quality, boron distribution and floral

AN ABSTRACT OF THE THESIS OF

Nancy Woodworth Callan for the degree of Doctor of Philosophy

in Horticulture presented on December 15, 1976

Title: EFFECT OF BORON SPRAYS ON FRUIT SET, FRUIT

QUALITY, BORON DISTRIBUTION AND FLORAL

MORPHOLOGY OF 'ITALIAN' PRUNE (PRUNUS

DOMESTICA L.)

Abstract approved: / ■ ; Maxine M. Thompson

The 'Italian1 prune (Prunus domestica L. ) is a high-quality

cultivar grown widely in the Willamette Valley of Oregon despite its

tendency for erratic production. Cool temperatures following bloom

slow pollen tube growth and thus limit fruit set.

In this study, 'Italian' prune trees which were not deficient in

B by August mid-'shoot leaf analysis, the standard nutritional index,

responded to fall foliar B application with increased fruit set.

Both pre- and postharvest B sprays were effective in increasing

set. A prebloom spray, however, did not increase fruit set. Mid-

summer, or "blue" drop, was unaffected by B treatment.

In an attempt to determine the basis of the fruit set increase,

the B concentration of various tissues was determined. Boron con-

tent of dormant bud and spur tissues, flower buds, flowers, and

immature fruit flesh was increased by 50 to 200% by the fall B spray.

August mid-shoot leaf B levels were increased by fall B in only one

of the two years of the study.

Various morphological and physiological changes resulted from

the fall B spray. Size of flower buds and of flowers at anthesis, as

well as style and pedicel length, was decreased by the fall spray, and

bloom was delayed slightly. Germinability of pollen from fall B-

treated trees was reduced. B levels of neither the pollen nor of the

pistil had an effect on pollen tube growth in the style.

Polyphenoloxidase activity of acetone powders prepared from

immature fruit was increased in fruits which had received fall foliar

B. This was in contrast to the observation that fruit from fall B-

treated trees browned more slowly when cut than fruit from control

trees. A decrease in the incidence of internal browning of mature

fruit after fall foliar B the previous year was also observed.

Speculations are made about possible relationships between the

observed responses of 'Italian' prune to fall B and the role of B in

the metabolism of phenolic compounds and in auxin oxidation.

Effect of Boron Sprays on Fruit Set, Fruit Quality, Boron Distribution and Floral

Morphology of 'Italian' Prune (Prunus domestica L. )

by

Nancy Woodworth Callan

A THESIS

submitted to

Oregon State University

in partial fulfillment of the requirements for the

degree of

Doctor of Philosophy

Completed December 1976

Commencement June 1977

APPROVED:

Associate Professor of Horticulture in charge of major

Head of Deparyraent of Horticultu re

Dean of Graduate School

Date thesis is presented O-^* ) S - ) f 7(a

Typed by Mary Jo Stratton for Nancy Woodworth Callan

ACKNOWLEDGMENTS

I would like to thank my adviser, Dr. Maxine Thompson, for

her advice, moral support and friendship offered during this study.

I also thank the members of my graduate committee. Dr. Michael

Chaplin, Dr. Warren Kronstad and Dr. Fred Rickson for their help-

ful suggestions and aid. Consultation with and assistance by Dr. Mel

Westwood, Dr. Robert Stebbins and Dr. Daryl Richardson has been

invaluable, as has the help given me by Harold Bjornstad and Fred

Dixon. Finally, I would like to thank the members of the Oregon

Processed Prune and Plum Growers Commission for the financial

support which made this work possible.

TABLE OF CONTENTS

Page

INTRODUCTION 1

REVIEW OF LITERATURE 3

Fruit Set 3 Boron and Pollen 8 Boron Levels and Plant Response 11 Mobility of Boron within the Plant 13 Fruit Quality 14 Postulated Roles of Boron in the Plant 16

FRUIT SET OF 'ITALIAN' PRUNE AS AFFECTED BY SPRING AND FALL BORON SPRAYS 20

Introduction 21 Materials and Methods 22 Results and Discussion 26

BORON DISTRIBUTION IN 'ITALIAN' PRUNE TISSUES FOLLOWING SPRING AND FALL BORON SPRAYS 39


THE EFFECT OF FALL FOLIAR BORON APPLICATION ON POLYPHENOLOXIDASE ACTIVITY AND INTERNAL BROWNING IN 'ITALIAN' PRUNE FRUIT 53


BIBLIOGRAPHY 62

LIST OF TABLES

Table Page

1 Fruit set of 'Italian' prune, 1975, following postharvest and prebloom B sprays. 33

2 Fruit set of 'Italian' prune, 1976, following fall foliar B sprays. 34

3 Fruit set of 'Italian1 prune in six orchards in 1976 following 492 ppm B applied postharvest. 35

4 Germinability of 'Italian' prune pollen in deionized water with 10% sucrose following 492 ppm B postharvest. 36

5 Dry weight of flower buds from seven orchards collected at various stages of bud development after 492 ppm B applied postharvest. 37

6 Style and pedicel length of 'Italian' prune in two orchards after 492 ppm B postharvest. 38

7 Boron in 'Italian' prune dormant bud and spur tissue and in flower buds in six orchards after 492 ppm B postharvest. 48

8 Boron in 'Italian' prune dormant bud and spur tissue and in floral tissues in two stages of bud development and full bloom, 1976. 49

9 Boron in 'Italian' prune flower parts at full bloom and at petal fall, 1975, as affected by 492 ppm B applied postharvest 1974 and prebloom 1975. 50

10 Boron in 'Italian' prune mid-shoot leaves in August as affected by B application. 51

11 Boron in 'Italian' prune leaves from four locations on the tree in August 1976 as influenced by 492 ppm B applied postharvest, 1975. 52

Table Page

12 Polyphenoloxidase activity and B content of immature 'Italian' prune fruit flesh, July 1976, after 492 ppm B applied postharvest, 1975. 60

13 Yield and fruit quality at harvest, 1976, as affected by fall foliar B applications. 61

EFFECT OF BORON SPRAYS ON FRUIT SET, FRUIT QUALITY, BORON DISTRIBUTION AND FLORAL

MORPHOLOGY OF 'ITALIAN' PRUNE (PRUNUS DOMESTICA L. )

INTRODUCTION

The 'Italian, ' or 'Fellenberg, ' prune (Prunus domestica L. ) is

a high-quality cultivar for drying, canning and fresh market. The

fact that it does not consistently produce a full crop of fruit is a major

defect of this cultivar. Erratic cropping of 'Italian' is not confined to

the Willamette Valley of Oregon; reports from Russia (160, 161) and

Italy (118) also indicate this tendency. Thompson and Liu (166) found

'Italian' to be self-compatible and not to benefit from supplemental

hand or bee pollination under Willamette Valley conditions. Fruit set

failure was related to temperature. When temperatures averaged

10 Cor below during the 3-week postbloom period pollen tube growth

was delayed and ovule breakdown began before fertilization could

stimulate fruit set (166).

In 1972, a year of generally poor cropping for 'Italian,' M.H.

Chaplin (personal communication) found that a fall foliar B application

resulted in 100% greater yield from trees which were not deficient

according to August mid-shoot leaf analysis, the standard index of

nutrition. Research which ensued had these objectives:

1. To reaffirm the increased yield following a fall foliar B applica-

tion of 'Italian' prune trees with adequate B nutrition and to

elucidate the basis of this yield increase by taking fruit set counts

during fruit development.

2. To determine optimum timing relative to harvest and optimum

concentration of the fall B spray.

3. To discover the effect of fall foliar B on factors which could

influence fruit set such as pollen tube growth in the style, pollen

germinability and date of bloom.

4. To determine B levels in buds, flowers and flower parts the

spring after B application and in leaves the following August.

5. To determine the effect of the fall B spray on the fruit itself.

This included B levels and polyphenoloxidase activity in imma-

ture fruit and harvest quality factors of mature fruit.

REVIEW OF LITERATURE

Fruit Set

Fruit set is a complex phenomenon which can be affected by a

wide range of cultural and climatic conditions (46, 112, 188). Tem-

perature appears to be a major factor limiting fruit set in 'Italian'

prune, as stated in the introduction to this thesis. While low tem-

peratures prolong ovule longevity (64, 65) as well as retard pollen

tube growth rate, the effect on pollen tube growth appears most

influential in determining fruit set in plum (58, 167).

Pollen tube growth rate varies with species and cultivar as well

as with temperature. Plum pollen tube growth rate was reported to

be slower than that of apple or pear (58, 158). Almond cultivars also

varied in pollen tube growth rate (75). In 'Bartlett' pear, pollen tubes

reached the ovary in 4 days at 10oC and in only 2 days at 15.5 C

(105). In 'Anjou' pear, 5 and 3 days respectively, were needed at the

above temperatures (114).

Fruit set can be reduced by abnormalities in pollen or ovule.

Irregularities in embryo sacs and their degeneration were responsible

for poor fruit set in apricot (66). Since ovule breakdown began at the

chalazal end near the vascular connection in peach, Harrold (81) pro-

posed that vascular disruption was responsible for fruit drop.

Competition between fruits or between fruits and shoots can

influence fruit set. Competition between flowers was proposed by-

Bradbury (24) to be a possible cause of fruit set reduction in sour

cherry. She found that degeneration of aborting flowers commenced

before pollination. Reducing competition by thinning clusters to one

flower increased percent fruit set in this study and in another study

with 'Cornice' pear (183). At the time of initial fruit set, when both

fruit and shoots are drawing on nutrients stored in the tree, competi-

tion between fruit and shoot can also be a determining factor in fruit

set (22, 137, 177).

Transfer of pollen from anther to stigma is essential for fruit

set in 'Italian, ' which does not normally set fruit parthenocarpically.

Thompson and Liu (166) found that 'Italian' prune, a self-fertile culti-

var, did not benefit from supplemental hand pollination or from the

presence of bees. This indicated that adequate pollen transfer occurs

by wind and by anthers touching the stigma. Style length was impor-

tant in positioning the stigma and anthers for efficient self-pollination

(76) and length of style could also determine the length of time neces-

sary for the pollen tube to travel from stigma to ovary to fertilize the

egg. The rootstock to which the cultivar was grafted had a marked

influence on style length in plum (76), peach and apricot (55).

Growth regulators, both endogenous and applied, influence fruit

set (45, 46). Although plums do not generally set fruit without

fertilization, parthenocarpy has been induced by gibberellin in plum

(89) as well as in peach (48, 49), almond and apricot (48). Both

gibberellin and auxin were required for parthenocarpy in sweet cherry

(47). Plant hormones may also influence fruit set by directing the

movement of assimilates within the plant. Assimilates will be

mobilized toward areas of high auxin concentration (22, 142, 177).

Competition for nutrients between developing fruits and shoots can

occur. Schneider (137) has proposed that a NAA spray to the entire

tree thins apples by creating vegetative sinks which compete with the

fruit for assimilates. Auxin sprays will also thin stone fruits.

Another synthetic auxin, 3-CPA (3-chlorophenoxy propionamide) is

used to thin peaches, plums and apricots (M.N. Westwood, personal

communication). It appears that the balance between growth regulators

such as gibberellin and auxin at each stage of fruit set is as important

as hormone levels per se. For example, while gibberellin is present

in stone fruits at bloom (112), auxin concentration is not detectable

until after about 2 weeks after bloom (46, 127).

A delay in the time of bloom can increase fruit set under cer-

tain conditions since chances of favorable temperatures for fruit set

increase as the season progresses. Boron levels may influence the

time of bloom in fruit and nut trees. Boron excess has been shown

to delay bud break in peach (38, 50, 83, 95, 186) and apple (80). In

Georgia, bloom date of pecans was advanced by B toxicity (147) but

the authors felt that this was probably due to the effect of injury on

rest. Growth regulators may also delay bud break. Ethephon (2-

chloroethyl phosphonic acid), applied the previous fall, was found to

delay bloom in sweet cherry (54, 130), sour cherry (31) and plum

(54). SADH (succinic acid 2, 2-dimethylhydrazide) delayed bloom of

'Delicious' apple (159) and azalea (93), while gibberellic acid retarded

bud break of peach (43).

Mineral nutrition, especially of the elements nitrogen and

boron, can affect fruit set. Williams (187) found that cell division in

the nucellus of the ovule continued later in the spring in apple flowers

which had received a summer soil nitrogen application. Also, fruit

set the next spring was increased when compared with trees which had

not received late summer N. Westwood et al. (182) also found N to

be important for fruit set of pear.

Boron is important for both flower development and initial fruit

or seed set. Boron is needed by ovules in vitro as well as by intact

flowers (16, 120). Higher levels of B in the nutrient solution were

needed for reproductive growth than for vegetative growth of cotton

(85). The B levels.of both the pollen and pistil were related to

seed-setting ability in clover. When no deficiencies existed, the B

content of the pollen-bearing plant appeared to be more important

than that of the pistil-bearing plant (115). In contrast, the B content

of only the female parent was important for seed set in another study

involving clover (15) and in rape (82). A decrease in seed or fruit set

is often one of the first signs of B deficiency. Batjer et al. ( 8 ) found

that an incipient B deficiency of pear caused by an inability of tree

roots to take up B from cold soil in early spring causes blossoms

to wilt and die, or "blast." Vegetative growth is normal during the

remainder of the season but set may be reduced on affected trees.

Boron for flower and fruit development can be provided by a

spray or soil application. Preharvest, postharvest and prebloom

sprays increased B levels in 'Bartlett' pear flower bud clusters (44).

Both fall-and spring-applied sprays were effective in correcting the

"blossom blast" condition of pear described above (8, 92) although fall

sprays were most consistent (92). Spring-applied B sprays increased

'Anjou1 pear fruit set in one study (9) but not in another (53). Fruit

set of 'Magness' pear was not increased by a spring spray of B (135).

Spring-applied B sprays increased fruit set of 'Stayman' apple (25)

but not of other cultivars (25, 86). Fruit and seed set increases

were also observed after prebloom B sprays on grape (123) and after

soil applications or the addition of B to the nutrient solution in many

crops (15, 82, 115,. 181, 184).

Boron excess, on the other hand, may or may not reduce fruit

set. Apricot trees with marked symptoms of B injury have been

observed to bear normal crops but prune trees, while they may flower

profusely, are sometimes reduced in set by excess B (63).

Boron appears to be important for flower development rather

than for initiation of flowers. No influence of B levels above the

deficiency level was seen on flower initiation in cotton (85).

Boron and Pollen

Boron has been reported to enhance in vitro pollen germination

in many plants. Stimulation by B is not universal among plant species,

however. Species such as pear with high endogenous pollen B levels

generally gave a greater response to B in the germination medium

than those such as pine with low levels (151). Boron had no effect

on or inhibited pollen of Capsicum annuum (170), Crotalaria juncea,

and Corchorus capsularis (174) while concentrations up to 1200 ppm

were optimal for other species (173). Boron was reported to improve

in vitro pollen germination in pea (100), onion (98), cherry (17, 165),

apple and apricot (165), trumpet vine (124), eggplant (170), chrysan-

themum (168), pear (17, 149, 165) and Seta via sphacelata (52).

Thompson and Batjer (165) reported a stimulation of plum pollen by

B while Blaha and Schmidt (17) found plum and prune pollen did not

respond to the addition of B to the germination medium. However,

only two prune pollen samples were examined by Thompson and

Batjer, and germination was very low both in the control (6%) and

when B was added to the germination medium (10%).

9

Conflicting reports of B requirements for pollen germination

may be reconciled when plant B supply is considered. According to

Vasil (169, 170) pollen is generally deficient in B. He states that

. . . a. relatively reliable bioassay technique. . .for B can be developed by employing the pollen germination test. Plants which are deficient in B do not show good germination of pollen but show considerably better germination after B has been supplied either through the roots or through cut ends of branches.

Pollen from B deficient pear trees was enhanced by the addition of B

to the germination medium but after 4 years of B fertilization it was

no longer necessary to add B to the medium (3). Likewise, response

of alsike clover (115) and Forsythia pollen (174) to the addition of B to

the germination medium was dependent on the plant's supply of B.

Boron sprays have also been reported to improve pollen germination.

Potted strawberry plants responded to boric acid applied to the flower

trusses by improved anther dehiscence and pollen germination (77).

Also, a foliar spray of B improved grape pollen germination (1Z3).

While endogenous or applied B appears necessary for optimum

pollen germination in many plants, an excess of B in the medium will

usually reduce pollen germinability. In most plants pollen germination

is enhanced by the addition of supra-optimal amounts of B to the

germination medium (171). Soil-induced B toxicity was reported to

reduce peach pollen germinability when compared with germination of

pollen from normal trees (95).

10

In contrast to the conflicting reports on the effect of B on pollen

germination, in nearly all plants studied pollen tube growth rate has

been accelerated by B in the germination medium. Visser (173)

stated that boric acid enhanced pollen tube growth rather than pollen

germination. Stanley and Loewus (152) agreed, adding that "pollen

tube membranes must be present for boron to exert a stimulatory

effect on the metabolism of glucose supplied exogenously to developing

pollen. "

The action of B in enhancing pollen tube growth may involve the

reaction of borate with compounds in the germination medium. Phe-

nolic compounds were shown to be deposited on the surface of the

pollen grain by the anther tapetum of Tulipa (162) and were found to

diffuse out of the pollen of several species into the germination medium

(150). Caffeic acid, a diphenol, inhibited pollen tube growth and

germination of several plant species (111). As B has been demon-

strated to form complexes with diphenols (192), it is possible that the

presence of B in the germination medium could prevent certain phe-

nolic compounds from inhibiting germination or pollen tube growth

in vitro.

Boron does not appear to influence pollen tube growth rate in the

style. The B content of neither the pollen nor the pistil was found to

influence the growth rate of clover pollen in the style (184).

11

Boron Levels and Plant Response

The standard technique of leaf analysis for determination of

plant mineral nutrition involves. sampling mid-shoot leaves in late

summer when levels of most elements fluctuate least. One of the first

workers to use mid-shoot leaves for B analysis was Burrell (34).

This practice arose out of convenience since these leaves were already

in use to index the nitrogen, magnesium and potassium status of fruit

trees. There is general agreement on the B levels desirable for opti-

mum growth in plum and prune trees. The Oregon State University

Fertilizer Guide for Prunes (153) states that 35-80 ppm B in mid-

shoot leaves is . optimal, below 25 ppm deficient and over 100 ppm

toxic. Leece (103) in Australia considers 25-60 ppm normal for plum

trees in midsummer with deficiency below 20 ppm. Hansen and

Proebsting (79) found that 'President' plum trees with 72-77 ppm B

in leaves showed injury. Leaf concentrations from 26-29 ppm were

found in both normal and deficient trees but below 25 ppm deficiency

symptoms were always found. Hernandez and Childers (83) found

toxicity symptoms in peach at levels of 100 ppm B or more.

It is difficult to specify B levels for optimum plant performance.

The greatest plant growth may occur at B concentrations which produce

mild toxicity symptoms on the leaves. Maximum plant response over-

lapped with onset of B toxicity symptoms when sunflower plants were

grown in nutrient solution (67). 'Valencia' orange trees suffered no

12

reduction in growth at B concentrations giving mild toxicity symptoms

(146). Optimum B concentration for reproductive growth may

generally be higher than for vegetative growth, as Montgomery (115)

found with alsike clover. Soybean growth was increased at B levels

producing leaf toxicity symptoms (42, 122). However, plant species

and cultivars vary widely in boron tolerance (68). Stone fruits are

less susceptible to boron toxicity than many other plants in that they

show fewer symptoms (179)- They also have an ability to divert B to

the stem and fruit (63).

Several studies have shown that the highest concentrations of B

occur in the reproductive organs. Boron accumulates in both male

and female tissues in apple (19), corn (21), lily (13), tobacco (14),

Qenothera (73), cherry, apple and pear (190, 191) and in buckwheat

(20). In Qenothera, the stigma contained higher B levels than the

style. In rye, more B was found in anthers and pollen than in female

tissues (104).

Seasonal variation in the B content of flower buds occurs.

Boron levels in buds change little during dormancy but as growth

begins in the spring B moves into the buds. At full bloom flowers

contained high concentrations of B (35, 74, 191). Pear, apple and

cherry fruit buds did not store sufficient B for their development but

drew on B from the soil or from reserves within the plant (191).

13

Mobility of Boron within the Plant

Many questions exist about the mobility of B within the plant

system. Boron has traditionally been grouped with the immobile

elements because deficiency symptoms first occur in the youngest

tissues (72). Under limited B supply most of the B was found in the

older, basal leaves of alfalfa (4, 56) and tomato (29). Boron did not

move out of senescing sugarcane leaves (116). Oertli and Richardson

(121) explained B immobility by a cyclic movement between leaf mar-

gin and leaf midrib and base, keeping B within the leaf. However,

some mobility of B within the plant has been reported. In broadbean

B was not normally transported to the radicle from cotyledons or the

epicotyl (117), but when cotyledons were provided with excess B it

was readily transported from this organ to the radicle (113). In

turnip (69), and in broccoli (12) B did not move out of the oldest

leaves of a plant but it was apparently translocated from younger but

mature leaves to apical tissues. Boron levels were similar in upper

and lower leaves in grapevines (140) and tobacco plants (176) deprived

of B. This would indicate redistribution within the plant. Stone

fruits are well known for their ability to transport B from the leaves

to the bark and then to the fruit under conditions of excess (62, 63).

When B was in limited supply the fruit exhibited B deficiency symp-

toms 'while the leaves contained adequate B (11). A loss of B from

14

the peach endocarp and a gain in flesh B occurred as fruits matured

(10).

Conflicting reports of B mobility can be more easily understood

when the solubility of the plant B is considered. Plants contain a

fixed amount of bound or insoluble B which remains constant under

conditions of varying B supply. There is little or no reutilization of

insoluble B. The soluble B content of plants, however, decreases as

B is withheld and drops to zero when the plants exhibit deficiency

symptoms (144). Differences in plant B solubility could be expected

among species and cultivars (Z, 113). Monocots, for example, have

a higher proportion of their B in soluble, or unbound form than dicots,

have a lower B requirement, and take longer to show B deficiency

symptoms (143). If the soluble B fraction, which is dependent on

plant B supply, is the only portion which is available for translocation,

conditions of plant B supply prior to an experiment as well as the

species or cultivar involved would have a considerable effect on

apparent B mobility.

Fruit Quality

Boron levels may affect fruit quality in various ways. Peaches

from trees suffering from B toxicity were insipid and flat in flavor (38,

186). Malformed peach fruits developed on trees given excess B

(83). Spring B sprays (25), as well as soil applications of borax (7),

15

reduced the storage life of apples. However, late summer B sprays

on 'Bartlett' pears had no effect on storage quality (44), nor did

spring sprays affect storage quality of 'Magness' pear (135). Foliar

sprays on 'Golden Delicious1 apple increased sugars and ascorbic

acid content (32). Boron application delayed maturity in 'Mclntosh'

(126) while 'Granny Smith1 apples remained green longer although

ripeness was not affected (30).

Boron sprays have also been useful in correcting some fruit

disorders. Spring B sprays reduced the incidence of bitter pit in

apple (60). Since calcium deficiency of the fruit has also been asso-

ciated with this disorder it is interesting to note Marsh's theory (108)

that B tends to keep Ca in an available condition. Boron sprays

applied in spring and summer did not decrease the amount of fruit

cracking, a disorder thought to be related to low calcium, in 'Italian'

prune (39)-

Internal browning of 'Italian1 prune is a complex physiological

disorder in which the fruit flesh near the pit breaks down and becomes

discolored (172). This disorder has shown variable response to B

applications. Spring B sprays reduced internal browning in one year

but not in another (172). A similar disorder of apricot was found to

be caused by B deficiency (33). Gibberellin sprays applied before

harvest to 'Early Italian' prune reduced internal browning (128, 129).

Summer applications of ethephon (2-chloroethyl phos phonic acid) and

16

SADH (succinic acid 2, 2-dimethylhydrazide) reduced browning in pro-

cessed peaches. Less discoloration of the pit cavity was found after

treatment with the above compounds alone or in combination (78).

Fresh or canned peaches from trees which had been treated with

SADH were found to be free from a benzaldehyde or "peach pit" odor

found in the control (141). SADH (36) and boron (101) have both been

proposed to direct glucose metabolism away from the pentose shunt,

from which phenolic acids arise.

Postulated Roles of Boron in the Plant

Many roles in plant metabolism have been proposed for B but

none have been established conclusively as its primary function.

Indeed, the ability of B to complex with hydroxyl-containing compounds

such as sugars, cellulose and polyphenols may allow it to enter into

many cellular associations. Briefly, some of the functions proposed

for B include carbohydrate translocation (70, 71, 72), starch-sugar

interconversions (59), cell wall metabolism (148), pectin synthesis

(189), plant osmotic balance (5), phosphate metabolism (133), calcium

metabolism (18, 94, 108), protein synthesis (175), synthesis of ATP

(132), RNA metabolism (90, 91), DNA metabolism (40), membrane

potentials (163), oxidative enzymes (107), metabolism of phenolic

compounds (1, 51, 101, 131), and regulation of auxin levels (41, 6l,

131, 145).

17

Since the developing flower bud is an area of active cell division

and growth, and since auxins may influence fruit set both alone and in

combination with other hormones, these aspects of B metabolism

will be considered.

Boron is extremely important for cell division and growth.

Several studies involving the early stages of B deficiency in roots

showed that cell division ceases at various intervals after B with-

drawal; in squash, 6.5 hours (40), in bean after 49 hours (185), and

in sunflower after 3 hours (97). In order to study DNA synthesis,

Cohen and Albert (40) examined tritiated thymidine uptake by squash

root meristems. Although mitotic figures were not seen after 6.5

hours without B, incorporation of label continued up to 20 hours.

When B was resupplied after B withdrawal, label uptake

resumed, indicating a temporary suspension of DNA synthesis during

the early period of deficiency.

Cessation of cell elongation is also an early feature of B

deficiency. Elongation stopped within 48 hours of B withdrawal in

Vicia faba (133) and after 6.5 hours in squash (40).

Boron appears to be involved in the metabolism of phenolic

compounds and, through these, auxin metabolism. The accumulation

of certain phenolic compounds in B deficiency has been well docu-

mented. Chlorogenic and caffeic acids accumulated in sunflower (51),

tomato (28), radish and lettuce (125); scopoletin and a glucose

18

derivative of gentisic acid in sunflower (178); and vanillic and ferulic

acids in oil palm (131).

Two explanations have been presented for the accumulation of

phenolic compounds in B deficiency. Rajaratnam and Lowry (131)

found a complete absence of one class of flavonoids, the leucoantho-

cyanins, in B-deficient tissues. They postulated that B was necessary

for leucoanthocyanin synthesis from phenolic precursors such as those

which accumulate in B deficiency. Gentisic acid is a simple phenolic

compound while the remainder of the above compounds represent

metabolic routes from cinnamic acid to compounds other than

flavonoids (134). Lee and Aronoff (101), on the other hand, presented

evidence for the role of B in partitioning glucose metabolism between

glycolysis and the pentose shunt. In B deficiency the pentose shunt is

favored and more phenolic acids are formed. Skol'nik (145) also felt

that fewer phenolic compounds were formed in the presence of B.

In 1968, Coke and Whittington (41) proposed that B deficiency

could be equivalent to a state of auxin toxicity. Boron deficiency

symptoms can be duplicated by auxin application (41, 97, 131, 133).

Auxin-like activity was high in extracts of 24-hour B deficient bean

root tips (41) and in extracts of primordia of oil-palm seedlings show-

ing little-leaf B deficiency symptoms (131).

Phenolic substances can be important in the regulation of IAA

oxidase (peroxidase) (138, 155, 157) and therefore in the control of

plant auxin levels (136, 164). In general, polyphenols, those with

19

ortho hydroxy groups, inhibit IAA oxidase while monophenols enhance

its activity (155). Chlorogenic and caffeic acids and scopoletin (87)

and ferulic and vanillic acids (138) inhibit IAA oxidase. Stonier et al.

(157) have isolated, "auxin protectors" of varying molecular weights.

These appear to be phenolic compounds which act as antioxidants

inhibiting the peroxidase-catalyzed oxidation of IAA. IAA was also

degraded in the presence of polyphenoloxidase and certain phenolic

substrates (27). The authors postulated that IAA was destroyed in a

reaction with intermediates of phenolic oxidation, since polyphenolox-

idase alone does not destroy IAA. Polyphenoloxidase was found to

eliminate "protector" activity (156).

In view of the abundance of phenolic compounds in Prunus

domestica (84) and the importance of auxin, both alone and in com-

bination with other hormones, for fruit set, this aspect of B meta-

bolism appears particularly relevant to a discussion of fruit set.

The remainder of the thesis is presented in the form of three

manuscripts, the first two to be submitted to J. Amer. Soc. Hort.

Sci, and the third to HortScience.

20

FRUIT SET OF 'ITALIAN' PRUNE AS AFFECTED BY SPRING AND FALL BORON SPRAYS1

Nancy W. Callan2

Department of Horticulture, Oregon State University Corvallis, Or. 97331

Additional index words. pollen, anthesis, Prunus domestica L.

Abstract. A fall foliar B application was found to increase

fruit set of 'Italian' prune (Prunus domestica L. ). A prebloom B

spray had no effect on fruit set. Neither fall nor spring applications

influenced the amount of fruit lost in the midsummer or "blue" drop.

All trees involved in the experiment had adequate B by the standard

index of tree nutrition, August mid-shoot leaf analysis. Incipient B

deficiency, in which cold, heavy soil prevents B uptake in early

spring, did not appear to be involved.

Several morphological and physiological effects of the fall B

spray were observed. Among these were a decrease in the size of

flower buds and mature flowers accompanied by reduction of style and

Received for publication . Published with the approval of the director of the Oregon State University Experiment Station as Journal Series No. . From a dissertation submitted by the senior author in partial fulfillment of the requirements for the PhD degree at Oregon State University.

2 Gratitude is expressed to Harold Bjornstad and Fred Dixon for technical assistance. We also thank the Oregon Processed Prune and Plum Growers Commission for financial assistance in this study.

21

pedicel length, a delay in the time of bud break and a decrease in

pollen germinability. B level of pistil and pollen had no effect on

in vivo pollen tube growth rate.

The postulated roles of B in phenolic metabolism and auxin

oxidation are discussed in relation to the observed effects of fall B.

Introduction

The 'Italian' prune is noted for its erratic bearing in the

Willamette Valley and in other areas of the world (118, 160, 161).

Low temperatures at anthesis and during the post-bloom period delay

fertilization and thus limit fruit set. Thompson and Liu (167) found

that when the average temperature for the 3 weeks immediately follow-

ing bloom was below 10 C a poor crop resulted.

In 1972, a year of generally poor cropping for 'Italian,' M.H.

Chaplin (personal communication) found that 'Italian' prune yields

could be increased by application of a fall foliar B spray. A crop of

115.5 kg fruit per tree was produced on trees which had received fall

B while adjacent untreated trees bore 57.6 kg per tree. These trees,

with 29 and 27 ppm B respectively, were not deficient in B by August

mid-shoot leaf analysis, the standard index of B nutrition for prunes.

According to the Oregon State University Fertilizer Guide for Prunes

22

(153), below 25 ppm B indicates deficiency while 35-80 ppm is con-

sidered optimal.

The purpose of this study was to confirm the yield increases

after fall foliar B application of trees which are apparently not

deficient in B and to compare the effects of a fall B spray on fruit set

with those of a prebloom B application. We wished to discover whether

the increased yields were due to a greater initial fruit set or to a

decrease in the amount of midsummer or "blue" drop. We also

wished to learn whether the B spray affected in vivo pollen tube growth

rate. During the course of the investigation, differences due to the

fall B spray in factors such as pollen germinability, flower size,

including style and pedicel length, and date of bloom were noted.

In the years of this study, .1975 and 1976, most 'Italian' prune

orchards in the Willamette Valley bore good crops of fruit. Unlike

1972, temperatures did not appear to be limiting fruit set. The

3-week average postbloom temperatures at the OSU experimental

orchard were 12.3 C in 1975 and 12 C in 1976.

Materials and Methods

Experiments were conducted at the Oregon State University

Experimental Orchard near Corvallis, Oregon (OSU) and in six com-

mercial orchards in the Willamette Valley. All orchards were

'Italian' prune on seedling peach (OSU and Orchards 1, 2, 3, 4 and

23

5) or myrobalan plum (Orchard 6) rootstocks. A range of nutritional

conditions was represented by the orchards selected. OSU and

orchards 1, 3, 4 and 6 had adequate levels of both B and N by August

mid-shoot leaf analysis, while orchard 2 was deficient in both B and

N and orchard 5 was deficient in N. Boron was applied as a com-

mercial product containing 20.5% B (78% Na Bo0...MH-O and 20%

Na B O *5H O). Three hundred ppm wetting agent (alkyl aryl poly-

oxyethylene glycol and free fatty acids in isopropanol) was added in

1976. Application was made with a hand sprayer and trees were

sprayed to the point of drip. Randomized block design was used with

eight replications in the OSU orchard and five replications in each

of the commercial orchards.

In 1975, B treatments at OSU included: (1) 4 92 ppm B (2 lb

product/lOO gal) 2 weeks after harvest and (2) 492 ppm B at first white

stage, when petals were first visible in the flower bud (22).

In 1976, timing of the fall B spray relative to harvest and con-

centration of the s pray were varied. As brown rot, Monilinia sp.,

may be a problem in the Willamette Valley, wettable sulfur was added

to one preharvest spray to determine compatibility of B and S when

applied together. Boron treatments at OSU in 1976 were: (1) 492 ppm

B 1 week before harvest, (2) 492 ppm B + 4798 ppm S 1 week before

harvest, (3) 492 ppm B 2 weeks after harvest, (4) 246 ppm B 2 weeks

24

after harvest, and (5) 123 ppm B 2 weeks after harvest. In the com-

mercial orchards 492 ppm B was applied within 2 weeks after harvest.

In each orchard approximately 8, 000-10, 000 flowers were

counted. Four to six limbs, each bearing approximately 300-400 flow-

ers, were selected on each tree. Blossom counts were made pre-

bloom. At OSU, initial set counts were made 4-5 and 7 weeks after

full bloom, and preharvest counts were made after the "blue" drop in

mid-August. In the commercial orchards, fruits were counted 5-7

weeks after bloom and after the "blue" drop. Fruit set data were

analyzed as a split plot, with dates at which set counts were made the

main plots and B treatments the subplots.

Nutritional status of all trees involved in the experiment was

determined by sampling mid-shoot leaves in August before B treat-

ment. Boron determinations were made on a 3/4-meter direct read-

ing spectrometer using the method of Chaplin and Dixon (37). Nitrogen

was measured using an automated Kjeldahl technique (139)-

To measure in vivo pollen tube growth, pollen from trees receiv-

ing 492 ppm B as a postharvest spray was placed on stigmas of control

flowers and of flowers from trees which had received the spray.

Likewise, control pollen was placed on both kinds of stigma. Pollen

was obtained from anthers removed in the orchard the previous day

and allowed to dehisce on glassine paper overnight. All pollen was

checked for germinability. Flowers to be used for pistils were picked

25

from the orchard in early morning just before petals unfolded.

Approximately 50 flowers per treatment were pollinated. Pollinated

flowers were placed on moist filter paper in petri dishes and incu-

o bated at 10 C for 2 days. This temperature was chosen because it

has been found to limit fruit set (166). Emas culation was unnecessary

because, due to the high humidity in the petri dish, anther dehiscence

did not occur. Pollen tubes were measured using Martin's (110)

fluorescence technique. Styles were frozen in the aniline blue stain

for later examination. Tubes fluoresced much more brightly when

styles were preserved in this manner than when a fixative or soften-

ing solution was used. After freezing a softening solution is not

needed. Styles were squashed in stain under plastic coverslips and

the length of the longest tube recorded. A completely randomized

design was employed in data analysis.

o In vitro pollen germination was also tested at 10 C. Large

populations of pollen were used to eliminate differences due to the

"population effect, " a phenomenon whereby large numbers of pollen

stimulate germination (26). Pollen was allowed to germinate in hang-

ing drops of 10% sucrose with and without 10 ppm boric acid. One

hundred to 500 pollen grains for each of six replications were counted.

Data were analyzed as a split plot, with B treatments the main plots

and B in the germination medium subplots.

26

Styles and pedicels were measured for 25 full bloom flowers

from each of five trees which had received 492 ppm B as a post-

harvest spray and from each of five control trees. The OSU orchard

and orchard 6 were sampled. Randomized block design was used for

data analysis. Average bud or flower size was obtained from dry

weight measurements of samples of 200 buds or flowers collected at

various stages of floral development. Flowers and buds of uniform

external stage of development were taken at each sampling date.

Treatments were replicated by location and data analyzed by t-test.

Results and Discussion

In 1975 initial fruit set at the OSU orchard was increased after

a fall B application (Table 1). These initial set increases following the

fall spray carried through to harvest. There was no significant dif-

ference due to treatment in the amount of fruit lost in the midsummer

or "blue" drop. A spring spray had no effect on initial, blue drop, or

final fruit set.

In 1976 initial fruit set at the OSU orchard was increased by all

preharvest B sprays and by all concentrations of postharvest sprays

applied (Table 2). The addition of wettable sulfur to a preharvest

spray for brown rot control did not interfere with the effect of B on

fruit set. Initial set increases were again sustained through the

season and, as in 1975, "blue" drop was not influenced by B treatment.

27

Four of the six commercial orchards also responded to the fall

B spray with increased fruit set in 1976 (Table 3). Of these, orchards

2 and 5, which were deficient in N, experienced very low fruit set.

This would be expected due to the need for N for fruit set (182, 187).

The other two orchards in which fruit set was increased after fall B,

orchards 1 and 3, had sufficient levels of both B andN. Fruit set

after fall B in these orchards was very good. Orchards 4 and 6,

which also had adequate B and N levels, had ample fruit set regard-

less of treatment. Apparently there were no limiting factors for

optimum fruit set in these orchards.

The presence of green leaves in the fall at the time of spraying

appears to be essential in 'Italian' for maximum effectiveness of B

application. Spring sprays have been found effective in correcting B

deficiencies of pear (8, 92) but even here a fall spray often proved the

most consistent (92). However, the increased fruit set in 'Italian'

prune in this study does not appear to be the result of correcting an

incipient B deficiency as in the "blossom blast" condition of pear. In

the latter case cold, heavy soil suppresses B uptake in the spring

and deprived flowers "blast" or shrivel and die. Control trees in this

study showed no signs of blossom dieback and at the OSU orchard had

adequate fruit set though lower than sprayed trees. Furthermore,

we would expect low flower bud B levels in control trees suffering

from an incipient B deficiency (8). Control flower bud levels were no

28

lower in orchards which responded to the fall B spray with increased

set than in orchards which had good fruit set regardless of B treat-

ment (data to be presented in subsequent paper).

The observed increase in fruit set after fall B does not appear

to be the result of accelerated pollen tube growth rate. Pollen tube

growth in 'Italian' prune styles at 10 C was unaffected by either the

B nutrition of the pollen-bearing plant or of the pistil-bearing plant.

This is in agreement with a report by Whittington (184), who found

that parental B level did not influence in vivo pollen tube growth rate

in red clover.

Pollen from trees which had received the 492 ppm B posthar-

vest spray was poorer in germinability than pollen from untreated

trees (Table 4). No difference was seen in germinability due to the

addition of 10 ppm boric acid to the germination medium. Thompson

and Batjer (165) reported a stimulation of plum pollen germination by

the addition of B to the medium, while Blaha and Schmidt (17) found

no such response in plum and prune pollen. Conflicting reports of B

requirements for pollen germination may be reconciled when plant B

supply is considered. Several authors have found that germination of

pollen from plants low in B is enhanced by the addition of B to the

medium but when the plants have adequate B there is no longer a

benefit from this addition (3, 115, 174). In contrast, soil-induced B

toxicity in peach trees was reported to reduce pollen germination

29

when compared with pollen from normal trees (95). In this study of

'Italian' prune, pollen germinability was also decreased but this was

apparently not sufficient to offset the beneficial effects of fall B on

fruit set.

Time of bloom was also affected by fall foliar B. On treated

trees in the orchard at OSU, bloom was delayed 3-4 days in 1975 and

1-2 days in 1976. When branches were brought into the laboratory in

early March and forced at 20 C, flowers on branches given all fall B

treatments opened 1 day after the control. When flower buds and

flowers at comparable stages of external development were compared,

buds from trees receiving fall B applications were found to be smaller

on a dry weight basis than those from control trees (Table 5). This

is reflected in a decrease in style and pedicel length of mature

flowers (Table 6). The effect on floral morphology appears to be

general. The actual amount of decrease in style length, though

statistically significant, amounts to only about 3% of the total style

length. It is doubtful that this alone would be responsible for increas-

ing initial fruit set; that is, by shortening the length of time from

pollination to fertilization. In addition, in both 1975 and 1976

temperatures were not low enough to limit fruit set because of slow

pollen tube growth. Style length as influenced by rootstock was found

to be important in 'Italian' prune fruit set in Poland (76). The authors

proposed that shortened styles positioned anthers and stigmas for

30

more effective self-pollination. Thompson and Liu (166) found no

increase in fruit set with supplemental pollination in orchard 6 and

in five other 'Italian' prune orchards in the Willamette Valley, indi-

cating that pollen transfer is not a limiting factor.

The observed morphological and developmental effects induced

by high B levels reflect alterations in physiology which probably in-

clude hormonal balances. One of the postulated roles of B in the plant

is regulation of auxin levels. Coke and Whittington (41) and others

(131, 145) proposed that B deficiency is actually a state of auxin

toxicity. Auxin-like activity was shown to be high in extracts of

B-deficient plant materials, and B deficiency symptoms could be

duplicated by applying auxins (41, 97, 131, 133). One of the means by

which auxin levels may be controlled is the IAA oxidase (peroxidase)

system (136, 138, 157, 164). Several of the polyphenolic compounds

which accumulate in B deficiency in sunflower such as chlorogenic and

caffeic acids (51) will inhibit IAA oxidase (87). Chlorogenic acid is

one of the most abundant phenolics in Prunus domestica (84). By

controlling the synthesis (101, 145) or metabolism (131) of phenolic

compounds which will inhibit IAA oxidase, B may decrease auxin

levels in plant tissue.

Although the mechanism of the fruit set increase following fall

B is not known, balance between auxin and other hormones such as

gibberellins and cytokinins at specific stages in fruit set may be

31

involved (46, 88, 112). Stone fruits do not readily set fruit without

fertilization, but gibberellin induced parthenocarpy in plum (89).

peach (48, 49), almond and apricot (48). Both gibberellin and auxin

were required for parthenocarpic fruit set in 'Bing' cherry (47). An

alteration of auxin balance between shoot and fruit could also influence

fruit set. Boron accumulates in both leaf and flower buds after a fall

foliar B application (data not presented), so a decrease of auxin

levels might be expected to occur in developing leaves as well as

fruits. At the time of initial fruit set both developing shoots and

fruits are metabolic sinks for assimilates stored in the tree. Move-

ment of assimilates appears to be directed toward areas of high auxin

concentration (22, 142, 177). A decrease in leaf auxin levels could

cause leaves to be less competitive with flowers for available nutrients

and allow more nutrients to be directed to the flower, thereby increas-

ing fruit set. Schneider (137) has proposed that naphthaleneacetic acid

(NAA), when applied to the entire tree after bloom, thins apples by

creating vegetative sinks which compete with the fruit for nutrients.

Another synthetic auxin, 2-(3 chlorophenoxy)-propionamide (3-CPA), is

effective as a post-bloom thinner for peach, plum and apricot (M.N.

Westwood, personal communication).

A role for auxin can also be envisioned in some of the other

observed responses to fall B. It is not known whether style and pedi-

cel length is reduced due to a decrease in cell elongation or cell

32

division, but higher auxin levels in the controls could conceivably

cause styles and pedicels to be longer through stimulation of cell

elongation. Also, higher auxin levels in control trees could lead to

an advance in the time of bud break by inducing renewed cambial

activity (99). Interaction between auxin and other hormones such as

ethylene or cytokinin is also possible. Measurement of auxin and

other hormone levels in developing flowers and leaves after a fall B

spray would aid greatly in elucidating the physiological basis of the

observed fruit set increase, and would also contribute to our under-

standing of the role of B in the plant.

33

Table 1. Fruit set of 'Italian' prune, 1975, following postharvest and prebloom B sprays. Oregon State University Experimental Orchard, Corvallis, Or.

% Fruit set Treatment _, , A„„l „ , „ ,_„ ,_ , A ^„ Treatment

4 wk AFB 7 wk AFB 17 wk AFB means

Control 26 21 11 19

492 ppm B postharvest 1974 35** 31** 17** 27**

492 ppm B prebloom 1975 28 20 10 19

AFB = after full bloom.

Significantly different from control at 1% level.

34

Table 2. Fruit set of 'Italian' prune, 1976, following fall foliar B sprays. Oregon State University Experimental Orchard, Corvallis, Or.

Treatment % Fruit set

„• , A^„i _ , A ^„ ,„ , . „„ Treatment SwkAFB1 7 wk AFB 17 wk AFB means

Control 23 22 18

492 ppm B preharvest 32** 31* 13** 25**

492 ppm B + 4798 ppm S preharvest 33** 31** 12** 25**

492 ppm B postharvest 35* 32** 15** 27*=:

246 ppm B postharvest 30 = 2 9" 13" 24**

123 ppm B postharvest 32** 30** 12** 25**

1 AFB = after full bloom.

** Significantly different from control at 1% level.

35

Table 3. Fruit set of 'Italian' prune in six orchards in 1976 following 492 ppm B applied postharvest.

Treatment Leaf B, ppm

Aug 1975

% Fruil t set Orchard

Initial F 'inal Treatment

means

1 Control 32 6 4 5 Postharvest B 16** Q** 13**

2 Control 21 5 2 4 Postharvest B 6* 3* 5*

3 Control 29 12 8 10 Postharvest B 19** 13** 16**

4 Control 38 39 23 31 Postharvest B 44 21 33

5 Control 41 7 3 5 Postharvest B 11* 5* Q -v

6 Control 37 36 11 23 Postharvest B 31 12 22

Significant at 5% level.


36

Table 4. Ge rminability of 'Italian' prune pollen in deionized water with 10% sucrose following 492 ppm B postharvest. Oregon State University Experimental Orchard, Corvallis, Or.

Treatment of Germination « ^ 1 % Germination

pollen source medium

Control no added B 69

Control 10 ppm B 65

Postharvest B no added B 42

Postharvest B 10 ppm B 37

1 Effect of pollen source significant at 5% level. Effect of B in germination medium not significant.

37

Table 5. Dry weight (mg/bud) of flower buds from seven orchards collected at various stages of bud development after 492 ppm B applied postharvest.

Orchard

1

2

3

4

5

6

OSU

T rea tment Control Pos tharvest B

6.2 D . 1 ^ '^

6.7 6.3

7.0 6.3*

7.6 6.2* *

10.9 10.3*

13.4 11.5 **

10.3 9.5**

**



38

Table 6. Style and pedicel length of 'Italian' prune in two orchards after 492 ppm B postharvest.

Orchard Treatment Style, mm Pedicel, mm

OSU Control 11.7

Postharvest B 11.3**

Control 11.2 15.4

Postharvest B 10.9** 12.8**

r for style and pedicel = 0.4152.


39

BORON DISTRIBUTION IN 'ITALIAN' PRUNE TISSUES FOLLOWING SPRING AND FALL BORON SPRAYS1

Nancy W. Callan2

Department of Horticulture, Oregon State University Corvallis, Or. 97331

Additional index words. Prunus domestica L., flowers, flower buds, anther, ovary, style

Abstract. Boron levels in 'Italian' prune (Prunus domestica L. )

tissues following a fall foliar B application and a prebloom B applica-

tion were compared. Fall foliar B increased B levels in dormant bud

and spur tissues and in flower buds and flowers. A prebloom B spray

increased B levels of floral tissues to a lesser degree. The highest

B concentration was found in the ovary. Boron concentration of flow-

er buds in April following a fall B spray was as much as five times

the amount ordinarily found in mid-shoot leaves in August. August

mid-shoot leaf analysis revealed higher levels in leaves from trees

treated the previous fall in one year of the study but not in the other

year. In fall-treated plants, B appeared to be uniformly distributed



40

in leaves from spur, shoot tip, mid-shoot and shoot base. In control

plants, B was higher in mid-shoot leaves than in leaves from spur,

shoot tip and shoot base. The suggestion is made that while August

mid-shoot leaf analysis is a useful indicator of the vegetative nutri-

tion of 'Italian' prune trees, analysis of bud or flower tissue might be

a better index of the B needs of the tree for reproductive growth.

Introduction

A fall foliar B application has been found to increase initial

fruit set of 'Italian' prune trees which were not B deficient by

August mid-shoot leaf analysis, the standard index of B nutrition.

Several morphological and physiological changes such as a decrease

in flower bud size, a delay in bloom and a decrease in pollen germin-

ability accompanied the set increase (Callan, first chapter of thesis).

The purpose of this study was to determine the distribution in

the plant of the applied B by analyzing B levels in various plant

tissues. Of special interest was the B concentration of floral tissues,

as these tissues often contain some of the highest B levels found in

plants (21, 191). The effect of B sprays on leaf levels the following

August was also of interest.


Experiments were conducted at the Oregon State University

41

Experimental Orchard near Corvallis, Oregon (OSU) and in six com-

mercial orchards in the Willamette Valley. All orchards were

'Italian' prune on seedling peach (OSU and orchards 1, 2, 3, 4 and 5)

or myrobalan plum (orchard 6) rootstocks. A range of nutritional

conditions was represented by the orchards selected. OSU and

orchards 1, 3, 4 and 6 had adequate levels of both B and N by August

mid-shoot leaf analysis, while orchard 2 was deficient in both B and

N and orchard 5 was deficient in N. Boron was applied as a com-

mercial product containing 20.5% B (78% Na Bo0..^H-O and 20% 2 8 13 2

Na B O *5H O). Three hundred ppm wetting agent (alkyl aryl

polyoxyethylene glycol and free fatty acids in isopropanol) was added

in 1976. Application was made with a hand sprayer and trees were

sprayed to the point of drip. Randomized block design was used with

eight replications in the OSU orchard and five replications in each of

the commercial orchards.

In 1975, B treatments at OSU included: (1) 492 ppm B (2 lb

product/100 gal) 2 weeks after harvest and (2) 492 ppm B at first

white stage, when petals were first visible in the flower bud (22).

In 1976, timing of the fall B spray relative to harvest and con-

centration of the spray were varied. As brown rot, Monilinia sp.,

may be a problem in the Willamette Valley, wettable sulfur was added

to one preharvest spray to determine compatibility of B and S when

applied together. Boron treatments at OSU in 1976 were: (1) 492 ppm

42

B 1 week before harvest, (2) 492 ppm B + 4798 ppm S 1 week before

harvest, (3) 492 ppm B 2 weeks after harvest, (4) 246 ppm B 2 weeks

after harvest, and (5) 123 ppm B 2 weeks after harvest. In the com-

mercial orchards 492 ppm B was applied within 2 weeks after harvest.

Boron determinations were made on a 3/4-meter direct reading

spectrometer using the method of Chaplin and Dixon (37). In 1975,

at OSU, eight replicates of 40 flowers each were sampled at fall bloom

and at petal fall. Anthers, ovaries and styles were analyzed sepa-

rately. Data from each sampling date were analyzed separately as

split plots, with flower parts the main plots and B treatments the

subplots. Short spurs (less than 2 cm) with 4-7 buds attached were

sampled in all orchards in Feb 1976. Ten spurs were collected

from each tree and samples composited for analysis. At OSU,

flower buds were collected at the tip green stage at which time

florets become visible within the bud scales, at the first white stage

when sepals separate and at full bloom (22). In the commercial

orchards, flower buds were collected at various stages before bloom.

Sixteen buds or flowers were collected from each tree and samples

composited for each, treatment. Bud weight was calculated from the

dry weight of the composite sample. Mid-shoot leaves were sampled

in August of 1975 and 1976. At OSU, 20-30 mid-shoot leaves were

taken per tree from 16 replications in 1975 and eight replications in

1976. In 1976 the two basal leaves on the shoot, the terminal shoot

43

leaf and two spur leaves were sampled from trees which had received

492 ppm B postharvest and from control trees in four replications.

Data were analyzed as a split plot, with B treatments the main plots

and leaf location the subplots.


Boron levels of dormant bud and spur tissues were greatly-

increased by fall foliar B sprays (Tables 7 and 8). When different

concentrations of B were applied, the amount of B found in bud and

spur tissue was roughtly proportional to the amount applied. At OSU,

applied before or after harvest, the highest concentration spray,

492 ppm B, approximately doubled the B concentration of these

tissues (Table 8). In the commercial orchards (Table 7), the 492

ppm B postharvest spray increased bud and spur B 50% to 200% over

the control. Boron concentration was higher in the bud than in the

spur. In control trees at OSU after leaf fall, buds contained 55 ppm B

and spur tissue 30 ppm B. At this time trees which had received fall

B as a 492 ppm postharvest spray contained 187 ppm B in buds and

95 ppm B in spurs. Differences due to tissue and to treatment are

significant to the 1% level. Since B appears to increase proportion-

ately in bud and spur tissues after fall B, combined bud and spur

tissue analysis should be a good indicator of the B status of dormant

tissue.

44

With the approach of anthesis B apparently moves into the flow-

er buds from subtending tissues. Little change occurred in B con-

centration of the buds despite the considerable increase in dry

weight which takes place between the green tip stage and full bloom

(Table 8). This is in agreement with reports by Woodbridge et al.

(191) who found that pear, apple and cherry buds did not store suffi-

cient B for their development but drew on B from the soil or from

reserves within the tree.

Boron levels in flower buds were increased considerably by the

fall B sprays, especially those of higher concentration. In each

orchard, after fall B treatment, the amount of increase in flower bud

B was approximately the same as that seen in bud and spur tissue.

Boron levels in anther, ovary and style, measured at full

bloom and at petal fall, were found to be higher after a fall or a

spring B spray than in the control (Table 9). A fall spray increased

B levels in the flower parts to a greater extent than did a prebloom

spray. Boron concentration of the ovary was higher than that of the

anther or of the style.

Young shoot B levels at the time of full bloom were also

increased by the fall B spray. Developing shoots 4-5 cm long from

orchard 6 had 44 ppm B in the control buds and 94 ppm after fall B.

Much of this B may be utilized in current season growth, however, as

B levels of mid-shoot leaves in August were increased by fall B in

45

only one of the two years studied (Table 10). In 1975 the amount of

increase in mid-shoot leaf B concentration after fall B was 6 ppm.

In 1976 none of the fall B applications tested increased mid-shoot

leaf B over that of the control. However, differences in B levels

due to the fall B spray were found in leaves from locations other

than mid-shoot, i.e., from spur, shoot base and shoot tip (Table 11).

In fall B-treated plants, B appeared to be uniformly distributed in

leaves from spur, shoot tip and shoot base (Table 11). In control

plants, B was higher in mid-shoot leaves than in leaves from spur,

shoot tip and shoot base. The cause of this is not known. Perhaps

the lower values of spur and shoot base leaves in control trees is a

result of B being drawn from them to the fruit. However, Leece

(10Z) found no difference in B content of leaves from fruiting and non-

fruiting wood in peach. It may also be that under more limited B

supply, as in the control trees, B content of leaves may reflect B

availability at the time they are developing. Spur and shoot base

leaves develop while the soil is still cold in early spring. Boron

uptake may have been hampered at this time and only trees which

had received fall B contained a large reserve of B readily available

for bud growth.

Preliminary studies show that B in the fall sprays is readily

taken up by the leaf and translocated to the bud and subtending tis-

sues. Boron is taken up quickly and passively into the intercellular

46

spaces in roots (23) and it is possible that this mechanism of uptake

can function in the leaf. Once in the leaf, B can move in both xylem

and phloem (121). Boron may be transported out of the leaf to the

bud and woody tissues in a complex with sugars or other polyhydroxy

compounds (70). The B, applied as a spray, apparently becomes

part of the "free" or "unbound" B pool. Shive (143) found that this

fraction of the plant B varied with B supply, dropping to zero as

deficiency symptoms arose. The amount of "bound" B in the plant

remained relatively constant. Mobility of B in stone fruits is well

known. Under conditions of excess B stone fruits will transport B

from the leaves to the bark and also to the fruit (63).

Fall B sprays are a means to achieve high bud and flower B

concentrations which stimulate fruit set in the spring. Due to the

dilution effect with growth, vegetative injury which would result from

high B levels later in the season is avoided. It is interesting to note

that, while response to fall foliar B could hardly be considered

toxicity because of the fruit set increase, two of the features of the

fall B response, a delay in bloom date and a decrease in pollen

germinability (Callan, first chapter of thesis), are characteristic of

B toxicity in peaches (38, 90, 186).

August mid-shoot leaf analysis is the standard index of 'Italian'

prune B nutrition. It indicates the B level, for best growth, perform-

ance and general tree health. However, these experiments and

47

studies by Montgomery (115) with alsike clover and Holley and Dulin

(85) with cotton indicate that optimum B levels for reproduction are

higher than those for vegetative growth. It may be possible to define

a B level for optimum reproductive growth, perhaps by utilizing

dormant bud and spur tissue or floral tissue as indicators.

48

Table 7. Boron (ppm) in 'Italian' prune dormant bud and spur tissue and in flower buds in six orchards after 492 ppm B postharvest.

Orchard Treatment Bud and spur Bud, first white

Jan 1976 April 1976

Control 26 71 Postharvest B 66 166


Control 22 6l Postharvest B 45 150


Control 47 121 PostharvestB 68 171


49

Table 8. Boron (ppm) in 'Italian' prune dormant bud and spur tissues and in floral tissues in two stages of bud development and full bloom, 1976. Oregon State University Experimental Orchard, Corvallis, Or.

Bud and spur, Flower Treatment , Green First

Jan . , . Full bloom tip white

Control 37 100 88 87

492 ppm B preharvest 66 175 172 165

492 ppm B + 4798 ppm S preharvest 76 168 175 170

4 92 ppm B postharvest 66 205 185 186

246 ppm B postharvest 62 149 131 153

123 ppm B postharvest 49 118 105 138

Table 9. Boron (ppm) in 'Italian' prune flower parts at full bloom and at petal fall, 1975, as affected by 492 ppm B applied postharvest 1974 and prebloom 1975.

Full bloom Petal fall

Treatment Flower part Flower part

... _ _. . Treatment Treatment Anther Ovary Style Ovary Style means means

Control 47 59 52 53 64 50 57

Postharvest B 120 171 133 141 138 89 113

Prebloom B 63 98 79 80 90 69 80

Flower part means 77 109 88 97 69

Full bloom: LSD for flower part: 5% = 9, 1% = 12. LSD for B treatment: 5% = 11, 1% = 15.

Petal fall: LSD for flower part: 5% = 10, 1% = 14. LSD for B treatment: 5% = 18, 1% = 24.

o

51

Table 10. Boron (ppm) in 'Italian' prune mid-shoot leaves in August as affected by B application. Oregon State University Experimental Orchard, Corvallis, Or.

Treatment 1975 1976

2 3 Control 36 35

492 ppm B prebloom 38

492 ppm B preharvest 37

492 ppm B + 4798 ppm S preharvest 38

492 ppm B postharvest 42** 33

246 ppm B postharvest 31

123 ppm B postharvest 33

Data supplied by Dr. M.H. Chaplin, Horticulture Dept., OSU. 2

Mean of 16 replications. 3 Mean of 8 replications.

** Significantly different from control at 1% level.

52

Table 11. Boron (ppm) in 'Italian' prune leaves from four locations on the tree in August 1976 as influenced by 492 ppm B applied postharvest, 1975. Oregon State University Experimental Orchard, Corvallis, Or.

r . L. Treatment Leaf location

Control Postharvest B

Spur 28 33

Shoot base 27 34

Mid-shoot 36 34

Shoot tip 25 31

Mean 29 33

Treatment effects not significant.

LSD for leaf location within a given B treatment: 5% = 3, 1% = 4.

53

THE EFFECT OF FALL FOLIAR BORON APPLICATION ON POLYPHENOLOXIDASE ACTIVITY AND INTERNAL BROWNING IN 'ITALIAN' PRUNE FRUIT1

2 Nancy W. Callan Department of Horticulture, Oregon State University Corvallis, Or. 97331

Additional index words. Prunus domestica L.

Abstract. Polyphenoloxidase activity of acetone powders prepared

from immature 'Italian' prune (Prunus domestica L. ) fruit was higher

in fruits from trees treated with fall foliar B the preceding year than

in fruit from control trees. This was in contrast to the observation

that green fruits from fall B-treated trees browned more slowly when

cut than did controls. Further, after fall B there was a decrease in

the amount of internal browning, a physiological disorder involving

breakdown and darkening of the fruit flesh next to the stone. The

role of B in phenolic metabolism is discussed in relation to both of

these observations.



54

Introduction

A fall foliar B application has been found to influence reproduc-

tive development of 'Italian' prune. Initial fruit set was increased by

fall B (Callan, first chapter of thesis). High levels of B were found

in buds and flowers the spring following a fall B spray (Callan, second

chapter of thesis).

Boron also appears to influence browning reactions. When flesh

of immature prune fruits was prepared for B analysis, the cut sur-

faces of fruit from trees which had received fall foliar B did not brown

as rapidly as those of fruit from control trees. Polyphenoloxidase

(PPO) is active in promoting browning in many fruits. Dilley (57)

states that "browning is the result of phenol oxidation and eventual

non-enzymatic polymerization of the quinones formed into tannins or

'melanins. ' " Polyphenoloxidase has been found to increase during B

deficiency in sunflower (107), tomato (96), and potato (154). In

addition, borate has been shown to be a competitive inhibitor of PPO

(180). Therefore, I wished to determine if a correlation existed

between the B content of immature prune fruit and its PPO activity.

I also wished to determine the effect of fall B on fruit quality, in

particular the complex physiological disorder called inte rnal brown-

ing. This disorder involves breakdown of the flesh and a darkening

next to the pit in mature fruit (172). A similar disorder of apricots

was caused by B deficiency (33), but attempts to prevent internal

55

browning in prunes by the use of B sprays have given variable

results (172).


Experiments were carried out at the Oregon State University

Experimental Orchard near Corvallis, Oregon. The orchard is

'Italian' prune on seedling peach rootstock. Boron was applied as a

commercial product containing 20.5% B (78% Na B00 _*4H_0 and 2 o 13 2

20% Na Bo0 -SH O). Three hundred ppm wetting agent (alkyl aryl 2 o 7 2

polyoxyethylene glycol and free fatty acids in isopropanol) was added.

Application was made with a hand sprayer and trees were sprayed to

the point of drip. Randomized block design with eight replications

was used. As brown fruit rot, Monilinia sp., may be a problem in the

Willamette Valley, wettable sulfur was added to one preharvest spray

to determine compatibility of B and S when applied together. Boron

treatments were: (1) 492 ppm B (2 lb product/100 gal) 1 week before

harvest, 1975 and again in 1976, (2) 492 ppm B + 4798 ppm S (4 lb /

100 gal) 1 week before harvest, 1975, and 984 ppm B 1 week before

harvest, 1976, (3) 492 ppm B 2 weeks after harvest, 1975, (4) 246

ppm B 2 weeks after harvest, 1975, and (5) 123 ppm B 2 weeks after

harvest, 1975i

Flesh of immature prune fruits from control trees and from

trees which had received a spray of 492 ppm B 2 weeks after harvest

56

were analyzed for B content and PPO activity on July 5, 1976, just

after the midsummer or "blue" drop. Boron determinations were

made on a 3/4-meter direct reading spectrometer using the method

of Chaplin and Dixon (37). Twenty fruits from each of seven trees

of each treatment we re sampled for B content. Acetone powders

were prepared from 20 fruits from each of two trees receiving each

treatment. Fruit flesh was homogenized with 100 ml cold acetone,

filtered three times with 100 ml cold acetone and the residue air-

dried overnight. To assay enzyme activity, 0.1 g acetone powder was

mixed with 10 ml O.OZ M pH 6.9 phosphate buffer and allowed to stand

10 min before filtering. Two ml filtrate and 1 ml 0. 1 M catechol

were mixed rapidly in a spectrophotometer cell and the OD at 240 nm

recorded after 1/4, 1, 2, 3, 4 and 5 min. A standard containing only

buffer and catechol was used. Average change in OD/min was cal-

culated. This method is a modification of that described by Zocca

and Ryugo (193). All acetone powder preparations were run in dupli-

cate and the experiment was performed twice. Soluble N content of

acetone powder filtrate was calculated in triplicate using Kjeldahl

analysis (139) of freeze-dried samples. The soluble protein was

then estimated by dividing by 6.25%. The amount of soluble N in the

filtrate did not differ due to B treatment. Average change in OD/min/

mg soluble protein was calculated. Data were analyzed as a split plot,

57

with B treatments the main plots and the individual acetone powder

preparations the subplots.

At harvest in 1976, eight replicates of 25 fruit per sample were

weighed and examined for fruit quality. Fruits were sliced length-

wise and transversely and internal browning was recorded. To meas-

ure firmness, a Magness-Taylor pressure tester with an 8 mm

(5 /l6 in) head was used on a peeled cheek of each fruit. To measure

color, fruits were separated into two groups and recorded as red or

purple fruit. Percent soluble solids was measured with a hand

refractometer. Two drops of juice from the stylar end of 10 prunes

were composited for each reading.


Boron content of immature fruit flesh was increased by a fall

foliar B application. Fruit from B-treated trees was higher in B

than fruit from control trees (Table 12). Acetone powders from fall B

fruits also contained more B than those from control trees. Although

the flesh of fruits from B-treated trees appeared to brown less and

acetone powders were lighter in color than those of controls, PPO

activity in acetone powders prepared from these fruits was greater.

The observed difference in PPO activity may not be due to an

increase in the amount of PPO as a result of fall foliar B but rather to

a decrease in phenolic compounds. These are the substrate of PPO

58

and they polymerize to form the pigments in browning of cut surfaces.

Prunus domestica has been reported to be high in phenolics (84).

These compounds are known to interact with proteins and can cause

much difficulty in the isolation of active enzymes (106). Phenolic

compounds accumulate in B-deficient plants (Z8, 51, 125, 131, 178).

B has been reported to shift plant metabolism away from the pro-

duction of phenolic acids (101). It also may be involved with the

synthesis of flavonoids from their phenolic precursors (131). There-

fore, a reduction in the amount of phenolics could be expected with an

abundant supply of B. The lower PPO activity in acetone powders of

control fruit may have been due to a polymerization and binding of the

more abundant phenolic compounds to the enzyme during acetone pow-

der preparation. An inverse relationship between acetone powder

PPO activity and substrate levels exists in apple. As 'Golden

Delicious1 apples mature, activity of acetone powders increased as

the amount of phenolic substrate in apple flesh decreased (193).

The reduction in the incidence of internal browning of mature

fruit after fall B likewise may reflect a decrease in the amount of

substrate available for browning reactions. Internal browning of

mature fruit was decreased by all concentrations of fall foliar B

tested, whether applied before or after harvest the preceding year.

Trees receiving preharvest B sprays in 1975 were sprayed again

before harvest in 1976. However, as less browning was seen in fruit

59

from trees which had received only a single postharvest spray, it

appears that B application the previous fall was responsible for the

observed reduction in internal browning. Yield and/or fruit size was

increased by some concentrations and timings of fall B but color,

soluble solids and firmness were not affected by B treatment (Table

13).

Additional research should be done to determine whether the

observed differences in PPO activity and internal browning were due

to a difference in the amount of PPO present or to the amount of

phenolic substrates available for browning. This could be accom-

plished through the use of phenolic binding agents in enzyme

preparation (106). Research should also involve determination of

differences, qualitative or quantitative, in the phenolic compounds of

'Italian' prune after fall foliar B.

Table 12. Polyphenoloxidase activity and B content of immature 'Italian' prune fruit flesh, July 1976, after 492 ppm B applied postharvest, 1975.

B (ppm) in B (ppm) in mg Soluble ^^ . / ' „ ._ \ . 1 , AOD/ Polyphenoloxidase

Treatment fruit Tree acetone protein/ml i ,. .. ? „ , ,., mm1 activity^

flesh powder filtrate

Control 22 1

Postharvest B 38*

29 .375 .0052

27 .469 .0066

33* .613 .0254

34* .425 .0162



.0146

.0395**

AOD/min/mg soluble protein. /N = 420 nm. 2 Each figure is the mean of two trials in each of two experiments.

o

Table 13. Yield and fruit quality at harvest, 1976, as affected by fall foliar B applications.

„. , , Internal Purple Soluble _ _ . . Yield, Size, Firmness, Treatment ,, i browning, color, solids, t/ha % g % % lb

Control 27.3 33 29.2 89 21 2.2

492 ppm B preharvest, 1975 and 1976 32.3 17** 29.2 92 20 2.5

492 ppm B + 4798 ppm S preharvest, 1975; 984 ppm B preharvest, 1976 34.0* 4** 29.8 87 20 2.4

492 ppm B postharvest, 1975 34.0* 3** 30. 1 74 18 2.6

246 ppm B postharvest, 1975 37.3* 10** 32.5* 82 17 2.4

123 ppm B postharvest, 19.75 28.4 13** 31.9* 85 18 2.3

Yield data supplied by Dr. M.N. Westwood, Horticulture Dept., Oregon State University.



•c^

62

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