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A SEARCH FOR TOLERANCE TO BLACK ROOT ROT IN STRAWBERRY
BY

Chrislyn Ann Particka

A DISSERTATION
Submitted to
Michigan State University

in partial fulfillment for the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Department of Horticulture/Plant Breeding and Genetics

2005

ABSTRACT
A SEARCH FOR TOLERANCE TO BLACK ROOT ROT IN STRAWBERRY
By
Chrislyn Ann Particka
Black root rot (BRR) is a widespread disease of strawberry (Fragaria

xananassa Duchnesne) that causes the death of feeder roots and the
degradation of structural roots. The major causal organisms of black root rot
include Rhizoctonia fragan'ae Husain and WE. McKeen, Pythium Pringsh. spp.
and Pratylenchus penetrans (Cobb) Filipjev and Schuurrnans Stekhoven. The
current method of control for black root rot is methyl-bromide fumigation;
however, methyl bromide is scheduled to be phased out in 2005, and its effects
are short-lived in matted-row systems. The objectives of the first study were to
measure levels of tolerance to black root rot in 20 strawberry genotypes and to
determine which pathogens were present in the soil. The genotypes were
planted in four blocks each of methyl-bromide fumigated and nonfumigated soil,
and were evaluated for crown number, number of flowers per crown, yield, and
average berry weight over two years. The results showed that all three
pathogens were present in the field, and that there was a significant genotype x
fumigation interaction for yield and crown number in both years. The cultivars
Bounty, Cabot, and Cavendish, all released from the breeding program in Nova
Scotia, displayed tolerance to the pathogens that cause BRR. The objective of
the second study was to determine the heritability of BRR tolerance. Nine

genotypes were chosen from the previous study to use as parents: three that

displayed high tolerance to BRR (‘Bounty’, ‘Cabot’, and ‘Cavendish’), three that
displayed intermediate tolerance (‘Guardian’, ‘Midway', and ‘Winona’), and three
that displayed little or no tolerance (‘Jewel’, LH50-4, and ‘Mesabi’). The progeny
from a diallel cross were grown on fumigated and nonfumigated soil and
evaluated for crown number, flower number, and yield. Results showed no
interaction between treatment and family, indicating that breeding for increased

tolerance to BRR will be difficult.

ACKNOWLEDGEMENTS

First and foremost, I would like to thank Dr. James Hancock for the
opportunity to earn my PhD under his direction. His support, guidance, humor,
and wealth of knowledge that he has so willingly shared has made this a
wonderful learning experience. I also thank the other members of my committee,
Dr. Amy lezzoni, Dr. James Kelly, and Dr. Annemiek Schilder for their helpful
input and advice.

Pete Callow also deserves an enormous thank-you, for all of his help in
every aspect of my research, and for answering the multitude of questions I had.
Somehow, Pete (and Jim) managed to make even the most mundane tasks fun.
I would also like to thank all of the other members of the Hancock lab, especially
Sedat Serge and Cholani Weebadde, for your friendship and camaraderie.
Sedat deserves a special thank-you for all of his help in analyzing my data.

I would also like to thank all of my friends and colleagues, including Mercy
and Jim Olmstead, Audrey and Chris Sebolt, Roberto Lopez, Liz Driscoll, Nat
Hauck, and Angie Durhman. Special thanks also goes to the support staff,
including Lorri Busick, Bill Chase, Joyce Lockwook, Kristi Lowrie, Sherry
Mulvaney, Sharon Roback, and Gary Winchell.

Finally, I thank my family, especially my parents, Steve and Julie Drake,
for your love and support, and my husband, Michael Particka, for making my life

complete.

TABLE OF CONTENTS

List of Tables .................................................................................................... vii
List of Figures ................................................................................................... ix
CHAPTER ONE
LITERATURE REVIEW .................................................................................... 1
Introduction ............................................................................................ 1
Biotic factors associated with BRR ........................................................ 2
Rhizoctonia ................................................................................. 2
Pythium ....................................................................................... 3
Other fungi .................................................................................. 4
Root lesion nematode ................................................................. 5
Other nematodes ........................................................................ 6
Rhizoctonia and P. penetrans complex ...................................... 7
Abiotic factors associated with BRR ...................................................... 8
Control of BRR ...................................................................................... 9
Chemical control ......................................................................... 10
Non-chemical control .................................................................. 1 1
Genetic control ........................................................................... 13
Objectives .............................................................................................. 16
CHAPTER TWO
FIELD EVALUATION OF STRAWBERRY GENOTYPES FOR TOLERANCE TO
BLACK ROOT ROT ON FUMIGATED AND NONFUMIGATED SOIL ............. 17
Introduction ............................................................................................ 17
Materials and Methods .......................................................................... 21
Results and Discussion ......................................................................... 24
Pathogens present ...................................................................... 24
Overall cultivar performance ....................................................... 25
Effects of fumigation on yield components of genotypes ............ 26
Potential for breeding new cultivars resistant to BRR ................. 28
CHAPTER THREE
BREEDING FOR INCREASED TOLERANCE TO BLACK ROOT ROT lN
STRAWBERRY ................................................................................................ 43
Introduction ............................................................................................ 43
Materials and Methods .......................................................................... 47
Results and Discussion ......................................................................... 51
Analysis of all the families ........................................................... 51
Analysis of the complete diallel ................................................... 53

APPENDIX
EVALUATION OF STRAWBERRY PEDIGREES TO DETERMINE IF BLACK
ROOT ROT TOLERANCE CAN BE TRACED TO A COMMON ANCESTRY.. 64

Introduction ............................................................................................ 64
Materials and Methods .......................................................................... 65
Results and Discussion ......................................................................... 66
LITERATURE CITED ....................................................................................... 74

vi

LIST OF TABLES

Table 1. Strawberry genotypes grown on fumigated and nonfumigated soil at
Holt, Mich. from 2002-2004 to evaluate tolerance to black root rot. ................. 30

Table 2. ANOVA and variance components for yield, crown number, flower
number, and fruit weight of strawberry genotypes in Holt, Mich. on fumigated and
nonfumigated soil. Plants were set in 2002 and evaluated in 2002-2003 (Year 1)
and 2003-2004 (Year 2). .................................................................................. 31

Table 3. Strawberry genotypes with high, medium, and intermediate tolerance to
black root rot used as parents in a diallel mating scheme ................................ 57

Table 4. Diallel mating scheme showing the number of progeny generated from
each cross. Note that there were no selfs, and that reciprocal crosses were
grouped into the same family. .......................................................................... 58

Table 5. Analysis of variance and variance components with all families for
crown number, inflorescence number, flowers per inflorescence, fruit weight, and
total yield of strawberry families grown in Holt, Mich., on fumigated and

nonfumigated soil. Plants were set in 2004 and evaluated over one year ....... 59

Table 6. Analysis of variance and variance components with the smaller set of
families for crown number, inflorescence number, flowers per inflorescence,
fruit weight, and total yield of strawberry families grown in Holt, Mich., on
fumigated and nonfumigated soil. Plants were set in 2004 and evaluated over
one year ........................................................................................................... 61

Table 7. Number of and significance for plant pathogenic nematodes in root and
soil samples from strawberries grown in Holt, Mich., on fumigated and
nonfumigated soil. Plants were set in 2004 and evaluated over one year ....... 62

Table 8. Percentage of root samples containing plant parasitic fungi from
strawberries grown on fumigated and nonfumigated soil in Holt, Mich. Plants
were set in 2004 and evaluated over one year. ............................................... 63

Table 9. Strawberry cultivars included in the pedigree study to determine if
tolerance to black root can be traced to a specific origin. These same cultivars
were evaluated in a field study to determine their level of tolerance to black root
rot (Chapter 2) .................................................................................................. 68

Table 10. Origin and mean genetic contribution (60) of the founding clones to

the 19 North American strawberry cultivars included in the pedigree study to
determine if tolerance to black root rot can be traced to a specific origin ......... 69

vii

Table 11. Cultivars and the genetic contribution of the founding clones included
in the pedigree study to determine if tolerance to black root rot can be traced to a
specific origin ......................................................................................... ’ .......... 71

viii

LIST OF FIGURES

Figure1. Plot showing yield and relative fumigation effect (percent reduction on
nonfumigated soil compared to fumigated soil) in year 1 for 20 genotypes grown
at Holt, Mich. Yield is expressed as the amount/surviving mother plant in a plot.
Positive values for percent reduction indicate the value was lower on
nonfumigated soil, while negative values indicate the value was higher on
nonfumigated soil. The genotypes were 1) ‘Allstar’, 2) ‘Annapolis', 3) ‘Bounty’, 4)
‘Brunswick’, 5) ‘Cabot’, 6) ‘Cavendish’, 7) ‘Chandler’, 8) ‘Earliglow’, 9)
“Evangeline”, 10) ‘Governor Simcoe’, 11) ‘Guardian’, 12) ‘Honeoye’, 13) ‘Jewel’,
14) ‘Kent’, 15) LH50-4, 16) ‘Mesabi’, 17) ‘Midway’, 18) ‘Mira’, 19) ‘Surecrop’, 20)
‘Winona’ ........................................................................................................... 33

Figure 2. Plot showing crown number and relative fumigation effect (percent
reduction on nonfumigated soil compared to fumigated soil) in year 1 for 20
genotypes grown at Holt, Mich. Crown number is expressed as the
amount/surviving mother plant in a plot. Positive values for percent reduction
indicate the value was lower on nonfumigated soil, while negative values indicate
the value was higher on nonfumigated soil. The genotypes were 1) ‘Allstar’, 2)
‘Annapolis’, 3) ‘Bounty’, 4) ‘Brunswick’, 5) ‘Cabot’, 6) ‘Cavendish’, 7) ‘Chandler’,
8) ‘Earliglow’, 9) ‘Evangeline’, 10) ‘Governor Simcoe’, 11) ‘Guardian’, 12)
‘Honeoye’, 13) ‘Jewel’, 14) ‘Kent’, 15) LH50-4, 16) ‘Mesabi’, 17) ‘Midway’, 18)
‘Mira’, 19) ‘Surecrop’, 20) ‘Winona’ .................................................................. 35

Figure 3. Plot showing average berry weight and relative fumigation effect
(percent reduction on nonfumigated soil compared to fumigated soil) in year 1 for
20 genotypes grown at Holt, Mich. Positive values for percent reduction indicate
the value was lower on nonfumigated soil, while negative values indicate the
value was higher on nonfumigated soil. The genotypes were 1) ‘Allstar’, 2)
‘Annapolis’, 3) ‘Bounty’, 4) ‘Brunswick’, 5) ‘Cabot’, 6) ‘Cavendish’, 7) ‘Chandler’,
8) ‘Earliglow’, 9) ‘Evangeline’, 10) ‘Governor Simcoe’, 11) ‘Guardian’, 12)
‘Honeoye’, 13) ‘Jewel’, 14) ‘Kent’, 15) LH50-4, 16) ‘Mesabi’, 17) ‘Midway’, 18)
‘Mira’, 19) ‘Surecrop’, 20) ‘Winona’ .................................................................. 37

Figure 4. Plot showing yield and relative fumigation effect (percent reduction on
nonfumigated soil compared to fumigated soil) in year 2 for 10 genotypes grown
at Holt, Mich. Yield is expressed as the amount/surviving mother plant in a plot.
Positive values for percent reduction indicate the value was lower on
nonfumigated soil, while negative values indicate the value was higher on
nonfumigated soil. The genotypes were 3) ‘Bounty’, 5) ‘Cabot’, 6) ‘Cavendish’,
11) ‘Guardian’, 13) ‘Jewel’, 14) ‘Kent’, 15) LH50-4, 16) ‘Mesabi’, 17) ‘Midway’, 20)
‘Winona’ ........................................................................................................... 39

Figure 5. Plots showing crown number and relative fumigation effect (percent
reduction on nonfumigated soil compared to fumigated soil) in year 2 for 10

genotypes grown at Holt, Mich. Crown number is expressed as the
amount/surviving mother plant in a plot. Positive values for percent reduction
indicate the value was lower on nonfumigated soil, while negative values indicate
the value was higher on nonfumigated soil. The genotypes were 3) ‘Bounty’, 5)
‘Cabot’, 6) ‘Cavendish’, 11) ‘Guardian’, 13) ‘Jewel’, 14) ‘Kent’, 15) LH50-4, 16)
‘Mesabi’, 17) ‘Midway’, 20) ‘Winona’ ................................................................ 41

Figure 6. Cluster dendrogram based on the genetic contribution of the founding
clones to the 19 strawberry cultivars included in the pedigree study to determine
if tolerance to black root rot can be traced to a specific origin .......................... 73

ANOVA
BRR

PDA

ABBREVIATIONS

Degrees Celsius
Analysis of Variance
Black root rot
Centimeter

Day

Hectare

Gram

Kilogram

Liter

Microgram

Meter

Millileter

Potato Dextrose Agar

xi

CHAPTER ONE
LITERATURE REVIEW
Introduction

Black root rot (BRR) is a serious disease of strawberry (Fragaria
xananassa Duchnesne) that causes the death of feeder roots, the degradation
and blackening of structural roots, and an overall decrease in plant vigor and
productivity (Maas, 1998). BRR has been reported in strawberry in the United
States, Canada, Europe, and Australia (Raski, 1956). One of the earliest reports
of BRR in Michigan was by Coons (1924), who noted that infected roots
blackened and the cortex peeled off. Many believe that BRR has now replaced
red stele as the most serious disease of strawberry in the Northeast (Pritts and
Wilcox, 1990).

As BRR does not affect the crown of the plant, it can be distinguished from
diseases that do, such as Phytophthora cactorum (Lebert & Cohn) Schrbt. and
Colletotn’chum fraganae A. N. Brooks (Wing at al., 1994). BRR can be separated
from Verticillium wilt (Verticillium albo-atmm Reinke & Berthier) as Verticillium wilt
is most severe in the first year of growth and the outer leaves of the infected
plants wilt and die while the inner leaves remain healthy (Maas, 1998). BRR-
infected plants, by contrast, rarely show symptoms during the first year of growth,

and leaves of all ages wilt (Maas, 1984).

Biotic factors associated with BRR
Rhizoctonia

Many biotic factors have been associated with BRR. Rhizoctonia DC.
spp. are some of the major fungi associated with BRR, and were first associated
with the disease by Zeller (1932). Zeller isolated Rhizoctonia solani Klihn as well
as other fungi from root lesions, but R. solani was the only one that demonstrated
pathogenicity. Many other early researchers identified or suspected R. solani or
other Rhizoctonia species as the cause of BRR (Coons, 1924; Hildebrand and
Koch, 1936; Katznelson and Richardson, 1948; Miller, 1948; Rich and Miller,
1963)

A new species of Rhizoctonia, R. fragariae Husain and WE. McKeen, was
described in the mid-1960s (Husain and McKeen, 1963a). They believed R.
fragariae had been overlooked in the past because of its close resemblance to R.
solani, which was common and well-known at that time. The new species was
differentiated from R. solani as well as other Rhizoctonia species as it did not
produce any sclerotia. Pea (Pisum sativum L.), bean (Phaesolus vulgaris L.),
tomato (Lycopersicon esculentum Mill.), carrot (Dacus carota L.), and sunflower
(Helianthus annuus L.) were found to be hosts of R. fragariae. Husain and
McKeen (1963b) showed that strawberry roots exude a substance that contains
many amino acids (glycine, threonine, alanine, serine, and tyrosine) which is
stimulatory to R. fragariae.

Since Husain and McKeen’s report describing R. fragariae, it has been

associated with BRR in strawberries in many areas of the United States and

other countries (Abad et al., 1999; D’Ercole et al., 1989; LaMondia and Martin,
1988; Szczygiel and Profic-Alwasiak, 1989.). In a survey of strawberry fields in
Massachusetts, Drozdowski (1987) found that binucleate Rhizoctonia-like fungi
(such as R. fragariae) were the predominant fungal pathogens in the root
systems. Maas (1998) stated that R. fragariae may be the most widespread
pathogen of BRR. In Michigan field surveys, R. fragariae was the only fungus
consistently isolated from plants showing symptoms of BRR and is currently
believed to be the main fungal pathogen that causes BRR (C. Osborn, personal
communication).

Although R. fragariae is found in most strawberry plantings, infected plants
do not always show symptoms of BRR. Ribeiro and Black (1971) sampled plants
from fields in West Virginia, Maryland, Pennsylvania, and Arkansas that showed
symptoms of BRR as well as adjacent plants showing no symptoms of the
disease. Although R. fragariae was isolated from all symptomatic plants, it was
also isolated from 80% of the healthy-looking plants. The authors concluded that
R. fragariae exists with strawberry plants as an endophytic mycorrhizal fungus,
and only when certain environmental or nutritional conditions occur does the
fungus become pathogenic.

Pythium

Many Pythium species have also been associated with BRR, with Pythium
ultimum Trow being the most common (Wilhelm, 1998). One of the first reports
of Pythium in strawberries was in 1930, when Plakidas isolated nine different

strains of the fungus from strawberry roots in Louisiana and found that one strain

was more pathogenic than the rest. However, none of the strains were identified.
In Ontario, Hildebrand and Koch (1936) and in England, Berkley and Lauder-
Thomson (1934), isolated Pythium (in addition to other pathogens) from field-
grown strawberries as well. Nemec and Sanders (1970) surveyed strawberry
fields in Illinois in order to identify the different Pythium species associated with
strawberries there. Eight different species were found, with P. imegulare
Buisman, P. pemiciosum Serbinow, and P. sylvaticum W.A. Campbell and J.W.
Hendrix being isolated most frequently. A study in Japan found P. sylvaticum, P.
ultimum, P. spinosum Sawada, and P. oedochilum Drechs. to be the most
common Pythium species in strawberry fields, although Rhizoctonia species
were more prevalent (Watanabe et al., 1977). In a Massachusetts survey
(Drozdowski, 1987), Pythium was only occasionally found in the wettest areas.
Other fungi

Over the years that BRR has been studied, many other fungi besides
Rhizoctonia and Pythium have been identified as possible causes of the disease.
Strong and Strong (1931) indicated through isolation and inoculation studies that
Coniothyn’um fuckelli Sacc. and Hainesia lythn' (Desmaz.) HOhn were common
causal organisms of BRR in Michigan. Isolation studies by Hildebrand (1934)
showed that Fusan’um spp., Ramulan'a spp., and Pythium spp. were most often
found in diseased strawberry roots in plantings in the Niagara Peninsula. In
Britain, Berkley and Lauder-Thomson (1934) found five fungi capable of
damaging strawberry roots, each of which caused very similar damage. They

were: C. fuckelli, H. Iythn', Cylindrocarpon radicicola WollenWeb., Fusan'um

on‘hoceras Appel. and Wollenweb., and Pachybasium candidum. Miller (1948)
discovered that Fusan’um Wr. and Ramulan'a Unger non Roussel were two of the
most prevalent and pathogenic fungi of strawberry roots in Oregon. In Italy,
Verticillium dahliae Kleb., ldn'ella Iunata P.E. Nelson and K. Wilh., and
Cylindrocarpon destructans (Zinssmeister) Scholten were shown to be
components of BRR in addition to Rhizoctonia and Pythium (D’Eroole et al.,
1989)
Root lesion nematode

Nematodes, specifically the root lesion nematode [Pratylenchus penetrans
(Cobb) F ilipjev and Schuurrnans Stekhoven], have been associated with
strawberries since Steiner first reported finding them in strawberry fields in
Florida in 1931. Berkley and Lauder-Thomson (1934) and Hildebrand and Koch
(1936) were some of the earliest researchers to link nematodes to BRR, although
the species of nematode was not mentioned. In the mid-1950s through the early
1960s, researchers worldwide found P. penetrans as well as other species of
Pratylenchus in strawberry fields having problems with BRR including
Klinkenberg in the Netherlands (1955), Goheen and Bailey in Massachusetts
(1955), Chapman in Kentucky (1956), Riggs et al. in Arkansas (1956), and
Townshend in Ontario (1962). P. penetrans has also been found in the roots of
wild plants of the alpine strawberry, F. vesca alpina (Klinkenberg, 1955), the
wood strawberry, F. vesca L. (T ownshend, 1958), and the Virginiana strawberry,

F. Virginiana Duch. (Goheen and Braun, 1956).

In the late 19505 and early 19605, researchers established the
pathogenicity of P. penetrans but were not able to clearly link it with the
symptoms of BRR. Townshend (1962) found that while P. penetrans did
considerable damage to strawberry roots, it was not the same sort of damage
attributed to BRR. He suggested that the symptoms of BRR were caused by
fungi and bacteria, and that the nematode was a precursor of the root rot. A later
study by Townshend (1963) proved that P. penetrans was pathogenic to
strawberry by following Koch’s postulates. A soil fumigation study found that
high populations of P. penetrans were associated with low plant vigor, while low
populations were associated with high plant vigor, but BRR symptoms were not
described (Braun and Keplinger, 1960). Goheen and Smith (1956) found that
high numbers of root lesion nematodes can produce injury and that it is a primary
parasite of strawberry. Raski (1956) found that even very high levels of
nematodes did little damage to roots, and concluded that P. penetrans was not
an important factor in BRR.

Other nematodes

A number of nematodes other than P. penetrans have been associated
with BRR. Chapman’s (1956) survey of strawberry fields in Kentucky found
many nematode species (Meloidogyne hapla Chitwood, Tylenchorhynchus
claytoni Steiner, T. dubius Cobb, Xiphinema amen’canum Cobb, Paratylenchus
spp., and Helicotylenchus nannus Steiner), although Pratylenchus spp. were
found most often. However, only M. hapla was a known pathogen of strawberry

other than Pratylenchus spp., and no pathogenicity tests were conducted for the

other nematodes. In Massachusetts, the root-knot nematode, Meloidogyne spp.,
was found to live in strawberry roots, although it did not thrive (Bailey, 1956). A
later study by Edwards et al. (1985) found that M. hapla parasitized and
reproduced in the 12 cultivars tested, with a wide range of effects on root growth.
Some of the cultivars (‘Apollo’, ‘Catskill’, ‘Delite’, ‘Earliglow’ and ‘Prelude’) were
unaffected by the nematode.
Rhizoctonia and P. penetrans complex

Although nematodes and fungi have been separately implicated as the
cause of BRR, most researchers now believe that BRR is caused by a disease
complex between the fungi and the nematodes. Chen and Rich (1962) were the
first to conduct studies to determine if such a relationship exists. They found that
fungi infected areas of the root damaged by P. penetrans more readily than
healthy tissue and that P. penetrans moved away from roots as fungi invaded the
tissue. LaMondia and Martin (1989) conducted a study to determine the effect of
P. penetrans, R. fragariae, and temperature on the severity of BRR, and found
that root infection by P. penetrans consistently raised the severity of BRR caused
by R. fragariae. The fungus alone caused 25-36% root rot at 10 °C and 30-38%
at 20 °C. Feeding by nematodes, however, increased root rot to 36-52% at 10 °C
and 70-82% at 20 °C.

The root lesion nematode has also been shown to interact with Verticillium
wilt. A study by Abu-Gharbieh et al. (1962) showed that ‘Dixieland’, which is
highly susceptible to Verticillium, developed disease symptoms more quickly and

more severely when inoculated with both Verticillium and P. penetrans than

when inoculated with Verticillium alone. However, an interaction was not
apparent when cultivars were used that were moderately or highly resistant to
Verticillium.

Interactions between nematodes and fungi have been shown in many
other crops besides strawberry. In cotton, studies have shown a link between
F usarium wilt and the sting nematode (Belonolaimus gracilis Steiner) (Holdeman
and Graham, 1954), the reniforrn nematode (Rotylenchulus reniformis Linford
and Oliveira) (Neal, 1954) and Meloidogyne incognita var. acn'ta (Kofoit and
White) Chitwood (Martin et al., 1955). In a study where tobacco plants
inoculated either alone with fungi that were either considered non-pathogens of
tobacco or were not important on plants beyond juvenile stage or in combination
with M. incognita showed that none of the pathogens [Pythium ultimum,
Curvulan'a tn'folii (Kauffm) Boedijn, Botrytis cinerea Pers.:Fr., Aspergillus
ochraceus K. Wilh., Penicillium martensii Biourge, and Trichoderma harzianum
Rifai] caused disease unless the nematode was present (Powell et al., 1971).
Some plants inoculated with T. harzianum and nematodes were so damaged that
they were near death at the end of the study.
Abiotic factors associated with BRR

Many different abiotic factors have been associated with BRR in addition
to the biotic factors. Fletcher (1917) attributed most BRR to winter injury, but
also indicated that poor culture, lack of fertility, plant crowding, insufficient mulch,
and wet soils were partial causes of the disease. Smith and Home (1922)

believed BRR was caused by waterlogged soil or sudden drying of the soil.

Miller (1948) thought BRR was due to desiccation of the roots during
transplanting.

While the abiotic factors have since been found to not cause BRR alone,
they may affect disease development. The establishment and growth of
pathogens that cause the disease can be influenced by certain abiotic factors.
For example, Zeller (1932) found that Rhizoctonia spp. were more common in
light soils, but disease symptoms were more severe in clay loam soils. Pythium
spp. are also favored by fine-textured soils (Hendrix and Campbell, 1973). In
contrast, Klinkenburg (1955) found that the root lesion nematode was more
common in sandy soils. Soil moisture also affects fungi and nematodes.
Pythium spp. are most often associated with very wet soils (Watanabe et al.,
1977). Wing at al. (1995a) found that soil compaction, fine-textured soils, age of
the planting, successive years of strawberry monoculture, flat beds, use of the
herbicide terbacil (3-tert-butyl-5-chloro-6-methyluracil), and non-use of the
fungicide metalaxyl (methyl-N-(2,6-dimethylphenyl)-N-(2-methoxyacetyl)-DL-
alaninate) all increased incidence of BRR. A recent study by Mervosh and
LaMondia (2004), however, found that the use of terbacil at up to four times the
maximum amount allowed per year did not increase incidence of P. penetrans or
R. fragariae, nor did it reduce the health of perennial, structural, or feeder roots.
Control of BRR

Researchers have been struggling to find an effective way to control BRR
in matted row systems since it was first described in the early 1900s. Early

researchers recommended a variety of cultural control measures. Coons (1924)

suggested rotation with grain crops, as they seemed to reduce Rhizoctonia
infestation in soils. He also suggested selecting healthy planting stock,
protecting plants from winter damage, incorporating mulch, and ensuring
adequate drainage, but seemed to imply that such measures were somewhat
futile. Strong and Strong (1930) also suggested selecting vigorous plants with
white roots as planting stock and careful handling of plants during setting to keep
roots from drying out. In another article in 1931, they included crop rotation and
breeding for resistance as additional control measures.
Chemical control

Even today, about 80 years after BRR was first reported, no long-lasting,
effective control measures have been developed for matted row culture.
Fumigation with methyl bromide and chloropicrin have been reported to give
good control in annual systems, especially where Rhizoctonia predominates
(Maas, 1998). However, the effects of fumigation wear off in perennial matted
row systems as pathogen populations reestablish over time. Some have even
speculated that fumigation could ultimately result in an increase in pathogen
pressure in perennial matted row systems, as fumigation kills beneficial soil
microbes, leaving nothing to compete with pathogens that either survive in low
numbers or are reintroduced to the field. Methyl bromide is scheduled to be
phased out (USDA, 2000), and while cost-effective chemical alternatives to
methyl bromide have been identified, none provide the full spectrum of control

that methyl bromide does (Fennimore et al., 2003; Shaw and Larson, 1999).

10

Non-chemical control

Many researchers have looked to crop rotation and cover crops to help
mitigate the effects of BRR. Morgan and Collins (1964) studied the effect of
different cover crops and organic soil amendments on P. penetrans populations.
They found that while actively growing timothy sod resulted in the highest
nematode populations, composted timothy hay resulted in the lowest. Peat moss
also significantly lowered nematode populations, and manure and coniferous
sawdust had a slight effect as well. LaMondia et al. (2002) found that rotation to
‘Saia’ oats (Avena strigosa Schreb.) and Triple S sorghum-sudangrass [Sorghum
bicolor Durra x S. sudanense (Piper) Stapf.] suppressed both R. fragariae and P.
penetrans, while ‘Garry’ oats (Avena sativa L.) suppressed R. fragariae but
increased P. penetrans. In a related study, Elmer and LaMondia (1999)
combined ‘Saia’ oats, ‘Garry’ oat, or ‘Triple 8’ sorgho-sudangrass with (NH4)
2804 or C8(N03) 2. The combination of ‘Saia’ oats and (NH) 2304 resulted in
less root damage, larger plants, and earlier harvest than if ‘Saia’ oats were
combined with Ca(N03) 2 or if another crop was combined with (NH4) 2804.
Application of sorgho-sudangrass in combination with (NH4) 2SO4didn’t affect
disease severity or yield, but reduced nematode numbers. Use of ‘Garry’ oat
reduced disease severity and R. fragariae infection, but did not affect yield or
plant growth. Recent studies conducted at Cornell University found that
fumigation with methyl bromide resulted in the highest yield, but a rotation of kale
(Brassica oleracea L.)/sweet corn [Zea mays L. var. saccharata (Sturt.)

Baileyjlrye (Secale cereale L.) also proved to be effective (Seigies, 2004).

11

Overall, rotations involving multiple species fared much better than rotations
involving single species.

Elmer and LaMondia (1995) also studied the effect of mineral nutrition
alone on BRR. They compared (NH4) 2804 and Ca(NOs) 2 supplemented with
KCI, CaClz, K2804, or 03804 combined with and without a slow-release
micronutrient product. Overall, plants fertilized with (NH4) 2SO4 had less disease
and higher yields, but no differences were seen in nematode densities. The use
of K or Cl salts and the use/non-use of micronutrients had no effect on disease
severity or yield.

Researchers in Israel studied the use of biological control agents in
commercial strawberry fields and nurseries (Elad, et al., 1981). In the study, they
investigated the effectiveness of the mycoparasitic fungus Tn'choderma
harzianum in controlling R. solani. In nursery plots, T. harzianum reduced the
disease severity of R. solani by 18-46% and reduced the infestation of the soil by
up to 92%. In commercial fields, plants treated with T. harzianum before planting
resulted in a 21-37% increase in early yield, and when treatments were
combined in the nursery and fruiting fields, a 20% increase in yield was
observed.

Current research at Michigan State University has shown promising
results from use of biocontrol agents in both greenhouse and field studies (A.
Schilder, personal communication). A controlled greenhouse study tested a
number of commercial biocontrol agents as well as isolates of Paenibacillus

macerans Schardinger Ash from cranberry fruit and Tn'choderma spp., and found

12

that one of the Tn'chodenna spp. isolates, T-10, was the most effective as it had
a positive significant impact on all parameters measured. Field studies tested a
number of different products, including Quadris (a strobilurin-type fungicide), in
two different sites, and showed mixed results. In the first site, there were no
significant differences between the untreated control and any of the products
used. At the second site, five products, Quadris, Polyversum (a beneficial
Pythium oligandrum Drechs.), T-10, Primastop (a commercial formulation of
Gliocladium catenulatum Gilman & Abbott), and DiTera [a nematicidal fungus
called Mynothecium vermcan'a (Albertini & SchweinitzzFr.) Ditmar] produced
significantly higher yields than the untreated control.

Genetic control

The most effective way to control BRR would be to identify or to develop
tolerant strawberry cultivars. A few studies have been undertaken to identify
genotypes with tolerance to the pathogens causing BRR, but most new eastern
cultivar releases have not been screened.

Potter and Dale (1994) conducted studies to determine if resistance to P.
penetrans alone could be found in strawberry, and showed that ‘Guardian’ had
the highest resistance overall. Further studies showed considerable variation
among 19 cultivars, with the four most resistant being ’Pajaro’, ‘Chandler’,
Annapolis’, and ‘Glooscap’ (Dale and Potter, 1998). Interestingly, all these have
the California cultivar Lassen in their pedigree.

Recent studies by Pinkerton and F inn (2005) evaluated the resistance and

tolerance of a number of strawberry cultivars and wild genotypes (F. chiloensis

13

and F. Virginiana subspecies) to both P. penetrans and M. hapla (northern root
knot nematode). The results showed that both cultivated and wild genotypes had
considerable resistance to M. hapla, and that resistance to P. penetrans was less
common. However, they did note that using wild genotypes as sources of
resistance would be of little value as resistance to both nematodes can be found
in Fragan'a xananassa.

Wing et al. (1995b) found that ‘Tristar’ ‘Earliglow’ and ‘Midway’ had the
healthiest roots in a field infected primarily with Pythium spp. However, disease
incidence in this field was relatively low as no above-ground symptoms were
observed, and Rhizoctonia spp. and the root lesion nematode were absent.

Studies in California on fumigated and nonfumigated soils that measured
leaf number, plant diameter, yield, fruit weight, and fruit appearance did not
identify any genotypes with strong tolerance to sublethal levels of soil pathogens
(Larson and Shaw, 1995; Shaw and Larson, 1996). In the first study, the
interaction between genotype and fumigation was not significant, but this
interaction was significant in the second study. The authors pointed out,
however, that the interaction explained only 2% to 5% of the variance, and
concluded that the lack of fumigation affected all cultivars similarly. In the first
study, only California genotypes were tested, and in the second study, nine
genotypes from outside California (six of which were from the USDA program in
Maryland) and nine California genotypes were used, which represented only a

small fraction of the gerrnplasm grown.

14

Studies in Michigan identified only modest differences in tolerance based
on yield, fruit weight, crown and runner number, and root and crown appearance
in fields infested with Pythium spp., Rhizoctonia spp., ldn'ella lunata P.E. Nelson
& K. Wilh., Meloidognye hapla, and P. penetrans (Hancock et al., 2001). This
study included four California cultivars, 11 eastern US cultivars, and 12 F.
Virginiana Duchnesne F1 hybrids. Although no significant genotype x fumigation
interactions were found, there was a significant species source x fumigation
interaction for fruit weight at P 5 0.01, and for yield and runner number at P 5
0.10. Also, the F. Virginiana hybrids performed better overall than the eastern or
California cultivars, and the authors concluded that some tolerance might exist in
F. Virginiana, which could be used to improve current cultivars.

Further studies using F. Virginiana and F. chiloensis wild selections as well
as F. xananassa cultivars showed positive results (C. Osborn, personal
communication). Field studies in methyl bromide fumigated and nonfumigated
soils did not find any genotypes with high levels of tolerance to BRR. However,
the wild genotypes performed better overall for a number of yield parameters
than did the cultivars. ln controlled greenhouse studies using the same
genotypes, plants were inoculated with Rhizoctonia spp. and P. penetrans alone
and in combination. The results showed that that Frederick 9 and NC 95-1-1
(both F. Virginiana genotypes) were resistant to P. penetrans and that NC 96-48-

1 (another F. Virginiana genotype) was resistant to the mixed infection.

15

Objectives

1) Measure the levels of tolerance to BRR in 20 diverse strawberry
genotypes [Chapter 2, Published in the Journal of the American Society for
Horticultural Science, 130(5):688-693.]

Nineteen old and new cultivars from six different breeding programs
across the United States and Canada along with one F. Virginiana selection from
Montana were screened for tolerance to BRR. The genotypes were evaluated
for crown number, number of flowers per crown, yield, and average berry weight
over two years. Genotypes were considered tolerant if the percent reduction
between fumigated and nonfumigated soil was low for most parameters, and if
they performed well overall on nonfumigated soil.

2) Determine the heritability of tolerance to BRR (Chapter 3).

Nine genotypes were chosen that represented the range of tolerance to
BRR—three that were identified as being highly tolerant, three that were highly
intolerant, and three that were intermediate. The genotypes were mated in a
diallel crossing scheme and each of the resulting progeny were evaluated on
fumigated and nonfumigated soil for crown number, number of inflorescences per
crown, number of flowers per inflorescence, yield, and average berry weight over

one year.

16

CHAPTER TWO
FIELD EVALUATION OF STRAWBERRY GENOTYPES FOR TOLERANCE TO

BLACK ROOT ROT ON FUMIGATED AND NONFUMIGATED SOIL
Introduction

Black root rot (BRR) is a widespread disease of strawberry (Fragaria
xananassa) that causes an overall decrease in productivity due to the death of
feeder roots and the degradation of structural roots (Maas, 1998). One of the
earliest reports of BRR was by Coons (1924), who noted that the roots of
infected plants were blackened and the cortex peeled off. By the 1950s, BRR
had been reported in the United States, Canada, Europe, and Australia (Raski,
1956). Many strawberry researchers now believe that BRR has replaced red
stele (Phytophthora fragariae Hickman) as the most serious root disease of
strawberry in the Northeastern USA (Pritts and Wilcox, 1990).

Many biotic factors have been associated with BRR. Rhizoctonia
fragariae Husain and WE. McKeen was first described as a causative organism
of BRR in 1963, and since then, it has been associated with BRR in many areas
of the United States and other countries (Abad et al., 1999; D’Ercole et al., 1989;
Husain and McKeen, 1963a). Maas (1998) concluded that R. fragariae is the
most widespread pathogen that causes BRR, although many Pythium species
have also been associated with the disease (Nemec and Sanders, 1970).
Pythium ultimum Trow is considered to be the most common one (Wilhelm,
1998). The root lesion nematode [Pratylenchus penetrans (Cobb) F ilipjev and

Schuurrnans Stekhoven] was first associated with strawberry in 1931, and was

17

linked to BRR by many researchers in the mid-19505 to early 19605
(Klinkenberg, 1955; Steiner, 1931; Townshend, 1962).

While R. fragariae, Pythium spp., and P. penetrans have been separately
implicated as the cause of BRR, the disease is often caused by a complex of all
three. Chen and Rich (1962) found that fungi infected areas of the root damaged
by P. penetrans more readily than healthy tissue, and that the nematodes moved
away from the roots as fungi invaded. LaMondia and Martin (1989) determined
that infection by P. penetrans consistently raised the severity of BRR caused by
R. fragariae.

Several abiotic factors have also been associated with BRR. Fletcher
(1917) attributed BRR to winter injury, poor culture, lack of fertility, plant
crowding, insufficient mulch, and wet soils. Wing et al. (1995a) found that soil
compaction, fine-textured soils, age of the planting, successive years of
strawberry monoculture, use of the herbicide terbacil, and non-use of raised beds
and of the fungicide metalaxyl all increased incidence of BRR.

Currently, there are no effective, long-lasting control measures for BRR in
perennial matted row culture. F umigation with methyl bromide and chloropicrin
has been reported to give good control in annual systems (Maas, 1998);
however, its effects gradually wear off in perennial matted row systems as
pathogen populations reestablish over time. Some have even suggested that
fumigation could ultimately result in an increase in BRR in perennial systems, as
fumigation kills beneficial soil microbes, leaving nothing to compete with the

pathogens that either survive in low numbers or are reintroduced to the field

18

(Pritts and Wilcox, 1990). Methyl bromide is scheduled to be phased out (USDA,
2000), and while cost-effective chemical alternatives to methyl bromide have
been identified, none provide the full spectrum of control that methyl bromide
does (Fennimore et al., 2003; Shaw and Larson, 1999).

Many researchers have looked to crop rotation and cover crops to help
mitigate the effects of BRR. Morgan and Collins (1964) studied the effect of
different cover crops and organic soil amendments on P. penetrans populations
and found that composted timothy hay (Phleum pretense L.) was most effective
in reducing nematode numbers. LaMondia et al. (2002) found that rotation to
‘Saia’ oats (Avena strigosa Schreb.) and Triple S sorghum-sudangrass [Sorghum
bicolor Durra x S. sudanense (Piper) Stapf.] suppressed both R. fragariae and P.
penetrans, while ‘Garry’ oats (Avena sativa L.) suppressed R. fragariae but
increased P. penetrans. Recent studies conducted at Cornell University found
that fumigation with methyl bromide resulted in the highest yield, but a rotation of
kale (Brassica oleracea L.)/5weet corn [Zea mays L. var. saccharata (Sturt.)
Bailey]lrye (Secale cereale L.) also proved to be effective (Seigies, 2004).
Overall, rotations involving multiple species fared much better than rotations
involving single species.

The most effective way to control BRR would be to identify or to develop
tolerant strawberry cultivars. A few studies have been undertaken to identify
genotypes with tolerance to the pathogens causing BRR, but most new eastern

cultivar releases have not been screened.

19

Potter and Dale (1994) conducted studies to determine if tolerance to P.
penetrans alone could be found in strawberry, and showed that ‘Guardian’ had
the highest resistance overall. Further studies showed considerable variation
among 19 cultivars, with the four most resistant being ‘Pajaro’, ‘Chandler’,
Annapolis’, and ’Glooscap’ (Dale and Potter, 1998). Interestingly, all these have
the California cultivar Lassen in their pedigree.

Wing et al. (1995b) found that ‘Tristar’ ‘Earliglow’ and ‘Midway’ had the
healthiest roots in a field infected primarily with Pythium spp. However, disease
incidence in this field was relatively low as no above ground symptoms were
observed, and Rhizoctonia DC. spp. and the root lesion nematode were absent.

Studies in California on fumigated and nonfumigated soils that measured
leaf number, plant diameter, yield, fruit weight, and fruit appearance did not
identify any genotypes with strong tolerance to sublethal levels of soil pathogens
(Larson and Shaw, 1995; Shaw and Larson, 1996). In the first study, the
interaction between genotype and fumigation was not significant, but this
interaction was significant in the second study. The authors pointed out,
however, that the interaction explained only 2% to 5% of the variance, and
concluded that the lack of fumigation affected all cultivars similarly. In the first
study, only California genotypes were tested, and in the second study, nine
genotypes from outside California (six of which were from the USDA program in
Maryland) and nine California genotypes were used, which represented only a

small fraction of the germplasm grown.

20

Studies in Michigan identified only modest differences in tolerance based
on yield, fruit weight, crown and runner number, and root and crown appearance
in fields infested with Pythium spp., Rhizoctonia spp., ldn'ella lunata P.E. Nelson
& K. Wilh., Meloidognye hapla Chitwood, and P. penetrans (Hancock et al.,
2001). This study included four California cultivars, 11 eastern US cultivars, and
12 F. Virginiana Duchnesne F1 hybrids. Although no significant genotype x
fumigation interactions were found, there was a significant source x fumigation
interaction for fruit weight at P _<_ 0.01, and for yield and runner number at P 5
0.10. Also, the F. Virginiana hybrids performed better overall than the eastern or
California cultivars, and the authors concluded that some tolerance might exist in
F. Virginiana, which could be used to improve current cultivars.

Herein we describe a field screen for BRR tolerance that included both old
and new cultivars from six breeding programs, as well as one wild genotype. We
focused on yield and vigor of the above-ground portion of the plants evaluated
with and without fumigation, and a number of cultivars developed in Nova Scotia
were shown to have considerable tolerance.

Materials and Methods

We evaluated 19 cultivars and one F. Virginiana genotype (Table 1). The
planting was established in June of 2002 at the Horticulture Teaching and
Research Center in Holt, Mich., on soil that had been in strawberries for over five
years. Plants were obtained from commercial nurseries, and were planted in a
split-plot design with four plots each of nonfumigated soil and soil that had been

treated with a mixture of 2 methyl bromide : 1 chloropicrin (weight : weight)

21

injected at a rate of 392 mm". Three plants of each genotype were planted in
each plot, with 46 cm within-row spacing and 61 cm between-row spacing. The
plots received supplemental irrigation as needed, and weeds were controlled
through use of pre-emergence herbicides and mechanical methods. At the end
of the first fruiting season, we selected 10 genotypes that appeared to represent
the range of variation for tolerance to BRR (Table 1), and the rest of the
genotypes were removed to simplify plot maintenance.

The plants were allowed to runner freely, with sufficient training to keep
plants of different genotypes separated. In the fall of year one (2002-2003
season) and year two (2003-2004 season), the total number of crowns was
recorded in each plot (8 Nov. and 24 Oct, respectively). Individual plants were
not counted, but most were represented by one or two crowns. In the spring of
year one and year two, the number of flowers per crown was counted (27 May
and 21 May, respectively). During the fruiting season each year (11 June-9 July
2003 and 3 June-29 June 2004), fruit that were at least 60% ripe were harvested
and weighed weekly. A random 25-berry sample was taken from each genotype
in each plot to determine average fruit weight. Crown and yield data were
divided by the number of surviving mother plants for analysis, as in a few
instances, not all of the original plants survived.

In both years, root samples were taken from randomly-selected plants to
determine presence of plant parasitic nematodes (14-21 July 2003 and 12-17
May 2004) and fungal pathogens (14 July 2003 and 28 Sept. 2004). For

nematode isolation, two plants were randomly selected from the 10 genotypes

22

included in both years of the study from each of the plots (for a total of 160
plants). Plants were dug using a hand spade and were placed in plastic bags on
ice and then stored at 1.5 °C until they were processed the following day.
Nematodes were extracted from root tissue following the flask-shaker method
(Bird, 1971). To make up a 1-g sample, approximately 0.5 g of roots were
selected from the two plants from each plot, for a total of 80 composite samples.
For fungal isolation, approximately 100 plants were randomly selected from the
same 10 genotypes in both fumigated and nonfumigated plots, but samples were
not kept separate for genotype or treatment. Plants were dug using a hand
spade and were placed in plastic bags on ice and stored at 1.5 °C until they were
processed later in the day or the following day. Fungi were isolated from roots by
washing the root systems in running water, and selecting root segments that had
visible lesions. The root segments were cut into 1-cm long sections, surface-
sterilized in 1% NaOCl for three minutes, rinsed three times in sterile distilled
water, and were blotted dry on sterile filter paper. At least 80 segments were
used both times fungi were isolated. The root pieces were placed on selective
medium [1/4 strength acidified PDA supplemented with ampicillin (50uglml) and
streptomycin (20pglml)] and were transferred to fresh media as needed for
identification. Fungi were identified based on the morphology of hyphae and of
spores (Barnett & Hunter, 1998; Maas, 1998).

Model variance components due to fumigation, genotype, and fumigation
x genotype treatment interaction and error were estimated as in Larson and

Shaw (1995). The analysis of variance was conducted using the GLM function of

23

SAS (SAS Institute, Cary, NC), with years being analyzed separately, as half
the genotypes were removed after the first year.

Results and Discussion

Pathogens present

Over 90% of the nematodes isolated were the root lesion nematode,
Pratylenchus Filipjev spp. F umigation with methyl bromide was effective at
killing the root lesion nematode, and they did not move back into fumigated areas
during the study. In samples from fumigated soil, 5% contained the root lesion
nematode in year one, and only 8% did in year two; numbers ranged from two to
54 per gram of root tissue across years, with an average of two. One hundred
percent of the samples from nonfumigated soil contained the root lesion
nematode in year one, while 85% of the samples contained nematodes in year
two. Root lesion nematode numbers ranged from two to 152 nematodes per
gram of root from nonfumigated soil, with an average of 28. No significant
differences in nematode number were observed across genotypes in either year
(P = 0.200, df=9 in year 1; P = 0.249, df=9 in year 2).

Rhizoctonia spp. and Pythium spp. were the most common pathogenic
fungi present in the soil. In year one Pythium spp. were found in many more
samples than Rhizoctonia spp., but unfortunately, the relative percentages were
not recorded. In year two, Rhizoctonia spp. was isolated from 43% of the root
segments, while Pythium spp. was isolated from only one sample.
Cylindrocarpon destructans (Zinnsmeister) Scholten, which has also been

associated with BRR (Wilhelm, 1998), was also isolated from three root

24

segments in year two. Differences in the most common type of pathogen found
between year 1 (samples were collected on 14 July) and year 2 (samples were
collected on 28 Sept.) is most likely due to the difference in sampling date. R.
fragariae is not parasitic on strawberry roots under warm temperatures, and is
therefore replaced by other pathogens during the spring and summer (Husain
and McKeen, 1963a).
Overall cultivar performance

In year one, mean yields across treatments were highest for ‘Mesabi’,
‘Cabot’, ‘Bounty’, ‘Brunswick’, ‘Annapolis’, and ‘Cavendish’, all of which produced
over 900 g per plant (Table 2). Each of these genotypes produced their high
yields in different ways. ‘Mesabi’ ranked in the top third of all genotypes for
crown number, number of flowers/crown, and fruit weight. ‘Cabot’ ranked only in
the upper half of all genotypes for crown number and number of flowers/crown,
but had the largest fruit. ‘Bounty’ ranked in the bottom third for fruit weight, but
was in the top third for crowns number and number of flowers/crown. ‘Brunswick’
was intermediate for number of flowers/crown and fruit weight, but ranked in the
top third for crown number. ‘Annapolis’ was only intermediate for number of
flowers/crown, but ranked in the top third for crown number and fruit weight.
’Cavendish’ was intermediate for crown number, but ranked in the top third for
number of flowers/crown and fruit weight.

In year two, ‘Brunswick’ and ‘Annapolis’ were dropped from the study, but
‘Mesabi’, ‘Cabot’, Bounty’, and ‘Cavendish’ remained among the highest

producers, joined by ‘Jewel’ and ‘Winona’. Again, their high yields were achieved

25

in different ways. ‘Bounty’ remained near the bottom for fruit weight, but was one
of the highest for crown number and number of flowers/crown. ‘Cabot’ had the
largest fruit and was in the top third for crown number, but had only intermediate
number of flowers/crown. ‘Cavendish’ ranked high for number of flowers/crown
and fruit weight, but was low for crown number. ‘Mesabi’ was intermediate for all
three yield components. ‘Jewel’ and ‘Winona’ had intermediate crown numbers
and numbers of flowers/crown, but were in the top third for fruit weight.

Effects of fumigation on yield components of genotypes

F umigation resulted in increased yield in both years of the study; yield on
fumigated soil was 46% higher (P < 0.001) in year one and 33% higher (P <
0.001) in year two (Table 2). Crown number was also significantly higher on
fumigated plots. In year one, fumigation resulted in 47% more crowns (P <
0.001), and in year two, crowns were increased by 41% (P < 0.001). Individual
fruit weight was significantly higher on nonfumigated plots in year two (P =
0.019), but by just 10%. The number of flowers per crown was not significantly
different between fumigated and nonfumigated plots in either year.

The genotype x fumigation interaction for yield was significant in both
years, indicating that some genotypes are more tolerant to BRR than others
(Table 2). The interaction explained 46% of the variance in year one, and 26% in
year two. In year one, the highest overall producers, ‘Bounty’, ‘Brunswick’,
‘Cabot’, and ‘Cavendish’, had high yields (866-790 g) on nonfumigated soil, and
their yields were reduced by only 25-35% without fumigation (Figure 1). In

contrast, ‘Annapolis’, and ‘Mesabi’ had only moderate yields on nonfumigated

26

soil (572 and 4839, respectively), and their yields were reduced by 60 to 70%. In
year two, ‘Cabot’, ‘Bounty’, and ‘Cavendish’ remained the highest producers on
nonfumigated soil (over 8009) and their yields were reduced by less than 20%
without fumigation (Figure 4). Yields of ‘Mesabi’, ‘Jewel’, and ‘Winona’ were
again less than 6009 on nonfumigated soil and were reduced by over 50%
without fumigation. This indicates that while ‘Mesabi’, ‘Jewel’, and ‘Winona’ are
vigorous and have high yield potentials, they have little tolerance to the
pathogens that cause black root rot. ‘Bounty’, ‘Brunswick’, ‘Cabot’, and
‘Cavendish’ all have high yield potentials and are tolerant to black root rot.

The genotype x fumigation interaction was significant for crown number in
both years, mirroring the yield data (T able 2). The interaction explained 61% of
the variance in year one, and 26% in year two. In year one, ‘Cavendish’ had
among the highest crown numbers (11) on nonfumigated soil, and had only 7%
fewer crowns than fumigated soil (Figure 2). ‘Bounty’, ‘Brunswick’, ‘Cabot’, and
‘Annapolis’ also had very high crown numbers (9-13), but their numbers were
reduced by 40-50% without fumigation. ‘Mesabi’ had a rather low number of
crowns on nonfumigated soil (6), and the crown numbers were reduced by 68%.

The number of flowers per crown was not significant for the genotype x
fumigation interaction in either year (Table 2). This suggests that the number of
flowers per crown is not involved in the yield reductions seen on nonfumigated
soil.

The fumigation x genotype interaction was significant for fruit weight in

year one (Table 2); however, the interaction explained only 6% of the variance,

27

and fumigation did not appear to have a consistent effect across genotypes.
Average berry weight was higher on nonfumigated soil for seven genotypes and
lower for 13 genotypes (Figure 3). Many authors have reported a significant
decrease in berry size on nonfumigated soil that was strongly associated with
yield reductions (Hancock et al., 2001; Larson and Shaw, 1995; Shaw and
Larson, 1996;). Our results indicate that while berry weight can be reduced on
nonfumigated soil, the effect is modest (10% or less) and variable across
genotypes, making reductions in crown number much more important in effecting
yields on nonfumigated fields of matted row cultivars.
Potential for breeding new cultivars resistant to BRR

Based on our studies, we feel there is sufficient variability to breed for
increased tolerance to BRR. Especially impressive are the cultivars from the
breeding program of Agriculture and Agri-Food Canada in Nova Scotia—all eight
of those cultivars included in the study were in the top half for yield on
nonfumigated soil in year one (Figure 1), and in the second year of the study, the
four that were included were ranked first, second, third, and fifth (Figure 4).
These cultivars also were the most tolerant to BRR, being among the lowest in
percent reduction on nonfumigated soil. This means there is opportunity to
combine vigor and tolerance to increase overall performance in soils infested
with the pathogens that cause BRR.

In previous studies conducted in California and Michigan, little genetic
diversity was found that could be utilized in increasing tolerance to soil

pathogens (Hancock et al., 2001 ; Larson and Shaw, 1995; Shaw and Larson,

28

1996), but the top-performing cultivars from Nova Scotia (‘Bounty,’ ‘Brunswick,’
‘Cabot,’ and ‘Cavendish’) were not used in these studies. In examining the
pedigrees of the cultivars from Nova Scotia, it was not evident that there was a
single source of tolerance; no one genotype was present in these cultivars that
was not also present in cultivars from other programs. However, methyl bromide
has not been used in the Nova Scotia breeding plots (A. Jamieson, personal
communication), as it has in most other breeding programs, suggesting that

tolerant genotypes can be selected in the presence of pathogen pressure.

29

Table 1. Strawberry genotypes grown on fumigated and nonfumigated soil at
Holt, Mich. from 2002-2004 to evaluate tolerance to black root rot.

 

 

Genotype ”:21; d Parentage Origin

Allstar 1981 US 4419 x MDUS 3184 USDA-Beltsville, Maryland
Annapolis 1984 K74-5 x Earliglow AAFC’, Kentville, N.S.
Bountyz 1972 Jerseybelle x Senga Sengana AAFC, Kentville, N.S.
Brunswick 1999 Cavendish x Honeoye AAFC, Kentville, N.S.
Cabot‘ 1998 K78-5 x K86-19 AAFC, Kentville, NS.
Cavendish‘ 1990 Glooscap x Annapolis AAFC, Kentville, N.S.
Chandler 1983 Douglas x Cal 72.361-105 University of California, Davis
Earliglow 1975 MDUS 2359 x MDUS 2713 USDA-Beltsville, Maryland
Evangeline 1999 Honeoye x Veestar AAFC, Kentville, N.S.

Gov. Simcoe 1985 Holiday x Guardian HRIO", Simcoe, Ontario
Guardianz 1969 NC 1768 x Surecrop USDA-Beltsville, Maryland
Honeoye 1979 Vibrant x Holiday NYSAES", Geneva, New York
Jewelz 1985 NY 1221 x Holiday NYSAES, Geneva, New York
Kent2 1981 K 68-5 x Raritan AAFC, Kentville, N.S.
LH50-4z - - Native F. Virginiana from Montana
Mesabiz 2000 Glooscap x MNUS 99 University of Minnesota, St. Paul
Midwayz 1960 Dixieland x Temple USDA-Beltsville, Maryland
Mira 1995 Scott x Raritan AAFC, Kentville, N.S.
Surecrop 1956 Fairland x MDUS 1972 USDA-Beltsville, Maryland
Winonaz 1995 Earliglow x MNUS 52 University of Minnesota, St. Paul

8. USDA-Beltsville, Maryland

 

z Included in both years
’ Agriculture and Agri-Food Canada
" Horticulture Research Institute of Ontario

w New York State Agricultural Experiment Station

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32

Figure1. Plot showing yield and relative fumigation effect (percent reduction on
nonfumigated soil compared to fumigated soil) in year 1 for 20 genotypes grown
at Holt, Mich. Yield is expressed as the amount/surviving mother plant in a plot.
'Positive values for percent reduction indicate the value was lower on
nonfumigated soil, while negative values indicate the value was higher on
nonfumigated soil. The genotypes were 1) ‘Allstar’, 2) ‘Annapolis’, 3) ‘Bounty’, 4)
‘Brunswick’, 5) ‘Cabot’, 6) ‘Cavendish’, 7) ‘Chandler’, 8) ‘Earliglow’, 9)
‘Evangeline’, 10) ‘Governor Simcoe’, 11) ‘Guardian’, 12) ‘Honeoye’, 13) ‘Jewel’,
14) ‘Kent’, 15) LH50-4, 16) ‘Mesabi’, 17) ‘Midway’, 18) ‘Mira’, 19) ‘Surecrop’, 20)

‘Winona’.

33

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34

Figure 2. Plot showing crown number and relative fumigation effect (percent
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amount/surviving mother plant in a plot. Positive values for percent reduction
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‘Annapolis’, 3) ‘Bounty’, 4) ‘Brunswick’, 5) ‘Cabot’, 6) ‘Cavendish’, 7) ‘Chandler’,
8) ‘Earliglow’, 9) ‘Evangeline’, 10) “Governor Simcoe’, 11) ‘Guardian’, 12)
‘Honeoye’, 13) ‘Jewel’, 14) ‘Kent’, 15) LH50-4, 16) ‘Mesabi’, 17) ‘Midway’, 18)
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35

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Figure 3. Plot showing average berry weight and relative fumigation effect
(percent reduction on nonfumigated soil compared to fumigated soil) in year 1 for
20 genotypes grown at Holt, Mich. Positive values for percent reduction indicate
the value was lower on nonfumigated soil, while negative values indicate the
value was higher on nonfumigated soil. The genotypes were 1) ‘Allstar’, 2)
‘Annapolis’, 3) ‘Bounty’, 4) ‘Brunswick’, 5) ‘Cabot’, 6) ‘Cavendish’, 7) ‘Chandler’,
8) ‘Earliglow’, 9) ‘Evangeline’, 10) ‘Governor Simcoe’, 11) ‘Guardian’, 12)
‘Honeoye’, 13) ‘Jewel’, 14) ‘Kent’, 15) LH50-4, 16) ‘Mesabi’, 17) ‘Midway’, 18)
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37

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Figure 4. Plot showing yield and relative fumigation effect (percent reduction on
nonfumigated soil compared to fumigated soil) in year 2 for 10 genotypes grown
at Holt, Mich. Yield is expressed as the amount/surviving mother plant in a plot.
Positive values for percent reduction indicate the value was lower on
nonfumigated soil, while negative values indicate the value was higher on
nonfumigated soil. The genotypes were 3) ‘Bounty’, 5) ‘Cabot’, 6) ‘Cavendish’,
11) ‘Guardian’, 13) ‘Jewel’, 14) ‘Kent’, 15) LH50-4, 16) ‘Mesabi’, 17) ‘Midway’, 20)

‘Winona’.

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genotypes grown at Holt, Mich. Crown number is expressed as the
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indicate the value was lower on nonfumigated soil, while negative values indicate
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42

CHAPTER THREE
BREEDING FOR lNCREASED TOLERANCE TO BLACK ROOT ROT IN
STRAWBERRY
Introduction

Black root rot (BRR) is a widespread disease of strawberry (Fragaria
xananassa) that causes the death of feeder roots and the degradation of
structural roots, resulting in an overall decrease in productivity (Maas, 1998). By
the 1950s, BRR had been reported in many areas of the world (Raski, 1956), and
most strawberry researchers now believe that BRR has replaced red stele
(Phytophthora fragariae Hickman) as the most serious root disease of strawberry
in the Northeastern USA (Pritts and Wilcox, 1990).

Many biotic factors have been associated with BRR. Rhizoctonia DC.
spp. was first linked to BRR in the early 19305 (Zeller, 1932), and Rhizoctonia
fragariae Husain and WE. McKeen was first described as a causative organism
of BRR in 1963 (Husain and McKeen, 1963a). Since then, R. fragariae has been
associated with BRR in many areas of the United States and other countries
(Abad et al., 1999; D’Ercole et al., 1989; Szczygiel and Profic-Alwasiak, 1989).
Although R. fragan’ae is the most widespread pathogen that causes BRR (Maas,
1998), various Pythium species have also been identified as causal organisms
(Nemec and Sanders, 1970). Pythium ultimum Trow is considered to be the
most common one (Wilhelm, 1998). The root lesion nematode [Pratylenchus

penetrans (Cobb) F ilipjev and Schuurrnans Stekhoven] was first associated with

43

strawberry in the early 19305 (Steiner, 1931), and Townshend (1963) proved it
was pathogenic to strawberry by following Koch’s postulates.

Although the three pathogens have been separately implicated as the
cause of BRR, it is now believed to be a disease complex between the fungi and
nematodes. Fungi are able to infect areas of the root damaged by P. penetrans
more easily than healthy tissue, and nematodes move away from roots after
fungi invade (Chen and Rich, 1962). LaMondia and Martin (1989) found that the
severity of R. fragariae infection is raised due to infestation by P.3p'enetrans.

In addition to biotic factors, several abiotic factors have been associated
with BRR. Wing et al. (1995a) found that soil compaction, fine-textured soils, age
of the planting, successive years of strawberry monoculture, use of the herbicide
terbacil, and non-use of raised beds and of the fungicide metalaxyl all increased
incidence of BRR. A recent study by Mervosh and LaMondia (2004), however,
found that use of Terbacil at up to four times the maximum recommended rate
each year did not increase incidence of P. penetrans or R. fragariae, nor did it
reduce the health of perennial, structural, or feeder roots.

No completely effective chemical control measures have been identified
for BRR in matted row culture. In annual systems, fumigation with methyl
bromide and chloropicrin is highly effective, especially where Rhizoctonia
predominates (Maas, 1998). However, pathogens ultimately reestablish in
perennial systems, and some believe that fumigation can result in an increasein
BRR as fumigation also kills beneficial soil microbes, leaving nothing to compete

with invading pathogens (Pritts and Wilcox, 1990). Methyl bromide is being

44

phased out by the end of this year (USDA, 2000), and no cost-effective
chemicals have been identified that provide the same level of control as methyl
bromide (Fennimore et al., 2003; Shaw and Larson, 1999).

Developing strawberry cultivars with tolerance to BRR would be an
effective control measure. Potter and Dale (1994) showed that ‘Guardian’ had a
high level of resistance to P. penetrans, and further studies showed considerable
variation among 19 cultivars, with the four most resistant being ‘Pajaro’,
‘Chandler’, Annapolis’, and ‘Glooscap’ (Dale and Potter, 1998). Interestingly,
those four cultivars have ‘Lassen’, a California cultivar, in their background.

In a field primarily infected with Pythium spp., ‘Tristar’, ‘Earliglow’, and
‘Midway’ had the healthiest roots (Wing et al., 1995b). However, Rhizoctonia
spp. and P. penetrans were not in the field and no disease symptoms were
observed on above-ground portions of the plants, indicating that disease
pressure was low.

Two studies in California that measured leaf number, plant diameter, yield,
fruit weight, and fruit appearance on fumigated and nonfumigated soils did not
identify any genotypes with strong tolerance to sublethal levels of soil pathogens
(Larson and Shaw, 1995; Shaw and Larson, 1996), although a significant
interaction between genotype and fumigation was observed in the second study.
The authors concluded that lack of fumigation affected all cultivars similarly as
the interaction only explained 2% to 5% of the variance. However, only a small
fraction of the strawberry germplasm grown in the USA was represented in these

studies. Only California genotypes were tested in Larson and Shaw (1995), and

45

in Shaw and Larson (1996), nine genotypes from outside California (six of which
were from the USDA program in Maryland) and nine California genotypes were
used.

Another study in Michigan using a broader array of germplasm (four
California cultivars, 11 eastern US cultivars, and 12 F. Virginiana Duch. F1
hybrids) also identified only modest differences in tolerance based on yield, fruit
weight, crown and runner number, and root and crown appearance. Fields used
in the study were infested with Pythium spp., Rhizoctonia spp., Idriella lunata
P.E. Nelson & K. Wilh., Meloidognye hapla Chitwood, and P. penetrans
(Hancock et al., 2001). Although no significant genotype x fumigation
interactions were found, there was a significant source x fumigation interaction
for fruit weight at P 5 0.01, and for yield and runner number at P _<_ 0.10. The
authors concluded that some tolerance might exist in F. Virginiana that could be
used to improve cultivars, as F. Virginiana hybrids performed better overall than
the eastern or California cultivars.

In the most recent study conducted in Michigan, 20 strawberry genotypes
were screened for field tolerance to BRR, including old and new cultivars from six
North American breeding programs as well as one wild genotype (Chapter 2).
The genotypes were evaluated for crown number, number of flowers per crown,
yield, and average berry weight in both fumigated and nonfumigated soil for two
years. A significant genotype x fumigation interaction was observed for crown
number and yield in both years of the study, explaining 46% and 26% of the

variance for yield in years 1 and 2, respectively, and 61% and 26% of the

46

variance for crown number in years 1 and 2, respectively. Interestingly, ‘Bounty’,
‘Cabot’, and ‘Cavendish’, all from the breeding program in Nova Scotia, displayed
the highest level of tolerance to BRR. These cultivars had not been previously
tested for their tolerance to BRR.

In the experiments outlined here, progeny populations were created to
determine the amount of genetic variability for BRR tolerance, where the Nova
Scotia cultivars were used as parents along with six other genotypes. Duplicate
daughter plants of the progeny population were grown on fumigated and
nonfumigated soil and were evaluated for crown number, flower number, and
yield.

Materials and Methods

We chose nine genotypes from the previous study (Chapter 2) to use as
parents: three that displayed high tolerance to BRR (‘Bounty’, ‘Cabot’, and
‘Cavendish’), three that displayed intermediate tolerance (‘Guardian', ‘Midway’,
and Winona’) and three that displayed low tolerance (‘Jewel’, LH 50-4, and
‘Mesabi’) (Table 3). The genotypes were crossed in diallel mating scheme with
no selfs, and reciprocal crosses were grouped into the same family. Thirty-two
out of 36 possible families were generated, as four crosses were not successful
and did not produce any progeny (Table 4).

Crosses were made on greenhouse-grown plants in the spring of 2003 by
removing stamens with a pair of sharp tweezers then transferring pollen to the
stigma with a camel hair paint brush or fingernail. Pollinated flowers were

covered with cheesecloth for 48 hours to reduce the chance of outside

47

pollination. In most instances, fresh pollen was collected from open flowers for
crosses, but occasionally pollen was used that had been previously collected and
stored in 1.5 mL Eppendorf tubes at -16 °C. Fruit were allowed to ripen on the
plant, then were harvested for seed collection. Seeds were sown on moist,
sterilized potting soil and placed in a growth chamber at 4 °C with continuous
inflorescent light until they began to germinate. The seedlings were then
transferred into growth chambers at ~ 20 °C until they reached the 4 - 6 leaf
stage, when they were potted in 10 x 10 x 12-cm pots. These were held in a
greenhouse in Holt, Mich. under natural daylengths and temperatures (5.5 - 38
°C) until they were planted into a field at the Horticulture Teaching and Research
Center in Holt.

On 2 June 2004, the field was fumigated with Vapam (metam sodium) in
1.2-m strips, alternating between fumigated and nonfumigated bands, at a rate of
89 L'ha". The field was planted on 21 June 2004. Plants in each family were
evenly divided into five replications, and the plants (hereafter referred to as
mother plants) were set on the border between the fumigated and nonfumigated
strips, with 1.07 m between plants. Beginning on 24 June 2004, two runners
from each of the mother plants were trained into the middle of the fumigated and
nonfumigated strips, and daughter plants were established. The runners
connecting the mother and daughter plants were cut beginning on 1 Aug. 2004,
and the mother plants were dug up and removed from the field on 31 Aug. and 1

Sept. 2004. The field received supplemental irrigation as needed, weeds were

48

controlled through use of preemergence herbicide and mechanical methods, and
fertilizer was applied on 10 Sept. at a rate of 1.8 kg‘ha‘1 N.

Plants were allowed to runner freely, with sufficient training to keep the
runners of each daughter plant separate from the runners of other daughter
plants in the field. The number of runner plants produced by each daughter plant
was counted on 21-28 Oct. 2004, after the first killing frost of the year. Crowns
were also counted on each daughter plant and all of the runner plants (crown
number), but crown counts and plants counts were essentially as over 90% of the
plants had only one crown due to the young age of the planting. The number of
infiorescences per daughter plant and all of the runner plants was counted on 23-
31 May 2005 before flowers were fully open. This number was divided by the
crown number to obtain mean number of infiorescences/crown. The number of
flowers per inflorescence were counted on five randomly-selected runner plants
from each mother plant on 9 and 10 June after all flowers were open and fruit
development had begun and an average flower number/inflorescence value was
calculated. Ten fruit were harvested from each daughter plant and the runner
plants on 20-27 June 2005 and weighed to calculate mean fruit weight. Total
yield was calculated by multiplying crown number, number of infiorescences/
crown, number of flowers/inflorescence, and mean fruit weight.

Root samples were taken from randomly-selected plants to determine
presence of fungal pathogens on 15 Aug. 2005 and plant parasitic nematodes on
29 Aug. 2005. For fungal isolation, z 10 plants were randomly selected from

both fumigated and nonfumigated soil from each of the five blocks and kept

49

separate for treatment and block. Plants were dug using a hand spade and were
placed in plastic bags on ice then stored at 1.5 °C until they were processed the
following day. Fungi were isolated from roots by washing the root systems in
running water and selecting root segments that had visible lesions. The root
segments were cut into 1-cm long sections, surface-sterilized in 1% NaOCl for
three minutes, rinsed three times in sterile distilled water, and were blotted dry on
sterile filter paper. The root pieces were placed on selective medium [1/4
strength acidified PDA supplemented with ampicillin (50pg/ml) and streptomycin
(20pg/ml)] and were transferred to fresh media as needed for identification.
Fungi were identified based on the morphology of hyphae and of spores (Barnett
& Hunter, 1998; Maas, 1998). For nematode isolation, four plants were randomly
selected from the front, middle, and back area of each of the five blocks, and
were kept separate for treatment, block, and area of the field, for a total of 120
plants. Soil was also collected from each of these areas for a total of 30
samples. Plants and soil were dug using a hand spade and were placed in
plastic bags on ice and stored at 1.5 °C until they were processed the following
day. Nematodes were extracted from root tissue following the flask-shaker
method, except that a 0.05% NaOCl solution was used in place of the ethyl-
mecuric-chloride dihydrostreptomycin sulfate solution (Bird, 1971). To make up a
1-g sample needed, ~0.259 of roots were selected from each of the four-plant
samples, for a total of 30 composite samples. Nematodes were isolated from
roots using a modified centrifugal flotation procedure with nested sieves (Jenkins,

1964).

50

Model variance components were estimated due to fumigation, family, and
family x genotype treatment interaction and error as in Larson and Shaw (1995).
The analysis of variance was conducted using the GLM function of SAS (SAS
Institute, Cary, NC). DIALLEL-SASOS (Zhang et al., 2005), a program based on
Griffing’s and Gardner-Ebhart Analyses, was used to calculate GCA and SCA
effects. Three parents, ‘Guardian’, LH 50-4, and ‘Mesabi’ were not included in
this analysis, as they did not produce sufficient progeny for a full diallel analysis.
Results and Discussion
Analysis of all the families

In the ANOVA of all the families, significant differences were observed for
both treatment and family, but not for the interaction between treatment and
family. Overall means for most of the parameters measured were higher on
fumigated soil than nonfumigated soil (Table 5). Crown number was 20% higher
(P < 0.001), mean individual fruit weight was 9% higher (P = 0.012), and total
yield was 26% higher (P < 0.001). Only the number of inflorescences/crown and
flowers/inflorescence were not significantly different between fumigated and
nonfumigated soil (P = 0.992 and P = 0.876, respectively). In our previous study
in Michigan, we found that mean yield reduction due to lack of fumigation was
46% in year one, and 33% in year 2. Average berry weight was smaller only in
the second year by just 10%, and crown number was reduced by 47% in the first
year and 41% in the second year. (Chapter 2). The lower reduction in berry
weight than crown number in both studies suggests that losses in runner

production (and therefore daughter plants and crowns) plays a more important

51

role in yield reduction associated with the lack of fumigation than loss of berry
size.

Significant differences (P < 0.001) were also observed between families
for all the parameters measured (Table 5). Mean crown numbers varied from 9.1
crowns for ‘Cabot’ x LH 50—4 to just 1.6 crowns for ‘Bounty’ x ‘Guardian’. The
rest of the eight families that had LH 50-4 as a parent had higher crown numbers
than all other families. This is not surprising, as LH 50-4 is a wild F. Virginiana
selection and produces a large number of new daughter plants from runners
(Hancock et al., 2001).

Mean individual fruit weight varied widely from the largest fruit of 16.1 g for
‘Cabot’ x ‘Cavendish’ to a low of 2.7 g for LH 50-4 x ‘Midway’. The eight families
with the lowest fruit weight each had LH 50-4 as a parent, which is expected
because of the small fruit size of LH 50-4.

The number of inflorescences/crown varied significantly across families,
but showed less variability than crown numbers or fruit weight. The number of
flowers/inflorescence ranged from a high of 9.3 for ‘Bounty’ x LH 50-4 to a low of
6.4 for ‘Guardian’ x ‘Winona’.

Total yield varied across families from a 1419.9 9 average for the progeny
of ‘Bounty’ x ‘Cabot’ to just 241.4 g for ‘Mesabi’ x ‘Midway’. Other high-yielding
families were ‘Cavendish’ x ‘Jewel’, ‘Bounty’ x ‘Cavendish’, ‘Bounty’ x ‘Winona’,
and ‘Cabot’ x ‘Cavendish’. Fruit weight appeared to be the factor that contributed
most to high yield, as three of the high-yielding families ranked high for fruit

weight (‘Bounty’ x ‘Cabot’, ‘Cavendish‘ x ‘Jewel’, and ‘Cabot’ x ‘Cavendish’) and

52

the other two were in the middle (‘Bounty’ x ‘Cavendish’ and ‘Bounty’ x
‘Winona’). Crown number contributed more modestly to high yield, with ‘Bounty’
x ‘Cabot’, ‘Bounty’ x ‘Cavendish’, and ‘Bounty’ x ‘Winona’ all having mid-range
crown numbers. The high number of flowers/inflorescence found in ‘Bounty’ x
‘Cavendish’ and ‘Bounty’ x ‘Winona’ also contributed to high yield.

Analysis of the complete diallel

In the ANOVA used to calculate general combining ability (GCA) and
specific combining ability (SCA) among the smaller set of families, treatment
effects were significant for crown number (P < 0.001) and total yield (P < 0.001),
but were not for infiorescences/crown (P = 0.757), flowers/inflorescence (P =
0.737) or mean individual fruit weight (P = 0.085) (Table 6). Similar to the
analysis with all of the families, crown number was 26% higher, and yield was
28% higher on fumigated soil. There were also significant differences (P < 0.01)
among families for crown number, berry weight, and total yield which mirrored
the full family analysis.

GCA was significant (P 5 0.01) for all parameters, and SCA was
significant for crown number (P = 0.014), fruit weight (P < 0.001 ), and total yield
(P = 0.007), but not for the number of inflorescences/crown (P = 0.987) or the
number of flowers/inflorescence (P = 0.399) (Table 6). These results are as
expected, as numerous studies on strawberries have uncovered a great deal of
genetic variability for most yield components (Hancock, 1999).

While there was considerable genetic variability observed among families

for most of the yield components, there appeared to be little genetic variability for

53

resistance to BRR. Treatment x family interactions were not significant in the
diallel analysis, as was the case in the analysis with the complete set of families.
Similarly, all of the treatment X GCA and treatment x SCA interactions were not
significant, except for a treatment x GCA interaction for berry weight (P = 0.049).
This result was surprising as we previously observed significant differences in
tolerance among the cultivars used as parents in this comparison (Chapter 2).

It may be that the difference in disease pressure between fumigated and
nonfumigated plots was not as great in this study as in the previous one, and as
a result made it more difficult to recognize tolerant types. Average yield was
46% higher due to fumigation in the first year after fumigation in the previous
study, as opposed to 26% in this study. The fumigated strips in this study were
only 1.2-m wide and bordered by nonfumigated strips on each side, so
pathogens did not have to move very far in order to reestablish in fumigated soil.
In the previous study, 11.5 x 4.5-m split plots (of 11.5 x 9-m plots) were
fumigated, so there was a larger barrier to pathogens moving into fumigated soil
from nonfumigated soil. We can also not rule out the possibility that Vapam,
which was used in this study, is a less-effective fumigant than methyl-bromide,
which was used in the previous study (Shaw and Larson, 1999).

Overall, the number of nematodes found in fumigated and nonfumigated
areas was not significantly different (P = 0.2848), nor were the number found in
soil alone (P = 0.4786) or roots alone (P = 0.2295) (Table 7). The only significant
difference between fumigated and nonfumigated areas was for the number of

root lesion nematodes found in root samples (P = 0.0127). The majority of

nematodes from soil samples were ring nematodes (Cn'conemella xenoplax De
Grisse & Loof), while the majority of nematodes found in root samples were root
lesion nematodes. When the root and soil samples were combined, 93% from
fumigated areas contained nematodes, while 100% from nonfumigated areas
contained nematodes. In the previous field studies using methyl bromide and
chloropicrin, very few samples from fumigated soil contained nematodes (5% and
8% the first and second year after fumigation, respectively), and they were found
in very low numbers (Chapter 2).

Rhizoctonia sp. was the most common pathogenic fungi found in root
samples, and was found equally in both fumigated and nonfumigated soil (Table
8). Fusarium sp. were also commonly found, both from fumigated and
nonfumigated samples. Fusarium sp. is generally considered a weak pathogen
in strawberries (A. Schilder, personal communication), and has been shown to
interact with P. penetrans in the BRR complex (Maas, 1998). Overall, 48% of the
root samples containing fungi were from fumigated areas, and 54% were from
nonfumigated areas. In the previous study, both Rhizoctonia sp. and Pythium sp.
were found, but Rhizoctonia sp. was more common (Chapter 2).

In conclusion, it appears that pathogen pressure had equalized in the
fumigated and nonfumigated plots by the end of the first fruiting season, even
though positive fumigation effects were observed on most yield components.
Apparently, the plants in this study were under less pathogen pressure than
those in our previous one. The data obtained in this study suggest that genetic

improvement in tolerance to BRR will be difficult to achieve, expect perhaps

55

under heavy pathogen pressure. Part of the difficulty in identifying tolerant types
is likely due to the fact that BRR is a disease complex involving three different
pathogens and the relative pressure from these pathogens can vary from year to
year and site to site (Wing et al., 1994). Several other studies have also reported
limited genetic variation in levels of tolerance to BRR (Hancock et al., 2001;
Larson and Shaw, 1995; Shaw and Larson, 1996). The key to finding cultivars
that will perform well in BRR-infested soils will be to select in soils with a long

history of no fumigation.

56

Table 3. Strawberry genotypes with high, medium, and intermediate tolerance to

black root rot used as parents in a diallel mating scheme.

 

Year

released Ofig'"

Genotype Parentage

 

High tolerance

Bounty 1972 Jerseybelle x Senga Sengana AAFC‘, Kentville, N.S.

Cabot 1998 K78-5 x K86-19 AAFC, Kentville, N.S.

Cavendish 1990 Glooscap x Annapolis AAFC, Kentville, N.S.
Intermediate tolerance

Guardian 1969 NC 1768 x Surecrop USDA-Beltsville, Md.

Midway 1960 Dixieland x Temple USDA-Beltsville, Md.

Winona 1995 Ear li9'0W " MNUS 52 Univ. of Minnesota, St. Paul

and USDA-Beltsville, Md.

Low tolerance

Jewel 1985 NY 1221 x Holiday NYSAES’, Geneva, NY

Mesabi 2000 Glooscap x MNUS 99 Univ. of Minnesota, St. Paul

Native F. Virginiana from

LH 50'4 -.. ..- Montana

 

2 Agriculture and Agri-Food Canada.

y New York State Agricultural Experiment Station

57

Table 4. Diallel mating scheme showing the number of progeny generated from
each cross. Note that there were no selfs, and that reciprocal crosses were

grouped into the same family.

 

Genotype Cabot Cavendish Guardian Jewel LH 50-4 Mesabi Midway Winona

Bounty 50 10 19 50 50 8 28 8
Cabot 50 50 8 50 0 32 41
Cavendish 0 50 50 0 50 50
Guardian 0 14 24 10 5
Jewel 50 23 39 50
LH 50-4 50 50 50
Mesabi 8 1 1
Midway 50

 

58

Table 5. Analysis of variance and variance components with all families for
crown number, inflorescence number, flowers per inflorescence, fruit weight, and
total yield of strawberry families grown in Holt, Mich., on fumigated and

nonfumigated soil. Plants were set in 2004 and evaluated over one year.

 

Crown Inflorescence Flowers per Fruit Total

 

Source number per crown inflorescence weight yield

Fumigation
Fumigated 5.0 2.4 7.8 8.6 711.0
Nonfumigated 4.0 2.4 7.7 7.8 526.3

Family
Bounty x Cabot 4.3 2.9 7.8 14.9 1419.9
Bounty x Cavendish 4.1 2.7 8.3 9.2 870.3
Bounty x Guardian 1.6 3.1 8.0 7.4 341.0
Bounty x Jewel 2.4 3.0 8.5 9.0 541.9
Bounty x LH50-4 7.0 2.5 9.3 3.4 580.8
Bounty x Mesabi 2.3 3.5 7.8 7.9 443.8
Bounty x Midway 2.6 3.1 8.2 7.1 510.8
Bounty x Winona 5.0 2.3 8.5 9.4 840.7
Cabot x Cavendish 2.8 2.4 7.0 16.1 804.6
Cabot x Guardian 5.0 1.6 6.6 11.1 575.8
Cabot x Jewel 2.2 2.5 7.3 12.9 445.9
Cabot x LH50-4 9.1 2.1 8.3 4.6 761.4
Cabot x Midway 1.8 2.9 7.0 8.6 365.9
Cabot x Winona 4.2 2.0 6.4 13.8 794.4
Cavendish x Jewel 3.3 2.8 7.8 13.2 979.2
Cavendish x LH50-4 6.6 2.7 8.3 4.1 578.3
Cavendish x Midway 1.9 2.9 6.7 9.2 398.8
Cavendish x Winona 3.7 2.4 7.0 11.2 727.4
Guardian x LH50-4 8.5 1.9 7.6 2.8 334.6
Guardian x Mesabi 2.7 2.5 7.5 8.6 490.3
Guardian x Midway 1.6 4.3 7.3 6.3 315.8
Guardian x Winona 2.6 3.0 6.4 9.4 412.4
Jewel x LH50-4 8.4 2.3 9.1 3.1 579.8
Jewel x Mesabi 3.5 2.3 7.9 11.3 795.6
Jewel x Midway 2.4 3.0 7.1 7.1 449.5
Jewel x Winona 2.5 2.6 7.9 9.9 544.4

59

LH50-4 X Mesabi 6.3 2.9 8.3 3.6 564.0

LH50-4 x Midway 5.5 3.0 8.3 2.7 376.5
LH50-4 x Winona 6.2 2.5 7.6 3.2 363.8
Mesabi x Midway 1.8 3.4 6.8 6.4 241.4
Mesabi x Winona 3.3 2.6 6.8 11.1 665.6
Midway x Winona 3.4 2.6 7.2 8.3 482.7
Mean 4.0 2.7 7.6 8.3 581.2
ANOVA
Significance (P)
Treatment (T) 0.001 0.992 0.876 0.012 < 0.001
Block/F 0.009 0.002 < 0.001 < 0.001 0.081
Family (F) < 0.001 < 0.001 < 0.001 < 0.001 < 0.001
T x F 0.928 0.966 0.576 0.362 0.527

 

‘ Crown number and total yield are expressed per daughter plant and all of her

runner plants.

60

Table 6. Analysis of variance and variance components with the smaller set of
families for crown number, inflorescence number, flowers per inflorescence, fruit
weight, and total yield of strawberry families grown in Holt, Mich., on fumigated

and nonfumigated soil. Plants were set in 2004 and evaluated over one year.

 

 

Crown Inflorescence Flowers per Fruit
Source df number per crown inflorescence weight Total yield

Treament (T) 1 26.1 ** 0.05 0.1 17.0 13466996“
Block/T 8 1.8 0.74 1.0 16.0“ 1674455
Family (F) 14 9.4" 0.95 4.5“ 79.6“ 8110997“

GCA 5 19.3“ 2.34" 11.0" 193.7“ 13893291"

SCA 9 4.1" 0.16 1.0 15.0” 5003594“
T x F 14 1.4 0.44 1.0 7.5 73205.1

T x GCA 5 2.5 0.46 1.2 128" 1228679

T x SCA 9 1.1 0.42 1.0 5.8 49319.0
Error 110 1.7 0.56 0.9 5.5 1055035

 

2 Crown number and total yield are expressed per daughter plant and all of her

runner plants.

61

 

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62

Table 8. Percentage of root samples containing plant parasitic fungi from
strawberries grown on fumigated and nonfumigated soil in Holt, Mich. Plants

were set in 2004 and evaluated over one year.

 

 

 

Treatment

Pathogen FumiLated Nonfumigated
Rhizoctonia 9.8 1 1.8
Epicoccum 9.8 7.8
Fusarium 1 1.8 13.7
Trichodenna 2.0 -
Alteman'a 2.0 -
Unknown 11.8 19.6

 

63

APPENDIX
EVALUATION OF STRAWBERRY PEDIGREES TO DETERMINE IF BLACK
ROOT ROT TOLERANCE CAN BE TRACED TO A COMMON ANCESTRY
Introduction

The most effective way to control BRR would be to identify or to develop
tolerant strawberry cultivars. Previous studies identified little genetic variability in
tolerance to BRR in field screens (Hancock et al., 2001; Larson and Shaw, 1995;
Shaw and Larson, 1996), but a very limited amount of the strawberry germplasm
was evaluated. Therefore, we screened 20 different strawberry genotypes from
six breeding programs for field tolerance to BRR (Chapter 2). The genotypes
were evaluated for crown number, number of flowers per crown, yield, and
average berry weight in both fumigated and nonfumigated soil for two years. The
results showed that there was a significant genotype x fumigation interaction for
crown number and yield in both years of the study. Interestingly, ‘Bounty’,
‘Cabot’, and ‘Cavendish’, all released from the breeding program in Nova Scotia,
displayed the highest level of tolerance to BRR.

A study by Sjulin and Dale (1987) evaluated the genetic diversity of
strawberry cultivars released from North American breeding programs from 1960
to 1985. They traced the pedigrees of the cultivars back to the founding clones,
calculated the genetic contribution each founding clone made to the cultivar, and
clustered them using Ward’s method (Ward, 1963). The cultivars clustered into

nine groups that were highly related to geographic origin, most strongly between

64

cultivars developed within California (two groups) and those developed outside of
California (seven groups).

In order to determine if tolerance (or lack thereof) to BRR can be attributed
to a specific origin, we traced the pedigrees of the 19 cultivars included in the
field screen (Chapter 2) and conducted analyses similar to Sjulin and Dale’s
(1987)

Materials and Methods

The 19 cultivars included in the study were all released from breeding
programs in North America from 1960-2000 (Table 9). The pedigrees of the
cultivars were traced using Brooks and Olmo (1997), Darrow (1937, 1966), Etter
(1920), Hedrick (1925), internet sources and published cultivar descriptions.
Personal communication from strawberry researchers was used as well.
Pedigrees were traced to a point where the parentage of a genotype was either
not known or differed between sources, and those genotypes were referred to as
founding clones (Table 10).

The genetic contribution was calculated as in Sjulin and Dale (1987).
However, unlike Sjulin and Dale, we did not assume that genotypes selected
from open-pollinated populations were self-pollinated, and instead designated the
unknown parent as the pollen parent of that particular genotype (Table 10). We
also did not sum together and enter as a single variable those founding clones
whose genetic contributions were always equal, and Excel® (Microsoft

Corporation, Redmond, Wash.) spreadsheets were used to calculate the genetic

65

contribution. The “multivariate” and “cluster observation” functions of MINITAB
were used for the cluster analysis.
Results and Discussion

The three BRR tolerant cultivars, ‘Bounty’, ‘Cabot’, and ‘Cavendish’
(Chapter 2), did not cluster together and in fact were quite distant in the cluster
analysis (Figure 6). Therefore, we were not able to identify a source of BRR
tolerance. The genes for tolerance likely come from a source in the background
of most cultivars.

Cluster analysis also did not indicate that cultivars grouped according to
geographic origin (Figure 6). For example, the cultivars from the Nova Scotia
breeding program did not group closely together, nor did the cultivars from the
USDA program in Maryland. The lack of distinct cluster groups can partially be
explained because 18 of the 57 founding clones appeared in at least 18 of the 19
cultivars included in the study (Table 11). These 18 founding clones accounted
for approximately 50 - 90% of the genetic contribution of each cultivar. Another
possible reason for the lack of cluster groups could be because of the small
number of cultivars included in the study. In Sjulin and Dale’s (1987) study, the
pedigrees of 134 cultivars were traced.

Some cultivars were found on distinct branches, separated from most
other cultivars (Figure 6, Table 11). ‘Bounty’ did not loosely group with any other
cultivars because ‘Markee’ accounts for 25% its genetic composition. ‘Chandler‘
fell onto a distinct branch because ‘Aberdeen’, which accounts for 11 — 25% of

the genetic contribution of all other cultivars, is absent from it. Also, ‘Chandler’

66

has a large amount of ‘Middlefield’ and the pollen parent of ‘Nich Ohmer’ in its
background. MN 2374 accounts for 13% of the genetic contribution of ‘Mesabi’,
causing it to be on a distinct branch. MN 2374 is a selection from the University
of Minnesota breeding program from 19605 when Wayne Wilcox was selfing
cultivars such as ‘Dunlap’ and ‘Trumpeter’ for up to six generations (J. Luby,
personal communication). Unfortunately, the exact background of MN 2374 is
unknown. ‘Kent’ fell onto a distinct branch due to the unique contributions from
Seedling T08 and ‘Bubach’. ‘Jewel’ fell into its own branch due to a 13%
contribution from ‘Markee’, as well as small contributions from other founding

clones such as ‘Early Jersey Giant’ and ‘lnepuisable’.

67

Table 9. Strawberry cultivars included in the pedigree study to determine if
tolerance to black root can be traced to a specific origin. These same cultivars

were evaluated in a field study to determine their level of tolerance to black root rot

 

 

(Chapter 2).

(“WWW ”'12:; d Parentage Origin

Allstar 1981 US 4419 x MDUS 3184 USDA-Beltsville, Maryland
Annapolis 1984 K74-5 x Earliglow AAFC’, Kentville, N.S.
Bounty 1972 Jerseybelle x Senga Sengana AAFC, Kentville, N.S.
Brunswick 1999 Cavendish x Honeoye AAFC, Kentville, N.S.
Cabot 1998 K78-5 x K86-19 AAFC, Kentville, N.S.
Cavendish 1990 Glooscap x Annapolis AAFC, Kentville, N.S.
Chandler 1983 Douglas x Cal 72.361-105 University of California, Davis
Earliglow 1975 MDUS 2359 x MDUS 2713 USDA-Beltsville, Maryland
Evangeline 1999 Honeoye x Veestar AAFC, Kentville, N.S.
Gov. Simcoe 1985 Holiday x Guardian HRIO", Simcoe, Ontario
Guardian 1969 NC 1768 x Surecrop USDA-Beltsville, Maryland
Honeoye 1979 Vibrant x Holiday NYSAES", Geneva, New York
Jewel 1985 NY 1221 x Holiday NYSAES, Geneva, New York
Kent 1981 K 68-5 x Raritan AAFC, Kentville, N.S.
Mesabi 2000 Glooscap x MNUS 99 University of Minnesota, St. Paul
Midway 1960 Dixieland x Temple USDA-Beltsville, Maryland
Mira 1995 Scott x Raritan AAFC, Kentville, N.S.
Surecrop 1956 Fairland x MDUS 1972 USDA-Beltsville, Maryland
Winona 1995 Earliglow x MNUS 52 University of Minnesota, St Paul &

USDA-Beltsville, Maryland

 

2Agriculture and Agri-Food Canada

Y Horticulture Research Institute of Ontario

" New York State Agricultural Experiment Station

68

 

Table 10. Origin and mean genetic contribution (GC) of the founding clones to

the 19 North American strawberry cultivars included in the pedigree study to

determine if tolerance to black root rot can be traced to a specific origin.

 

 

Clone no. Founding clones (igln Mean GC (%)
1 Belmont Massachusetts, 1880 4
2 British Queen England, 1840 <1
3 F. Virginiana N.J. Scarlet New Jersey, 1868 9
4 Jucunda England, 1854 1
5 Methven Scarlet England, 1815 5
6 Sharpless Pennsylvania, 1872 6
7 White Carolina England, before 1806 2
8 Wilson New York, 1851 3
9 Pollen parent of Keen's Seedling - 4
10 Pollen parent of Keen's Imperial - 2
11 Pollen parent of Hoffman - 1
12 Pollen parent of Goliath - <1
13 Pollen parent of Neunan - <1
14 Pollen parent of Clyde - 3
15 Pollen parent of Vicomtesse - 2
16 Aberdeen New Jersey, before 1917 18
17 Missionary Virginia, 1900 13
18 Pollen parent of Black Prince - 2
19 William Belt Ohio, 1888 1
20 Pollen parent of Portia - 1
21 Chesapeake Maryland, 1903 2
22 Middlefield Connecticut, 1890 2

Pollen parent of unnamed
23 seedling in pedigree of Rose - <1

Ettersburg
24 F. chiloensis (unnamed) Unknown, before 1905 <1
25 F. chiloensis (unnamed) Peru, before 1905 <1
26 Frith Scotland, 1918 2
27 Glendale Ohio, 1971 <1
28 Jersey Queen New Jersey, 1878 <1
29 Parry New Jersey, 1880 <01
30 Chair's Favourite England, <01
31 Banner Massachusetts, 1890 <1
32 Ettersburg121 California, 1905 1
33 Pollen parent of Nich Ohmer - 2
34 Marshall Massachusetts, 1890 <1
35 Teutonia Germany (7) <1
36 Unser Fritz Germany, 1872 <1
37 Pollen parent of KOnig Albert von __ <1

Sachsen
38 Cassandra Ontario, 1906 <1
39 Markee Germany, before 1942 3
40 Pocomoke Maryland, 1902 1
41 Pollen parent of Aroma —- <1
42 Pearl New Jersey, 1889 <1

 

43
44
45
46
47

48

49
50
51
52
53
54
55
56
57

SeedlingTDB

Bubach

Cal. 1021

Cal. BH14

Early Jersey Giant

F. chiloensis from Cape
Mendocino

F. Virginiana glauca
Inepuisable

Longworth

Lucie Boisselot

MN 2374

Streamliner

Pollen parent of US 235 op
Pollen parent of St. Joseph
Pollen parent of Louis Gauthier

70

Scotland, before 1945
lllinios, 1882
California, before 1930
California, before 1930
New Jersey, 1907

California, before 1905

Unknown, before 1905
France, 1870

Ohio, 19848

France, before 1930
Minnesota, before 1970
Oregon, 1938

 

Table 11. Cultivars and the genetic contribution of the founding clonesz included

in the pedigree study to determine if tolerance to black root rot can be traced to a

specific origin.

 

Cultivar
“009 Bou Cha Cab Ann Bru Ear Mir Cav Mid Win All Gov Grd Sur Eva Hon Mes Knt le

 

Founding

 

4

444344444

4

4

7

8

106

10 10 10 10 10 10 11

91097

9

9

4

3

5566

5 5

5

5
6 6 6 7 5 5 6 5
2 2 2 2 2 2 2 2
4 2 3 3 2 2 2 2

5554

5
6
2
2
4
2

7
2
3
5
2

5
2
2
4
2

6
2
2
4
2

6

11

44444454
22222232

10
11
12
13
14
15
16
17
18
19
20
21

4

4
2

44434444

2 2 2 2 2 2 2 2
17 22 20251517 25 241620192524 23

4
2

4
2

3

11 20 16

13

11 21

19913261913816 8

12121318 7

15

6

2

2
2
2
8

22222222
322
322

2

1
1

1
1
1

34144

13

22
23

24
25
26
27
28

13

6

29
30

314

243121136

.3
2637211
5
2241 .
1321111 3
2
1421
111 111 2
1
63.1158
5
123456789
333333333

71

41....1.1.......22

47 . . . . . . . . . . . . . . . . . . 2
49 . . 1

50

53 . . . . . . . . . . . . . . . . 1 3

55 . 1 . . . . . . . . . . . . . . . . .
56 . . . . . . . . . . . . . . . . . . 1
57 . . . . . . . . . . . . . . . . . . 1

‘ K

7' See Table 10 for the names of the founding clones.

 

72

 

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Phytopathol. 89(6):S1 (Abstr.)

Abu-Gharbieh, W., E.H. Varney, and W.R. Jenkins. 1962. Relationship of
meadow nematode to Verticillium wilt of strawberries. Phytopathol. 52: 921.
(Abstr.)

Bailey, J.S. 1956. Does the root-knot nematode (Meloidogyne sp.) thrive in the
roots of strawberry plants in Massachusetts? Plant Dis. Rptr. 40(1):44. (Abstr.)

Barnett, H.L. and BB. Hunter. 1998. Illustrated genera of imperfect fungi. 4th
Edition. APS Press, St. Paul MN.

Berkeley, G.H. and l. Lauder-Thomson. 1934. Root rots of strawberry in Britain:
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246.

Bird, G.W. 1971. Influence of incubation solution of the rate of recovery of
Pratylenchus penetrans from cotton roots. J.Nematol. 32378-385.

Braun, A.J. and J.A. Keplinger. 1960. The pathogenicity of meadow nematodes
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Phytopathol. 50:239. (Abstr.)

Brooks, RM. and HP. Olmo. 1997. The Brooks and Olmo Register of fruit and
nut varieties. ASHS Press, Alexandria, Va.

Chapman, RA. 1956. Plant parasitic nematodes associated with strawberries in
Kentucky. Plant Dis. Rptr. 40:179-183.

Chen, TA. and Rich, A.E. 1962. The role of Pratylenchus penetrans in the
development of strawberry black root rot. Plant Dis. Rptr. 46(12):839-843.

Coons, G.H. 1924. Black root rot of strawberry. Mich. Agr. Expt. Sta. Quarterly
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