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GENETIC DIFFERENCE IN LEUCOSTOMA CANKER
RESISTANCE IN A DIVERSE PEACH POPULATION

by

Loong-sheng Chang

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Horticulture
Plant Breeding and Genetics Program

1989

f?
5

(00500;-

Abstract

Genetic Difference in Leucostoma Canker
Resistance in a Diverse Peach Population

by
Loong-sheng Chang

Progress was made towards identifying and
characterizing resistance to Leucostoma canker caused by
Leucostoma persoonii in peach. High levels of resistance to
both field and excised shoot inoculation were identified in
open-pollinated progeny of the Russian Plant Introduction
"Yennoh" and "NJ672017002".

Eight peach clones that represented 3 distinct groups,
susceptible, intermediate, and resistant in terms of
Leucostoma infection, were selected to measure the levels of
xylem dysfunction induced by infection. The resistant clones
were able to maintain adequate water transport through the
canker infection zone during the summer in contrast to
susceptible clones. Surviving vascular cambium evident in
resistant clones subsequently differentiated new xylem and
phloem to replace the damaged tissues.

The quantity of lignin formed in response to wounding
of the bark and 3-4 mm depth of wood was measured using an
assay of ligninthioglycolic acid (LTGA). The lignin content
in the resistant clones was twice that of susceptible clones
providing evidence that lignification was the mechanism of

disease resistance in dormant trees.

Heritability of resistance to Leucostoma canker was
quantified on a diverse range of peach genotypes by (1)
partitioning the variance components in the least squares
statistical method, and (2) determining parent-offspring
regression. The two methods provided very similar estimates
of the narrow sense heritability for canker necrotic length;
0.65 and 0.72, respectively. The estimates indicate that
superior resistant genotypes can be identified in a breeding

population to increase Leucostoma resistance.

ACKNOWLEDGEMENT

I would like to express my sincere appreciation to my
major advisor, Dr. Amy Iezzoni, for her financial and
educational support and guidance. The credit for initiating
the program for peach resistance to Leucostoma canker and
for collecting the broad base germplasm is owed to her.
Without Amy's efforts, there would not have been available
adequate sources of germplasm to breed peach for Leucostoma
resistance.

Dr. Gerry Adams gave me all kinds of help including
inoculation, evaluation, and discussion of the disease
progress in the field and in the lab. I appreciated his
helpful comments during the study.

My guidance committee, Drs. Adams, Hancock, Ewarts, and
Everson, corrected and commented on my thesis work. I thank
all of you very much.

Additionally, I am in debt to Drs. Ewers, Cress and
Isleib. Dr. Ewers helped me and let me use his lab equipment
to conduct the hydraulic conductance experiment. Dr. Cress
helped me concerning statistical analysis. I thank Dr.

Isleib for his comments on calculating the heritability

iv

estimate.

I wish to express my appreciation for the gang in Amy's
lab and Gerry's lab, and Sping Wang. Without their help, the
job would have been more difficult to complete.

Finally, I sincerely thank my wife, Ru-fen, my daughter,
Eileen, and my son, Kevin, for their patience, love and
unwavering support. Also, my parents, sisters, brothers ,
and Ru-fen's parents gave me extra financial support which

made living more decent.

Guidance committee:

The journal paper format was chosen for this thesis in
accordance with departmental and university regulation. The
thesis is divided into five chapter. Chapter 1 was published
in the Journal of the American Society of Horticultural
Science. Chapter 2 has been published in HortScience.
Chapter 3 and Chapter 4 will be submitted to Plant Disease.

Chapter 5 is intended for publication in HortScience.

vi

TABLE OF CONTENTS

Page

LIST OF TABLES..........OOOOOOOOOOOOOOOOO ....... O ..... ix

LIST OF FIGURESOOOOOOOOOOOOOOOOOOO0...... ...... O ...... Xi

INTRODUCTIONOOOOOOOOOOO......OOOOOOOO ......... 00...... 1

CHAPTER 1: LEUCOSTOMA PERSOONII TOLERANCE AND COLD-
HARDINESS AMONG DIVERSE PEACH GENOTYPES .............. 4

Abstract ..................... ................ 5
Introduction ........................ ...... ... 5
Materials and Methods ........................ 7
Results and Discussion ....................... 10
Literature Cited ............................. 18

CHAPTER 2: EXCISED-SHOOT ASSAY FOR TOLERANCE OF PEACH TO
LEUQOSTOMA PERSOONII...’......OCOOOOOOO0.0.0.0000...O. 20

Abstract ................. ....... . ............ 21
Introduction ..................... ........... . 21
Materials and Methods............... ..... ..... 23
Results and Discussion................. ....... 27
Literature Cited.............................. 30

CHAPTER 3. HYDRAULIC CONDUCTANCE IN SUSCEPTIBLE VS.
RESISTANT PEACH CLONES INFECTED WITH LEUCOSTOMA PERSOONII

...0.0.0.0000......0....O....O......IOOOOOOOOOOOOOOOOO 32

Abstract .............................. ....... 33
Introduction ................................. 34
Materials and Methods ........................ 35
Results and Discussion .............. ......... 38
Literature Cited .. .............. . ............ 50

vii

TABLE OF CONTENTS (continued)

CHAPTER 4. QUANTIFICATION OF LIGNIN CONTENT IN WOUNDS OF
PEACH CLONES SELECTED FOR THEIR RESISTANCE TO LEUCOSTOMA
PERSQQNII 0.0.0.0000.........OOOOOOOOOOOOOOOOOO0...... 53

Abstract ..................................... 54
Introduction ................................. 54
Materials and Methods ........................ 56
Results and Discussion ....................... 59
Literature cited ............................. 65

CHAPTER 5. HERITABILITY OF LEUCOSTOMA PERSOONII CANKER
RESISTANCE AMONG DIVERSE PEACH GENOTYPES ............. 68

Abstract...................................... 69
Introduction ................................. 69
Materials and Methods ........................ 71
Results and Discussion ........... ....... ..... 75
Literature Cited . ............................ 84

viii

LIST OF TABLES

Table Page
Chapter 1
1. Parents of the 18 clones used in the Leucostoma/cold-

hardiness screening program........................ 8

Correlation coefficients between Leucostomainfection
canker necrotic length and canker rating following
inoculation on 22 Oct. 1985 and cold hardiness in a
population of 693 open-pollinated peach seedlings from
18 cultivars. Cold hardiness was evaluated on the
following dates: 20 Nov. 1985, 9 Jan., 24 Feb., and 3
Apr. 1986 .................................. ...... 11
Mean values and coefficients of variation for L.
persoonii canker necrotic length and combined canker
ratings and winter hardiness ratings from artificial
inoculations in the fall of 1985 and of 1986 of 20
open-pollinated progeny of 15 peach selection .... 13

A comparison of published Leucostoma‘ankings from
wound-freeze inoculated peach cultivars compared to
rankings from open-pollinated progeny ............ 15

QQQEEEI 2

A comparison of canker necrotic lengths for 8 peach
seedlings following field inoculation with those on
excised-shoots inserted into cultures inoculated

with Leucostoma persoonii ........................ 28

Chapter 3

Mean values and coefficients of variation for L.
persoonii canker length and canker ratings following
artificial inoculations in the fall of 1986 and 1987
of open-pollinated progeny of 8 peach selection.... 39

ix

LIST OF TABLES (continued)

Specific _hydraul c conductance per unit length (specific
Kn) in 10'5 ma‘hMPa "' and percent specific Kh of
experiment/control Kh (% specific K" for Leucostoma
canker infection from artificial inoculations in the
fall of 1986 and 1987 of open-pollinated progeny of
peach .............................. ....... ........ 40

Percent infection of open-pollinated progeny of peach as
calculated as re-isolation of Leucostoma from the wounds
of artificial inoculation ......................... 43

Chapter 4

Relative lignification detected in wounded bark and wood
two days after wounding compared with mean disease
symptom and canker necrotic length rating .. ....... 60

Relative lignification of unwounded and wounded tissues
sampled 2, 4, 8, 14, and 21 days postwounding (DAW) for

five peaCh families ......OOOOOOOOOOOO ...... O ...... 61
Chapper 5

Parents of the 18 clones used in the heritability

estimates of Leucostoma resistance ............... 72

 

The analysis of variance of the linear statistical model
of heritability of canker resistance among half-sib
families ......... .. ........ ...... ................ 76

Estimates of variance components of the canker necrotic
length rating of a diverse peach seedling population
inoculated during fall of 1985, 1986 and 1987 and
evaluated during spring of 1986, 1987, and 1988 ... 79

The mean values of the canker necrotic length on diverse
peach genotypes ............. ..... . ................ 80

Narrow-sense heritability (hz) by parent-offspring
regression over 2 years data ..................... 82

TABLE OF FIGURES

Figure Page
Chappep ;
1. Cross-sections of a pair of the disease susceptible Can.

Harmony (3-17) peach stems used for hydraulic
conductance measurements. The stem on the left was
inoculated with Leucostoma, and the stem on the right
was the control. Red areas indicated when safranin-O
(0.5%) moved through the functional xylem area in the
middle of inoculated

wound ............................................ 44

Cross-sections of a pair of the disease resistant
NJ672017002 (1-8) peach stems used for hydraulic
conductance measurements. The stem on the left was
inoculated with Leucostoma, and the stem on the right
was the control. Red areas indicated when safranin-O
(0.5%) moved through the functional xylem area in the
middle of inoculated wound ....................... 46

xi

INTRODUCTION

Lepgpgpppa canker has been recognized as the most
serious disease reducing peach tree life in Michigan and
other northern peach growing areas. The fungal pathogens
involved, Lgugospomg pepsponii Fr. (Nits.) Hohn and
Lgucospoma pincta (Pers, ex Fr.) Hohn, infect through dead
or damaged tissues. Symptoms include cankering of the trunk
and branches, branch dieback, progressive weakening, and
ultimately death of the tree. Usually the combined influence
of low temperature stress and Leucostoma canker is greater
than the effect of either acting alone.

Although peach cultivars differ in their resistance to
Lgpppgpppg canker, no highly resistant selections have been
identified in previous studies. This is due to the lack of a
large-scale effort to identify resistance to Leucostoma in a
broad-based population of peach. Therefore, we initiated a
program to identify resistant selections which could be used
for peach breeding. Because cold injury predisposes peach
trees to Lgucospoma infection, cold hardy cultivars might
not only decrease the Leucostoma infections but also
increase the capacity to limit disease progression.

Chapter 1 describes an evaluation of a broad

2
collection of peach germplasm, for Lgpgostoma resistance
following inoculation, and for cold hardiness in the
Michigan climate. The goal was to identify useful genetic
material for breeding peach for Lgpgostoma resistance.

The screening procedures described in chapter I
required an effort spanning 7 months. To avoid this long-
term time commitments, and concomitant introduction of
copious infections into the plantings, a quick screening
technique to evaluate Leucostoma canker resistance in-vitro
was developed. This is described in chapter II.

Attempts have been made to understand the causes of
death of branches following Leucostoma infection. The levels
of xylem dysfunction due to the Leucostoma invasion into the
wood were measured in the study described in chapter III.
The susceptibility to Leucostoma was related to the plants
failure to maintain sufficient water transport though the
zone of infection. this restriction on transport
consequently caused wilting and death of branches.

The cultivars differed in their levels of resistance
to Leppospoma. However, the mechanism of host resistance in
the highly resistant cultivars was unknown. Therefore an
investigation was undertaken of selections of Yennoh (1-39)
and NJ672017002 (1-8) which had been identified as highly
resistant to Leucostoma infection. These genotypes were

examined to quantify the accumulation of lignin in response

3

to wounding. Significant differences in lignin content in
selected clones and peach cultivars were measurable during
cold temperature acclimation. Chapter IV describes these new
findings indicating that lignification is the mechanism of
host resistance functioning during the peach dormant season.

The purpose of the study described in Chapter V was to
estimate the heritability of Leucostoma resistance on this
diverse peach germplasm collection. The estimate of the
narrow sense heritability was about 0.65 which could be used
to discriminate superior genotypes resistant to Leucosgoma

infection and select useful breeding materials.

CHAPTER ONE

Leucostoma pepgoopii Tolerance and Cold

Hardiness among Diverse Peach Genotypes

Abstract

Open-pollinated progeny from 15 peach (Prunus persica)
cultivars, two peach x P. kanspensis hybrids, and one peach
almond (g. apygdalps) hybrid were evaluated for their cold
hardiness and for tolerance to Cytospora canker following
artificial inoculation with Leucostoma persoonii. Winter
hardiness was negatively correlated with canker necrotic
length (r = -0.26**) and positively correlated with canker
rating (r = 0.26**), as indicated by qualitative ratings.
The half-sib families differed for canker necrotic length
following fall inoculation, indicating that individuals with
increased tolerance to L. persoonii canker could be selected
from the population. Progeny from the cultivar 'Yennoh'
exhibited the shortest canker necrotic length following fall
inoculation, and all the inoculated branches were visually
healthy. 'Yennoh', a plant introduction from Russia, may
have a higher tolerance to Leucostoma than has previously

been found in U.S. germplasm.

Introduction
Qytospora canker, caused by Leucostoma persoonii Fr.

(Nits.) Hahn. and Leucostpma cincta (Pers. ex Fr.) Hohn., is

6
the most serious disease reducing peach life in Michigan,
New York, New Jersey, and Colorado (7). Symptoms include
cankering of the trunk and branch dieback, progressive
weakening, and, ultimately, death of the tree. Leucostoma is
a pathogen that enters through dead and damaged tissues.
Pruning cuts that do not callus properly are ideal
Lepgpsppma entry sites. Winter injury that results in dead
and damaged tissue predisposes that tree to Leucostoma
invasion (1). Usually the combined influence of low
temperature stress and Cvtospora canker is greater than the
effect of either acting alone.

There has been no large-scale effort to identify
genetic resistance or tolerance to Leucostoma in a broad-
based population of peach (4). Cultivars do, however, differ
in their level of tolerance to Leucostoma, but no highly
tolerant selections have been identified (2,6). Various
inoculation techniques have been investigated for screening
peach for Leucostoma tolerance, but these techniques have
not been used to screen a diverse peach germplasm collection
(5,8).

Because cold injury predisposes peach trees to
Leucostoma infection, it is important to determine the
hardiness of the trees inoculated in a screening program.
Well-acclimated, hardy cultivars not only should have

reduced infection, but also have increased capacity to

7
combat disease progression. The objectives of this study
were to evaluate a broad collection of peach germplasm for
L. pgpgpppii disease development following inoculation and
for cold hardiness in the Michigan climate to identify

useful genetic material to breed for L. persoonii tolerance.

Materials and Methods

In Spring 1984, open-pollinated peach seedlings from
15 peach clones of diverse background, two peach x 2.
Kansuepsis hybrids, and one peach almond (2. amygdalus)
hybrid (Table 1) were planted in a completely randomized
design at the Horticultural Research Center, East Lansing,
MI. Open-pollinated progeny, instead of clones, were used to
maximize the genetic diversity in the population to be
screened. These seedlings represent half-sib families with
progeny numbers ranging from 3 to 73. On 22 Oct. 1985, a 2—
year-old branch on each seedling was inoculated with 20 ul
of a suspension of 107 Leucostoma persoonii conidia per
milliliter derived from isolates collected from cankers on
peach at Clarksville and Hartford, MI. A wound-freezing
inoculation technique developed by Scorza and Pusey (8) was
followed. A second branch on each seedling was inoculated
the following day.

On 21 Nov., two l-year-old shoots were collected from

each seedling and placed in a freezing chamber and chilled

Table 1. Parents of the 18 clones used in the Leucostoma/cold-
hardiness screening program.

 

Clone

Parents

 

Babygold 8

Canadian Harmony

Elberta
Glohaven
Harken
Loring
Reliance
Red Hale
Yennoh
88-11-147
88-20-171
88-21-20
C2-28-89
C4-11-97
NJ257

NJN69

NJ672017002

RR37-15

P135201 x Ambergen

Redskin x Sunhaven

Chinese Cling (open-pollinated)

(J.H. Hale open—pollinated) x Kalhaven
Redskin x Sunhaven

Frank x Halehaven

(Minn. PH04559 x Meredith) open-pollinated
Unknown 1

Plant Introduction from Russia

(K82 x Sunrise) x [(Red C x NJ191) x Okinawa]
(5110417 X Ta Tao 3) x C2R31T45

Orange Cling x RR65-l

Kasna Dupnishka open-pollinated

peach x almond

Honeydew Hall x Jefferson

(NJN55 x NJC68) x Marzochella

(P135321 x Cherryred) x Prunuscansuensis

NJ174 x Prunuskansuensis

 

9
to a critical temperature of -22C at a cooling rate of
4C/hr. The critical temperature was determined the previous
week as the temperature required to cause cambium death of
50% of two 1-year-old shoots from 13 'Redhaven' trees. One
week later, the cambium from each shoot was examined and
rated as dead (0) or alive (1). Three additional hardiness
evaluations were made on 9 Jan., 24 Feb., and 4 Apr. 1986,
with critical temperatures of -29.5C, -28C, and -23C,
respectively.

On 19 May, when the trees were beginning to leaf out,
all the inoculated branches were measured for canker
necrotic length (length of necrotic area distal to the point
of inoculation) and branch diameter. Additionally,
inoculated branches were rated visually for canker using a
scale where l = dead, 2 = severe wilting of expanding
leaves, 3 = weak growth and slight wilting of expanding
leaves and 4 = healthy.

On 26 May 1986, two branches on each seedling were
inoculated with L. persoonii as described earlier. The
resulting cankers were evaluated for canker necrotic length
and canker necrotic length/branch cross-sectional area on 19
Sept. 1986.

A second fall inoculation was made on 8 Oct. 1986 and
evaluated on 30 apr. 1987. A cold-hardiness evaluation using

two 1-year-old shoots per seedling as described earlier was

10
performed on 18 Feb. 1987 (critical temperature = -25C). A
second spring inoculation was made on 26 Apr. 1987 and
evaluated on 21 Sept. 1987.

For the inoculation and hardiness experiments, all
the progeny from all 18 families were evaluated; however,
because equal progeny numbers are required to statistically
conduct mean comparisons, from each family with 20 or more
progeny, 20 progeny were chosen at random for use in the
statistical analysis, which was a nested design. Since the
canker and cold-hardiness rating was nonparametric data, the
Kruskal and Wallis test (9) was used to test for significant
differences among families. Mean comparisons of these

nonparametric data are not appropriate.

Result and Discussion

Canker necrotic length was positively correlated with
canker necrotic length/branch cross-sectional area (r =
0.60**). for the 693 open-pollinated peach seedlings
following inoculation on 22 Oct. 1985 (Table 2). Therefore,
only canker necrotic length data will be presented in Tables
3 and 4. Both canker necrotic length and canker necrotic
length/branch cross-sectional area were negatively
correlated with canker rating. Canker necrotic length and
canker length/branch cross-sectional area were also

negatively correlated with cold hardiness on all four

1.1

Table 2. Correlation coefficients between Lgppggggpg infection canker necrotic length and canker ratingy
following inoculation on 22 Oct. 1985 and cold hardiness in a population of 693 open-pollinated peach seedlings
from 18 cultivars. Cold hardinessx was evaluated on the following dates: 20 Nov. 1985, 9 Jan., 24 Feb., and 3
Apr. 1986.

 

Date of cold treatment
Canker Canker necrotic
Length/branch symptom necrotic section Nov. Jan. Feb. Apr.
Trait rating length area 1985 1986 1986 1986

 

 

Canker rating

Canker necrotic

it:
length -0.42
Canker necrotic
length/branch .. '*
x section area -0.34 0.60
t.
Nov. 1985 0.05 -0.14 -0.06
t. it fit
Jan. 1986 0.15 -0.16 -0.06 0.11
it it it it
Feb. 1986 0.19 -0.16 -0.06 0.09 0.34
it fit it it it
Apr. 1986 0.18 -0.17 -0.19 0.04 0.16 0.14
X over fwr Gald it it it it it 9* 1..
treatments 0.26 -0.26 -0.15 0.35 0.70 0.70 0.61

 

x The critical temperatures were: ~22C, -29.5C, -za.5c, -23C, respectively; Osdead, 1=alive.

Y Visual rating: indeed, Zasevere wilting, 3-week growth and slight, bshealthy.

2 “Significant at P80.05 and 0.01%, respectively.

12
sampling dates, confirming the negative association between
canker development and cold hardiness. The sum of the cold
hardiness values from the four dates was positively
correlated with cold-hardiness values on each of the
individual dates. The correlations obtained from the Feb.
1987 hardiness evaluation and the 9 Oct. 1986 inoculation
(data not presented) were similar to those presented in
Table 2.

There were significant differences between half-sib
families for canker necrotic length and canker rating
following fall inoculation, and for cold hardiness (Table
3). The data are presented as means over 2 years, since the
year by half-sib family interaction was not significant.
Progeny half-sib family mean values for canker necrotic
length following the October inoculations ranged from 8.8 to
14.4 cm. Progeny from C2-28-89 and 'Loring' had the longest
mean canker necrotic length and one of the lowest cold-
hardiness ratings. Progeny from 'Baby-gold 8' had the
shortest mean canker necrotic length and the largest number
of healthy branches following inoculation. The data
presented represent half-sib family means; however, within
some of the more-tolerant families, individuals with small
canker necrotic lengths and healthy branches following
inoculation could be selected. For example, 'Babygold 8'

open-pollinated progeny number 1-30 had a mean canker

13

Table 3. Mean values and coefficients of variation for L. persoonii
canker necrotic length and combined canker ratings and winter
hardiness ratings from artificial inoculations in the fall of 1985
and of 1986 of 20 open-pollinated pregeny of 15 peach selection.”z

 

 

 

Canker

necrotic

length (cm) Cold

Canker hardiness
Trait x cv rating“" rating“'v

C2-28-89 14.4 58 1.64 0.38
Loring 15.2 62 1.61 0.57
NJN69 13.1 65 1.88 0.56
NJ257 11.8 62 1.71 0.67
Elberta 10.8 40 1.61 0.77
Harken 10.8 63 1.91 0.92
Canadian Harmony 10.4 30 1.79 0.61
88-21-20 10.1 60 2.05 0.66
RR37-15 9.5 52 2.21 0.68
Red Hale 9.8 36 1.97 0.72
NJ672017002 9.4 65 2.72 0.91'
C4-11-97 9.2 75 2.33 0.89
88-20-171 9.1 38 2.13 0.82
88-11-147 8.8 37 2.66 0.84
Babygold 8 8.8 81 2.88 0.82
LSD 5% 1.8

 

z inoculation dates and harvest dates were respectively; Year 1, 22-
23 Oct. 1985 and 19-20 May 1986; year 2, 6 Oct. 1986 and 12 Apr.
1987.

y The data represent identical trees inoculated both years.

‘ Data for 2 years were combined. Rating scale: 1=dead; 2=severe
wilting; 3=weak growth; 4=healthy.

" Means were significantly different at the 1% level using the
Kruskal and Wallis test (9).

V Hardiness tests were performed in February. Data for 2 years were
combined. Rating scale: 0=dead: 1=alive.

14
necrotic length for 1986 and 1987 of 4.6 cm and a canker
rating of 4.0.

For the spring inoculation date, the half-sib families
and progeny within half-sib families responded similarly in
both years for canker necrotic length and canker necrotic
length/branch cross-sectional area (data not presented).
However, the peach seedlings in half-sib families responded
differently to the two inoculation times (spring vs. fall).
Following spring inoculations, host callus tissue was
deposited and mean canker necrotic lengths were
significantly smaller (ranging from 4.8 to 6.6cm) than
following fall inoculation. Cytospora canker development on
peaches has been observed as being restricted during the
summer (3). In northern peach growing areas, delaying
pruning until trees are near bloom is recommended (7). At
this time, trees are actively growing and pruning wounds
will heal more rapidly, allowing less time for invasion by
Leucostoma spp.

When the half—sib families include 'Yennoh',
'Reliance', 'Glohaven', 'Harken', 'Canadian Harmony',
'Elberta', and 'Loring' were ranked for mean canker necrotic
length, the rankings generally agreed with a previous
ranking of some of the parent clones using the wound-
freezing technique (8) Table 4. In general, 'Reliance' and

'Harken' are most to tolerant to Leucostoma infection,

15

Table 4. A comparison of published Leucostomarankings from wound-
freeze inoculated peach cultivars compared to rankings from open-
pollinated progeny.

 

 

 

Open-pollinated
Progeny Data of
Scorza and
No. Mean Pusey (8)"'x
Cultivar trees length3(cm) (cm)
Yennoh 3 6.2
Reliance 5 8.7 2.2 a
Glohaven" 3 9.0 --
Harken 22 10.4 3.8 ab
Canadian Harmony 22 10.1 5.5 bc
Elberta 20 10.8 10.2 d
Loring 22 13.1 7.3 c

 

’ Length of necrosis on 2-year-old limbs, values represent 2-year
means (1986 and 1987).

Y Length of necrosis of inoculated wounds minus that of control
wounds on young budded trees.

‘ Means within a column sharing a letter in common are not
significantly different (P = 0.05).

“ Only 1986 data.

16
'Canadian Harmony' is intermediate, and 'Elberta' and
'Loring' are the most susceptible. The similarity in ranking
between the open-pollinated progeny means plus the parent
cultivar data suggests that the differences in Leucostoma
tolerance observed in this study are heritable.

Because the 'Reliance' and 'Yennoh' families had only
five and three progeny respectively, these progenies were
only included in Table 4. Progeny from 'Reliance' had a
small canker necrotic length; however, many of the branches
were wilting and would eventually die. Although 'Reliance'
is considered to support less canker growth than other
cultivars, cankers do develop under field conditions. A
canker rating of 2.58, indicating wilting and eventual death
of the inoculated branches, confirms that the destructive
effect of L. persoonii on 'Reliance' is also observed on its
progeny.

In contrast, although there were only three progeny of
'Yennoh', the six branches that were inoculated had the
smallest mean canker necrotic length (Table 4) and showed no
symptoms of wilting, having a canker rating of 4.0,
'Yennoh', a plant introduction from Russia, may have a
higher tolerance to Leucostoma canker development than
previously reported for peach. 'Yennoh' ripens late-season
and has white-fleshed fruit.

In conclusion, fall inoculations with L. persoonii

17
permitted detection of genetic differences between open-
pollinated half-sib families for L. persoonii tolerance. The
host-pathogen response differed between spring vs. fall
inoculations: however, it is the fall inoculation that
resulted in the best discrimination among the seedlings for
L. pepsopnii tolerance. Genetic material useful for breeding
for L. persoonii tolerance has been identified in this
study. However, further evaluations are being conducted to
determine which - 'Babygold 8' seedling, 'Yennoh' seedling,
or the 'Yennoh' cultivar itself - would be the best parent
for incorporating genes for L. persoonii tolerance and cold
hardiness into a breeding program. Additionally, this
tolerance material must be screened for its reaction to the

other Leucostoma species, L. cincta.

18

Literature Cited

1.Dhanvantari, B.N. 1978. Cold predisposition of dormant
peach twigs to nodal cankers caused by Leucostoma §ppL
Phytopathology 68:1779-1783.

2.Dhanvantari, B.N. and V.A. Dirks. 1983. An evolution of
peach cultivars and selections for resistance to
Leugostoma cincta. Can. J. Plant Sci. 63:307-310.

3.Jones, A.C. and N.S. Luepschen. 1971. Seasonal
development of Cytospora canker on peach in Colorado.
plant Dis. Rpt. 55:314-317.

4.Layne, R.E.C. 1984. Breeding peaches in north America for
cold hardiness and perennial canker (Leucostoma spp.).
Resistance -review and outlook. Fruit Var. J. 38:130-
136.

5.Luepschen, N.S. 1981. Criteria for determining peach
variety susceptibility to Cytospora canker. Fruit Var. J.
35:137-140.

6.Luepschen. N.S. and K.G. Rohrback, A.C. Jones, and L.E.
Dickens. 1975. Susceptibility of peach cultivars to
Qytospora canker under Colorado orchard conditions.
HortScience 10:76-77.

7.Rosenberger, D.A. 1982. Biology and control of Cvtospora
fungi in peach plantings. New York food & Life Sci. Bul.

92.

19
8.Scorza, R. and P.L. Pusey. 1984. A wound-freezing
inoculation technique for evaluating resistance to
gyppspppa leucpstoma in young peach trees. Phytopathology
74:569-572 .
9.Steel, R.G.D. and J.H. Torrie. 1960. Principles and

procedures of statistics. McGraw-Hill, New York.

CHAPTER TWO

Bxcised-shoot Assay for Tolerance

of Peach to Lepcpstoma persoonii

20

21

Abstract

Dormant, excised shoot segments from peach [Prunus
pgppigg (L.) Batsch] seedlings previously identified as
tolerant, intermediate, or susceptible to L. persoonii
(Nits) Hohn. were evaluated for longitudinal canker necrotic
length after incubation in contact with a culture of L.
pepsponii growing on clarified oatmeal agar. The differences
in seedling canker necrotic lengths were significant and
corresponded with field ratings of disease susceptibility.
Seedlings Yennoh 1-39 and NJ672017002 1-8 were the most
tolerant, whereas Loring 14-20 and Elberta 8-25 were the
most susceptible. The excise shoot assay is sufficiently
quick, reliable, and related to field disease reaction to be
used as a screening procedure in the breeding of peach

cultivars tolerant to L. persoonii.

Introduction
L. persoonii (Nits) Hohn. [-Valsa leucostoma (Pers. ex
Fr.) Fr.], one causal agent of Cytospora canker in peach,
usually colonizes cold-injured or dead tissue during cool
weather when the trees are not actively growing. In the

spring, the fungus progressively invades adjacent healthy

22
tissue, forming cankers and eventually plugging the xylem
vessels and causing wilting and death of the infected twigs,
ranches or limbs (Willison, 1933; Tekauz and Patrick, 1974:
Biggs, 1984: Wisniewski et al., 1984).

Previous work (Chang et al., 1989) has shown that
open-pollinated progenies from 'Yennoh', a peach
introduction from the Soviet Union, and NJ672017002, a peach
x Prunus kansuensis Rehd. hybrid, are tolerant of L.
persoonii based on 2 years of field evaluation using the
screening technique of Scorza and Pusey (1984). Since
fungicides and cultural practices inadequately control
canker caused by Leucostoma spp., the best control approach
is through breeding and selection for host resistance
(Luepschen, 1981). The field screening procedure used to
identify seedlings of 'Yennoh' and NJ672017002 as L.
pepsoonii tolerant was time consuming in the inoculation and
evaluation, and required 7 months (October to May) for
symptom development to occur. Moreover, large amounts of
inoculum were introduced into the seedling plot during
inoculation. Therefore, the objective of this work was to
develop an excised-shoot assay for L. persoonii tolerance in
peach which would be efficient, reliable, less destructive
to the seedling plot and correlated with disease reaction in
the field.

Excised twig assays have been used previously with

23
apple to determine the pathogenicity of isolates of
Epypppnppppg spp. (Jeffers et al., 1981) and for studying
the infection of apple rootstocks with different isolates of
Ehyppppppppa cggpopum (Sewell and Wilson, 1959). Sewell and
Wilson (1959) suggested that dormant twig assays in the
laboratory could substitute for field-inoculated screening

trials.

Materials and Methods

In the spring of 1984, open-pollinated peach seedlings
from 15 peach clones, 2 peach x Prunus kansuensis hybrids,
and one peach x almond (21 dulcis Webb) hybrid were planted
in a completely randomized design at the Horticultural
Research Center, East Lansing , Mich. Open-pollinated
seedlings were used to maximize the genetic diversity in the
population to be screened. These seedlings, representing
half-sib families with progeny numbers ranging from 3 to 73,
were evaluated twice for canker necrotic length following 2-
year-old branch inoculation with L. persoonii using the
technique developed by Scorza and Pusey (1984). Branches for
the 2 experiments were inoculated in October, 1985 and 1986,
and evaluated in May, 1986 and 1987, respectively. Two
branches per seedling were each inoculated with 20 ul of a
suspension of 107 L. persoonii conidia per milliliter

derived from a mixture of isolates collected in Hartford and

24
Clarksville, Mich. (Hammer, 1988). Canker necrotic length
was measured as a length of the necrotic area distal to the
point of inoculation. Dhanvantari and Dirks (1983) found
with Lepcpstoma that linear canker extension following
artificial inoculation was related to visual ratings of the
incidence of naturally induced cankers over 1 10-year
period.

Following the field inoculation experiments with L.
persoonii, 8 peach seedlings which were identified as being
tolerant (Yennoh 1-39, NJ672017002 1-8), intermediate
(Reliance 3-1, Babygold 8 16-11, Canadian Harmony 6-19, BB-
20-171 17-45) or susceptible (Loring 14-20, Elberta 8-25) to
L. persoonii were selected (Chang et al., 1989). These open-
pollinated seedlings are identified by the maternal parent,
i.e. 'Yennoh', and the seedling clone number, i.e. 1-39.
These seedlings were sampled on December 10, 1987 for
excised-shoot experiments I and II, and on January 22, 1988
for experiment III. One-year-old peach branches were
collected from each seedling.

Clarified oatmeal agar was prepared as follows: 75 g
rolled oats were autoclaved for 5 min in 1 liter water,
homogenized for 5 min in a Waring blender, and filtered
through 3 layers of cheesecloth. The liquid was then
centrifuged at 20,000 g for 30 min. The upper 200 ml of

liquid was added to 800 ml H20 with 20 g agar and 1 ml of

25
vitamin solution (Adams et al., 1987) and autoclaved for 30
min. The medium was cooled to 45C and 1 ml pure lactic acid
and 200 mg streptomycin sulfate were added per liter prior
to dispensing. The agar was dispensed into sterile 6.5 x 6.5
x 9.5 on tissue culture boxes (Magenta Corp., Chicago, IL)
to a depth of 1.5 cm.

The medium in each culture box was inoculated with a
0.7 cm diameter agar plug with mycelium of L. persoonii cut
from a culture growth on clarified oatmeal agar. The L.
pepsoonii culture was derived from the same mixture of
isolates used for the field inoculation experiments. The
control treatment consisted of non-inoculated medium. The
mycelium covered the oatmeal agar surface of the inoculated
boxes in approximately 14 days.

Shoot segments 10 cm long were cut from the lower
central portion of the current seasons' growth, disinfected
for 5 minutes in 0.6% NaOCl, rinsed in distilled sterilized
water 3 times, and blotted dry. A 1-cm segment was cut off
each end of the shoot segments to removed portions which may
have absorbed the NaOCl from solution. A sterilized razor
blade was used to make one tangential 2.0 cm cut on the end
of each shoot segment to expose the cambium and vascular
tissue. One shoot segment from each of the 8 seedlings was
inoculated in each test box with the cut end of each segment

inserted to a depth of 1.5 cm in the culture medium.

26

For those shoots collected on December 10, 12 boxes
(10 inoculated and 2 control) were placed at 25C under cool-
white fluorescent lights (35 umoles s'1m2) and evaluated 24
days later (experiment I). Another set of 12 boxes (10
inoculated and 2 control) were placed in the dark at 4C for
60 days and then moved under the same lights at 25C for 10
days until evaluation (experiment II). For those twigs
collected on January 22, 14 boxes (12 inoculated and 2
control) were placed at 25C under the same lights for 20
days until evaluation (experiment III).

For evaluation, the shoot segments were removed from
the boxes and the bottom 1.5 cm portion which has been below
the agar surface was cut off. The bark adjacent to the
tangential cut was removed and the longitudinal length of
the necrotic tissue was measured. When the excised shoot
segments were completely necrotic , tissue was surface
sterilized for 1 min. in 0.6% NaOCl and the fungi were
reisolated into Difco potato dextrose agar (Difco Lab.,
Detroit, MI) to verify the pathogens. Tissues rotted by
fungi other than L. persoonii were treated as missing data.
Three twig segments in experiment II (2 Loring 14-20 and 1
Elberta 18-25) were colonized by other fungi (Rhizoctonia
Fomes-like organism) and treated as missing values. Canker
necrotic lengths from previous field screening test (Chang

et al., 1989) were compared with the results from the

27
excised twig assays.

The zonate longitudinal necrosis on most shoot
segments was similar to that observed on shoots following
field-inoculations with L. pepsoonii. L. persoonii was
reisolated from the zonate necrotic margins confirming the
association between the necrosis and this fungus. No

necrosis developed on the shoots in the control boxes.

Results and Discussion

Data was analyzed by analysis of variance. Where a
significant F-test was observed (5% level), means were
separated with Duncan's multiple range test. The 2 field
experiments were treated as replications.

The difference in canker necrotic length among the 8
seedlings was highly significant and the ranking of the
seedlings for canker necrotic length was similar for all 3
experiments (Table l). Excised-shoot canker necrotic lengths
correlated well with the canker necrotic lengths following
field inoculation (r=0.84", 0.95", and 0.89", for
experiments I, II, and III, respectively).

Although the shoots for experiment III were collected
a month later in the winter than those for experiment I, the
results were very similar. In experiment II, the canker
necrotic lengths were generally longer than in the 2 other

experiments. The shoots in experiment II were kept in the

28

Table 1. A comparison of canker necrotic lengths for 8 peach
seedlings following field inoculation with those on excised-shoots
inserted into cultures inoculated with Lepgpsppma pepsopni'.

 

Seedling

Field Excised Shoot Assays
inoculation
mean canker mean length of necrotic zone (cm)y
necrotic

length (cm)‘Expt. I Expt. II Expt. III

 

 

 

Loring (14-20)
Elberta (8-25)
Reliance (3-1)
Babygold 8
Canadian Harmony
38-20-171 (17-45)
NJ672017002

Yennoh (1-39)

11.0ax 5.05a 6.14a 4.91a
10.83b 3.79a 5.28a 3.52b
8.6ab 3.27ab 3.84b 3.11bc
8.0ab 4.53bc 3.66b 2.3lc
7.5ab 2.58cd 3.29b 2.36c
4.9ab 2.60cd 2.79b 2.36c
5.0c 1.66d 1.60c 0.85d
4.8c 1.75d 1.28c 0.91d

 

x Each value is the mean of 4 observations.

y Each value is the mean of 10 observations for experiment I and II
and 12 observations for experiment III.

2

0.05).

Mean separation in columns by Duncan's multiple range test (P-

29
cold longer than those in the other experiments; however, L.
perspopii did not cause measurable canker necrosis
development during the period at 4C.

In summary, the excised-shoot assay for tolerance of
peach to L. pepsoopii is rapid, reproducible, and gave
relative responses to those in the field. The laboratory
evaluation was completed in one month in contrast to the 7
months required for the field screen. The laboratory method
avoids the introduction of L. persoonii into the orchard.
There was good agreement between the results of laboratory
and field experiments suggesting that Yennoh 1-39 and
NJ672017002 1-8 are tolerant to L. persoonii. The
identification of a high degree of tolerance to L. persoonii
and the development of a rapid screening technique will

facilitate the breeding of peach cultivars tolerant to L.

persoopi .

30

Literature cited
Adams, 6., N. Johnson, J.F. Leslie, and P. Hart. 1987.
Heterokaryons of gipbgrella zeae formed following hyphal
anastomosis or protoplast fusions. Experimental Mycology
11:339-353.
Biggs, A.R. 1984. Boundary-zone formation in peach bark
in response to wounds and Cytospora leucostoma
infection. Can. J. Bot. 62:2814-2821.
Chang, L.S., A. Iezzoni, G. Adams and 6.3. Howell. 1989.
Leucostoma tolerance and cold hardiness among diverse
peach Genotypes. J. Amer. Soc. Hort Sci. 114 (accepted).
Dhanvantari, B.N. and V.A. Dirks. 1983. An evaluation of
peach cultivars and selections for resistance to
Leucostoma gincta. Can. J. Plant Sci. 63:307-310.
Hammer, S. 1988. Virulence and hypovirulence and
Leugostoma spp. MS Thesis, Mich. State Univ., East
Lansing, Mich.
Jeffers, S.N., H.S. Aldwinckle, and P.A. Arneson. 1981.
Excised twig assay for the study of apple tree crown rot
pathogens in vitro. Plant Disease 65:823-825.
Luepschen, N.S. 1981. Criteria for determining peach
variety susceptibility to Cytospora canker. Fruit Var.
J. 35:137-140.

Scorza, R. and P.L. Pusey. 1984. A wound-freezing

31
inoculation technique for evaluating resistance to
gypospppa leucostoma in young peach trees.
Phytopathology 74:569-572.

9. Sewell, G.W.F. and J.F. Wilson. 1959. Resistance trials
of some apple rootstock varieties to Phytophthora
pappoppm (L. & C.) Schroet. J. Hort. Sci. 34:51-58.

10.Tekauz, A. and Z.A. Patrick. 1974. The role of twig
infections on the incidence of perennial canker of
peach. Phytopathology 64:683-688.

11.Willison, R.S. 1933. Peach canker investigations. I.
Some notes on incidence, contributing factors and
control measures. Scientific Agric. 14:32-47.

12.Wisniewski, M., A.L. Bogle and C.L. Wilson. 1984.
Histopathology of canker development on peach trees
after inoculation with Cytospora leucostoma. Can. J.

Bot. 62:2804-2813.

CHAPTER THREE

Hydraulic Conductance in susceptible vs. Resistant Peach Clones

infected with Leugogtoma persoonii

32

33

Abstract

Eight open-pollinated peach families (Prunus persica.L)
were selected from a germplasm collection which was screened
for resistance to Leucosppma pezsoonii following field
inoculation (Chang et al., 1989). The eight peach families
could be divided into three distinct groups: susceptible,
intermediate, and resistant to L. persopnii infection based
on canker length measurements. Following artificial
inoculation, measurements of hydraulic conductance per unit
length (K11) were made on the branch segments from the eight
clones, and safranin dye was used to mark the conductive
xylem pathways. For the peach clones resistant to L.
persoonii , the K1. of the canker branch segments was
significantly greater than that for the susceptible peach
clones. The inoculated branch segments from the resistant
peach clones maintained about 20-30% of water transport and
infected seedlings survived in the field. Safranin dye
ascents and descents indicated that the conductive xylem
tissue was almost completely blocked in the susceptible
infected peach segments. However, branch segments from the
resistant peach clones infected with L. persoonii often had

an incomplete xylem growth ring with part of the cambium

34
surviving and the fungus could not be isolated from the wood
beneath the cambium. It is concluded that xylem dysfunction
is correlated with reduction in.B% and that the resistant
peach clones are better able to maintain water
transportation. Additionally, the surviving vascular cambium
present in resistant peach clones is capable of producing

new xylem vessels and phloem.

Introduction

In the 19503, peach (Prunus persica) orchards in the
northeastern United States were expected to remain
productive for at least 30 years. Now, the life of a peach
orchard averages 15 years or less. Cytospora canker, caused
by Leucostoma persoonii and L. cincta, is the most serious
disease reducing peach tree life in central and northern
United States (Hildebrand, 1947; Gairola and Powell, 1970;
Jones and Luepschen, 1982: Luepschen, 1981) and in eastern
peach growing regions of Canada (Cline, 1982; Dayne, 1976;
Wensley, 1964).

Histopathological investigations of Leucostoma
classified the canker pathogen as a facultative bark
parasite (Wisniewski et al., 1984: Biggs, 1984, 1986) or as
a sapwood parasite (Tekauz and Patrick, 1974; Banko and
Helton, 1974). These histopathological investigations

concentrated on fungal growth in the bark; if, however, the

35

pathogen grew beyond the bark and into the xylem, there
could be a noticeable reduction in water transport measured
as hydraulic conductance. Hydraulic conductance is the
measured rate of a fluid divided by the pressure
differential. Reductions in hydraulic conductance have been
associated with death of branches in American chestnut
blight (Ewers et al., 1989) and peach phony disease (Evert,
1987). Hampson and Sinclair (1973) inoculated five peach
cultivars grown in a greenhouse with a canker causing
pathogen and reported that there were no significant
differences among these peach cultivars for xylem
dysfunction following pathogen invasion. However, the
pathogen infection reduced the xylem function in the
inoculated trees in comparisons with healthy ones.

Recently, peach clones resistant to L. persponii
following field inoculation were identified (Chang et al.,
1989). The objective of this work was to determine if the
level of xylem dysfunction were associated with resistance
or susceptibility to L. persoonii. In addition, stem
segments were sectioned and L. persoonii isolations were
made to determine the depth of invasion of the fungus into

the xylem and wood.

Materials and Methods

Peach (Prunus persica (L.) Batch) seedlings were

36
planted in 1984 at the Horticultural Research Center,
Michigan State University, East Lansing, MI. In 1985, two-
year-old branches on healthy trees were randomly selected
for inoculation with spores of Leucostoma persoonii (Nits.)
Hohn. Following the procedure described previously (Chang et
al., 1989). these open-pollinated peach seedlings were
evaluated twice for canker necrotic length following 2-
year-old branch inoculation with L. persoonii using the
technique developed by Scorza and Pusey (1984).

For hydraulic conductance measurements, eight open-
pollinated peach families were selected based on the
previous studies as susceptible, intermediate, or resistant
(Chang et al., 1989), and three seedlings within each of
these peach families were chosen for the experiments. Two
branches per seedling were each inoculated with 20 ul of a
suspension of 106 L. persoonii conidia per milliter as
previously described (Chang, et al, 1989). Two healthy
branches within each tree were used as the non-inoculated
control.

To measure hydraulic conductance, the branches were
trimmed to similar lengths (approximately 0.25m) with a
bandsaw and stored under water. Both ends of each stem
segment were shaved until smooth with a fresh razor blade.
Vinyl tubing was then firmly clamped to the distal end. The

stem surface was vacuum infiltrated at -87 Kpa for 5 minutes

37
prior to the conductivity measurement. Pressure was applied
with a distilled water column and the rate of flow measured
with a stopwatch and pipet (Zimmermann, 1978). The hydraulic
conductance per unit stem length (3%) was calculated as the
rate of (ma/s) divided by the applied pressure gradient
(MPamd). Filtered oxalic acid (0.1M, PH 2.0) was used to
minimize possible artifacts caused by swelling of pit
membranes (Sperry et al., 1988). Three readings were used to
calculate a mean RT of each stem segment. Data was recorded
as specific conductivity and percent specific conductivity.
Specific conductivity (specific K%)*was calculated as Kh
divided by cross-sectional area. The percent specific Kl‘was
the specific Kh of the experimental stem segments divided by
the specific Kh of the control stem segments.

0.5% Safranin-O dye was poured into the vinyl tube
immediately after finishing Kn measurements. The height of
the dye in the tubes was kept constant for each stem segment
for a 1.5 hour period. The dye was used to mark the
functional xylem vessels as a comparison of the tissue area
capable of water transport.

Various precautions were taken to minimize the error in
the hydraulic conductance measurements. To reduce the
lateral transport of water, lateral twigs were removed from
the branches and wounds were immediately sealed with nail

polish. To minimize the introduction of embolisms, branches

38
were harvested from the trees at 7-8 am, the basal ends
immediately immersed in water, and recut under water. All
hydraulic conductance measurements were done within 12 hours
of the initial collection time.

Attempts were then made to isolate L. persoonii from
the wood to determine the depth of the fungal invasion. The
stem segments containing the inoculated wound were trimmed
to 6 mm lengths with a bandsaw, de-barked, and surface
sterilized with 95% ethanol. 1 mm deep sections of wood were
excised with a razor blade from stem segments beginning at
the surface margins of necrosis and proceeding downward to
the pith and beyond through the stem in 1 mm increments. The
1 mm sections of tissue layers were incubated on Leonian
medium (Leonion, 1921). The culture plates were stored at
25°C under cool-white fluorescent light for 2 weeks and

scored for the presence or absence of L. persoonii.

Results and discussion
Following field inoculation in the fall of 1986 and
1987, there were significant differences in response to L.
persoonii infection among the diverse peach populations
(Chang et al., 1989). The open pollinated Yennoh seedlings
numbered 1-31, 1-39 and 4-11, and open pollinated
NJ672017002 seedlings numbered 1-8, 2-32, and 4-16 had the

shortest longitudinal length of necrosis (cankers) following

39

Table 1. Mean values and coefficients of variation for L. persoonii
canker length and canker ratings following artificial inoculations
in the fall of 1986 and 1987 of open-pollinated progeny of 8 peach
selection.u

 

 

 

Canker length (cm) Canker
ratings
Selection 1986 c.v. 1987 c.v. 1986 1987
Loring 15.7av 19 19.0a 23 0.0 1.7
(2-7: 11-27, 11-32)
Elberta 17.0a 33 11.8ab 50 0.0 1.0
(6-32, 11-34, 11-38)
Canadian Harmony 13.1abc 18 8.7cd 27 0.5 1.7
(3-17, 3-20, 5—9)
Harken 11.6abc l9 15.0ab 54 0.5 1.7
(2-6, 2-29, 2-44)
Reliance 13.8ab 46 7.8cde 27 0.3 0.8
(1-33, 1-41, 15-16)
Babygold 8 12.8abc 43 11.2bc 50 0.5 1.0
(2-13, 2-14, 3-10)
NJ672017002 7.00 71 5.4de 71 2.3 3.0
(1-8, 2-32, 4-16)
Yennoh 7.1bc 51 3.2e 39 2.5 3.0

(1-31, 1-39, 4-11)

 

Data is the mean of three progeny per selection.

V Different letters are significantly different according to LSD
at 0.05 level.

40

Table 2. Specific hydraulic conductance per unit length (specific
Kn)” in 10's szPa'1s'1“ and percent specific K1, of experiment/control
Kh (% specific Kn)” for W canker infection from artificial
inoculations in the fall

of 1986 and 1987 of open-pollinated progeny of peach.’

 

 

 

Specific Kn“ % Specific Kh
Selection 1986 c.v. 1987 c.v. 1986 c.v. 1987 c.v.
Susceptible seedlings
Loring 0.58cz 49 1.02b 48 5.9cd 49 8.8bc 48
(2-7; 11-27, 11-32)
Elberta 0.92bc 76 1.52ab 69 6.0cd 65 17.9bc 70
(6-32, 11-34, 11-38)
Canadian Harmony 1.81abc 68 0.78b 44 14.2abcd 42 5.9c 47
(3-17, 3-20, 5-9)
Harken 0.63c 20 0.82b 73 5.4d 41 11.4bc 80
(2-6, 2-29, 2-44)
Intermediate seedlings
Reliance 1.18bc 85 0.93b 69 10.9de 41 11.8bc 70
(1-33, 1—41, 15-16)
Babygold 8 1.81abc 56 2.81ab 34 20.3a 67 20.7ab 47
(2-13, 2-14, 3-10)
Resistant seedlings
NJ672017002 3.103 54 3.44a 80 15.2abc 101 21.4ab 62
(1-8, 2-32, 4-16)
Yennoh 2.48ab 48 3.16a 61 18.5ab 29 33.9a 38

(1-31, 1-39, 4-11)

 

Hydraulic conductance (KL) = volume of water'(n9) x length of
segment (cm) / seconds (5) x height of segment (cm) x gravity
gradient (MPa/m). The S.I. unit of Kh is m‘s'1MPa'1. Specific Kh =
Kh /cross sectional area of segment (m2) = X (mzs'1MPa'1) .
Percent Specific Kn = (Specific Kh of experimental stem
segment/specific Kr. of control stem segment) x 100.
y Data is the mean of three progeny per selection.

Different letters are significantly different according to LSD at
0.05 level.

41
inoculation and the highest canker rating (Table 1). These
seedlings were classified as resistant clones. Likewise the
open-pollinated progeny from Loring, Elberta, Canadian
Harmony, and Harken vs those from Reliance and Babygold 8
were classified as susceptible and intermediate tolerance,
respectively.

The specific Kh calculated as Kh divided by cross-
sectional area was significantly different among the
seedlings of the 8 peach clones (Table 2). For the resistant
peach clones, the % specific K1. was 20-30% of the healthy
control branches while in susceptible peach clones, the %
specific Kh was 5-18%. The variation in specific Kr. and %
specific K1, was quite large and probably attributed to the
variation among the stems which could be due to size, age,
vigor of the plants plus summing over the 3 seedlings which
are genetically different in the experiment. Despite the
variation in Table 3, Rq‘was clearly reduced by Leucostoma
infection and the resistant plants were capable of
transporting more water through the infected zone compared
to the susceptible plants. This is probably why the
resistant branches remained healthy 7 months after
inoculation and infection. Also, the canker necrotic length
and canker rating were correlated with % specific Kh
(r=0.74, 0.75 respectively). The longer the canker extension

and the less water transport and more severely wilting.

42

Vessel elements which stained with Safranin-O were
visibly reduced in the inoculated branches compared to the
non-inoculated control indicating that infection decreased
the amount of conductive xylem tissue (Fig. 1). However, in
the tolerant clones the extent of xylem damage following
inoculation was less than that for the susceptible clones.
In the susceptible clones the outer xylem ring was no longer
functional while in the tolerant clones the outer and some
of the inner xylem vessels remained functional (Fig. 2).
Since the branches had been inoculated in October and
evaluated in May, the presence of these functional xylem
vessels would be critical during the growing period.

Above the necrotic zone of L. persgpnii infection,
there was no difference in KL, specific Kh and quantity of
dyed functional xylem vessels between the control and
inoculated branches. This indicated that the vessel damage
and subsequent reduction in.B% and specific K5 is fairly
localized around the infected canker area.

The dramatically decreased water transport through the
localized necrotic longitudinal zone following L. persoonii
infection was associated with disease susceptibility. Where
the vascular cambium was not damaged, new xylem and phloem
could be produced to replace the damaged tissues and keep
the plants alive. This is important to the survival of

infected branches. It is clear that the resistant reaction

43

Table 3. Percent infection of open-pollinated progeny of peach as
calculated as re-isolation of Leuggstoma from the wounds of
artificial inoculation.u

 

Percent
Selection infection Coefficient
of progeny of variation

Loring 90aV 17
(2- : 11-27; 11-32)

Elberta 71° 52
(6-32: 11-34: 11-38)

Canadian Harmony 97a 6
(3—17; 3-20: 5-9)

Harken .95a 10
(2-6: 2-29: 2-44)

Reliance 70' 35
(1-33: 1-41; 15-16)

Babygold 8 20a 12
(2-13; 2-14: 3-10)

NJ672017002 9b 133
(1-8: 2-32; 4-16)

Yennoh 5b 109
(1-31: 1-39: 4-11) .

Data is the mean of three progeny per selection.

V Different letters are significantly different according to LSD
at 0.05 level.

44
Figure l. Cross-sections of a pair of the disease susceptible Can.
Harmony (3-17) peach stems used for hydraulic conductance
measurements. The stem on the left was inoculated with
Leucostoma, and the stem on the right was the control.
Red areas indicated when safranin-O (0.5%) moved through
the functional xylem area in the middle of inoculated

wound.

45

 

Figure 1.

46

Figure 2. Cross-sections of a pair of the disease resistant
NJ672017002 (1-8) peach stems used for hydraulic
conductance measurements. The stem on the left was
inoculated with Leucostoma, and the stem on the right was
the control. Red areas indicated when safranin-O (0.5%)
moved through the functional xylem area in the middle of

inoculated wound.

 

47

 

Figure 2.

48
of the clones investigated is in part due to the production
of new xylem and phloem, and in part due to the capability
of the inner xylem to maintain water transportation.

To understand the involvement of the L. persoonii
fungus in disrupting xylem flow, attempts were made to
determine the depth of L. pepsoonii invasion into the xylem
area. In susceptible clones, it was possible to isolate the
fungus from the margin of the surface necrosis down to the
pith area. For the resistant clones, the fungus was isolated
only as deep as the secondary layer (approximately 2mm)
beneath the margin of the surface necrosis. Percent
infection was determined by isolation of Leucostoma from the
wounds following artificial inoculation. Yennoh and
NJ672017002 seedling clones had less than 10% infection,
however, the susceptible ones had more than 90% infection
(Table 3). The lack of penetration of the fungus through the
wood of the resistant clones suggests that Yennoh and
NJ672017002 seedlings may be limiting the advance of the
pathogen.

In conclusion, xylem dysfunction following L. persoonii
infection of susceptible clones is associated with wilting
and death of branches. The canker pathogen invaded the wood
xylem in the susceptible peach seedlings and significantly
reduced the water transport through the infection zone. In

the resistant clones, the xylem remains functional following

49

branch inoculation and the vascular cambium is able to
continue to differentiate new xylem and phloem to replace
the damaged tissue, perhaps because of reduced penetration
by the fungus. Therefore, the tolerant clones were able to
maintain adequate water transport through the necrotic
canker zone during the summer following inoculation.

A determination of the actual cause of branch death in
susceptible clones still needs further study because both
xylem and phloem are damaged by the pathogen and we know

little of the role of phloem in the disease.

Literature Cited
Banko, T.J. and A.W. Helton. 1974. Cytospora-induced
changes in stems of Prunus persica. Phytopathology
64:899-901.
Biggs, A.R. 1984. Boundary-zone formation in peach bark
in response to wounds and Qytospora leucostoma
infection. Can. J. Bot. 62:2814-2821.
Biggs, A.R. 1986a. Wound age and infection of peach bark
by Qytospora leucostoma. Can. J. Bot. 64:2319-2321.
Biggs. A.R. 1986b. Comparative anatomy and host response
to two peach cultivars inoculated with Leucostoma cincta
and L. persooni'. Phytopathology 76:905-912.
Biggs, A.R. and L.W. Stobbs. 1986. Fine structure of the
suberized cell walls in the boundary zone and
necrophylactic periderm in wounded peach bark. Can. J.
Bot. 64:1606-1610.

Chang, L.S., A. Iezzoni, G. Adams and G.S. Howell. 1989.

Leucostoma tolerance and cold hardiness among diverse

peach genotypes. J. Amer. Soc. Hort Sci. 114:482-485.
Chang, L.S., A. Iezzoni, and G. Adams. 1989. Excised

twig assay for tolerance of peach Lgucostoma canker.

HortSci. (in press).

Cline, R.A. 1982. Cultural practices in peach canker

development in Ontario. Proc. Stone Fr. Decline

10.

11.

12.

51
Workshop. E. Lansing, MI. pp. 30-33.
Evert, D.R. 1987. Influence of phony disease of peach on
stem hydraulic conductivity and leaf xylem pressure
potential. J.A.S. H.S. 112-1032-1036.
Ewers, F.W., P.S. McManus, A. Goldman, R. Guice, and
D.W. Fulbright. 1989. The effect of virulent and
hypovirulent strains of Endotpia parasitica on hydraulic
conductance in American chestnut blight. Can. J. Bot.
(in press).
Gairola, C. and D. Powell. 1970. Cytospora canker in
Illinois. Pl. Dis. Rep. 54:832-835.
Hampson, M.C. and W.A. Sinclair. 1973. Xylem disfunction
in peach caused by Cytospora leucostoma. Phytopathology

63:676-681.

13.J6nes, A.C. and N.S. Luepschen. 1971. Seasonal

14.

15.

16.

development of Cytospora canker on peach in Colorado.
Pl. Dis. Rep. 55:314-317.

Hildebrand, E.M. 1947. Perennial peach canker and the
canker complex in New York with methods of control.
Cornell Univ. Agr. Exp. Stn. Mam. 276:61.

Layne, R.E.C. 1976. Influence of peach seedling
rootstocks on perennial canker of peach. HortScience
11:509-511.

Leonion, L. H. 1921. Studies on the Valsa apple canker

in New Mexico. Phytopathology 11:236-243.

17.

18.

19.

20.

21.

22.

23.

52
Luepschen, N.S. 1981. Criteria for determining peach
variety susceptibility to Cytospora canker. Fruit Var.
J. 35:137-140.
Luepschen. N.S. and K.G. Rohrback, A.C. Jones, and L.E.
Dickens. 1975. Susceptibility of peach cultivars to
Cyppspppa canker under Colorado orchard conditions.
HortScience 10:76-77.
Sperry, J.S., J.R. Donnelly, and M.T. Tyree. 1988. A
method for measuring hydraulic conductivity and embolism
in xylem. P1. Cell Environ. 11:35-40.
Tekauz, A. and Z.A. Patrick. 1974. The role of twig
infections on the incidence of perennial canker of
peach. Phytopathology 64:683-688.
Wensley, R.N. 1964. Occurrence and pathogenicity of
Valsa (Cytospora) species and other fungi associated
with peach canker in southern Ontario. Can. J. Bot.
42:841-857.
Wisniewski, M., A.L. Bogle and C.L. Wilson. 1984.
Histopathology of canker development on peach trees
after inoculation with Cytospora leucostoma. Can. J.
Bot. 62:2804-2813.
Zimmermann, M. H. 1978. Hydraulic architecture of some

diffuse-porous trees. Can. J. Bot. 56:2286-2295.

CHAPTER FOUR

Quantification of lignin content in wounds of

peach clones selected for their resistance to

Leuco m persoonii

53

54

Abstract

Two peach cultivars and three selected peach clones
that vary in their resistance to Lepgpstoma persoonii from
highly susceptible to resistant were examined for their
response to wounding. Field grown trees undergoing cold
temperature acclimation were wounded to a depth of 2mm and
lignin deposition in the wounded bark and wood to a 4mm
depth was quantified using a thioglycolic acid assay. The
measured constitutive lignin quantity was correlated with

resistance in the field.

Introduction

Perennial canker of peach (Prunus persica (L.) Batch)
which is caused by Leucostoma persoonii (Nits.) Hohn.
(imperfect stage Leucocvtospora leucostoma (Pers.) Hohn) and
L. cincta (Fr.) Hohn. (imperfect stage Leucocytospora cincta
(Sacc.) Hohn.), is the most serious disease reducing peach
tree life in the colder areas of peach production. The
disease is characterized by perennial cankers that may kill
large branches and weaken scaffold resulting in the eventual
splitting of branch crotches under heavy crop loads.

Successful invasion by the pathogen requires the presence of

55
wounds or dead tissues on the host plants (Willison, 1933).
Various histopathological investigations of perennial canker
classified Lgucospoma as a facultative bark parasite
(Wisniewski et a1, 1984; Biggs, 1984, 1986). The mode of
pathogenesis of Leucostoma involved necrosis of the bark
tissues. Alternatively, Lgucpspoma was described as a
sapwood parasite with necrosis first progressing through
xylem tissues prior to invading the bark (Banko and Helton,
1974; Tekauz and Patrick, 1974). Recently, it has been shown
that the pathogen invades xylem tissues significantly
reducing water transport (Chang, 1989).

The formation of lignin and wound periderm has been
recognized as an important mechanism of disease resistance
in many plant species (Vance et a1, 1980; Hammerschmidt,
1984). Wound-induced lignification in almond bark has been
associated with slower expansion of cankers caused by
Phytophthora species (Doster and Bostock, 1988). Wilson
(1982) suggested that efficient xylem compartmentalization
would increase peach longevity. Previous data further
indicated that the ability to heal wounds may play a part in
the ability of different peach cultivars and clones to
resist Leucostoma (Willison, 1933: Wensley, 1966; Weaver,
1963). Biggs (1989) also demonstrated that increases in the
rate of suberin accumulation following wounding were

correlated with observations of resistance of peach

56
cultivars in the field to infection by Leucostoma.

After screening over 700 peach seedlings in a
germplasm population, we identified several individuals
which exhibited a higher tolerance to Leucostoma than had
previously been reported in the United States (Chang, et a1,
1989). It was found that L. persoonii invaded the wood
tissue reducing the hydraulic conductance in susceptible
genotypes: whereas, in the resistant genotypes the xylem was
not invaded (Chang, 1989).

The purpose of this research was to determine relative
lignin content following wounding in several peach clones
identified as susceptible and resistant to Leucostoma.
Lignin deposition was quantitatively assayed using
thioglycolic acid. This assay was adapted by Doster and
Bostock (1988) for investigating lignification in inner bark

of almond trees.

Materials and Methods
Plant materials:
In spring 1984, open-pollinated peach seedlings from
15 peach clones,two peach * 2. kansuensis hybrids, and one
peach * almond (2. dulcis Webb.) hybrid were planted in a
completely randomized design at the Horticultural Research
Center, East Lansing, Michigan. Open-pollinated seedlings

were used to maximize the genetic diversity in the

57
population to be screened. These seedlings , representing
half-sib families with progeny number ranging from three to
73, were evaluated twice for canker necrotic length
following inoculation with L. persooni'. Two 2-year-old
branches were inoculated using the techniques developed by
Scorza and Pusey (1984). Branches for the two experiments
were inoculated in October 1985 and 1986 and evaluated in
May 1986 and 1987, respectively. Two branches per seedling
were each inoculated with 20 ul of a suspension of 107.L-
persoonii conidia/ml derived from a mixture of two isolates
collected in Hartford and Clarksville, MI. Canker necrotic
length was measured as the length of necrotic area distal to
the point of inoculation (canker necrotic rating).
Additionally, inoculated branches were visually rated for
canker using a scale where 1=dead, 2=severe wilting of
expanding leaves, 3=weak growth, 4=healthy (Chang et a.,
1989).

In 1987, eight peach seedlings that were identified
as being resistant (Yennoh 1-39, NJ672017002 1-8, B8-20-171
17-45), intermediate (Babygold 8 16-11, NJ257 2-15, Reliance
3-1), or susceptible (Loring 18-30, Elberta 10-40) were
selected for the lignin analysis. The open-pollinated
seedlings are identified by the maternal parent, i.e.
Yennoh, and the seedling clone number, i.e. 1-39.

In 1986, two resistant and one intermediate seedling

58
plus the cultivars Yennoh and Loring were grafted on Lovell
rootstock and then planted in 1988 at Horticultural Research

Center adjacent to the seedling block.

Wounding and Lignin analysis:

Two 2-year-old healthy branches per seedling which
were chosen for wounding, were wounded by insertion of a 4
mm diameter cork borer thrbugh the bark to the xylem. Two
days after wounding, the bark and 2 mm depth of wood was
removed by a 10 mm diameter cork borer. This procedure was
repeated four times on each branch from Sept. 6 to Sept. 12,
1987. The dead outer bark and browned tissue were carefully
peeled or cut away from the periderm of each 10 mm disk
prior to lignin extraction. The tissue sample were then
extracted in methanol and the lignin was quantified as
ligninthioglycolic acid (LTGA) (Doster and Bostock,1988).

Additionally, four trees of Loring and Yennoh, plus
the seedlings Yennoh (1-39) and NJ672017002 (1-8) were
selected for wounding on September 20, 1989. Two 2-year-old
branches, approximately 10 mm in diameter were wounded by
the triggered impact with the impeller of an empty staple
gun. Samples were removed by a 10 mm cork borer to the 3 to
4 mm depth of wood then analyzed for lignin content (LTGA).
Comparisons were made with samples taken from unwounded

controls at day 0 and 2, 4, 8, 14, and 21 days after

59

wounding.

Result and Discussion
Since LTGA production can be viewed as an accurate
representation of the relative lignin content in the tissue
(Freudenberg, 1968), the LTGA data will be discussed as
such.

The methanol-extracted tissues from eight peach
seedlings yielded significantly different relative values of
lignin (Table 1). The resistant Yennoh 1-39, NJ672017002 1-
8, and B8-20-171 17-45 produced twice as much lignin as the
susceptible seedlings Loring 18-30, Reliance 3-1, and
Elberta 10-40. Babygold 8 16-11 which had intermediate
resistance to L. persoonii, also had intermediate lignin
levels. For the resistant clones (Yennoh 1-39, NJ672017002
1-8), lignin was significantly higher than in the
susceptible cultivar Loring and intermediate clone NJ257 2-
15 at all sampling times (Table 2). For the resistant
clones, both the unwounded and wounded tissues had
significant amounts of lignin content. Despite the increase
in lignin in Yennoh, NJ257 (2-15) and Loring in response to
wounding, the lignin content of these selections was still
less than that produced by the resistant clones.

Resistance of peach to Leucostoma persoonii in our selected

clones was previously characterized by the apparent failure

60

Table 1. Relative lignification" detected in wounded bark and
wood two days after wounding compared with mean disease symptom
and canker necrotic length rating.

 

 

Meanx mean

disease canker RelativeV
Seedling symptom necrotic length lignification
NJ672017002 1-8 4.0 5.0 16.18az
Yennoh 1-39 4.0 4.8 15.09a
88-20-171 17-45 4.0 4.9 15.01a
Babtgold 8 16-11 2.5 8.0 11.45ab
NJ 257 2-15 2.0 7.2 9.00b
Loring 18-30 1.0 12.0 8.89b
Reliance 3-1 2.5 8.6 8.83b
Elberta 10-40 1.0 13.2 7.79b

 

Relative lignification was measured in bark and wood tissue
with 2mm depth.

1986 and 1987 data combined. Canker rating: 1=dead, 2=severe
wilting, 3=weak growth, 4=healthy.

V LTGA yield expressed as A280 nm per gram tissues in 5 ml of
0.5N NaOH.

2 Different letters are significantly different according to
L.S.D. at 0.05 level.

61

Table 2. Relative lignification of unwounded and wounded tissues"
sampled 2, 4, 8, 14, and 21 days postwounding (DAW) for five
peach families.

 

Relative lignin contentx

 

 

 

Yennoh NJ672017002 Yennoh NJ257 Loring

Treatment (1-39) (1-8) (2-15)

Unwound 20.07aV 19.37a 8.47bc 11.48b 6.09c
2 DAW 18.76a 20.06a 9.54b 11.89b 5.46c
4 DAW 20.13a 20.13a 7.93b 7.30b 4.97b
8 DAW 17.25b 20.92a 13.05c 9.17d 6.07e

14 DAW 20.99a 20.42a 19.11a 9.13b 8.08b

21 DAW 22.21a 20.16a 19.76b 14.18c 13.47c
Mean 20.50a 19.90a 12.98b 10.19b 7.34b

 

w Tissues included bark and wood from 0 to 4 mm deep into the
wood.

x Relative lignification was measured as LTGA yield and expressed
as spectrometer absorption at A280 nm per gram tissue in 5ml of
0.5N NaOH.

y Different letters are significantly different according to
L.S.D. at 0.05 level.

62

of the fungus to penetrate into wood even though there was
some growth on the surface of the wood (Chang, 1989). This
observation may be attributed to the resistant peach clones
containing significant amounts of lignin: the lignin
preventing further penetration by the fungus. Biggs (1984)
found that Lgucostoma could penetrate wound periderm of the
Loring cultivar of peach. If Leucostoma became established
in a wound in a susceptible host such as Loring, the small
amount increase in lignin deposition might represent an
insufficient defense mechanism. In contrast, in our
resistant peach seedlings, the lignin may be able to
prevent damage the xylem vessels so they remained functional
seven months after inoculation and the vascular cambium
continued to differentiate new xylem and phloem during the
growing season to replace the damaged tissues (Chang, 1989).

Although a good correlation between constitutive
lignin quantification and resistant performance in the field
has been demonstrated, the ability of lignin to wall-off the
pathogen has not been tested because the wounding was not
followed by inoculation. However, Doster and Bostock (1988)
found that lignin deposition in almond inner bark appeared
to be a response to wounding and infection by Phytophthora.

During the first eight days after wounding, the
cultivar Yennoh did not have a significantly increased

amount of lignin, and the major accumulation occurred two

63
weeks after wounding. It is interesting that its open-
pollinated progeny Yennoh 1-39 has higher lignin production.

Previous literature associating lignin with disease
resistance discusses the rate of lignin or suberin
accumulation in response to wounding and/or inoculation
(Doster and Bostock, 1988: Biggs, 1989). However, our time-
course data indicated that our resistant selections, Yennoh
1-39 and NJ672017002 1-8, initially contained high lignin
amounts that did not change in response to wounding. Even
the unwounded resistant controls have significantly higher
amounts of constitutive lignin than that found to accumulate
in susceptible clones 21 days postwounding. If lignification
is to play a major role in restricting pathogen development,
it must occur early.

Previous studies have shown that host resistance to
Leucostoma differs, and these differences might be due to
time of defoliation (Weaver, 1963), rate of wound healing
(Wensley, 1966), and rate of suberization (Biggs, 1989).
These host resistant mechanisms might be expected during the
rapid growing season. However, these mechanisms might be of
little value during the dormant period when little formation
of wound periderm occurs (Layne, 1984). Our studies of
resistance to Leucostoma in peach and lignin deposition
differ fundamentally from similar studies on suberin

deposition by other researchers because of differences in

 

64
physiology of peach during the dormant season. In initial
field trials, a diverse peach population was inoculated in
spring and fall with L. persoonii (Chang et a1, 1989).
Spring inoculation lead to callus formation in susceptible
and resistant genotypes and no statistically significant
difference were measurable. In contrast, fall inoculations
caused rapid necrosis of functional xylem in early spring
followed by rapid death of inoculated branches in
susceptible genotypes and survival of inoculated branches in
resistant genotypes (Chang et al, 1989).

While other workers have studied suberin formation in
peach that were wounded during the growing season (Biggs and
Miles, 1988), or rate of suberin accumulation in wounded and
inoculated seedlings growing in the greenhouse (Biggs,
1989), here we purposely conducted wounding experiments
during the dormancy in the field. The constitutive lignin
quantity measured in our study of resistant genotypes
apparently is present year-round and continues to be
expressed during the dormant period when other disease
defense mechanisms are likely non-functional. By inoculating
the seedling populations following fall acclimation, we were
imposing a severe disease pressure on the trees, and may
have been sufficiently severe to have inadventantly selected
for those genotypes which accumulate large amounts of lignin

during acclimation.

65

Literature cited
Banko, T. J. and A. W. Helton. 1974. Cytospora-induced
changes in stems of Eppppg pepsiga. Phytopathology
64:899-901.
Biggs, A. R. 1984. Boundary-zone formation in peach bark
in response to wounds and gytospora leucostoma
infection. Can. J. Bot. 62:2814-2821.
Biggs, A. R. 1985. Prediction of lignin and suberin
deposition in boundary zone tissue of wounded tree bark
using accumulated degree days. J. Amer. Soc. Hort. Sci.
111:757-760.
Biggs, A. R. 1986. Wound age and infection of peach bark
by Cytospora leucostoma. Can. J. Bot. 64:2319-2321.
Biggs, A. R. 1989. Temporal changes in the infection
court after wounding of peach bark and their association
with cultivar variation in infection by Leucostoma
persoonii. Phytopathology 79:627-630.
Biggs, A. R. and N. W. Miles. 1988. Association of
suberin formation in uninoculated wounds with
susceptibility to Leucostoma cincta and L. persoonii in
various peach cultivars. Phytopathology 78:1070-1074.
Chang, L. S. 1989. Hydraulic conductance in susceptible
vs. resistant peach clones infected with Leucostoma
persponi'. Ph.D. Dissertation, Michigan State

University, East Lansing, MI.

10.

11.

12.

13.

14.

15.

66
Chang, L. S., A. Iezzoni, G. Adams, and G. S. Howell.
1989. Lppgpspppa tolerance and cold hardiness among
diverse peach genotypes. J. Amer. Soc. Hort. Sci.
114:482-485.
Doster, M. A. and R. M. Bostock. 1988. Quantification of
lignin formation in almond bark in response to wounding
and infection by Phytophthora species. Phytopathology
78:473-477.
Hammerschmidt, R. 1984. Rapid deposition of lignin in
potato tuber tissue as a response to fungi non-
pathogenic on potato. Physiol. Pl. Pathology 24:33-42.
Layne, R. E. C. 1984. Breeding peaches in north America
for cold hardiness and perennial canker (Leucostoma
species) resistance-review and outlook. Fr. Var. J.
38:130-136.
Tekauz, A. and z. A. Patrick. 1974. The role of twig
infections on the incidence of perennial canker of
peach. Phytopathology 64:683-688.
Vance, C. P.,T. K. Kirk, and R. T. Sherwood. 1980.
Lignification as a mechanism of disease resistance.
Annu. Rev. Phytopathology 18:259-372.
Weaver, G. M. 1963. A relationship between the rate of
leaf abscission and perennial canker in peach varieties.
Can. J. Plant Sci. 48:37-47.

Wensley, R. N. 1966. Rate of healing and its relation to

67

canker of peach. Can. J. Plant Sci. 46:257-264.

16. Wilson, C. L. 1982. Peach tree wounds and decline. Proc.

Stone Fruit Decline Workshop (18-20 Oct.), Michigan

State University, East Lansing, MI.

I7.Willison, R. S. 1933. Peach canker investigation. 1.

18.

some notes on incidence, contributing factors, and
control measures. Sci. Agr. 14:32-47.

Wisniewski, M., A. L. Bogle, and C. L. Wilson. 1984.
Histopathology of canker development on peach trees
after inoculation with gytospora leucostoma. Can. J.

Bot. 62:2804-2813.

CHAPTER FIVE

Heritability of Leucosgppg pgpsoonii canker resistance

among diverse peach genotypes

68

69

Abstract

Heritability of resistance to the canker causing
pathogen, Leucostoma ersoon", was estimated in a
population of diverse peach genotypes. Disease resistance
was measured as the amount of necrotic tissue, i.e. canker
length, following artificial inoculation in the field. The
heritability was estimated by (1) partitioning the variance
components in the least statistical model, and (2) parent-
offspring regression by regressing the average performance
of the seedlings on the mean performance of its female
parent. The two estimates of heritability for canker

necrotic length were very similar, 0.65, 0.72, respectively.

Introduction
Perennial canker, incited by Leucostoma persoonii or
L. c' ta, is recognized as a severe disease limiting peach
longevity and productivity in the colder areas of peach
production (Cline, 1982; Gairola and Powell, 1970:
Hildebrand, 1947; Jones and Luepschen, 1971: Layne, 1976.
1984: Luepschen, 1981: Wensley, 1964). Since both cultural

practices and chemical treatments do not adequately control

70
the disease, the ultimate approach is through host plant
resistance. Studies of peach susceptibility to Leucostoma
infection based on either natural infection or artificial
inoculation have been conducted. However, almost all
commercial peach cultivars failed to have resistance to the
canker pathogen. (Dhanvantari and Dirk, 1981; Dhanvantari,
1978; Gairola and Powell, 1970, Hildebrand, 1947; Layne,
1984; Luepshen, 1981, Palmiter and Hickey,1970). Where some
level of resistance was found, the reaction tended to be
inconsistent among experiments. Possibly this lack of
resistance to Leucostoma can be attributed to the narrow
genetic base of peach cultivars in North America (Layne,
1976, 1984). Following the inoculation of a diverse peach
population and subsequent evaluation of disease resistance
and cold hardiness, we reported that some peach genotypes
may have a higher tolerance to Leucostoma canker than has
been previously found in U.S. germplasm (Chang et al. 1989a:
Chang et al. 1989b).

Studies of various tree crops including sweet cherry,
peach, nectarine, walnut, and plum have shown considerable
additive genetic variability for commercially important
traits (Hansche, Beres and Brooks, 1966; Hansche, Hess and
Beres, 1972: Hansche, Beres, and Forde, 1972; Hansche,
Hesse, and Beres, 1975: Hester, Hanche, Beres, and Asay,

1977: Hansche, 1986, 1986). A knowledge of heritability

71
estimates is useful as a guide for breeders in improving
tree crops and in maximizing the breeding efficiency.
This study was undertaken to estimate the genetic and
environmental parameters and to examine the heritability of

Lepcpstpma resistance traits in a diverse peach population.

Materials and Methods

Heritability estimates were obtained by two methods.
The first method used the linear statistical model. Least
squares estimates were calculated for the variance among the
peach families, year, year and peach family interaction,
progeny within peach family, year and progeny within peach
family interaction, sampling branches within progeny within
peach family, and year and sampling branches within progeny
within peach family interaction. The second method involved
a linear regression of offspring performance on the average
performance of its female parent.

(1) Linear Statistical Model

 

In Spring 1984, open-pollinated peach seedlings from
diverse background were planted in a completely randomized
design at the Horticultural Research Center, East Lasing, MI
(Chang et al., 1989). Open-pollinated peach progeny, instead
of clones, were used to increase the genetic diversity of
the population to be screened. From this population, 14

peach families with at least 6 progeny were chosen to

Table 1. Parents

72

of the 18 clones used in the heritability

 

 

estimates of Lgpppsppma resistance

Parents used
Clone in
Parent the model
Babygold 8 P135201 x Ambergen LSy
Canadian Harmony Redskin x Sunhaven PORx
Elberta Chinese Cling (open-pollinated) POR
Glohaven (J.H. Hale open-pollinated) x Kalhaven
Harken Redskin x Sunhaven POR LS
Loring Frank x Halehaven POR LS
Reliance (Minn. PH04559 x Meredith) open-pollinated
Red Hale Unknown LS
Yennoh Plant Introduction from Russia
B8-1l-147 (K82xSunrise)x[(Red CxNJ19l)kainawa] POR LS
B8-20-171 (5110417 x Ta Tao 3) x C2R31T45 LS
B8-21-20 Orange Cling x RR65-l LS
C2-28-89 Kasna Dupnishka open-pollinated POR LS
C4-11-97 peach x almond POR LS
NJ257 Honeydew Hall x Jefferson LS
NJN69 (NJNSS x NJC68) x Marzochella POR LS
NJ672017002 (P135321 x Cherryred) x Prunuétansuensis LS
RR37-15 NJ 174 x Prunuskansuensis LS

 

X

Parent-offspring regression

V Least square model

73
estimate the heritability of L. persoonii resistance (Table
1).

Two 2-year-old branches of about 17 mm in diameter on
each seedling were inoculated with 20ul of a suspension of
longmppstpma,pgp§pppii conidia per milliliter derived from
isolates collected from cankers on peach at Clarksville and
Hartford, Mich. A wound-freezing inoculation technique
developed by Scorza and Pusey (1984) was followed. The trees
were inoculated in early October of 1985, 1986, and 1987,
then evaluated in the spring when the trees began to leaf
out. All the inoculated branches were measured for length of
canker necrosis, distal to the point of inoculation.
Additionally, inoculated branches were rated visually for
disease symptoms using a scale: 1=dead, 2=severe wilting of
expanding leaves, 3=weak growth and 4=healthy (disease
symptom rating). Fourteen peach families with 6 progeny were
inoculated and evaluated for canker infection over three
years. They were chosen to estimate the component variance
and used to estimate the heritability in the seedling
population of resistance to Leucostoma infection. The
seedlings in this study represented open-pollinated half-
sib families.

Phenotypic variability of all observations of canker
necrotic length on this diverse collection of peach progeny

over all 3 years is described by the following model:

74

YUH=U + F, + Pij + Sm + Y. + (Fir)il + (Pym, + EU.kl
where Yum represents k sample branch with jth progeny
within ith peach family in the 1th year. The effects in this
model stand for the overall population mean (u); a random
effect contributed to each family (F3): the effect of
progeny within family (PU); the effect of sampling branches
within progeny within peach family (8”,): the year effect
(Y;): the interaction of family * year ((FY)“); the error
due to the year * Sijk (Emu) .
(2) Parent-offspring Regression

Eight randomly selected female parents which had been
grafted on Tennessee peach rootstock were planted in blocks
adjacent to the orchard of diverse peach progeny in 1985.
Ten grafted trees per clone were selected for artificial
inoculation. The two randomly selected healthy branches of
each grafted tree and each progeny were inoculated as
described above the same day. Using data from 1986 and 1987
inoculations, the performance of the female parent was rated
as the mean canker length over two years of 10 replicated
clones. The performance of the progeny from the eight
families was rates as the mean disease response over two
years of inoculation.

These data were based on unadjusted data for the year-

effect, because no significant variation doe to year effect

has been evident in previous studies (Chang et al., 1989).

75
The heritability was based on the regression of the mean of
the performance of peach offspring on the average

performance of its female parents.

Results and discussion
(1) zppippgg Components

The expected and actual mean squares of the linear
statistical model are present in Table 2. The year variation
over three years data did not dramatically influence the
peach genotypic performance rather performance was based on
the canker necrotic length rating or on the disease symptom
rating following artificial inoculation in the field. Since
the year variability is relatively small, it does not
significantly decrease the efficiency of selection of the
more disease resistant genotypes among the diverse peach
population. However, statistically estimating the
variability due to year, and statistically removing the
yearly environmental effect would increase the selection
efficiency and the rate of genetic gain.

The mean squares of year * sampling branches within
progeny within family (BUN) and mean squares (Sin) of
sampling branches within progeny within family are
attributable to the environmental effect. The year *
sampling branches within progeny within family is quite

large, which might be due to environmental effects on cold

76

Table 2. The analysis of variance of the linear statistical model
of heritability of canker resistance among half-sib families

 

Sources of Mean Expected Mean
Variation d.f. Squares Squares of

 

Among peach families (15'i ) 13 179.03 V2+yV2b+bV2~+byV2H+bnyvzf
Progeny within family (PH) 68 38.48 V2+yV2b+bV2W+byV2u
Year (in) 2 6.28 V2+bV2YH+an2Yf+ban2y
Year * peach family (FY)il 26 32.96 v2+bv2w+bnvzyf
Year * progeny

within family (109”) 136 25.02 V2+bV2W
Sampled branch within progeny

within family (Sijk) 82 8.54 V2+yv2b

y * sijk (Emu) 163 13.55 v2

 

V: Standard deviation

y: no. of years

b: no. of sample branches

n: no. of sibs/half-sib families

f: no. of half-sib families

77

hardiness in different years or on different sampling
branches within each tree. The inoculated branches were
removed from the trees after canker evaluation each year.
The error variance probably is attributable to the
variations among branches such as in size, age, year, and
orientation as well as to branch response to different
environmental effects in different years. Error due to
sampling branches within progeny within family was negative
and thus is ignored. If combining two error terms to one,
the actual values for the error was 11.87. In the estimated
variance components, variability due to year was rather
small of negative value,if assuming 0 instead of negative
values. However, the interaction between year and peach
family was relatively low and was not significant. This
indicated that performance was stable among the peach clone
families over the tested 3 years. The estimated variance
component, due to the progeny within family, was relatively
large. Doubtless, this error term for peach families was a
combined effect of genetic and environmental variability.
Therefore, it was not appropriate to estimate how much
variability due to the environmental effect without clonal
propagation.

The component variance due to the family effect is the
covariance of half-sib families and this is equal to one

quarter of the additive genetic variance (Falconer, 1980).

78
The summation of all the variance components represents the
phenotypic variance,‘v'>2 in this diverse peach seedling
population (see statistical model, Table 3). The narrow
sense heritability was calculated as the additive variance
over the phenotypic variance. The heritability of canker
necrotic length was 0.65.
(2) Parent-offspring Regression

The parent-offspring regression is commonly used to
estimate the heritability of quantitative traits in
different crop species. Progeny mean values are usually
regressed on values from one or two parents, depending on
pollination control. Peach presumably is highly self-
pollinated (Hesse, 1976: Hansche 1986). The performance of
the female parent has been used to determine the mid-parent
performance (Hansche 1986a, 1986b). According to a report by
Falconer (1981), use of the parent performance will cause
the heritability estimate to be biased downward by about 5%.
The linear regression coefficient gives the estimate of
heritability as the ratio of additive genetic variance to
phenotypic variance of the parents.

Generally, the mean canker necrotic length rating was
lower in the seedling population compared to the parental
performance over the 2 years of observations (Table 4). The
apparent increase in resistance to Leucostoma infection seen

in the progeny may have occurred by self-pollination

79

Table 3. Estimates of variance components of the canker necrotic
length rating of a diverse peach seedling population inoculated
during fall of 1985, 1986 and 1987 and evaluated during spring of
1986, 1987, and 1988.

 

Actual values
in estimating

 

Variance components Values heritability
Error (v2) 13.55 11.87"
Sample branch (Vzb) -1.69

Year * progeny within family (Van 5.73 5 73

Year * Family (Vad) 0.66 0.66

Year (V2,) -0.16 0.00
Progeny within family (vzu) 2.48 2.48
Half-sib family (Va) 4.00 4.00

 

V: Standard deviation

‘ Summation of error variance

 

80
Table 4. The mean values of the canker necrotic length on diverse

peach genotypes.

 

 

Parent canker Offspring canker
Clonene crotic lengthx necrotic lengthV
(cm) (cm)
NJN69 13.48 11.13
Elberta 13.08 12.76
Harken 11.02 9.36
Loring 10.34 9.58
Can. Harmony 9.67 8.28
C2-28-89 9.26 9.43
88-11-147 8.26 7.40
C4-11-97 5.06 5.68

 

‘ mean of 10 clones over 2 years, the performance of female

parent was estimated by the mean of the necrotic length ratings
from 10 replicated clones following inoculations in fall of
1986 and 1987.

V mean of 20 progeny per clone over 2 years.

81
increasing the additive gene effect, or alternatively a
grafted propagation effect or age difference. The
heritability estimate and the standard deviation of the
estimate, based on parent-offspring regression of the canker
necrotic length rating, are listed on Table 5.

Since the heritability estimate in this parent-
offspring regression probably is biased by about 5%, the
heritability of disease resistance in the diverse progeny
probably is calculated to be 0.76 * 0.95 = 0.72. This
adjusted estimate agrees well with the heritability estimate
of 0.65 obtained from the variance components. It may well
represent the true value of heritability for canker
resistance in this peach population.

Several factors influence the breeder's selection of a
breeding scheme to improve their genetic stocks. These
include (1) quantity and types of genetic variance, (2)
environmental effects, (3) the interaction between
environment and genotype, and (4) linkage effects. Genetic
and environmental effects, and interactions between genotype
and environment were estimated in this population. The
estimate of heritability for canker length were high. The
year and year by peach family interactions were relatively
small. This will assist the breeder in discriminating
between genotypes for superior resistance to Leucostoma

persoonii in this segregating population without a masking

82

Table 5. Narrow-sense heritability (he) by parent-offspring
regression over 2 years data.

 

 

 

Female parent Progeny
mean of mean of Standard
necrotic Standard necrotic Standard 2 deviation
length deviation length deviation h of h2

 

10.02 2.7 9.20 2.2 0.76 0.11

 

83
effect from year to year. The highly resistant progeny from
the most resistant families could be readily identified and
maintained in the breeding program. If the highly resistant
trees were often associated with undesirable horticultural
traits, resistant.F3 individuals could be intermated to
produce a large F2 population. Selection could be practiced
for desirable recombinant F2 plants having both disease
resistance and horticulturally important commercial
traits. Then the superior F2 plants could be backcrossed to
elite commercial peach cultivars, then selfed 1 or 2
generations to fix the desirable traits. A logical breeding
scheme for canker resistance will be a combined approach of
recurrent selection with backcrossing, or mass selection

with backcrossing.

84

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Chang, L. S. 1989. Hydraulic conductance in susceptible
vs. resistant peach clones following infection with
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87
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