Development of a Phytophthora-Resistant and Blackline

DEVELOPMENT OF A PHYTOPHTHORA-RESISTANTAND BIACKLINE-TOLERANT ROOTSTOCK
Interim
Report
Gale McGranahan,
Chuck
Ingels,
Keith Woeste, John Mircetich, Adib Rowhani, Herb Phillips,
and Ron
Snyder
ABSTRACT
project is designed to provide superior clonal rootstock for California
This
walnut growers.
The project began in 1988 when over 13,000 seed from 17
different Paradox sources was planted in a randomized complete block design in
the field.
Plots were inoculated with Phytophthora-infested
soil in the
second, third, and fourth growing seasons and were flooded every 2-3 weeks for
48 hours throughout the growing seasons.
Trees were measured for vigor
(height and diameter) at the end of the first and second growing season and
were rated for survival after the third and fourth seasons.
Thirty-five
seedlings were selected at the end of the second growing season on the basis
of vigor and are undergoing
tests for response to cherry leafroll virus
(black1ine).
Of these six have now died presumably due to Phytophthora,
fourteen have been shown to be hypersensitive
to CLRV, and three have already
been selected as both vigorous and tolerant to CLRV.
Our goal is to provide
rootstocks
for field trials in 1995.
OBJECTIVE
Phytouhthora
root and crown rots and b1ackline disease are two of the most
serious problems confronting walnut growers in California.
The objective of
this project is to develop a clonal rootstock that combines an acceptable
level of resistance to PhytoDhthora
(similar to Paradox walnut) with tolerance
to the cherry leafro11 virus (like English walnut).
Because these rootstocks
will be c10nally propagated,
rootability is also being evaluated.
This
project was initiated i~ 1988 and will continue until 1995 when rootstocks
should be available for field testing.
In preliminary
experiments
the inheritance of response to cherry leafrol1 was
investigated
in a population
(black X English) X English walnut trees provided
to the Walnut Improvement
Program by Frank Van Konynenberg
and (black X
English) X black walnut trees collected locally.
The backcross to English
population
segregated 1:1 (tolerant:hypersensitive)
and the backcross to black
walnut were almost uniformly hypersensitive.
Both populations
were highly
variable in terms of vigor, leaflet number and shape, color and morphology.
This led to the hypothesis
that a vigorous tolerant rootstock with tolerance
to CLRV could be selected from among (black X English) X English (or BC1)
seedlings.
PROCEDURE
The location for this trial in the Pomo1ogy orchards was selected because John
Mircetich had identified PhytoDhthora
in the soil at this site in the past.
Seed were collected in fall 1987 from 17 different Paradox trees growing in
40
-
-
--
different locations in California
(Table 1). The male parent of these seeds
was unknown but it is expected that at least a portion of the seeds has an
English parent.
Seed were stratified and planted (in 1988) along with
standards (seedlings of English Eureka, black walnut Rawlins and wingnut) in a
randomized complete block design with eight blocks and equal number of trees
per family per block.
Each block consisted of nine 55 ft long beds.
The beds
were spaced 40in apart, with two rows per bed.
Seeds were planted 3-in deep,
5 in apart in the rows.
Seeds of individual families were planted down the
length of the rows in random assignments
in each block.
Because of poor field
germination
in the past, wingnut seed were germinated in the greenhouse and
transplanted
to their assigned spaces in the blocks when their first true
leaves emerged.
The beds were sprinkler irrigated for one hour every three
days for 6 weeks and then approximately
for 2-3 hours every 5 days until early
September and once a week until mid-October.
Weeds were controlled with Round
up before walnut germination and with RonStar (3#/1000sq ft) in September of
the first growing season.
PhytoDhthora
inoculum provided by John Mircetich was applied to six of the
blocks (two blocks serve as uninoculated
controls) in March, 1989; March,
1990;
October, 1990; and January, 1991.
The inoculum consisted of soil from
pots which had been part of greenhouse screens for response to PhvtoDhthora
citricola and l. cactorum.
Nine to 15 3-gallon buckets of infested soil were
evenly distributed
over each of the six blocks.
Berms were raised around each
block and blocks were flooded for 48 hours every 2-3 weeks between'May
and
September, 1989; March and November, 1990 and April and September, 1991.
Pears were placed in flooded blocks for 48 hours in May and July, 1990 to
serve as bait to determine. whether PhvtoDhthora was present.
Resulting
necrotic spots on pears were counted and samples were cultured using standard
procedures
(Mircetich, pers. com.).
Trees were measured for vigor (height and diameter)in
February, 1989 and
December, 1989, and rated for survival in August, 1990 and October, 1991.
Four to 5 superior trees were selected from each block and wood was collected
in January, 1990.
Selection was based on vigor but those that appeared to be
backcrosses
to black walnut were omitted.
For evaluation
of response to cherry leafroll virus, wood from selected trees
was grafted onto both English and black walnut seedling rootstock in 1990 and
1991.
The replications
on English rootstock were inoculated in the rootstock
with patches of CLRV-infected
bark 4-8 weeks after grafting.
Those on black
rootstock were inoculated in the wood of the selection about one year after
grafting.
Grafted and inoculated trees were examined for survival during the
growing season.
About one year after successful grafting and inoculation,
bark was removed from the graft unions to evaluate for presence of a blackline
and tissue was taken for ELISA.
Results of this screening procedure are
interpreted as follows: A b1ackline at the graft union between the English
rootstock and the selection indicates that the selection is hypersensitive.
No blackline and a positive ELISA in the selection wood indicates a tolerant
response.
A tolerant response is then confirmed when the selection on black
rootstock develops a blackline.
Rootability
of selections was assessed in apical shoot cuttings (3-5cm)
collected in 10/4/90 and dipped briefly in IBA solution (2500 ppm).
After
treatment cuttings were placed in moist 50:50 vermiculite:perlite,
sprayed
41
with Cap tan , and enclosed in plastic boxes to maintain high humidity.
Two
months later, cuttings were scored for presence or absence of roots and
callus.
RESULTS AND CONCLUSION
Percent germination ranged from very low (3-8%)
in two families (Eureka
seedlings and Paradox Park-E. Visalia seedlings) that were later discarded
from some analyses, to very high (92%) in wingnut seedlings.
The remainder
ranged from 26 to 66% germination
(Table 1). At the end of the first growing
season, the NC black family (which includes a substantial number of Paradox)
was tallest, but by the end of the second growing season the wingnuts were
larger (Table 2).
The backcross families were in general less vigorous but
more variable than the blacks and wingnuts.
The coefficient of variation was
greater in the Paradox offspring than in the blacks or wingnuts in both years.
This illustrates
the variability present in the Paradox offspring that allows
for selection of superior trees.
The percent survival in all families was higher than would be expected under
intense selection pressure from Phytouhthora
(Table 1). However, by the end
of the growing season in 1991 only 37% of the blacks were still alive, while
no wingnuts had been lost.
This corresponds
to results obtained in greenhouse
screens by John Mircetich (pers. com.) in which wingnuts are resistant and
black walnuts susceptible to Phytophthora.
The BCl families ranged from 75
to 97% survival.
The trees in uninoculated blocks were not significantly different in vigor
from those in the inoculated blocks but survival may have been related to
whether or not inoculum had been applied.
The two uninoculated blocks had
over 98% survival whereas inoculated blocks had survival ranging from 79 to
89% (Table 3).
Phytouhthora cactorum and citrico1a were recovered from all
blocks except control block 6. Other species of Phytophthora present included
l.
megasuerma,
l.
uarasitica
and
others
that
could
not
be
identified.
All selections were at. least two standard deviations above the mean in terms
of vigor at the time of selection.
Of the 35 original selections, six are now
sickly or have died in the trials, presumably due to Phytophthora;
14 were
found to be hypersensitive in response to CLRV; and 11 have not been confirmed
one way or the other.
The latte~ are likely to be tolerant because no
blackline has appeared at the graft union between the inoculated English
rootstock and the selection being tested, but ELISA tests must confirm that
the virus
rootable,
Three
is in fact present in the selection.
Six selections
but additional replicated tests are still needed.
selections
shown
easily
to be vigorous and tolerant to CLRV.
These
(Paradox Gridley North), 5-5-19, an
offspring of 87-50 (Paradox Matthew), and 7-2-26, an offspring of 87-26
(Paradox O'Farrell).
These will be grafted for clonal propagation
trials.
are 4-5-26,
have been
were
an offspring of 87-27
42
---'--
-
-- -
TABLE
1.
Origin,
genainetion
ORIGIN
10
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13_
14.
15.
16.
17.
18.
19.
20.
87-25
87-26
87-27
87-28
87-29
87-30
87-31
87-32
87-33
87-34
87-35
85-36
85-31
85-38
85-48
85-49
81-50
and survival
Paradox UC Phi losophy Oept.
Paradox Davis O'Farrell
Paradox Gridley North
Paradox Gridley south
Paradox UC Aquatic Weeds
Paradox Visalia MooneyBlYd.
Paradox Tulare 1528 E Sonora
Paradox Tulare 1516 E Sonora
Paradox Visalia Park's East
Paradox Visalia Park's West
Paradox Visalia MaddoxT6
Paradox Visalia MaddoxT7
Paradox Visal ia Sibbett
Paradox Modesto Driver/Vlatch
Paradox Matteson
Paradox Mark, Vine Monastery
Paradox Matthew, Vine Monast.
J. hindsii Rawlins
P. stenoptera Dairy Rd
J. regia Eureka Stuke
of Paradox
Seed
(n)
Plants
(n)
776
237
3816 2088
1056
616
1008
479
656
223
392
227
304
143
304
150
312
10
288
184
95
168
360 175
280 130
1424 120
440 291
160
42
2040 1291
1600 908
1600 1481
1600 132
43
----
--
offspring
(Be1.).
Gel'1llineted
(X)
30
55
58
48
34
58
47
49
3
64
56
49
46
51
66
26
64
51
92
8
Death. Cn)
1990 1991
14
88
26
27
30
5
5
4
0
4
4
1
8
31
4
0
35
97
0
14
18
111
31
20
24
1
12
6
0
1
10
3
3
9
6
2
49
469
0
1
Survival
eX)
1990 1991
94
95
95
94
86
97
96
97
100
97
95
99
93
95
98
100
91
89
100
89
86
90
90
90
75
97
88
93
100
97
85
91
91
94
96
95
93
31
100
89
TABLE
2.
Height end diameter of Plredox offspring
sel.on.
Ifter
'.t
end 2nd growing
1988
JO
Parent
Oi_ter
(11III) cv
Peredox
PI redox
Paredox
87-28 PI redox
87-29 PI redox
87-30 Plredox
87-31 Plredox
87-32 Plredox
87-33 Plredox
10. >87-34 Plredox
11. 87-35 Plredox
12. 85-36 Plredox
13. 85-37 Plredox
14. 85-38 Plredox
15. 85-48 Plredox
16. 85-49 Paradox
17. 87-50 PI redox
J. hindsi i
18.
19.
P. stenopterl
20.
J. regia
1.
2.
3.
4.
5.
6.
7.
8.
9.
87-25
87-26
87.27
TABLE
3.
8
6
6
7
7
7
7
7
9
7
6
7
6
6
7
8
6
10
6
5
.38
.33
.33
.29
.29
.29
.43
.29
.22
.29
.33
.29
.33
.33
.29
.25
.33
.20
.17
.60
Averlge height Ind survivil
Seed
(n)
Block
1
2
3
4 (control)
5
6 (control)
7
8
2123
2123
2123
2123
2123
2123
2123
2123
Plints
(n)
1270
1175
1217
1217
1173
1197
1220
1177
Germinated
(X)
59
55
57
57
55
56
57
55
1989
Oi_ter
(118) f:V
Height
(em) cv
37
32
20
33
35
33
36
35
37
34
24
33
25
29
33
33
33
64
42
21
.46
.47
.47
.45
.46
.45
.47
.43
.30
.41
.46
.42
.44
.48
.45
.38
.45
.25
.28
.62
21
20
20
22
20
21
20
21
26
20
15
21
17
21
19
25
20
27
21
18
of Plredox offspring
(11m) (em)
(11III) (ell)
44
-
-
---
20
22
20
21
21
21
21
21
132
130
134
135
121
126
141
131
111
131
79
129
95
123
132
133
138
206
223
106
.51
.41
.45
.42
.55
.46
.43
.40
.29
.41
.55
.45
.52
.47
.41
.44
.41
.18
.24
.64
by block.
1990
dia. height
36
34
31
36
36
33
37
36
f:V
.41
.40
.43
.39
.45
.48
.40
.41
.27
.38
.40
.43
.39
.50
.40
.48
.40
.29
.29
.58
1988
dia. height
8
7
7
8
7
7
8
8
Heiiiht
( CIII)
161
172
146
161
148
141
144
137
Deaths (n)
1990 1991
85
44
46
3
77
6
67
69
137
159
80
19
160
3
119
99
Survival eX)
1990 1991
93
96
96
99
93
99
94
94
82
82
89
98
79
99
84
85