1969 PROGRESS REPORTS ON HOPS RESEARCH
AND
BUDGET PROPOSALS FOR 1970
TO
UNITED STATES BREWERS ASSOCIATION
BY
HOP INVESTIGATIONS, UNITED STATES
DEPARTMENT OF AGRICULTURE, AGRICULTURAL
RESEARCH SERVICE, CORVALLIS, OREGON
December, 1969
Results
of research
reported herein are
preliminary and are
not for publication.
Table of Contents
Page no.
Introduction
1
Development and Evaluation of Experimental Hops
2
Breeding and Preliminary Evaluation
2
Crosses Made in 1969
Nursery Evaluation and Selection
2
2
4
Evaluation of Hop Lines at Prosser
Evaluation of Advanced Lines
5
Off-Station Production of Brewing Trials
Recommendations for Disposition of Off-Station Varieties
. .
[
Other Advanced Selections
5
6
7
Hop Genetics
11
Triploid Hops
Other Genetic Studies
'
'
Hop Diseases
11
n
12
Hop Downy Mildew
Verticillium Wilt
'
'
Hop Chemistry
Storage Stability
12
13
14
14
Trial Crosses for High Analysis
' 15
Development of a Pool of High Analysis Lines
' 19
Preliminary Crosses for Genetic Study of Alpha Acid Inheritance 21
Hop Physiology and Propagation
25
Propagation of Advanced Lines
!
25
Evaluation of New Lines
26
Evaluation of Hops in Seedling Year
30
Correlation of Plant Morphology and Yield .
30
Relationship of Floral Initiation, Growth Factors and
Supplemental Lighting
Determination of Insecticide Residues on Fresh and Dry Hops
31
...
33
Phorate
33
Disulfoton
33
Azodrin
34
Proposed Work and Budget for 1970
34
Page no,
Biosynthesis of Humulone in Hops
36
Genetic Study Proposal
42
Proposed Budget for Fiscal 1970-71
44
1969
REPORT OF HOP INVESTIGATIONS
This report covers cooperative research on hops sponsored by the
UNITED STATES BREWERS ASSOCIATION with the U. S.
DEPARTMENT OF AGRICULTURE
and the Oregon Agricultural Experiment Station. Cooperative work on variety
development between USDA and Washington State University, Agricultural
Research and Extension Center, Prosser is included also.
The first section of the report, DEVELOPMENT AND EVALUATION OF
EXPERIMENTAL HOPS, integrates all contributions to the breeding of new hop
varieties into a single section. Research not directly related to the
varietal improvement program is then presented according to the areas of
research, such as GENETICS, PESTICIDE RESIDUES, DISEASES, etc.
Lengthy and detailed data tables not immediately relev nt to the
text have been omitted. Clarification of minor points can be made either
at the USBA Hop Research Sub-Committee Meeting, January 1970, or in the
USDA Annual Report available about June, 1970.
The following research personnel contributed to this report
Oregon State
Washington State
U.S.D.A.
Dr.
W.
D.
Loomis
Mr.
Dr.
A.
Haunold
Dr.
L.
C.
Terriere
Dr.
C.
E.
Horner
Mr. U. Kiigemagi
Mr.
S.
T.
Likens
Miss Gail Nickerson
Mr.
C.
E.
Zimmermann
Mr.
J.
F.
Anderson
C.
E.
Nelson
DEVELOPMENT AND EVALUATION OF EXPERIMENTAL HOPS
(USBA Project 8 - C. E. Horner, S. T. Likens, C. E. Zimmermann and A. Haunold)
Breeding and Preliminary Evaluation
Crosses Made in 1969
Relatively few crosses were made in 1969.
Three of these involve
a combination of Yakima Cluster (genotype 65102) with males that have shown
good mildew resistance in the past (genotypes 64032M, 64033M, and 64037M).
Genotype 62013, a high a female, was crossed to several high a males (19039M,
6616-61, and 6669-09) in an effort to further capitalize on the apparent
potential of this experimental variety. Two crosses were made on genotype
19105, a female with good yield potential and downy mildew resistance, by
using two males that have shown good a-acid potential. Two crosses were
also made on tetraploid Fuggle in an effort to obtain triploids with a
high a-acid potential.
Good seed set was obtained in all crosses.
Greenhouse evaluation
for downy mildew resistance of seedling populations will be carried out in
1970. The high a-acid crosses may be moved directly to the field and mildew
testing in this material will be done at a more advanced stage in the selection
program.
Nursery Evaluation and Selection
Twenty five selections from the 1967 seedling nursery, representing
14 different crosses, will be planted at Prosser, Washington in 1970, based
on preliminary information on vigor, yield and a-acid potential obtained at
Corvallis in 1969 (Table 1). One selection was saved for the germ plasm
nursery at Corvallis.
The 1968 nursery, consisting of about 670 genotypes plus appro
priate check varieties (Early Cluster and Brewers Gold) was established in
the main yard in 1969. Most plants got well established and preliminary data
on growth, and sex expression were obtained.
Final evaluation and selection
from this material will be made in 1970.
Seven selections from the 1966 seedling nursery (see 1968 USBA report,
P.4, Table 1) were planted in the seedless yard at Corvallis for advanced
evaluation. Two of these were discarded after the 1969 season and the rest
(selectionsNo. 6503-25, 6517-46, 6517-47, 6527-17, and 6532-14) will be further
tested in 1970.
Eighteen lines from the 1965 seedling nursery, which had been
previously tested in the main yard (seeded observation block), were evaluated
for the first year under seedless conditions.
because of agronomic and quality shortcomings.
Nine of these were discarded
The remainder will be
evaluated for another year; these are: genotypes No. 65032, 64002, 64003,
64007, 64024, 64026, 65002, 65009, and 65011. One of these, genotype 64007,
was submitted this year to the USBA Hop Committee for evaluation.
Table 1.
Selections from the 1967 Seedling Nursery for Planting at
Prosser, Washington.
Date
Pedigree
Sel No.
crosswire flower
June
6621-01
h Bu; h genotype 51114M
6622-11
h Bu; h genotype 19043M
30
Good picker
"
"
, Bu type
High a, weak
20
-09
Remarks
June
11
28
-16
12
30
-27
2
25
Early, open cone, shatters
6623-01
2/4 Bu; % Fu; h unk.
15
22
6625-01
h Bu; % unk.
11
30
6626-01
3/4 Eur; \ unk.
3
20
Early
6628-01
8/16 Eur, 3/16 Fu; 5/16 unk.
3
26
Fuggle type
6630-05
h Eur; % unk.
7
20
6635-17
h Early CI.; % unk.
7/1
7/15
Vigorous, late
-22
20
7/10
Cluster type, late
-23
13
28
-25
4
20
Good CI., some deflowers
-34
5/30
27
Late
-35
5/29
7/1
Late
6636-05
-06
h Eur; h Wild Amer.
it
ii
8
20
9
29
Early, Fuggle type
High a
6642-07
5/16 Fu; 2/16 Serebr; 9/16 unk,
10
19
Fuggle type
6645-02
4/16 Fu; 3/16 Bel; 5/16 unk.
12
28
Fuggle type
6650-13
1/8 L. CI.; 4/8 Eur; 3/8 unk.
11
7/14
6656-04
h. Fu H; % unk.
6669-10
h Bullion; % unk.
6
28
-12
9
11
7/13
-28
6
20
Good cone type
Fuggle type
High a potential
"
" »
, late
Evaluation of Hop Lines at Prosser
A large amount of breeding material is also being evaluated annually
at Prosser, Washington. Some genotypes that may not show much potential at
Corvallis have looked quite promising at Prosser under different environmental
conditions.
One of these, selection 6443-14, is being submitted to the USBA
Committee for evaluation this year.
A partial listing of other promising
selections from Prosser appears in Table 2.
Table 2.
Hop Selections Grown in Five-hill Plots at Prosser,
Washington.
Sel.
or Ace.
No.
1969.*
Yield
ml oil
-6a
6402- 39
Good
7.7
4.2
6428- 07
Med.
5.3
3.4
6440- 07
Good
5.2
6443- 14
V.
5.2
Good
Comments
6.3
0.8
1.0
0.6
Good cone § lupulin
"
» , low lup.
Cock-hops, WA type
4.1
0.7
Sent to USBA-Eval.
1.5
0.5
Good lup.
Cont. type aroma
0.9
0.6
Large cone, poor lup,
Discard, poor cone
1.1
0.4
Ore. sample to USBA
poor cone
1.4
0.6
Large cone, good lup.
Good lup, Discard
6308- 25
Good
7.9
5.3
6314- 22
Med.
4.6
4.7
6344- 30
Med.
6.4
4.0
6345- 35
Poor
4.2
2.6
62013
Good
11,
4.4
63002
Good
5.
3.7
63006
Med.
9.
5.1
63019
Good
4.
3.4
* Plots were established in Spring of 1969,
Evaluation of Advanced Lines
Off-Station Production for Brewing Trials
Three advanced selections (56008, 56013, 58112) were grown
commercially in blocks of two acres each to produce hops for brewing
trials.
The pertinent data and observations on these selections are as
follows:
Variety 56008. This selection, grown by Stauffer Brothers, Hubbard,
Oregon, was discarded in July, 1969 because of its extreme susceptibility
to downy mildew infection of flowers and cones. The variety is resistant
to rootstock infection and could be grown in Washington or Idaho without
problems from mildew; however, it does not produce well in those areas.
Selection 56008 has been dug out and removed from our program.
Variety 56013. This selection now appears to be the best of the
three "Off Station" varieties, all things considered. 56013 is midseason
in maturity (about September 1) and was grown by Mission Bottom Farms,
Salem, Oregon. It has a strong Fuggle background in its parentage. It
has features of continental-type hops such as an alpha-acid, beta-acid
ratio near one, a low cohumulone ratio, and a mild but characteristic
aroma. 56013 requires refrigeration during storage. Brewers using con
tinental-type hops should be considered for test brews. This variety per
forms exceptionally well grown seedless. It has darker green color in sample
which does not indicate immaturity. It retains its green color during
harvest season better than other varieties and cones are not damaged or
discolored by wind and adverse weather.
It has downy mildew and Verticillium
wilt resistance.
Variety 56013 performed well in both 1968 and 1969.
In 1969 it
was exposed to both a severe downy mildew situation and a heavy spider mite
infestation.
The cones were not damaged from mildew or mites to the extent
other varieties were.
This ability to hold cone color for a long period
in spite of adverse conditions is characteristic of the variety. The
grower considered 56013 "a very good hop to grow; it is easy to train and
manage and it produces well and picks easily".
1968
Harvest date
8 Sept.
1969
30
Acres harvested
1.6
Yield per acre
Seed content (%)
Leaf § stem (%)
Alpha acid (%)
Beta acid (%)
Oil (ml/100g)
1,760 lbs,
2
12
4
2
1
Aug.
2
,100 lbs,
6
7.,2
5.4
6.,4
1.2
1.,4
Twenty two bales of the 1969 crop of 56013 are in cold storage at
the Washington State Hop Producers Warehouse, Yakima.
Variety 58112.
This is a late-maturing hop grown by Coleman
Ranch, St. Paul, Oregon. Its analysis is similar to Cluster varieties
with a medium-high alpha acid content with relatively high cohumulone
ratio. It has excellent storage characteristics and is resistant to downy
mildew and tolerant to Verticillium wilt. 58112 has several shortcomings
that might seriously limit its success as a variety. Spring growth is
delayed and sometimes uneven; new shoots are prostrate, therefore, subject
to damage before training; some years male flowers are produced abundantly.
In 1969 a virus disease greatly damaged 58112 in our OSU test plots, however,
it was not damaged in the commercial trial.
1968
Harvest date
14 Sept.
Acres harvested
1.7
Yield per acre
Seeds (%)
Leaf § stem (%)
Alpha acid (%)
Beta acid (%)
Oil (ml/lOOg)
1720
1969
1 Sept.
2
lbs
2
2,200 lbs
3
0
3
8.9
8.3
3.5
3.6
0.8
1.1
Twenty one bales of the 1969 crop of 58112 are in cold storage at
Washington State Hop Producers Warehouse, Yakima.
Recommendations for Disposition of "Off Station" Varieties
1.
Selection 56008 has been discarded from our tests and has been
removed from off station plots.
2.
Selection 58112 will be discarded unless one or more brewers
show special interest.
3.
We have better selections coming aJong
Selection 56013 is recommended for release as a new variety
provided one or more brewers or extractors become interested in ic.
If
a brewer could substitute hops of 56013 for Fuggle or other European types,
it has several advantages: Its yield potential is about double that of
Fuggle; it produces exceptionally well as a seedless hop; it has good
disease resistance; its ability to produce is not affected by adverse weather
to the extent that Fuggle is.
Plans for Selection 56013.
The three Oregon growers of Off Station
varieties are all interested in growing 56013 and promoting it as a new
variety. Their intention is to each grow a small acreage and provide
dealers, brewers, and extractors with test and promotional samples. One
Washington grower will begin commercial testing in 1970.
The contracts under which the Off Station varieties are grown
expire March 31, 1970. There is a further provision in the contracts which
states: "Notice of discontinuation of the trial must be given the Grower
prior to February 1 of the pending crop year - -".
Other Advanced Selections
Selection 62013 is a new and promising high analysis hop which we
have been evaluating for three years.
It is a vigorous, high-yielding,
late-maturing hop with a characteristic golden-green foliage which distin
guishes it from all other commerical varieties. Some analytical data are
as
follows:
Location
Corvallis
1967
1968
1969
Alpha
Beta
Alpha
Beta
Alpha
Beta
12.3
4.0
13.2
4.7
13.3
5.5
11.6
4.4
Prosser
Selection 62013 appeared to us to be of special interest to
extractors. Therefore, we provided major extractors with samples and have
received very favorable response. One extractor wishes us to proceed
with commercialization of the hop as rapidly as possible. Another describes
62013 as a remarkable hop with very good extract properties.
Selection 62013 was included in Brewer Inspection Samples sub
mitted in 1969 to the USBA sub-committee on Hop Research. We have
additional small quantities for persons especially interested in testing it.
Presently we have moved 62013 into a rapid propagation plan and
expect to have planting stock for commercial trials available by 1971.
In 1970 we will have 20 mature plants at Corvallis from which we expect
about 60 pounds of hops for further evaluation. Selection 62013 will again
be grown in Washington in 1970.
Agronomic evaluation of 62013 is far from complete.
We have
inadequate information on its resistance to downy mildew; however,it appears
to have good resistance in our test plots.
We have no information on its
reaction to Verticillium wilt or virus diseases.
be grown seedless.
Selection 62013 should
Its indicated yield potential is 2000 - 2600 pounds per
acre.
Selection 19110, a European type with good yield potential, has
been evaluated for several years. 19110 received consistently favorable
evaluation by the USBA sub-committee. The 1968 sample planned for brewer
inspection was damaged by a dryer fire at Prosser and the 1969 sample showed
an abnormally low alpha acid which we believe is not characteristic of the
variety. Thus our evaluation of 19110 has been delayed, but will continue.
It has less tolerance to Verticillium wilt than Fuggle, Bullion or Clusters.
Table 1 shows a summary of agronomic and chemical evaluations of
advanced lines.
Included are lines which will be evaluated by the USBA
Hop Research Sub-Committee in 1969. Also included are evaluations of varieties
used as controls or standards when samples were sent to the Committee.
TABLE 1 (over)
Table l.
Summary of 1969 Brewers Inspection Samples and other Advanced Hop Lines
Identification
Acc/Sel No.
Pedigree
Hop Lines Evaluated Previously:
19110
3/8 Belgian
3/8 Fuggle
56013
irewers Gold, 3/4
63018
3/4 Brewers Gold
63019
ii
North
Brew
ii
Br am.
n
Cross
1
—
Agronomic
Yield
Cone
B/A
type
1
Pickab ility'
Year
Maturity
Ore
1965
9/14 L
10
Ore
1967
L
10
Ore
1968
8-9
G
8-9
G
3
3
12-14
G
2
3
P
2
4
2
Location
Wash
1968
Ore
1969
9/17 L
9/15 L
9/5 L
Wash
1969
L
9-10
Cone
Vine
G
1-2
1-2
G
2
3
2
3
Ore
1965
L
9-10
G
2
Ore
1967
L
10
G
2
Wash
1967
10
G
2
Ore
1968
8/31 L
9/8 ML
9
M
2
3
Ore
1968
12SepL
10-12
G
2
2
Ida
1968
Ore
1969
Ore
L
-
G
_
_
_
_
10-12
G
2
2
1969
8/30 ML
9/5
L
12-14
G
1
2
Ore
1968
9/5
L
10-12
2
2
Ore
1969
--
Vines
slipped
Ore
1968
9/17 VL
Ore
1969
--
Wash
1967
8/29 M
Wash
1968
Wash
1967
Wash
1968
10
Possible virus
ME
8/29 M
G
down
string
M
3
4
§ we ak sidearms,
6
G
6-7
G
3
2
-
-
4
G
ME
5-6
G
E
6-7
G
2
3
VE
5-6
G
1-2
2
3
2
2
_
-
Hop Lines Evaluated by USBA, 1969:
21001
ii
Unknown
ii
JjUtah WA,%Su.
Ore
1968
Ore
1969
Ore
1967
it
ii
ii
Ore
1968
11
ii
ii
Ore
1969
II
ii
ii
Wash
1969
62013
8/21
8/1
_
9/10
9/11
L
10
G
2
L
10-12
G
2
3
L
12-14
G
2
3
L
64007
%EG,3/16Fu,l/8LGp
Ore
1969
9/15
L
10-12
G
2
2
E-2
Early CI. - Sel.
Ore
1969
8/15
E
9-10
G
2
2
Ida-40
h Cluster
9/5
ML
10-12
G
ML
10-12
G
L
9-10
G
L
9-10
G
L
9-10
G
it
ii
Late
Cluster
Ida
1968
Ida
1969
Ida
1969
6443-14
% Late Cluster
Wash
1969
L-l
Late Cluster-Sel
Wash
1969
-
-
_
_
_
-
-
2
-
10
Evaluated in 1967-68.
Chemical
%a
ml Oil
W
7.0
8.2
1.0
5.5
5.9
1.0
MC
-
Aroma
pi. spice
pi. good
6.2
7.8
1.3
11.1
mild,estery
4.5
5.8
0.4
10.6
off, smokey
5.4
6.1
0.8
11.3
3.9
4.3
0.3
7.4
mild, acetate
off, mild
6.2
8.1
2.4
7.4
6.1
2.2
5.2
5.4
0.8
6.0
5.4
1.2
6.1
6.2
1.9
6.2
5.4
1.7
7.2
6.4
1.4
-
-
sv.
cone
-
-
-
-
Good USBA Eval.
-
si. yellow fl.
-
-
s1.
-
_
V. good USBA Eval.
Storage test
_
floral
_
Variable cone
-
-
mild,
crown DM
Off-station, 12% seed
Storage test
Fair baby vigor
Off-station, 2% seed
floral
3
7.5
2.5
12.8
mild, pi.
slip-down
wilted, poor agronomic
5.6
5,,2
- P'oor
0.9
10.8
yellow fleck
agronomic
13.5
3,,7
1.8
10.0
3.,6
1.2
7.5
8.0
pleasant
str., good
Good USBA Eval.
Good USBA Eval.
aromatic, good
Good USBA Eval.
Good USBA Eval.
7.5
2,,7
1.0
2.,5
0.8
6.5
4.8
1.0
12.7
mi1d, pi.
si.
cone
5.4
5.2
0.8
11.3
estery, mild
s1.
crown DM
12.3
4.0
2.3
13.2
5.5
2.9
11.7
pi. WA
13.3
5.5
2.8
10.9
str., WA
2.5
4.3
0.9
13.2
estery
6.2
1.0
8.9
mild-floral-WA
0.6
6.3
mild-frag.
mild
5.4
7.0
5.3
6.5
7.1
5.7
7.7
6.8
4.1
4.8
DM
Good USBA Eval
USBA Code 0-1
_
6.5
5.2
Good USBA Eval.
Discarded
6.6
10.3
Good USBA Eval,
Discarded
bl. atypical
0.7
10.3
0.7
6.8
_
mod.
crown DM
-
cone § crown DM
-
mild,
off-WA
USBA Code 0-2
USBA Code 0-3
USBA-Standard
Fair USBA Eval,
USBA Code 1-1
-
-
cont.
-
|
-
slip-down
pi. cont.
-
DM
-
-
Comments
Fair USBA Eval.
Good USBA Eval.
-
floral
-
-
Disease
USBA-Standard
USBA Code W-l
USBA Standard
11
Hop Genetics
Triploid Hops
Over 700 plants from a genetic study involving crosses between a
tetraploid (40 chromosome) Fuggle and selected diploid (20 chromosome) males
(see 1968 report, p. 17) were evaluated for the second year in 1969. Most
of these plants had 30 chromosomes and were triploids. Detailed data were
taken on growth type and vigor, date of flowering, sex, maturity, yield per
plant, cone size and -weight, seed set under open pollinated conditions,
and diseases. About 350 different female plants were harvested individually.
About one third had an indicated yield potential substantially above Fuggle.
Morphologically many looked similar to Fuggle. A wide range in a-acid from
very low to very high was found among different selections. Many had the
"European" aroma characteristics, as well as the characteristic a/3 ratio
of Fuggle. Seed set under a heavy continuous pollen load throughout the
flowering period ranged from slightly over 1% to a little over 4%, with a
mean of about 2.7%, as compared to about 18% for diploid hop under identical
field conditions. Maturity among genotypes ranged from very early to late,
but a number of good yielding early types have been identified.
Some of the more promising selections from this material will
be grown in the seedless yard and also at Prosser, Washington in 1970.
Other Genetic Studies
Plants from the UV irradiation and the Ethyl methane sulfonate
experiment have been evaluated for mildew resistance in the greenhouse
(see 1968 report, p. 17).
Some clones that did not show much infection
in 1969 will be reevaluated in the greenhouse in 1970.
The remainder, which
was completely susceptible, was discarded.
Preliminary work on four crosses, involving parents with low and
high a-acid content, respectively, as a basis for our genetic proposal for
the inheritance of a-acid, was carried out in 1969.
A number of male and
female genotypes from each cross are currently being analyzed by Mr. Likens
and his staff. Contrary to our original plans, the high-low reciprocal
crosses were not repeated in 1969.
Rather, efforts were directed toward
obtaining quality data from our large collection of male genotypes.
work will be of considerable value in planning future crosses.
This
12
Hop Diseases
Hop Downy Mildew
Advanced selections must be thoroughly tested for resistance to downy
mildew before release as new varieties. This is an on-going program as new
selections are advanced in the breeding program. Table 1, below, shows mildew
reaction of most of our advanced selections to both the crown rot and foliage
blight phases of downy mildew.
Table 1.
Summary of Reaction of Advanced Hop Lines to Downy Mildew Inoculation
Crown
Genotype
infection
w
/a
Foliage
infection
/b
CI assification
(0-5)
Yakima Cluster (Susc.)
Fuggle (resistant)
92
4.0
Susc.
19
0.8
Res.
56013
28
1.8
Res.
56008
41
3.9
Susc.
58112
0
0.7
Res.
19110
20
3.3
21001
0
62013
20
/b
-
Intermed.
Res.
-
infection based on a minimum of 20 plants.
Fo liage infection scored on a scale of 0 to 5 meaning severe infection,
About 20 plants each of 17 other hop genotypes were tested in the
greenhouse for resistance to downy mildew. These were mostly selections in the
intermediate stages of advancement. Several were rejected from further
consideration because of susceptibility to downy mildew.
Another 250 plants of Early Cluster (strain E-2) were tested for mildew
resistance. These were the survivors of previous material which had been treated
with the mutagenic agents, ultra violet radiation and ethylene methane sulfonate,
for the purpose of inducing a resistant mutation in the mildew-susceptible Early
Clusters type hops. Among the 250 plants, 33 either were resistant or escaped
infection and will be retested in 1970. The normal "escape" percentage for
Early Clusters is about 10%, therefore, most of the survivors were probably
escapes, but if there is only one resistant plant, the UV and EMS experiments
will have been successful.
Seedlings from crosses are given a preliminary screening in the green
house to eliminate most of those highly susceptible to downy mildew crown
infection. In 1969, 1,217 seedlings were tested (Table 2). On the average, 80%
of the seedlings were susceptible and were discarded. The seedling test
eliminates much material that would otherwise require space and time in agronomic
tests. Further evaluation is required, however, because a portion of the
susceptible plants in a seedling population always "escape" infection. These
are eliminated in our evaluation of advanced selection at a later date.
13
Table 2.
Reaction of Seedlings from 1968 Crosses to Downy Mildew Inoculation
Purpose
Cross
No.
6811
No .
of cross
plants
tested
DM res.
No. plants
Percent
infected
infection
63
54
6812
ii
46
39
85
6814
ii
77
74
96
6815
H
13
10
77
6816
ii
8
6
75
6824
H
34
25
73
6817
Eur. type
86
44
33
75
ii
ii
420
274
65
6819
ii
ti
252
211
84
6820
ii
ii
260
239
92_
,217
965
79
6818
Totals
1
Verticillium Wilt
Our techniques for testing hops for resistance to Verticillium wilt
have been improved to the extent we feel we can place confidence in the results
In 1969, the advanced selections 19110, 21001, 63018, and Hallertau-S were
compared in our wilt nursery with Fuggle and Bullion varieties. In these tests
a minimum of 10 plants each are planted in plots separately infested with five
different strains of the wilt fungus.
Testing for resistance to Verticillium wilt is complicated by the
fact that various strains of the wilt fungus are present in most potential hop-
growing soils of the Pacific Northwest. Table 3 shows a summary reaction of the
varieties and lines tested in 1969 to five strains of the wilt fungus.
Table 3.
Genotype
Summary of Verticillium Infection
Average infection (%) /a
by all 5 Vert, strains-
Fuggle
36
Bullion
15
Hall.-S
41
19110
57
21001
55
63018
18
Z.a
1969
Average
/b
Propagules pergram stem
3,048
1,007
16,460
22,781
19,840
1,960
Average symptom /_c
severity (0-4)
0.68
0.13
0.73
0.71
0.49
0.31
Based on direct laboratory recovery of the wilt fungus from a minimum
of 50 assays per genotype.
Z.b
/c
Based on quantitative recovery of spores of the wilt fungus within the
stem tissue; minimum of 8 assays per genotype.
Symptoms based on: 0=none, l=light, 2=moderate, 3=severe, and 4=dead.
14
Hop Chemistry
Storage Stability
Evaluation of commercial varieties and developing lines: The
6-month, room-temperature storage stability trials described in the last
USBA report were completed and the results are given in Table 1.
Although
several more lines were tested, they have since been discontinued in the
program and are not reported.
Table 1.
Very Good
L-l(2)
C19110
Relative Storage Stability of Several Hop Varieties by Groups
Good
Fair
E-2 (3)
Talisman (1)
C58112 (2)
Alliance
C21001
Idaho 40
Storage Conditions:
Very Bad
Poor
Brewers Gold (2)
Bullion (2)
Fuggle (1)
Hallertau
Brewers Gold (1)
Bullion (1)
C56008 (1)
C56013 (2)
Talisman (1)
161021
6620-06
C63020
C56008 (1)
166030 (2)
L-l (1)
6619-08
Fuggle (2)
C62013
6 months, dark, 72° F. in % lb. bales
(11 lb./l cu. ft.) in polyethylene bags.
Spectrophotometric analyses made before and after storage period
on single samples.
3.
Grouping was on the basis of the fraction of a-acid remaining after
the storage period:
4.
5.
Very good
90-100% remaining
Good
Fair
80-90 % remaining
60-80 % remaining
Poor
Very bad
40-60 % remaining
< 40 % remaining
Numbers in parentheses indicate number of times (years) the variety
has fallen into that group.
L-l and E-2 are Clusters grown from certified rootstock provided by
C. B. Skotland, WSU, Prosser.
6.
Numbers preceded by C and I are experimental lines.
7.
All varieties except Idaho 40 were grown at Corvallis.
15
Development of techniques for screening populations including
male lines": Storage evaluations such as summarized above are satisfactory
for the evaluation of commercial varieties and developing lines, but are
wholly inadequate for screening large populations of female hops, and
obviously unsuitable for evaluating male lines. In order to prepare for
future genetic studies involving the heritability of storage stability, it
will be necessary to develop a relatively simple test which will apply
equally to males or females.
Storage stability must be associated with some property of lupulin,
and since lupulin is common to both male and female plants, we decided to
work with that botanical unit.
It was necessary to establish at the outset that isolated lupulin
deteriorates similarly to whole cones. Lupulin was dry-sieved through
60-mesh and collected on 100-mesh, from Brewers Gold and Yakima Cluster.
Cubes from baled samples of intact cones and 50-mg. samples of isolated
lupulin from each of the varieties were exposed to 60° C and analyzed
periodically. The ratio A275/325 was used as the index of stability ...
a low ratio indicating good storage. Results of this test (Fig. 1) showed
that lupulin from Brewers Gold deteriorated more rapidly than that from
Yakima Cluster and indicated that lupulin could probably be used for a
"quick-test" of large numbers of both males and females.
hours at 60
FIGURE
1.
C
Comparison of ^21S/Z2S Detween whole cones and
lupulin after 60o C storage.
16
On the chance that a simpler test could be found, several more
experiments were conducted to answer the more fundamental question of what
is responsible for the difference in the stability of a and g-acids in
different varieties. These disclosed that the pellicle or "shell" of the
gland is not involved; the content or type of oil associated with the
variety is not responsible; that the characteristic stability of a variety
is lost upon solution into a solvent and cannot be reliably regained after
removal of that solvent. In short, it appears that the fundamental and
biologically controlled structural status of the a and g-acid molecules may
be responsible for the array of storage-stabilities of different varieties.
It is interesting to note that, while Bullion and Brewers Gold
appear to have poor storage properties, some genotypes are far worse.
Three from the 1968 crop were so bad that they completely deteriorated
before an analysis could be made when handled in the usual routine. They
were given special handling this year and will be used in additional storage
studies, as well as added to our germ plasm block.
Survey of existing germ plasm: A simple technique was developed
for isolation of 20 to 100 mg of clean lupulin from either male or female
flowers based on water flotation and screening. Using this technique,
lupulin was collected from 900 to 1000 different genotypes, including both
male and female. Analyses revealed that relatively few had the necessary
analytical values (at least 40% a-acid or a/3 = 1.5 or more) to make them
suitable for commercial acceptance and, therefore, suitable for breeding
material. These, however, will be further tested for storage stability
and the good and poor will be used for genetic crosses at some future date.
Plans:
The 6-months sample-bale test of storage stability will
be applied to 7 commercial lines, 10 advanced lines, 22 high analysis
selections (>10% a-acid), and approximately 36 continental types. The
results, which will be available in June, will be used as a guide in
advancing lines from the 1970 crop.
Lupulin will be tested for storage-stability from about 200 male
and female hybrid lines. A range of storage from good to poor can be
expected. The best can be considered for inclusion into the breeding block
and a small number of the poorest will be retained for genetic studies. In
addition, many will also be tested by the r.t. bale storage method to verify
validity of the lupulin method.
Trial Crosses for High Analysis
Seven test crosses involving high-analysis males and females were
made in 1966. Seeds from these were planted in the field and analyses on
cones from selected females* were made in 1967. The selected plants were
transplanted and treated with Simazine for weed control. Chemical damage
occurred in the spring of 1968 but the planting produced a relatively heavy
crop that year and chemical data were again collected. The plants were
left in place and measurement of yield, vigor, chemical analyses, etc., were
made again in 1969.
* Selection was based on visual examination of the cones for lupulin.
other property of the plants was taken into consideration.
No
17
Table 2.
Parents involved in the crosses,
Cross No.
Female
Male
Abridged Pedigree of Progeny
1/2
3/4
7/8
3/4
1/2
5/8
5/8
6616-00
B.G.
x
6339-09
6617-00
B.G.
X
63013M
6618-00
B.G.
X
63023M
6619-00
B.G.
X
63025M
6620-00
B.G.
X
60013M
6659-00
63020
X
63025M
Table 3.
1/4 Fu, 1/4 WA (Colo 2-1)
1/4 WA (Utah 526-4)
1/16 EKG, 1/8 Bav-S
3/16 Fu
1/2 WA (Ariz. 1-2)
3/16 Fu, 1/16 EKG, 1/16 Bav-S
Analysis of parents (1965 and 1966 data)
Identity
Brewers
BG,
BG,
BG,
BG,
BG,
BG,
BG,
Sex
gold
%a (lup)
(lup)
%g (cone)
%a (cone)
a/i
Fern.
46
23
2.0
9.1
5.6
63020
Fern.
46
19
2.4
11.4
4.8
6339-09
Male
63013M
Male
53
15
3.7
63023M
Male
51
28
1.8
6 3025M
Male
47
30
1.6
Table 4.
--
—
- -
- -
Summary of selection for high analysis lines
No.
Cross
"
seeds
planted
No,
No.
Nurs.
plants
No.
select,
for "hi lup'
No.
Contain.
10% a (69)
No. to
6616-00
714
254
25
3
6617-00
102
53
2
2
1
6618-00
765
2 34
23
15
6
6619-00
408
253
16
7
5
6620-00
587
132
25
2
1
6659-00
510
142
16
1
1
3,086
1,068
107
30
17
/a
—
/_a
advance
3
After discarding lines with disease or unfavorable agronomic
characteristics.
Results:
The major objective of the crosses was to determine whether
the a-acid content of female progeny could be improved by using males selected
on the basis of the a-acid content of their lupulin.
1.
2.
The results indicate:
The a-acid content of the female parent can be improved,
The overall rate of improvement is about 1% of the seeds planted,
3. Some males appear to be better than others,
4. Physical evaluation for lupulin content of cones in the
seedling year appears to be a satisfactory criterion for reduction of the
nursery population.
Several incidental observations are worth noting:
1.
19 genotypes looked good in 1968 and were subjected to a
20-plant test for susceptibility to crown infection by downy mildew.
2 were worse than the female parent and a few were superior.
Only
2. A few (of the 19 examined in more detail in 1968) were
subjected to room temperature storage tests. Most were worse than the
female parent (BG). However, one appears to be much better.
3. Second year analyses (1968) were generally much lower
than either 1967 or 1969. This fact may have been the result of delayed
initial growth due to chemical damage from simazine (soil sterilizer).
4. Nearly all of the 107 plants produced cones of higher
density than Brewers Gold.
Summary:
It is probably useful to select male parents of high-
analysis crosses on the basis of chemical analyses in addition to agronomic
features. We can expect up to 1% of the seeds planted to result in a suitable
selection for advancement from the Nursery. Since three additional require
ments must be met (mildew resistance, Verticillium wilt resistance, and
storage stability), there should be 10 or 20 plants to advance from the
nursery. If a cross is to be expected to produce one to five commercially
potential high analysis genotypes, 1000 - 2000 seeds should be planted.
While physical evaluation of the seedlings appears to be suitable
for selecting potentially high analysis material, explicit evaluation for
yield, quality etc. should await the second year of production after the
seedling year.
If the parents have relatively good mildew resistance we can
probably expect a fair number of the progeny to retain this feature.
It may be much more difficult to retain good storage stability. Experimen
tal investigation of the inheritance of storage stability should be under
taken to clarify the requirements of parents.
Plans:
Sixteen selections will be advanced from the trial into a
high-quality reserve block.
One will be entered into an observation block
in Oregon and 8 into a similar block in Washington. Five will go into a
nursery in Washington. Two will go into the germ plasm bank. The
remainder will be discarded and the trial will be terminated.
19
Development of a Pool of High Analysis Lines
Plans for developing a large pool of high a-acid genotypes were
begun in 1961 at which time we had only few lines over 6% (Table 5). During
the interim until 1965, crosses were made by Dr. Brooks and additional infor
mation regarding potential parents was accumulated. The two years 1965 and
1966 saw a twofold increase in number of lines in our program containing more
than 6% a-acid.
In 1967 we increased the a-acid content required for labeling
as "high-analysis" from 6% to 8% and measured a few lines with 13%, while still
maintaining a pool of over 25 lines. In 1968 and 1969 the pool increased
substantially and the required a-acid content was raised to 10% (Table 6).
Two genotypes were noted which contained approximately 30% lupulin which is
necessary for development of a variety containing 18% a-acid (a long term goal)
One of these presently contains over 16% a-acid but is unsuitable for commer
cial development.
Both are to be used in future breeding.
Plans:
We probably have an adequate pool of high analysis lines.
The weakness of these, from a quality standpoint, will very likely appear as
a deficiency in storage stability. Consequently, we feel attainment of higher
concentrations of a-acid should be de-emphasized and our major effort should
be development of similar lines with superior stability.
Table 5.
Progress in incorporating high analysis lines into the breeding
program.
No.
Min.
Year
to
va lue
qual:ify
Max.
a-acid
content
found
genotypes available
Namesd
varieties
Exptl.
varieties
1961
6%
10%
1964
6
10
1965
6
12
2
29
1966
6
11
2
28
1967
8
13
5
26
1969
10
16
7
31
2
2
12
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21
"Preliminary" Crosses for Genetic Study of Alpha Acid Inheritance
This trial was initiated for four purposes:
1. To determine if the a-acid content (or a/3) of the lupulin of
male hops is indicative of their ability to transmit high quality to the
lupulin of their progeny.
2. To develop and test techniques for the isolation and evalu
ation of lupulin from large numbers of male and female plants of the progenies
3. To evaluate the reciprocal-cross technique for study of
inheritance of characterisitcs in hops.
4.
To evaluate and refine propagation techniques necessary to
evaluate genetic crosses most efficiently.
Results:
Contribution of the male: The high female x high male (a-acid)
resulted in a progeny whose lupulin averaged 45% a-acid and 26% 3-acid (a/3 =1.8)
as shown in Figure 2 and Table 7. Unfortunately, the male parent was much
lower than the female parent.
When the high female was crossed with the low male, a-acid in the
lupulin of the progeny dropped slightly and 3-acid was raised, to decrease
the a/3 to 1.4.
The low female crossed with the high male yielded a progeny whose a
and 3-acid content were similar, as in the high x low cross, (a/3 = 1.2)
Using both low females and low males gave a progeny with lupulin whose
a-acid content was low and whose 3-acid was high, ^'a/3 = 0.6)
An independent high x high cross was also evaluated.
This cross
(6806) employed a male with a higher a/3 and the progeny contained a superior
relationship of a-acid and 3-acid with a/3 =2.3.
From a practical standpoint, it would appear that our best chance
of producing varieties with particular a/3would be to select both the female
and male parents which were in the desired range.
Isolation of lupulin: A water flotation technique based on lowspeed blending of dried inflorescences, followed by wet sieving was found to
be fast, simple, economical (from the standpoint of sample size required), and
fairly reproducible.
Some maturity studies were made and these are being evaluated.
It appears that maturity of male flowers presents no difficulties.
The fractions to be collected may be of consequence, with the larger
lupulin having higher quality lupulin than the smaller lupulin. The a+3 was
22
found to range from 60% to 80% of the lupulin. This may reflect either
impure lupulin (debris) or variable dilution by essential oils or other
chemical components of the lupulin.
The method developed produced spectral curves in alkaline methanol
which were normal and without evidence of deterioration of a or 3-acids.
The method should require very little (if any) modification for
application to future studies.
Evaluation of the reciprocal-cross technique: This technique is
subject to the criticism that the results apply only to the parents
involved in the crosses and general statements regarding hop varieties in
general are questionable.
Techniques for future trials are under consider
ation by Dr. Haunold.
Evaluation of propagation techniques:
Hop seedlings grown from
seed provide a means to test large progenies in the field. Seedlings with
1 or 2 true leaves can be transplanted to the field in April and produce
a pound of hops on an eighteen foot vine by August. Potted seedlings are
transplanted in rows, covered with black plastic, and spaced at 10 inches.
Lower axillary buds are removed as they elongate to provide the plant with
maximum apical dominance. Three to four weeks after planting it is possible
to note and remove dwarfs and other abnormal plants in the progeny.
This technique of seedling planting provides an opportunity to
study large populations or progenies and to observe the growth of both normal
and abnormal plants. The technique also provides a method to collect data
on a large number of plants in a small area. An acre would support
approximately 7,000 plants, whereas at normal spacing, the same number of
plants would require 10 acres.
Plans: Data from this trial will be examined thoroughly and if
they are found to be sufficient, the trial may be terminated. However, if
if appears a second year's data would be useful, the trial will be continued
through 1970.
23
Figure 2.
Distribution of a and 3-acids
Table 7.
Data resulting in curve
shown in Figure 2.
in lupulin of progenies from
several crosses.
Hi x Hi
Cross
Obs.
6801
38
%a
%3_
Fem.
54
24
Male
41
35
Prog.
45
26
range (27-56) (12-39)
ii0
20*
30~
40
50
60
% a or 3-acid
o
c
<r>
3
cr
u
Hi x
J,
1
30-
i
i
>
Cross
Obs.
6802
131
Low
-6a
ft
a
20-
Fem.
54
24
Male
10
57
Prog.
42
32
range (25-53) (18-44)
/
/
/
10-
0
1
10
/
^
20
/ i
/ \
*
,
30
40
^
I
50
60
% a or 3-acid
x
o
53
3
cr
Low x Hi
o
30-
Cross
Obs,
6803
74
Fem.
6
45
Male
41
34
Prog.
38
36
range (23-54) (26-48)
20-
10-
/
V/\\
30
40
—r-
10
20
% a or 3-acid
50
24
Figure 2 cont.
Table 7 cont.
Low x Low
o.
Cross
Obs.
6804
-8a
Fem.
6
45
Male
10
56
Prog.
23
46
range (9-41) (26-62)
10
20
30
40
50
60
% a or 3-acid
Hi x Hi
Cross
Obs.
6806
73
%a
%6_
Fem.
50
20
Male
40
22
Prog.
47
21
range (37-61) (12-35)
20
30
% a or 3-acid
25
Hop Physiology and Propagation
Propagation of Advanced Lines:
Forty-five hop selections were increased to provide planting stock
for future testing. Based on the eight-year testing plan, hop root stock is
required the third year for evaluating a line's reaction to downy mildew,
Verticillium wilt, and viruses.
hop lines.
Approximately 50 to 300 roots are available from each of the 45
Twenty-five of the lines were planted to 10-hill observation
plots in 1969. These lines were evaluated for quality and vigor this year
and will be tested for disease reaction in 1970.
The other 19 lines which
were increased, are high quality selections used as a model for advancing
new hop lines.
plan as follows:
This program is in the third year of the 8-year testing
Evaluation
Stock
Make cross
3000 seed
Vigor, visual analysis,
1 hill each of
remove males, reject
1000 genotypes
abnormal lines and
selected 106 lines
1968
Yield, lab analysis,
1 hill each of
commercial potential,
cultural, storage, and
106 genotypes
selected 19 lines for
propagation.
1969
~
Yield, lab analysis
commerical potential
cultural, downy mildew,
virus
1970
10-hill observation
1 hill each of
106 genotypes plus
100 roots each of
the 19 lines.
10 hills each of
for yield, quality,Vert,
wilt and USBA hand'Eval.
selec. lines plus
1971
Yield, quality, virus
indexing
1000 roots
1972
Yield trial, off-sta.
5,000 roots
1973
Yield trial, off-sta.
50,000 roots
test brew.
300 roots.
26
The following hop lines were also increased: 21001, 61021, 62013,
63018, 63019, 63020, 56008, 56013, 58112, and Pride of Ringwood.
The 8-year testing program as outlined last year and listed above
is a satisfactory plan for a single progeny or cross. The plan has a
limited capacity for handling selections during and after the third year.
Technological and physical limitations for disease testing restrict the
number of lines. Also, the number of samples for hand evaluation should
be kept to a resonable number. We propose that the observation block be
limited to 30 lines with the remaining selections held in a 2 to 5 hill
reserve block. This plan would provide a continuous flow of lines into
the development plan, with the necessary evaluations being conducted as
scheduled, and would result in one or two lines being tested off-station
every year.
Evaluation of New Lines:
Twenty-five lines were evaluated in the seedless observation
block.
Thirty-one lines grown in observation plots at Prosser were
evaluated by Mr. C. E. Nelson and data were summarized on 12 lines with
commercial potential (see Table 2, under Breeding and Preliminary Evaluation)
A single-hill nursery of 130 genotypes was established at Prosser
from stock grown in the 1966 nursery at Corvallis. Nineteen lines were
selected for their vigor and harvested for chemical evaluation. The
following 10 lines were selected for further advancement at Prosser:
6502-06
6512-11
6530-12
6503-22
6517-24
6532-03
6503-25
6527-21
6532-04
6532-14
The following tables list the 23 lines from the 1967 nursery and
the 101 lines from the 1968 nursery at Corvallis which will be planted at
Prosser in 1970. A large number of selections going to Prosser, especially
from the 1968 nursery, are from crosses with "Cluster" varieties.
The
crosses were made in 1966 and 1967 and reflect our emphasis on improving the
"Cluster" types.
27
Table
1.
Selection
No.
Selections for Prosser from 1967 Nursery; Corvallis, 1969
1969
Location
Purpose-—
Comments
Cross
6625-01
2:52a
AR
Bullion x
6635-17
5:50a
a
Early Cluster x OP
5:53
ii
ii
"
-22
"
-25
6:50a
"
-34
7:50b
"
-35
6656-04
6669-10
late
MR
Fuggle x OP
a
Bullion x OP
-12
10:52b
-28
12:52b
6621-01
13:50a
14:50a
6645-02
14:52a
6622-11
16:51
aAMR
AR
aAMR
ii
6626-01
18:54a
AR
-27
Bullion x 51114M
ti
18:51
"
V.
8:52a
"
-09
-^Late Cluster
7:51
10:51
19164 x OP
Bullion x
ii
19043M
ii
19038 x 51101M
Bullion x
19:52b
aAMR
20:50a
AR
19038 x 54066M
6630-05
21:52b
AR
52043 x
OP
6636-05
23:54a
AR
62002 x
51114M
23:54b
ii
62002 x 51114M
-06
late
•~ Fuggle
hi lup.
late-hi lup
Two plants
good pick
good pick, hi lup
•"* Fuggle
hi lup, weak SA
early
early
19037M
6623-05
6628-01
"
Vig. Late
poor SA
ii
"
"
OP
"
Fuggle
early Fuggle
late, hi lup.
6642-07
25:54a
AR
19124 x OP
"•"•Fuggle
6650-13
27:54a
MR
19200 x 54066M
good cone
/l
AR = aphid resistance; MR = mite resistance;
Table 2,
Selections for Prosser from 1968 Nursery; Corvallis, 1969
Selection
1969
No.
Location
6701-01
30:42a
-02
42b
-12
49
-14
50b
-17
52b
-24
57
-27
59
-31
62a
-39
31:44a
-44
47
-54
54b
-55
55
-60
60a
-67
64a
-68
-111
hi alpha-acid content
Purpose
DMR
Cross
Comments
E2 (Cluster) x OP
64b
33:47
no a ?
Selections for Prosser cont,
Selection
No.
1969
Location
6702-07
34:50b
6703-02
34:53
-13
60b
-21
35:42b
-30
48b
6704-01
35:52b
-05
56a
-13
61
-33
36:51
-38
54b
-45
59
-58
-63
48b
50a
-66
50b
-93
38:46b
38:47
-105
54b
-120
64b
-124
39:44a
-138
54a
-147
60b
-153
64b
-158
40:44b
-164
48b
-167
50b
6705-07
Comments
SMR
E2 x
51114M
DMR
E2 x
63015M
DMR
Yakima Cluster x OP
DMR
Yakima Cluster x
OP
DMR
Yakima Cluster x
19040M
SMR
Yakima Cluster x 51114M
64a
37:44b
-65
6704-94
a
Cross
55
-06
-52
Purpose
40:58b
-11
61
-15
64a
6706-03
41:48b
-22
62a
-23
62b
6707-04
42:42b
-12
48a
6708-01
42:56a
a DMR
Yakima Cluster x 63012M
6709-01
43:43
a DMR
Yakima Cluster x 63013M
-08
43:48a
-10
50a
a DMR
ii
Yakima Cluster x 63014
ii
weak SA
29
Selections for Prosser from 1968 Nursery cont,
Selection
1969
No.
Location
Purpose
Cross
Comments
6709-20
43:56b
a-DMR
Yakima Cluster x 63013M
6710-01
43:58a
a-DMR
Yakima Cluster x
-19
44:46b
ii
it
-21
48a
ii
ii
it
-23
49
ii
-32
55
n
it
-38
59
ii
ii
-49
45:43
it
ii
6711-01
45:47
BC-ct
Brewers Gold x
it
ii
-03
6712-01
48b
BC-a
Brewers Gold x
-03
52b
it
ii
-06
55
n
ii
-10
58a
ii
ii
58b
ii
it
-13
60a
ii
ii
-21
46:42b
ii
ii
ii
ii
-11
-25
45:51
45
aphids
46:48b
50b
6715-02
46:58a
a
Bullion x
-05
60a
ii
ii
-11
64a
ii
ii
-13
47:42a
ii
ii
ii
Brewers
ii
-14
42b
ii
-15
43
M
11
6716-08
47:53
Gold x
a
Bullion x
-13
56b
ii
ii
-17
bOa
ii
ti
-28
48:44a
it
ii
-32
46b
ii
ii
-40
52a
ii
ii
6717-06
48:62b
a
Bullion x
-07
63
ii
ii
-08
64a
ii
ii
-11
49:42b
ii
ii
-13
44a
ii
ii
-15
46a
it
it
-16
46b
ii
ii
-18
48a
ii
M
-21
50a
ii
it
-22
50b
ii
H
63012M
suscept. to
-08
BC-a
63015M
no lup.
6714-05
ii
6301
63014M
63014M
lup on strig
63013M
63012M
hi aphid count
30
Selections for Prosser from 1968 Nursery cont.
Selection
1969
Location
No.
6717-26
49:54a
6733-02
49:64a
-05
50:42b
/!
Purpose
a
Genetic
ii
Cross
Comments
Bullion x 63012M
19151 x 19041M
ii
no lup.
DMR = downy mildew resistance
SMR = spider mite resistance
a = high a-acid
BC
= backcross
The following selections grown in a 10-hill observation block at
Corvallis will be established at Prosser in 1970:
63032
64026
64002
65002
64003
65009
64007
65011
6503-25
6517-46
6517-47
6527-17
64024
Hop genotypes selected from the high analysis selections, from the
6801, 6802, 6803, 6804, and 6806 progenies, and from the triploid nursery
will also be established at Prosser.
Evaluation of Hops in Seedling Year:
The evaluation and propagation techniques for evaluating hops
established in the field from seed has been successful.
The success is
reflected in different areas of research, but my enthusiasm (CEZ) rests
with the fact of working with larger populations in the field. Large
populations in the field are reduced by removing males, hermaphrodites,
dwarfs-abberrations, virus and other disease-like types. The remaining
population can then be evaluated for selecting germ plasm to be incorporated
into a breeding program, for determining the inheritance of the factor(s)
used in making the cross, and for selecting genotypes with commercial
potential.
This type of program was initiated in 1966 and repeated in 1969.
Results are reported under Hop Chemistry: Trial Crosses for High Analysis.
Correlation of Plant Morphology and Yield:
The following parents were used in a yield-quality study:
31
Male Parents
Female Parents
Genotype
Vigor
a/3
Genotype
Vigor
a/i
Hallertau
Low
1.2
19170
Low
0.6
Fuggle
Med
1.8
19173
Med
0.6
19105
High
0.2
19058
High
0.6
A combination of nine possible crosses was obtained and the progeny
evaluated.
Data were obtained on:
a)
Maturity
b)
c)
d)
e)
f)
Percentage of a and 3-acid in lupulin
Sidearm length
Nodes per sidearm
Flowers per node
Floral branching pattern/node
These data were collected on 106 different male and female genotypes
from the nine different crosses.
The information has not been evaluated at
this time, but preliminary observations indicate that this type of genetic
study would provide useful information for studying the inheritance of yield.
Relationship of Floral Initiation, Growth Factors and Supplemental Lighting:
Wye College in England has recently confirmed an earlier Japanese
report that hop is a short-day plant. Research workers believe that a
critical day length of 14 hours is necessary for floral initiation in hop.
It is also known that hop remains vegetative under long days and
undergoes dormancy in 8-hour days.
Hop plants, like many other flowering
plants, must attain a definite stage of growth prior to floral initiation;
therefore, this growth stage and critical day length occur during early June
with most hop varieties. Late flowering hop varieties may reach the
necessary growth stage near June 21 and require the shorter day lengths in
early July for floral initiation.
An application of gibberellin-A3 to hop, when vines are 5 to 8-feet
in length, increases the number of hop cones (flowers) similar to the effect
of supplemental illumination at the same stage of growth. The supplemental
lighting provides the same effect as a long day, hence, when hop plants reach
the stage of growth necessary to develop the "flowering stimulus", floral
development is halted until the plant is subjected to short days.
This flowering behavior of hop may be related to the effect of
training on yield,as noted by Mr. C. E. Nelson.
The physiological aspects of hop yield will be continued next year,
but without the financial support of the U. S. Brewers Association.
Private
industry has provided financial support for these studies in previous years,
due to their interest in plant growth regulation.
32
Mr. Zimmermann will not submit a proposal to the USBA for 1970;
therefore, he wishes to express his sincere appreciation to the USBA Hop
Committee for their continued support of our program and for the financial
assistance provided him to assist in the hop breeding and development
studies at Corvallis, Oregon.
33
DETERMINATION OF INSECTICIDE RESIDUES ON FRESH AND DRY HOPS
(USBA Project 19, L. C. Terriere)
PHORATE:
Two reports describing the fate of this insecticide on hops have
been submitted to the manufacturer of phorate (American Cyanamid Company).
These reports describe the results of analyses of fresh and dry hops from
Oregon and Washington and show that neither the parent insecticide nor its
metabolites were present in amounts greater than the sensitivity limits of
the analytical method. On the basis of these reports the manufacturer of
phorate submitted a petition to the Pesticides Regulations Division of U. S.
Department of Agriculture and to the Food and Drug Administration requesting
a label clearance and a residue tolerance for phorate on hops.
It was learned in summer of 1969 that the Pesticides Regulations
Division was ready to grant a label registration for phorate on hops and
that the Food and Drug Administration was willing to issue a negligible
residue tolerance of 0.5 ppm for the soil application. The petition for
the residue tolerance for the foliar application was rejected on the grounds
that the analytical method used to determine the metabolites was of insuf
ficient sensitivity. Upon learning this, new large scale plots were
immediately established in Washington where hops received three foliar
applications of 2.25 lbs. per acre. Dry hops were sampled and analyzed for
the parent compound and the metabolites using a method of improved sensi
tivity. The results of this work showed the absence of phorate residues
at 0.05 ppm and the presence of metabolites at about 0.1 ppm level. These
results confirm our previous findings that there is very little hazard to
the consumer associated with the use of this insecticide.
This new infor
mation has been used by the American Cyanamid Company to support their
request for a residue tolerance for the foliar treatment.
DISULFOTON:
A study of residues resulting from disulfoton treatment was initiated
in the summer of 1968. Field experiments were set up in 1968 in Corvallis,
Oregon and in 1969 in Corvallis and Prosser, Washington. The insecticide
was applied to the soil at both locations and was also used as a foliar spray
at Prosser. The analytical method for phorate was modified to permit the
detection of disulfoton and its metabolites. The sample was extracted with
acetonitrile and disulfoton partitioned into pentane. The pentane extract
was then purified by passage through a magnesia: celite column and the
eluate analyzed by gas chromatography using a phosphorus sensitive detector.
The disulfoton metabolites (five toxic metabolites are known) were
extracted into organic solvent, the solvent removed and the residue treated
with aqueous potassium permanganate.
This treatment destroyed some of the
hop extractives and oxidized the disulfoton metabolites to their sulfones.
A second oxidizing agent (m-chloroperbenzoic acid) was used to convert all
metabolites to their most oxidized form (disulfotoxon sulfone) which was then
determined by gas chromatography.
34
This analytical method is sensitive to 0.1 ppm of disulfoton and
0.3 ppm of disulfotoxon sulfone.
averaged 71%.
Recoveries of added insecticides
The results of the analyses of the 1968 samples showed the
absence of residues and have been submitted to the manufacturer of
disulfoton (Chemagro Corporation) who have used the data to petition the
USDA and FDA for a registration and residue tolerance for the soil appli
cation of disulfoton. The word has been received that USDA has approved
this use, however, the disposition of this case by the FDA is not yet known.
AZODRIN:
Azodrin has shown an excellent potential as a miticide on hops
and,therefore, the development of an analytical method was initiated during
the past year.
Gas chromatography of the parent compound (Azodrin) was studied
using a phosphorus sensitive detector.
Various columns tested include 2%
and 4% diethylene glycol succinate, 6% phenyl diethyleneamine succinate,
and 10% Dow 11.
Since Azodrin tends to break down in a gas chromatograph,
an entirely satisfactory column has not yet been found.
Best results have
been obtained with the 10% Dow 11 column using Chromosorb Q as the solid
support.
The analytical method will involve extraction by chloroform or
water and cleanup by solvent partitioning or by a celite column. Insuf
ficient removal of hop extractives is the most serious problem at this time.
Research with other plants indicates that only one toxic meta
bolite may be present in hops in sufficient quantities to warrant determina
tion. This metabolite is the glucoside of hydroxymethyl Azodrin. The
present analytical method involves extraction with methanol, hydrolysis of
the glucoside with hydrochloric acid and coupling of the liberated hydroxy
methyl Azodrin with sec.-butyl mercaptan.
The thioether formed was then
determined by thin layer chromatography using blood plasma cholinesterase
for detection. It was found that insufficient removal of plant materials
complicates the thin layer chromatography and makes quantitation difficult.
PROPOSED WORK AND BUDGET FOR 1970
OBJECTIVE
1. To develop analytical methods for the determination of pesticide
residues in hops and to gather residue data on experimentally or commercially
applied pesticides.
2. To prepare or to assist in preparation of requests for USDA
or FDA clearance for the use of such pesticides on hops.
35
JUSTIFICATION:
Pesticides are necessary for the production of good quality hops
in economically profitable yields.
However, before a pesticide can be
registered for use, residue data, showing the magnitude and persistence of
residues, must be submitted to the U. S. Department of Agriculture and to
the Food and Drug Administration.
Such data are difficult to obtain for
hops because interferences from extractives make it impossible to use
already existing analytical methods and new methods have to be developed.
This laboratory has been developing analytical methods for hops for
several years and because of the constantly changing pest problems, must
continue to do so.
PROCEDURE:
The progress of the petitions for residue tolerances for phorate
and disulfoton will be followed during the coming year and every assistance
will be rendered to the companies to secure the registration of these
pesticides for the use on hops. The disulfoton samples collected from Oregon
and Washington during the 1969 season will be analyzed using the already
developed methods. If additional residue data are necessary, new plots
will be established and the necessary data gathered.
Major attention will be given to the insecticide Azodrin which
has shown promise for the control of mites on hops. The work on analytical
methods for both the parent compound and the metabolites will be continued
and if progress permits field treated samples already collected analyzed.
It probably will be necessary to establish new plots during the 1970 growing
season for additional samples.
Gas chromatography will be the main analytical procedure used in
the study of Azodrin.
Attempts will be made to develop a gas chromatographic
procedure also for the metabolites which should be more satisfactory than
the present thin layer procedure.
BUDGET REQUESTS:
Salaries (1/2 technician)
Supplies
Overhead
$ 3,600
675
225
Total
$ 4,500
36
BIOSYNTHESIS OF HUMULONE IN HOPS (USBA
Project 24)
W. D. Loomis
The present report summarizes briefly the results from this
project since its inception.
There have been three major areas of
research: enzyme studies; ll4C-tracer studies; and methodology.
Enzymatic Studies with Isolated Secretory
Glands from Hops
W.
E.
Shine, J.
v
Vrkoc and W. D.
Loomis
Enzyme studies with isolated resin glands from hops were
described in our report of January 1969, and the details will not
be repeated here.
Briefly, the glands were isolated by agitating
hop cones at low speed in a Waring Blendor in cold acetone, and
sieving. Resin and essential oil were removed by acetone washing.
The isolated glands were shown to contain mevalonic kinase ,
geraniol kinase and alkaline phosphatase.
Silyl Esters of Isomeric Carboxylic Acids:
A Gas Chromatographic Separation of the Pyrolysis
Products of Alpha Acids from Hops
Mary Barnes and W. D. Loomis
The alpha acids of hops (Humulus lupulus L.) have three
principal constituents: humulone, cohumulone, and adhumulone,
which differ only in their acyl side chain (isovaleryl,
isobutyryl, and 2-methylbutyryl, respectively).
Pyrolysis
of the lead salts of the alpha acids yields the free carboxylic
acids corresponding to the acyl side chains. In studying the
biosynthesis of alpha acids in hops it is necessary to
separate these acids and determine the quantity of label
incorporated into each acid. Although many free acids have
been successfully separated by gas chromatography (glc) the
separation of saturated isomeric carboxylic acids is
difficult. Preliminary experiments showed that both methyl
and isopropyl esters of the three acids in question can be
separated by glc, but some overlapping occurred. Separation
of the acids improves with the increasing size of the
esterifying group, and silylation of the free acids greatly
enhances the separation of the acids by glc.
37
Methods
Following the procedure originally described by Rigby,
Sihto and Bars (1), 100 mg of the lead salt of the natural
alpha acid mixture, sealed under vacuum in a 6 mm x 30 0 mm
glass tube, was pyrolyzed for 10 minutes at 400° .
Approximately
25% of the theoretical yield of acid distilled over the rightangle bend of the pyrolysis tube and condensed in the trap
cooled in an ice-salt mixture.
After cooling, the tube was
opened and a 10-fold excess of hexamethyldisilizane (HMDS,
obtained from Pierce Chemical Co., Rockford, Illinois), and
The tube was
a few grains of acid-washed sand were added.
centrifuged, sealed over a flame, and heated for one hour in
a boiling water bath.
The esterified mixture was cooled and
opened, and 50 to 15 0 yl aliquots were immediately chromatographed.
The esters were separated with the gas chromatographyeffluent counting system on a 40 ft. x 1/8 in. stainless
steel column packed with 1% SF-96 on Chromosorb G (100/120
mesh). Both the stationary phase and the support were obtained
from Varian Aerograph, Walnut Creek, California.
The column
temperature was maintained at 70° until HMDS and the trimethylsilyl ester of isobutyric acid had eluted (approximately 30
minutes), then programmed for a rise of 100° in 38 minutes
(2.7° /min.). Retention times and quantitative yields were
determined with silyl esters of known acids.
Results
A number of silylation procedures were tested but the
procedure described above was the only one which completely
esterified the pyrolysis mixture.
The use of more than
catalytic amounts of TMCS (trimethylchlorosilane) or the
use of HMDS:pyridine:TMCS in the ratio 2:2:1 gave incomplete
esterification of the acids. The chromatographic procedure
described here was shown to give complete separation of the
silyl esters of n-butyric acid, isobutyric acid, isovaleric
acid and 2-methylbutyric acid. The peaks for these acids
are not contaminated by any of the minor components present
in the alpha acid pyrolysates. The esters are extremely
sensitive to hydrolysis and must be chromatographed as soon
as esterification is complete.
However, the method as
described is rapid and efficient and eliminates the washing
and extraction of the pyrolysate, with its corresponding
losses, necessary for the formation of isopropyl esters.
Literature Cited
1.
F. L. Rigby, E. Sihto, and A. Bars, J. Inst. Brewing
66,
242
(1960).
38
Biosynthesis of Humulone
R.
L. Gardner, Mary Barnes and W. D. Loomis
The resin glands on the oones of hops contain in
addition to mono- and sesquiterpenes a variety of compounds
which appear to fit the "polyacetate" or "polyketide"
pattern.
Such compounds are thought to arise, by pathways
similar to those involved in fatty acid biosynthesis, from
an acyl "starter" unit plus repeating "acetate" units.
There is considerable tracer evidence supporting this
hypothesis, both for fungal products and for products of
higher plants.
At the time this work was started there was
only one brief report of cell-free biosynthesis of a poly
acetate compound (1), to the effect that the biosynthesis
of 6-methylsalicylic acid by cell-free enzymes from
Penicillium patulum required both acetyl-CoA and malonyl-
CoA.
This work appeared not to have been continued.
Hop glands contain a number of fatty acids of both
odd and even chain length and several unusual ring
compounds with isoprenoid side chains, of which humulone
is the most abundant (Figure 1). A postulated biosynthetic
scheme for humulone is shown in Figure 2.
Replacing isovaleryl CoA in this scheme with isobutyryl CoA (from
valine), 2-methylbutyryl CoA (from isoleucine), n-butyryl
CoA or propionyl CoA, with or without the addition of a
third dimethylallyl group, would account for the biosynthesis
of the other alpha and beta acids which are found with
humulone.
Wright and Howard (2) showed that carboxyl labeled
acetate was incorporated into alpha acids when it was given
to a hop plant over a two-week period. In this length of
time there was extensive randomization of the label, and it
was found in all parts of the humulone molecule.
However, the
heaviest labeling was in the ring. It appeared that the hop
compounds might be a good model system for polyacetate
biosynthesis.
For this reason, the tracer studies described
here and the enzyme studies described elsewhere in this
report (Shine and Loomis) were undertaken.
Results
Label from all of the postulated precursors was
incorporat ed in good yield into the a lpha acid fraction
Since the uptake of substrate through
within thr ee hours.
the cut st em of the hop cones require d some time (about an
hour), the actual reaction time after the substrate reached
Thus the biosynthesis
the glands was less than three hours,
of these c ompounds is a much more rap id process than had
A prelimin ary degradation gave
been suppo sed previously.
Pyrolysis
nsistent
with
the
postulate d scheme.
results co
39
of humulone forms isovaleric acid from the isovaleryl group,
and isohexenoic acid from the lower isoprenoid group plus
the adjacent ring carbon, as well as unidentified products.
A sample of humulone containing label from leucine was
degraded by pyrolysis, and the products were esterified,
and separated by gas chromatography.
The isovalerate
contained -^C , while neither the isohexenoate nor any of
the unidentified products separated by the gas chromatograph were labeled.
In the course of this work it became apparent that
the alpha acids are much more sensitive to air and light
then had previously been recognized. All work is now
done in dim light, and much of it under nitrogen. From
very recent work it appears that light is more damaging
than oxygen, and if reasonable precautions are taken, it is
possible to work in the air with only moderate losses of
product.
It also became apparent that our purification methods
were inadequate, and that labeled samples we had thought
were ready for degradation required further purification.
Column chromatography on silica gel G yields what appear
to be essentially pure alpha acids. The incorporation of
l'+C-label from various precursors, calculated from the
specific activity of the chromatographically purified alpha
acids is shown in Table I.
The 11 purified samples
represented in Table I are awaiting degradation by pyrolysis
and the improved gas-chromatography-counting procedure
described elsewhere in this report (Barnes and Loomis).
Experimental
Freshly harvested hop cones (about 15 g per sample)
were placed with their cut stems in 0.2 ml of solution of
C-substrate in a sma 11 vial, and allowed to take up the
When the solution
solution in a lighted growth chamber.
had been taken up it w as chased in with 2x1 drop of
water.
The vials were then filled with water, and the cones
were left in the light for three hours total time,
Preliminary experiment s had shown that this was an
The cones were then frozen
appropriate exposure t ime.
for later extraction.
The radioactive c ones were ground in methanol and
The
extracted twice with m ethanol and once with ether.
combined extract was a cidified with H2S0|4 and extracted
was
evaporated at
with ether.
The ether extract
reduced pressure, and the oily residue was taken up in
filtered (0.6 yMillipore).
The resulting cle ar yellow extract was treated with
methanol and
0.26M methanolic
lead acetate.
The
lead humulonate
precipitate was washed with cold methanol and dried under
40
vacuum. Some samples were later converted to the free acids
and reprecipitated; some were washed several times with CH oC12 5
ether and methanol; some were not further treated at this
stage.
Separation by thin layer chromatography, combined with
autoradiography, showed that the samples at this stage
contained radioactive contaminants.
This was especially true
of the acetate and malonate samples, which appeared to contain
appreciable quantities of the labeled precursors.
Further purification was accomplished by column
chromatography on silica gel G. The silica gel was washed
successively with 10% HC1 in methanol, methanol, acetone,
anhydrous ether, and petroleum ether. Humulone samples,
freshly freed from the lead salt, in petroleum ether solution,
were placed on the column and eluted with petroleum etherethyl acetate-formic acid (80:18:2). Humulone was estimated
by U.V. analysis in 0.002N methanolic NaOH (3).
Radioactivity
was counted in a Packard Tri-Carb Liquid Scintillation
Spectrometer. Aliquots of each sample were analyzed by
thin-layer chromatography, and no radioactive impurities
could be found.
The bulk of each sample was reprecipitated
with lead acetate and stored in the freezer for future
degradation.
Literature cited
1.
F. Lynen S M. Tada, Angewandte Chemie 7_3_, 513 (1961).
2.
D. Wright and G. A. Howard, J. Inst. Brew. 67_, 236 (1961)
3.
Alderton et al, Analytical Chem. 26_, 983 (1954).
41
Table
Incorporation of
14
I
C-Precursors into
Alpha Acids by Hop Cones
(3-Hour Exposure)
% Incorporation
Precursor
of
Label
Acetate-l-ll4C
0.34
Acetate-2-14C
0
59
0
51
1
45
1
26
0
37
0
35
0
012
Malonate-2-14C
Glycolate-2-14C
L-Leucine-U-
14
C
D,L-Valine-4-14C
L-Isoleucine-U-
14
C
(±)Mevalonate-2-14C
0
29
0
42
0.023
42
Genetic Study Proposal
Title
Inheritance of Alpha Acid in Hops
Objectives:
The major objective will be to determine the genetic basis for
inheritance of alpha acid. This will be accomplished by making a genetic
analysis of the components of alpha acid yield. We feel the major
components are:
1.
Amount of alpha acid per lupulin gland
2.
Number
Number
Number
Number
3.
4.
5.
and weight of lupulin glands per node of the hop flower
of nodes per hop flower
of hop flowers per side-branch of the vine
of side-branches per plant.
Justification
Alpha acid is the most important constituent of hops. Hop
varieties and genotypes vary greatly in their yield of alpha acid, as well
as in their yield of total hops. Some varieties such as Bullion grown in
the U. S., and Northern Brewer, grown in Europe, have both high alpha acid
and high cone yields. Other varieties, such as Fuggle and Hallertau, have
lower alpha acid and lower yields. Some experimental varieties have shown
low alpha and high hop yield, while others have shown the reverse - high
alpha and low yield.
We believe that yield of alpha acid is genetically controlled.
Logically, size, number and richness of lupulin glands would influence
yield of alpha acid. Likewise, number of lupulin glands per flower and
number of flowers per plant would also be important. We propose to study
the genetic inheritance of the above factors to determine how they are
inherited and to what extent they influence alpha acid yield. Such infor
mation would provide a solid base from which to make crosses to obtain a
specific alpha acid composition.
Procedure:
The proposed study would follow the sequence below:
1.
Develop techniques required for the study.
Most of these
are known, but we would need to make a "trial run" with
some known varieties to establish the validity of our techniques
2.
Survey existing hop germ plasm, both males and females, for
the most suitable experimental material to use in making crosses,
3.
Make the crosses and genetically analyze their offspring
for inheritance of each of the components of alpha acid yield.
43
4.
Incorporate the information obtained into our breeding
program.
Proposed Target Dates:
1970 - Evaluate and perfect techniques
1971 - Survey germ plasm
1972 - Make crosses
1973 - Preliminary sexing of progenies
1974 - Genetic analysis
1975 - Genetic analysis
Expected Results:
At the end of the six year period indicated we would expect to know:
1.
What characteristics are important in determining alpha acid
yield.
2.
How at least some of these characteristics are inherited,
therefore:
3.
How to breed for alpha acid.
4.
We would have identified certain parents whose offspring would
give us a certain alpha acid level.
5.
We would have identified certain genotypes that could be moved
directly into our variety development program.
Our proposed budget for 1970 includes an item of $7500 for the
above genetic study.
In budgeting for the genetic study we have agreed to
drop Physiology and Propagation - $1,000, Special Travel - $500, Biosynthesis
of Humulone - $1,400, and to reduce Hop Diseases from $10,000 to $8,000.
44
Proposed Budget for Fiscal 1970-71
USBA
Project
OAES
Project
Project Title
Proposed
Budget
Development and evaluation
of hop varieties
F.C.
19
36-1
Breeding, testing and
yard management
$ 7,500
F.C.
36-2
Genetic studies
7,500
Bot.
36-1
Disease control
8,000
AC 36-1
Chemical evaluation
9,500
AC 36-7
Pesticide residues
4,500
Total budget request
$37,000