee meeting.
111

Mission:
The Cereal Crops Research Unit will support public sector barley breeders by providing
the malt quality analyses necessary to assist in making knowledgeable selections for
generation of high quality malting barleys.
Primary Objective:
Generate malts from barleys submitted to us from public sector breeders. Analyze the
malts and report the data back to the submitting researcher(s).
Materials and methods:
The methods used for the physical and chemical analyses of micro-malts are, for the
most part, ASBC procedures. Some automated versions of the standard methods are
used, in which case they have been standardized against the standard ASBC methods.
In order to ensure that the methods used and results produced at CCRU are
representative, we participate in three quality analysis collaboratives comparing our
analyses of common samples and standards with those of other analytical laboratories.
Micro-malts are prepared with 170 g (dw) samples, if sufficient grain is available.
Smaller samples are used with appropriately modified procedures if sample quantity is
insufficient for the full sample size. The samples are steeped to 45% moisture at 16 ◦ C,
germinated for five days at 17 ◦ C, with intermittent turning and kilned over a period of 24
hours to a final temperature of 85 ◦ C. These conditions have been shown to produce
malts with quality characteristics similar to commercially prepared malts.
All analytical data and evaluations of the micro-malts are summarized in individual
reports that are returned to the breeders submitting samples. An evaluation of the
malting quality of selected locations of the Mississippi Valley Regional Spring Barley
Nursery and the Western Regional Spring Barley Nursery are provided to interested
parties. At the end of the analysis year, the results are compiled into two annual
regional reports. Data from the pre-screen and pilot scale analyses were submitted to
AMBA for collaborative evaluation.
Changes in current funding year:
Personnel: Mr. Andrew Standish has replaced Mr. Donovan Craig. Andy is a graduate
of the Botany department at the University of Wisconsin – Madison. Andy started work
on March 5, 2007 and is 100% supported by the AMBA grant over his current 9 month
appointment.
Equipment: Our very old kiln was replaced with a new version from Standard Industries
Inc. of Fargo, ND. The new kiln allows us greater control and monitoring of kilning
parameters and provides increased sample throughput capabilities.
112

We recently secured an industrial chiller to cool the process water in the malting lab.
The incoming cold water at the new laboratory was several degrees higher than at the
old facility and a chiller was necessary to achieve the required temperature for steeping.
Results:
Barley Malting and Malting Quality Analysis
2005 Crop Summary
5578 samples were received for analysis:
Breeders’ line micro-malts (170 g) 4409
Western Regional Spring Barley Nursery 136
Mississippi Valley Nursery 140
AMBA Pilot/Plant Scale malting samples 82
In House experimental studies 111
Breeders’ experimental studies 700
Total 5578
2006 Crop Summary
4574 samples have been received for analysis (434 malted as of 3/8/07).
Breeders’ line micro-malts (170 g), 4106
Includes 1593 samples for Barley CAP
Western Regional Spring Barley Nursery 155
Mississippi Valley Nursery 190
AMBA Pilot/Plant Scale malting samples 73
In House experimental studies 8
System testing has generated 812 malts (not in total)
Protein only samples 50
Total 4582
113

Other Barley Research:
In House Osmolality Study 108
Sorghum Survey 142
Malt Genes Project 411
In House Expression Profiling (Green and Kilned Malt) 3
Barley CAP (Green and Kilned Malt) 48
Environmental Effects on Barley and Malt β-glucan content 187
Total 899
Personnel:
Mr. A.D. Budde, Plant Physiologist Mr. L. E. Hornbacher, Physical Science Tech
Mr. C.H. Martens, Biol. Science Tech. Mr. M.E. Marinac, Physical Science Tech.
Ms. L.M. Short, Laboratory Tech.* Mr. Andrew Standish, Biol. Science Tech.*
Dr. M Schmitt, Research Chemist Ms. Cynthia Zick, Secretary
Ms. D. K. Schaefer, Secretary Dr. Cynthia Henson, RL
*AMBA supported
Publications:
Budde, A.D., C. Martens, M. Schmitt and Staff. Mississippi Valley Uniform Regional
Nursery - 2005 Preliminary Quality <strong>Report</strong>. 2006 (Technical <strong>Report</strong>)
http://www.ars.usda.gov/Research/docs.htm?docid=10297
Budde, A.D., C. Martens, M. Schmitt and Staff. Western Regional Spring Barley
Nursery - 2005 Preliminary Quality <strong>Report</strong>. 2006 (Technical <strong>Report</strong>)
http://www.ars.usda.gov/Research/docs.htm?docid=10297
B. L. Jones and A. D. Budde. 2005. How various malt endoproteinase classes affect
wort soluble protein levels. J. Cereal Science 41:95-106.
Schmitt, M. R., A. D. Budde and L. A. Marinac. 2006. Research Mash: A simple
procedure for conducting Congress mashes using reduced quantities of malt. J. ASBC.
64(4):181-186.
Schmitt, M. R. and A. D. Budde. 2007. Improved methods for high-throughput
extraction and assay of green barley malt proteinase activity facilitate examination of
proteinase activity across large-scale barley populations. In press.
114

2006-2007 <strong>Progress</strong> <strong>Report</strong>
Characterization of Novel Genes Encoding α-Glucosidase in Malting Barley
Executive Summary
Stanley H. Duke 1 and Cynthia A. Henson 1,2
Department of Agronomy, University of Wisconsin-Madison 1
USDA-ARS-Cereal Crops Research Unit
1575 Linden Drive, Madison WI 53706 2
How this project helps meet AMBA’s mission and primary objective: AMBA’s mission
of encouraging and supporting an adequate supply of high quality malting barley is
addressed by this project’s primary goal which is the development of knowledge of
starch degrading enzymes and assays that measure their collective activity that will be
useful in selecting germplasm with improved malting quality. The malting quality
parameter this research addresses is the efficiency of starch conversion to fermentable
sugars. It is widely accepted that four enzymes (α-amylase, β-amylase, α-glucosidase
and limit dextrinase) work in concert to produce fermentable sugars from starch. The
ability to consistently maximize production of fermentable sugars from raw products,
both malt and adjunct grains, can only be obtained by having a thorough knowledge of
the enzymes involved. This project is contributing to AMBA’s mission by building a
more complete knowledge of starch degradation and measures of starch degradation
during malting and mashing which supports basic and applied scientists involved in the
development of malting quality barley.
The Primary Objective we are addressing is to remedy our lack of knowledge of the
novel α-glucosidase genes we discovered and reported on in the 2005-2006 progress
report.
115

Introduction
There is general agreement that in germinating barley, the degradation of starch
and production of fermentable sugars results from the concerted action of four
carbohydrases - α-amylase, β-amylase, α-glucosidase and limit dextrinase. Compared
to the extensive body of knowledge about barley α- and β-amylases, there is only
rudimentary knowledge available about α-glucosidase and limit dextrinase. This
research was conducted to increase our fundamental knowledge of α-glucosidases in
germinating barley seeds by examining the genes that encode these enzymes and by
characterizing the functional properties of the α-glucosidases that are expressed in
germinating barley seeds.
α-Glucosidases are a diverse group of enzymes that catalyze several reactions
and are capable of using a wide variety of substrates. One feature that all α-
glucosidases share is the ability to hydrolytically remove the terminal monosaccharide
from the non-reducing end of a carbohydrate. Plant α-glucosidases have been
reported to hydrolyze di, - oligo- and polysaccharides, soluble starch, starch granules
with intact crystalline structure, and the carbohydrate structures attached to
glycoproteins. They can hydrolyze α-D-glycosidic bonds with –1,1-, -1,2-, -1,3-, 1,4-,
and -1,6- linkages. Plant α-glucosidases can hydrolyze glycosidic bonds between
glucose residues, between glucose and fructose residues, between glucose and
mannose residues, and between xylose residues. Additionally, they can catalyze
hydrolytic, condensation and transglycosylation reactions.
All plant α-glucosidases sequenced as of this time are categorized as members
of the glycosyl hydrolase family 31. In most cases this categorization is based upon
sequence similarity alone as enzyme function has not yet been determined. All
members of the glycosyl hydrolase family 31 share the same active site sequence and
have at least one additional highly conserved region. The first α-glucosidase cloned
from a plant was in 1996 when Dr. Ron Skadsen’s lab cloned Agl1 from barley (Tibbot
and Skadsen, 1996). Since that time, other genes encoding α-glucosidases have been
identified in plants although, in almost all cases, more is known about the gene than the
116

enzyme it encodes.
We found that seven α-glucosidase genes are expressed in barley seeds that
have been germinated for three days (see 2004-2005 progress report). Only one of
these, Agl1, has been previously reported (Tibbot and Skadsen, 1996). Four of these
seven genes, including Agl1, were abundantly expressed and three had low levels of
expression. The potential roles of the novel α-glucosidases were unknown as were
any characteristics of their gene expression or of the expressed proteins. In this report
we present results of studies to characterize expression of Agl2 , named AglX in the
2004-2005 progress report, and compare its expression to that of Agl1.
Methods
Quantitative RT-PCR (qtRT-PCR)
Total RNA was extracted from 3 – 9 individual samples using Plant RNA reagent
from Invitrogen (Carlsbad, CA) according to the manufacturer’s instructions. Purified
RNA was treated with DNAse using RNeasy mini kits (Qiagen, Valencia, CA) according
to the manufacturer’s instructions. For cDNA synthesis, three independent reactions
were undertaken for each tissue, 1 µg of total RNA for each reaction was reverse-
transcribed using Superscript III (Invitrogen) and random hexamers according to the
manufacturer’s instructions. For qtRT-PCR, 1 µL of a 1:5 dilution of the cDNA was
used in a reaction which also consisted of 5 µL of SYBR premix ExTaq (Takara mirus
bio, Madison, WI), 0.4 µL each of the forward and reverse primers at 5 µM each, 0.2 µL
of ROX reference dye II, and 3 µL of water. Reactions were performed on an Applied
Biosystems 7300 Real-Time PCR system using the following schedule: 10 s at 95°C
followed by 40 cycles of 5 s at 95°C then 31 s at 60°C. A melting curve was obtained
from the product at the end of the amplification by heating from 70°C to 99°C.
Cloning of the putative promoters
Genomic DNA was purified from 7 day old seedling leaves using DNeasy Mini
kits (Qiagen) according to the manufacturer’s instructions. Purified genomic DNA was
digested with the restriction enzymes to create blunt-end fragments that were then
117

ligated to GenomeWalker (Clontech Laboratories, Mountain View, CA) adaptors to
produce GenomeWalker libraries. Using gene specific primers, based on the 5’
regions of cDNA sequences and two kinds of GenomeWalker adaptor primers, PCR
amplifications were performed using general conditions. PCR products were cloned
and sequenced. A search for putative cis-acting DNA elements with sequences
homologous to the sequences of the cloned products was performed using the PLACE
(Plant cis-acting Regulatory DNA Elements) database.
Preparation of peptides and antisera
Synthetic peptides were commercially prepared (Anaspec, Inc., San Jose, CA) to
limited regions (about 15 amino acids) of deduced amino acid sequences of the four
highly expressed α-glucosidases. We selected one region of each of the four proteins
that we determined to be unique to that protein and that we predicted to be antigenic
based on the extent of it’s hydrophobicity. Synthetic peptides were conjugated with
keyhole limpet hemocyanin then used to produce antibodies specific to the four
isozymes of α-glucosidase from barley. Polyclonal antibodies to those conjugated
peptides were produced in rabbits.
Western blots
Crude protein extracts were made from barley tissues using the protocol of
Tibbot et al. (1998). Briefly, the seeds or leaves were ground to a fine powder with
liquid nitrogen in a mortar. Approximately one gram of ground tissue was suspended
in 1 mL of buffer containing 50 mM Na2CO3/NaHCO3 (pH 9.0), 1 M NaCl, 1% Triton-
X100, 2 mM β-mercaptoethanol and 1% protease inhibitor cocktail for plant extracts
(Sigma, St. Louis, MO, USA). Suspensions were shaken gently for 1 hr at 4°C. The
homogenates were centrifuged at 12,000xg for 10 min at 4°C. Four mL of 50 mM
sodium succinate buffer (pH 4.5) were added to the supernatants, which were then
dialyzed against the same buffer. After dialysis, extracts were centrifuged again to
obtain samples for Western blot analysis. Proteins in these supernatants were
resolved by SDS-PAGE, transferred to polyvinylidene fluoride membranes and probed
with the rabbit antiserum. Detection of cross reacting proteins were determined using
118

horseradish peroxidase-conjugated goat anti-rabbit IgG (Cell Signaling Technology,
Beverly, MA, USA) and an ECL-plus Western blotting detection reagent (Amersham
Bioscience, Piscataway, NJ) on X-ray film (Roche Diagnostics) according to the
manufacturer’s instructions.
Results and Discussion
To determine in which tissues and developmental stages the four major α-
glucosidase genes were expressed, we investigated the temporal gene expression
patterns in seeds and leaves. To accomplish this we used quantitative reverse
transcriptase polymerase chain reaction (qtRT-PCR) to determine the relative
abundance of mRNA transcripts for individual genes. To ensure accurate expression
profiling, geometric averaging of transcripts of multiple (3 – 5) internal control genes has
been conducted which allows normalization of transcript abundance. We have
preliminary data for expression of three of the novel abundantly expressed genes, but
we only have complete data for Agl2, upon which we focus in this report, and Agl1,
which we have examined for comparative purposes. The results are that Agl1
expression is significantly higher in seeds than in leaves. In contrast, expression levels
of Agl2 are the same in seeds and leaves. Furthermore, expression of Agl2 in both
seeds and leaves is greatly reduced compared to Agl1 expression levels in seeds.
Differences in the levels of expression in seeds of these two genes may result
from interesting structural features of the genes or their promoters. We determined
that Agl1 contains an amylase element (AE) in its first intron and that Agl2 has no AE
anywhere in the structural gene. AE is a cis-element required for α-amylase
expression during sugar starvation (Hwang et al., 1998). This difference may influence
expression of Agl1 and Agl2 during early stages of germination when sugar levels are
low. Additional influence on gene expression may result from differences we’ve found
in the promoters of these two genes. The Agl1 promoter contained a gibberellic acid
response element (GARE). The promoter of Agl2 did not contain a GARE, which
renders it insensitive to the high levels of GA synthesized by the embryo during
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germination that is known to stimulate expression of many genes in the aleurone layers
surrounding the endosperm.
To analyze expression of the proteins encoded by the four abundantly expressed
Agl genes, we prepared four antisera. Each antiserum was designed to be specific to
only one of the four proteins encoded by these four genes. The specificities of these
antisera were determined by testing against extracts of E. coli transformed with one of
each of the four Agl genes we identified. Each of these cultures expressed only one of
the recombinant isozymes. Each antiserum recognized only the isozyme containing
the peptide to which it was designed. Again, in this report we will focus on comparing
expression of the proteins α-glucosidase1 and α-glucosidase2, which are encoded by
Agl1 with Agl2, respectively. Western blot analyses of germinated seed extracts
probed with antibodies that recognized only the α-glucosidase1 protein showed that this
antibody reacted with seed proteins having molecular masses of approximately 100, 95,
75 and 65 kD. These are data in agreement with those reported by Tibbot et al., 1998.
The anti-α-glucosidse1 probe cross reacted with proteins extracted from seedling
leaves with approximate molecular masses of 100, 95 and 80 kD. The Western blots
using the anti-α-glucosidase2 probe detected cross reacting proteins of approximately
100 and 80 kD in seedling leaves but no cross reacting proteins were detected in
germinated seeds. Possible explanations of why the Agl2 gene is transcribed yet the
protein is not detected in the seed extracts include: 1) the protein may be present but
below the limits of detection with the antibody; 2) the seed protein may be processed
differently from the leaf protein such that the epitope is cleaved from the seed protein; 3)
the seed protein may be insoluble possibly via membrane associations or aggregations;
or 4) RNA silencing may be occurring in the seeds that is not occurring in the leaves.
Purification and characterization of Agl2 demonstrated that it not only had
maltase activity (the definitive test for Agl), but also had α-xylosidase activity.
Xyloglucans were found to be better substrates than maltose for Agl2. This indicates
that although Agl2 can contribute to starch degradation, it could have a major role in cell
wall degradation during germination. Xyloglucans are the primary hemicelluloses
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found in plant cell walls. The degradation of xyloglucans would result in the production
of both glucose, which is fermentable, and xylose, which is usually not fermentable.
Xylose is a aldopentose that most yeasts are unable to use for fermentation. Maximal
expression of both Agl1 and Agl2 occurs at ca. five days of germination, about the time
that green malt is kilned. An experiment planned for this study that will soon be
conducted it to monitor Agl2 activity during mashing will be conducted using xyloglucans
as the substrate and end product detection/quantification will be done using our HPLC
equipped with a pulsed amperometric detector. Western blots using the Agl2 specific
antibody will be conducted to moniter the stability of the Agl2 protein during mashing.
Work conducted thus far to develop a modified diastatic power assay has been to
initiate a comparison of using boiled soluble starch, nonboiled soluble starch, and a
mixture of boiled malt and adjunct rice as substrates for the production of reducing
sugars from malt extracts that have been centrifuged at high speed to remove
endogenous starch. This work is continuing.
References
Coen ES, Romero JM,Soyle S, Elliot R, Murphy G, Carpenter F (1990) floricaula: A
homeotic gene required for flower development in Antirrhium majus. Cell
63:1311-1322.
Hwang YS, Karrer EE, Thomas BR, Chen L, and Rodriquez RL (1998) Three ciselements
required for rice α-amylase Amy3D expression during sugar starvation.
Plant Mol. Biol. 36:331-341
Jackson DP (1991) In situ hybridization in plants. In: Molecular Plant Pathology: A
practical approach, Bowles DJ, Gurr SJ, and McPherson M, eds. Oxford
University Press, England
Tibbot BK and Skadsen RW (1996) Molecular cloning and characterization of a
gibberellin-inducible, putative α-glucosidase gene from barley. Plant Mol. Biol.
30:229-241
Tibbot BK, Henson CA, Skadsen RW (1998) Expression of enzymatically active
recombinant α-glucosidase in yeast and immunological detection of α-glucosidase
from seed. Plant Mol. Biol. 38:379-391
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Other Barley Research and Future Direction of Program
The major long term objective of the PIs research is to provide sufficient
information to barley breeders that they can produce genotypes whose seeds contain
the best (e.g.- most active, most thermostable, most desirable substrate specificity)
combination of alleles of the enzymes that produce fermentable sugars from starch,
their promoters, and their regulatory DNA-binding proteins.
Henson’s prior research demonstrated that the endosperm specific β-amylase
from Steptoe was both higher in activity and thermostability than the endosperm specific
β-amylase from Morex and determined that the favorable characteristics of the Steptoe
enzyme were due to having an arginine in amino acid position 115, an aspartate in
position 165 and a valine in position 430 (Clark et al., 2003). The enzyme from Morex
had CEA in these three positions. The PIs are conducting collaborative research, with
others funded by NABGP or Barley CAP, to determine the distribution of the RDV and
CEA haplotypes in a collection of important barley germplasm known as MaltGenes and
to correlate these amino acid footprints with levels of diastatic power plus β-amylase
activities and thermostabilities in malts of these genotypes. This research is testing the
hypothesis that the RDV haplotype is higher in malt β-amylase activity/thermostability
and in DP values. The PIs are collaborating, with Prof. Patrick Hayes, to test this same
hypothesis in breeding lines of known ancestry containing three 115, 165 and 430
alleles and two intron III alleles. Based on data from our study of Steptoe and Morex
(Clark et al., 2003) and on data from the first year’s trials of the breeding lines, it
appears more likely than not that the null hypothesis will be accepted which forces us to
conclude that other regions of the genome either individually determine β-amylase
activity and DP or interact with bmy1, the gene encoding endosperm specific β-amylase,
to determine these two traits.
The PIs are collaborating to determine if the allelic variation in intron III of bmy1
regulates expression levels or thermostabilities of the enzyme. Numerous publications
correlate two intron variants with these enzyme properties, yet no mechanistic data
prove that structural features of intron III have any affect on gene expression.
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Establishing that sequence variations in this intron do regulate gene expression, via
recruiting transcription factors or other DNA-binding proteins, will provide additional
gene sequences to fix in malting barley germplasm as we proceed to assemble the best
package of alleles for the conversion of starch to fermentable sugars.
Henson’s lab has established that there are two amino acid positions in α-
glucosidase1, the product of Agl1, that, when the optimal amino acids are in those
positions, impart increased thermostability (Muslin et al., 2002; Clark et al., 2004). We
further demonstrated that α-glucosidase with one of these substitutions increases the
yield of fermentable sugars from malt starch during mashing (Muslin et al., 2003). We
created these mutant enzymes based upon sequences of α-glucosidases from other
plant species whose activities we demonstrated were more thermostable than the α-
glucosidase from barley. Because these thermostable α-glucosidases with these
sequences exist in plants, we’ve embarked upon an effort to sequence Agl1 from the
MaltGenes collection. To date we have sequenced 21 cultivars, analyzed 20 of the
sequences and found no sequence variation. Interestingly enough, we’ve shown that
the extractable α-glucosidase activities from 7 of these cultivars show considerable
variation in enzyme thermostability. As is the case for β-amylase, the emerging picture
is that sequence variation in the structural gene alone is insufficient to account for
variations in the phenotypes of α-glucosidases, the enzymes responsible for the
production of at least 30% of the glucose produced during mashing.
The PIs are working together to establish metabolic profiles that distinguish
between the best of elite malting barley cultivars and other commercially available
malting barley cultivars that produce less RDF under commercial brewing conditions.
The goal of this research is to identify 1 – 10 candidate metabolites for which we can
develop simple quantitative assays as novel measures to predict malt quality.
The PIs are working together to assess novel measures of malt quality. We are
testing the hypothesis that malt and seed osmolyte concentrations (OC) will be good
indicators of malt quality and pre-harvest germination. These hypotheses are based
on the knowledge that the degradation of storage compounds (starch and protein) that
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occurs during germination should reflect the degree of malt modification and seed
germination and the fact that OC measures every molecule that becomes soluble during
germination. In contrast, malt extract (ME) is based only on the mass of material in
solution, which determines specific gravity. This difference between OC and ME
means that, for example, the contribution of one maltopentaose to ME values will be
~five times greater than the contribution of one molecule of glucose whereas in the OC
assay each molecule in solution contributes an equal amount to the OC value. Hence,
the OC assay should more completely quantify the production of solutes as modification
increases during malting than do measures of ME. Our studies have shown that OC
values correlate positively and significantly with ME values for the first 5 days of malting.
Interestingly, malt OC values increased with days of germination for about 24 hours
longer than did ME values, suggesting that the OC assay reflects the production of
additional solutes not detected in the ME measurements. OC values of malt at 2 and 3
days of germination correlate positively and significantly with ME values of malts from
days 4 - 6 of germination indicating that OC values from early stages of malting may be
good predictors of the quality of malts produced from typical malting schedules.
Further studies indicated that green malt OC values correlated earlier and better than
finished malt measurements than did finished malt OC values. Further studies have
shown that the thermostabilities of malt α-amylase, α-glucosidase, β-amylase, and limit
dextrinase were positively and significantly correlated with OC, suggesting that malt
quality is related to the integrity of the complement of enzymes responsible for starch
degradation. OC values of barley seeds were found to positively and significantly
correlate with pearling values indicating that OC may be a non-subjective and
quantitative indicator of pre-harvest germination. This may be of particular value to
breeders and researchers with limited quantities of seed to analyze as the OC measure
can reproducibly be made on less than 10 seeds.
Current Project Personnel
Federal State
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Charles Karpelenia, technician Joe Dietrich, technician
Joel Phillips, technician Mariah Peronto, undergraduate
Recent Publications
Yoshio Tanaka, postdoctoral associate
Marcus Vinje, graduate student
125
Muslin, E.H., Clark, S.E., Henson, C.A. The effect of proline insertions on the thermostability of
a barley ∀-glucosidase. Protein Engineering. 15:29-33. 2002.
Muslin, E.H., Karpelenia, C.B., Henson, C.A. The impact of thermostable ∀-glucosidase on the
production of fermentable sugars during mashing. J. Amer. Soc. Brew. Chem. 61:142-
145. 2003.
Clark, S.E., Hayes, P.M., Henson, C.A. Effects of single nucleotide polymorphisms in ∃amylase1
alleles from barley on functional properties of the enzymes. Plant Physiol. and
Biochem. 41:798-804. 2003.
Clark, S.E., Muslin, E.H., Henson, C.A. Effect of adding and removing N-glycosylation
recognition sites on the thermostability of barley ∀-glucosidase. Protein Engineering,
Design & Selection. 17:245-249. 2004.
Havey, M.J., Galmari, C.R., Gokce, A.F., Henson, C. QTL affecting soluble carbohyrate
concentrations in stored onion bulbs and their association with flavor and healthenhancing
attributes. Genome. 47:463-468. 2004.
Schwarz, P., Henson, C., Horsley, R., McNamara, H. Pre-harvest sprouting in the 2002
Midwestern barley crop: occurrence and assessment of methodology. J. Amer. Soc.
Brew. Chem. 62:147-154. 2004
Clark, S.C., Hayes, P.M., Henson, C.A. Characterization of barley tissue-ubiquitous β-amylase
and the effects of single nucleotide polymorphisms on enzyme thermostability. Crop Sci.
45:1868-1876. 2005.
Henson, C.A., Duke, S.H. Osmolyte concentration as an indicator of malt quality. J. Amer.
Soc. Brew. Chem. 65: 59-62. 2007.
Henson, C.A., Duke, S.H., Schwarz, P., Horsley, R. Barley seed osmolyte concentration as an
indicator of preharvest sprouting. J. Amer. Soc. Brew. Chem. 65:in press. 2007.
Duke, S.H., Henson, C.A. Green malt osmolyte concentration as an early indicator of finished
malt quality. J. Amer. Soc. Brew. Chem. 65:in press. 2007
Henson, C.A., Duke, S.H. A comparison of standard and nonstandard measures of malt
quality. J. Amer. Soc. Brew. Chem. 65:accepted for publication 2007