DEVELOPMENT OF A PHYTOPHTHORA-RESISTANTAND BIACKLINE-TOLERANT ROOTSTOCKInterim Report

Gale McGranahan, Keith Woeste, John Mircetich, Adib Rowhani, Herb Phillips,

Chuck Ingels, and Ron Snyder

ABSTRACT

This project is designed to provide superior clonal rootstock for Californiawalnut growers. The project began in 1988 when over 13,000 seed from 17different 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 for48 hours throughout the growing seasons. Trees were measured for vigor

(height and diameter) at the end of the first and second growing season andwere 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 providerootstocks 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 ofthis project is to develop a clonal rootstock that combines an acceptablelevel 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. Thisproject 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 providedto the Walnut Improvement Program by Frank Van Konynenberg and (black X

English) X black walnut trees collected locally. The backcross to Englishpopulation 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 JohnMircetich 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

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different locations in California (Table 1). The male parent of these seedswas 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 arandomized 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 andtransplanted 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 earlySeptember and once a week until mid-October. Weeds were controlled with Round

up before walnut germination and with RonStar (3#/1000sq ft) in September ofthe first growing season.

PhytoDhthora inoculum provided by John Mircetich was applied to six of theblocks (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 wereevenly 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 andSeptember, 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. Resultingnecrotic spots on pears were counted and samples were cultured using standardprocedures (Mircetich, pers. com.).

Trees were measured for vigor (height and diameter)in February, 1989 andDecember, 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 bebackcrosses 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 and1991. The replications on English rootstock were inoculated in the rootstock

with patches of CLRV-infected bark 4-8 weeks after grafting. Those on blackrootstock were inoculated in the wood of the selection about one year after

grafting. Grafted and inoculated trees were examined for survival during thegrowing 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 areinterpreted 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 tolerantresponse. A tolerant response is then confirmed when the selection on blackrootstock 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

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with Cap tan , and enclosed in plastic boxes to maintain high humidity. Twomonths later, cuttings were scored for presence or absence of roots andcallus.

RESULTS AND CONCLUSION

Percent germination ranged from very low (3-8%) in two families (Eurekaseedlings 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 growingseason, 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 butmore 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 allowsfor 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 endof 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 greenhousescreens by John Mircetich (pers. com.) in which wingnuts are resistant and

black walnuts susceptible to Phytophthora. The BCl families ranged from 75to 97% survival.

The trees in uninoculated blocks were not significantly different in vigorfrom those in the inoculated blocks but survival may have been related towhether or not inoculum had been applied. The two uninoculated blocks had

over 98% survival whereas inoculated blocks had survival ranging from 79 to89% (Table 3). Phytouhthora cactorum and citrico1a were recovered from all

blocks except control block 6. Other species of Phytophthora present includedl. 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 werefound to be hypersensitive in response to CLRV; and 11 have not been confirmedone way or the other. The latte~ are likely to be tolerant because no

blackline has appeared at the graft union between the inoculated Englishrootstock and the selection being tested, but ELISA tests must confirm that

the virus is in fact present in the selection. Six selections were easilyrootable, but additional replicated tests are still needed.

Three selections have been shown to be vigorous and tolerant to CLRV. These

are 4-5-26, an offspring of 87-27 (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.

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TABLE 1. Origin, genainetion and survival of Paradox offspring (Be1.).

10 ORIGIN Seed Plants Gel'1llineted Death. Cn) Survival eX)(n) (n) (X) 1990 1991 1990 1991

1. 87-25 Paradox UCPhi losophy Oept. 776 237 30 14 18 94 862. 87-26 Paradox Davis O'Farrell 3816 2088 55 88 111 95 903. 87-27 Paradox Gridley North 1056 616 58 26 31 95 904. 87-28 Paradox Gridley south 1008 479 48 27 20 94 905. 87-29 Paradox UCAquatic Weeds 656 223 34 30 24 86 756. 87-30 Paradox Visalia MooneyBlYd. 392 227 58 5 1 97 977. 87-31 Paradox Tulare 1528 E Sonora 304 143 47 5 12 96 888. 87-32 Paradox Tulare 1516 E Sonora 304 150 49 4 6 97 939. 87-33 Paradox Visalia Park's East 312 10 3 0 0 100 10010. 87-34 Paradox Visalia Park's West 288 184 64 4 1 97 9711. 87-35 Paradox Visalia MaddoxT6 168 95 56 4 10 95 8512. 85-36 Paradox Visalia MaddoxT7 360 175 49 1 3 99 9113_ 85-31 Paradox Visal ia Sibbett 280 130 46 8 3 93 9114. 85-38 Paradox Modesto Driver/Vlatch 1424 120 51 31 9 95 9415. 85-48 Paradox Matteson 440 291 66 4 6 98 9616. 85-49 Paradox Mark, Vine Monastery 160 42 26 0 2 100 9517. 81-50 Paradox Matthew, Vine Monast. 2040 1291 64 35 49 91 9318. J. hindsii Rawlins 1600 908 51 97 469 89 3119. P. stenoptera Dairy Rd 1600 1481 92 0 0 100 10020. J. regia Eureka Stuke 1600 132 8 14 1 89 89

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TABLE2. Height end diameter of Plredox offspring Ifter '.t end 2nd growingsel.on.

1988 1989JO Parent Oi_ter Height Oi_ter Heiiiht

(11III)cv (em) cv (118) f:V ( CIII) f:V

1. 87-25 Peredox 8 .38 37 .46 21 .41 132 .512. 87-26 PI redox 6 .33 32 .47 20 .40 130 .413. 87.27 Paredox 6 .33 20 .47 20 .43 134 .454. 87-28 PI redox 7 .29 33 .45 22 .39 135 .425. 87-29 PI redox 7 .29 35 .46 20 .45 121 .556. 87-30 Plredox 7 .29 33 .45 21 .48 126 .467. 87-31 Plredox 7 .43 36 .47 20 .40 141 .438. 87-32 Plredox 7 .29 35 .43 21 .41 131 .409. 87-33 Plredox 9 .22 37 .30 26 .27 111 .2910. >87-34 Plredox 7 .29 34 .41 20 .38 131 .4111. 87-35 Plredox 6 .33 24 .46 15 .40 79 .5512. 85-36 Plredox 7 .29 33 .42 21 .43 129 .4513. 85-37 Plredox 6 .33 25 .44 17 .39 95 .5214. 85-38 Plredox 6 .33 29 .48 21 .50 123 .4715. 85-48 Plredox 7 .29 33 .45 19 .40 132 .4116. 85-49 Paradox 8 .25 33 .38 25 .48 133 .4417. 87-50 PI redox 6 .33 33 .45 20 .40 138 .4118. J. hindsi i 10 .20 64 .25 27 .29 206 .1819. P. stenopterl 6 .17 42 .28 21 .29 223 .2420. J. regia 5 .60 21 .62 18 .58 106 .64

TABLE3. Averlge height Ind survivil of Plredox offspring by block.

1988 1990 Deaths (n) Survival eX)Seed Plints Germinated dia. height dia. height 1990 1991 1990 1991

Block (n) (n) (X) (11m) (em) (11III) (ell)

1 2123 1270 59 8 36 20 161 85 137 93 822 2123 1175 55 7 34 22 172 44 159 96 823 2123 1217 57 7 31 20 146 46 80 96 894 (control) 2123 1217 57 8 36 21 161 3 19 99 985 2123 1173 55 7 36 21 148 77 160 93 796 (control) 2123 1197 56 7 33 21 141 6 3 99 997 2123 1220 57 8 37 21 144 67 119 94 848 2123 1177 55 8 36 21 137 69 99 94 85