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ALTERNATIVES TO METHYL BROMIDE FOR
CONTROLLING THE BLACK ROOT ROT DISEASE
COMPLEX OF STRAWBERRY

presented by
BENJAMIN W. GLASS

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ALTERNATIVES TO METHYL BROMIDE FOR CONTROLLING THE BLACK
ROOT ROT DISEASE COMPLEX OF STRAWBERRY

By

Benjamin W. Glass

A THESIS

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

MASTER OF SCIENCE
Plant Pathology

2008

ABSTRACT

ALTERNATIVES TO METHYL BROMIDE FOR CONTROLLING THE BLACK
ROOT ROT DISEASE COMPLEX OF STRAWBERRY

By
Benjamin W. Glass
The United States strawberry industry is heavily dependent on methyl bromide to
control soil-borne diseases and weeds. The continued phase-out of methyl bromide has
left a void in the arsenal growers have traditionally used to manage these pests.
Strawberry black root rot is a disease complex of Rhizoctoniafragariae Husain &
McKeen, Pythium species, and the nematode Pratylenchus penetrcms (Cobb) Filipjev and
Shuurmans Stekhoven that affects the productivity and longevity of strawberry plantings.
In a Michigan study, 13 commercial bio-control or reduced-risk fungicides, applied as
drenches or pre-plant dips, were evaluated for their efﬁcacy in the control of black root
rot at the time of planting in a naturally infested ﬁeld. This experiment was also
replicated in the greenhouse. A pre-plant root dip of azoxystrobin (Abound) with a
potassium salt (ProPhyt) has shown a measure of control. Experiments conducted to
evaluate combinations of crop rotations, in conjunction with two commercially available
biological control products, in an effort to create suppressive soils prior to the
establishment of strawberries, and a study to evaluate the ‘best practices’ for controlling
black root rot at the time of planting consisting of crop rotation, fungicide dip, compost, a
biocontrol product, and the use of resistant varieties have shown that while fumigation
resulted in the largest, healthiest plants, the use of the rotational crops squash, rye, and
Brassica spp. tended to cause healthier strawberry plants. Compost and the Abound +

ProPhyt pre-plant dip offered some improvement in plant health.

ACKNOWLEDGEMENTS

I would like to thank Annemiek Schilder for the opportunity to work on this
roject and the support her laboratory provided. It allowed me to work on a distinctly
ifferent system than I had been exposed to previously. In addition to her involvement,
ie input from Jim Hancock and Mary Hausbeck were invaluable to my development as a
:searcher and a person. I also appreciate the insights and knowledge contributed by
.oger Sysak and Jerri Gillett, which made my time at Michigan State University a
aluable experience. My work would not have been as complete without the assistance
fBill Chase and Gary Winchell at the Horticulture farm, Fred Warner and Angela
enney in the Diagnostic Services, and Jennifer Powers. I am also grateful to George
ird, John Davenport, Gerard Adams, Mursel Catal, Andrew Jarosz, and the laboratory
fFrances Trail for their assistance in the project. Finally, I would like to thank Gary
ardenhagen and Phil DeLange for the opportunity to work with them in their production

elds.

iii

TABLE OF CONTENTS
CHAPTER ONE: LITERATURE REVIEW ...................................................... I
Introduction ................................................................................... 1
Strawberry Production ...................................................................... 4
Black Root Rot Complex ................................................................... 8
Rhizoctonia spp. ........................................................................... 10
Pythium spp ................................................................................. 12
Nematodes .................................................................................. 14
Other Factors Inﬂuencing Black Root Rot .............................................. 18
Black Root Rot in Michigan ............................................................. 19
Current Control ............................................................................. 20
Methyl Bromide Fumigation ............................................................. 21
Biological Control ......................................................................... 23
Cultural Control ............................................................................. 26
Literature Cited ............................................................................. 29

CHAPTER TWO: BIOCONTROL AND REDUCED-RISK F UNGICIDE EFFICACY

TRIAL ................................................................................................ 37
Introduction ................................................................................. 37
Materials and Methods .................................................................... 40
Results and Discussion .................................................................... 48
Literature Cited ............................................................................. 64

CHAPTER THREE: GREENHOUSE BIOCONTROL AND REDUCED RISK

FUNGICIDE EFFICACY TRIAL .................................. , ............................ 66
Introduction ................................................................................. 66
Materials and Methods .................................................................... 69
Results and Discussion .................................................................... 82
Literature Cited ........................................................................... 104

CHAPTER FOUR: EVALUATION OF AN INTEGRATED APPROACH FOR THE

MANAGEMENT OF BLACK ROOT ROT .................................................. 106
Introduction ............................................................................... 106
Materials and Methods .................................................................. 109
Results and Discussion .................................................................. 1 17
Literature Cited ........................................................................... 131

CHAPTER FIVE: ESTABLISHMENT OF BIOCONTROLS IN ROTATIONS........133

Introduction ............................................................................... l 3 3
Materials and Methods .................................................................. 135
Results and Discussion ................................................................... 142
Literature Cited ........................................................................... 159

iv

CHAPTER SIX. SLOWING BLACK ROOT ROT IN DECLINING ESTABLISHED

STRAWBERRYFIELDS ......................................................................... 162
Introduction ............................................................................... I 62
Materials and Methods .................................................................. 163
Results and Discussion .................................................................... 172
Literature Cited ........................................................................... 187

CHAPTER SEVEN: EVALUATING RHIZOCTONIA VIRULENCE IN-VITRO.....I89

Introduction ............................................................................... I 89
Materials and Methods .................................................................. 190
Results and Discussion .................................................................. 192
Literature Cited ........................................................................... 195
APPENDIX A: INOCULATION PROCEDURE EVALUATION ........................ 196
Introduction ............................................................................... 196
Materials and Methods .................................................................. 196
Results and Discussion .................................................................. 202
Literature Cited ........................................................................... 207
APPENDIX B: INOCULATION OPTIMIZATION ......................................... 208
Introduction ............................................................................... 208
Materials and Methods .................................................................. 208
Results and Discussion .................................................................. 211

APPENDIX C: RISK RATINGS FOR MAJOR PARASITIC NEMATODES OF
STRAWBERRIES IN MICHIGAN ............................................................. 214

 

 

LIST OF TABLES

Table 1. Products, active ingredient, application method, and label rate used of strawberry
biocontrol and fungicide products applied at planting in an efﬁcacy trial in East Lansing,
MI from 2005-2007 ................................................................................... 41

Table 2. Plant vigor and bed ﬁll ratings used in the strawberry biocontrol and fungicide
products applied at planting efﬁcacy trial in East Lansing, MI during 2005-2007. . . . .....44

Table 3. Effects of different biocontrol and fungicide products applied at planting on
plant vigor and bed ﬁll of strawberry cv. Allstar in a black root rot infested soil in East
Lansing, MI, in 2005 and 2006 .................................................................... 50

Table 4. Effects of different biocontrol and fungicide products applied at planting on
plant vigor and bed ﬁll of strawberry cv. Allstar in a black root rot infested soil in East
Lansing, MI, in 2007 ................................................................................ 53

Table 5. Effects of different biocontrol and fungicide products applied at planting on
plant vigor, bed ﬁll, and yield of strawberry cv. Allstar in a black root rot infested soil in
East Lansing, MI, in 2005-2007. ................................................................. 55

Table 6. Effects of different biocontrol and fungicide products applied at planting on
individual plant parameters of strawberry cv. Allstar in a black root rot infested soil in
East Lansing, MI, in 2006. ........................................................................ 57

Table 7. Effects of different biocontrol and fungicide products applied at planting on
individual plant parameters of strawberry cv. Allstar in a black root rot infested soil in
East Lansing, MI, in 2007. ......................................................................... 59

Table 8. Root (1 g) and soil (100 cm3) nematode samples taken from efﬁcacy trial of
different biocontrol and fungicide products applied at planting on individual plant
parameters of strawberry cv. Allstar in a black root rot infested soil in East Lansing, MI,
in 2005, 2006, and 2007. ........................................................................... 61

Table 9. Frequency of fungal genera isolated from roots in different biocontrol and
fungicide products applied at planting to strawberry cv. Allstar in a black root rot infested
soil in East Lansing, MI, in 2005-2007 .............................................................. 62

TablelO. Potted plant experiments conducted, inoculum level used, pathogens tested
against, and number of blocks used in greenhouse trials evaluating efﬁcacy of strawberry
biocontrol and reduced-risk fungicide applied at planting in East Lansing, MI. . . . .74

vi

Table II. Treatments, manufacturer rates, active ingredient and application method used
in a greenhouse trial evaluating the efﬁcacy of biocontrol and reduced-risk fungicide
applied at planting to strawberry cv. Allstar for control of black root rot pathogens in East
Lansing, MI from 2005 ............................................................................. 76

Table 12. Treatments, manufacturer rates, and application method, used in a greenhouse
tn'al evaluating biocontrol and reduced-risk fungicide applied at planting in strawberry
cv. Allstar inoculated with black root rot pathogens in East Lansing, MI in 2006 and

2007 .................................................................................................... 77

Table 13. Effects of different biocontrol and fungicide products on plant biomass and root
health of strawberry cv. Allstar in a greenhouse study in soil with and without inoculation
with Rhizoctoniaﬁ'agariae in East Lansing, MI, in 2005. .................................... 85

Table 14. Effects of different biocontrol and fungicide products on plant biomass and
root health of strawberry cv. Allstar in a greenhouse study in soil inoculated with and
without Rhizoctoniafragariae in East Lansing, MI, in 2006 ................................. 89

Table 15. Frequency of fungi isolated from roots of strawberry cv. Allstar treated with
biocontrol and fungicide products applied to the in a greenhouse study in soil inoculated
with and without Rhizoctoniafragariae in East Lansing, MI, in 2006 ....................... 90

Table 16. Effects of different biocontrol and fungicide products on plant biomass and
root health of strawberry cv. Allstar in a greenhouse study in soil inoculated with and
without Pythium ultimum var. ultimum in East Lansing, MI, in 2006. . . . . . . . . . ...............92

Table 17. Frequency of fungi isolated from roots of strawberry cv. Allstar treated with
biocontrol and fungicide products applied to the in a greenhouse study in soil inoculated
with and without Pythium ultimum var. ultimum in East Lansing, MI, in 2006 ............ 93

Table 18. Effects of different biocontrol and fungicide products on plant biomass and
root health of strawberry cv. Allstar in a greenhouse study in soil inoculated with and
without Pythium ultimum var. ultimum + Rhizoctoniafragariae in East Lansing, MI, in
2006 .................................................................................................................................... 95

Table 19. Frequency of fungi isolated from roots of strawberry cv. Allstar treated with
biocontrol and fungicide products applied to the in a greenhouse study in soil inoculated
with and without Pythium ultimum var. ultimum + Rhizoctoniafragariae in East
Lansing, MI, in 2006. ................................................................................ 96

Table 20. Effects of different biocontrol and fungicide products on plant biomass and
root health of strawberry cv. Allstar in a greenhouse study in soil inoculated with and
without Rhizoctoniaﬁagariae in East Lansing, MI, in 2007 ................................ 100

vii

Table 21. Frequency of fungi isolated from roots of strawberry cv. Allstar treated with
biocontrol and fungicide products applied to the in a greenhouse study in soil inoculated
with and without Rhizoctoniafmgariae in East Lansing, MI, in 2007 .................... 101

Table 22. Effects of oat bran as an inoculum carrier on plant weights and root quality in
a greenhouse study using cv. Allstar in a greenhouse study using autoclaved soil
inoculated with and without Rhizoctoniafragariae in East Lansing, MI in 2005 and

2007 ................................................................................................... 102

Table 23. Main treatments, sub-treatments, and strawberry varieties used in the
integrated alternatives experiment conducted in a black root rot infested ﬁeld in East
Lansing, MI during 2006—2007. .................................................................. l 1 1

Table 24. Bed ﬁll and plant vigor ratings used in the integrated management practices
experiment conducted in a black root rot infested ﬁeld in East Lansing, MI during
2006-2007 using strawberry cvs. Allstar and Cavendish ...................................... 113

Table 25. Effects of cultural and chemical treatments applied at planting to strawberry cv.
Allstar and Cavendish on plant vigor, bed ﬁll, biomass, and yield in a black root rot
infested soil in East Lansing, MI, in 2006 and 2007 ........................................... 121

Table 26. Root lesion and general plant health ratings of strawberry cv. Allstar and
Cavendish grown under in different cultural and chemical treatments in a black root rot
infested soil in East Lansing, MI, in 2006 and 2007 .......................................... 122

Table 27. Plant parasitic nematode populations in 100 cm3 soil or 1 g roots from roots of
strawberry cv. Allstar and Cavendish grown under different cultural and chemical
treatments in a black root rot infested soil in East Lansing, MI, in 2006 and 2007. ..... 124

Table 28. Fungal genera isolated from roots of strawberry cv. Allstar and Cavendish
grown under different cultural and chemical treatments in a black root rot infested soil in
East Lansing, MI, in 2006 and 2007 ............................................................. 125

Table 29. Effect of fumigation and rotation crops on soil inoculum concentration of
Rhizoctom'a in a strawberry black root rot infested ﬁeld in East Lansing, MI in
2006. ................................................................................................. 127

Table 30. Effect of fumigation and rotation crops on soil inoculum concentration of
Rhizoctonia in a strawberry black root rot infested ﬁeld in East Lansing, MI in
2007. ................................................................................................. 128

Table 31. Rotations utilized in establishing biocontrols in rotations in a black root rot
infested ﬁeld in East Lansing, MI prior to planting strawberry cv. Allstar from 2005-
2007 ................................................................................................................................. 138

viii

Table 32. Bed ﬁll and plant vigor rating used in establishing biocontrols in rotations in a
black root rot infested ﬁeld in East Lansing, MI prior to planting strawberry cv. Allstar
from 2005-2007 .................................................................................... 139

Table 33. Effects of creating disease suppressive conditions using different rotations and
biocontrol products on plant vigor, bed ﬁll and total crown number of strawberry cv.
Allstar in a black root rot infested soil in East Lansing, MI, in 2006 ....................... 144

Table 34. Effects of creating disease suppressive conditions using different rotations and
biocontrol products on plant vigor, bed ﬁll and total crown number of strawberry
cv.Allstar in a black root rot infested soil in East Lansing, MI, in 2007 ................... 147

Table 35. Effects of creating disease suppressive conditions using different rotations and
biocontrol products on plant vigor, bed ﬁll and total crown number of strawberry cv.
Allstar in a black root rot infested soil in East Lansing, MI, in 2006-2007 ............... 148

Table 36. Effects of creating disease suppressive conditions using different rotations and
biocontrol products on plant vigor, bed ﬁll, total crown number, biomass and yield of
strawberry cv. Allstar in a black root rot infested soil in East Lansing, MI, in 2006 and
2007 .................................................................................................. 151

Table 37. Effects of creating disease suppressive conditions using different rotations and
biocontrol products on plant vigor, bed ﬁll, and total crown number of strawberry cv.
Allstar in a black root rot infested soil in East Lansing, MI, in 2006-2007 ................. 152

Table 38. Effects of creating suppressive soils using different rotations and biocontrol
products on individual plant biomass and root health of strawberry cv. Allstar in a black
root rot infested soil in East Lansing, MI, in 2007 ............................................ 154

Table 39. Effects of different rotations on nematode populations, by season sampled, in
100 cm3 soil or 1 g roots of strawberry cv. Allstar in a black root rot infested soil in East
Lansing, MI, in 2006 and 2007 ................................................................... 155

Table 40. Effects of creating suppressive soils using different rotations and biocontrols
on frequency of fungal genera isolated from roots of strawberry cv. Allstar in a black root
rot infested soil in East Lansing, MI, in 2007 ................................................... 156

Table 41. Cooperator application rates and dates in the effort to slow black root rot in
infested established strawberry ﬁelds in Ottawa (cv. ‘Jewel’) and Leelanau (cv.
‘Northeaster’), MI during 2007 ................................................................... 166

Table 42. Active ingredients of products used in effort to slow black root rot in infested
established strawberry ﬁelds in Ottawa (cv. ‘Jewel’) and Leelanau (cv. ‘Northeaster’), MI
during 2007 ......................................................................................... 167

ix

Table 43. Application method, rate, and schedule for products used in effort to slow
black root rot in infested established strawberry ﬁelds in Ottawa (cv. ‘Jewel’) and
Leelanau (cv. ‘Northeaster’), MI during 2007 .................................................. 168

Table 44. Preliminary fungal isolations from 40 strawberry root pieces from infested
established strawberry ﬁelds in Ottawa (cv. ‘Jewel’) and Leelanau (cv. ‘Northeaster’), MI
prior to study evaluating products in an effort to slow black root rot during 2007 ....... 173

Table 45. Preliminary nematode presence at the Ottawa site (cv. ‘Jewel’) prior to the
study to evaluate products in an effort to slow black root rot in infested established
strawberry ﬁelds during 2007 and the risk rating for needle nematode at this location. 1 74

Table 46. Effects of fungicide or fertilizer treatments on bed ﬁll of cv. ‘Jewel’ or
‘Northeaster’ strawberries in a study to slow black root rot decline conducted in
Leelanau and Ottawa counties in Michigan in 2007 .......................................... 177

Table 47. Effects of fungicide or fertilizer treatments on yield and plant volume of cv.
‘Jewel’ or ‘Northeaster’ strawberries in a study to slow black root rot decline conducted
in Leelanau and Ottawa counties in Michigan in 2007 ......................................... 178

Table 48. Effects of fungicide or fertilizer treatments on plant weights and root necrosis
in cv. ‘Jewel’ or ‘Northeaster’ strawberries in a study to slow black root rot decline
conducted in Leelanau and Ottawa counties in Michigan in 2007 .......................... 181

Table 49. Frequency of fungal genera isolated from diseased roots of cv. ‘Jewel’ or
‘Northeaster’ strawberries from fungicide or fertilizer treatments in a study to slow black
root rot decline conducted in Leelanau and Ottawa counties in Michigan in 2007. .......182

Table 50. Average number of nematodes present in 100 cm3 of soil and l g of roots at
the Ottawa county site in a study evaluating products in an effort to slow black root rot in
Hudsonville, MI in September and October 2007 ........................................ , ..... 183

Table 60. Percent necrosis caused by Rhizoctoniafragariae isolates from strawberries in
an in-vitro evaluation of virulence and anastomosis group at Michigan State University,
East Lansing, MI ................................................................................... 193

Table 61. Inoculation method, soil treatment, and pathogen inoculated in greenhouse
experiment to develop an inoculation protocol for creating black root rot conditions in
greenhouse experiments using strawberry cv. Allstar in East Lansing, MI in 2005 ....... 198

Table 62. Effects of inoculation, soil treatment, and pathogen on root health of cv. Allstar
strawberries in a greenhouse experiment to develp an protocol for creating black root rot
conditions in East Lansing, MI in 2005 ........................................................... 203

Table 63. Fungal genera recovered from roots of cv. Allstar strawberries in a greenhouse

experiment to develp a protocol for creating black root rot conditions in East Lansing, MI
in 2005 .............................................................................................. 205

Table 64. Effects of varying amounts of bran and inoculation with Rhizoctoniafragariae
on plant biomass and root health of cv. Allstar strawberries in a greenhouse study to
develop an inoculation protocol for black root rot in East Lansing, MI in 2007.... .. .....213

Table 65. Risk ratings of major parasitic nematodes found on strawberries grown in

Michigan. (Data furnished by Fred Warner, Michigan State University Diagnostic
Services) ............................................................................................. 214

xi

LIST OF FIGURES

Figure 1. Rating scale used for root quality ratings for strawberry black root rot studies
conducted in infested ﬁelds or greenhouse trials at Michigan State University, East
Lansing, MI during 2005-2007. This image is presented in color ........................... 201

xii

SYMBOLS AND ABBREVIATIONS
ANOVA: Analysis of variance

BRR: Black root rot

ITS: Internal transcribed spacer

PDA: Potato dextrose agar

PDAamp: Potato dextrose agar amended with 50 ul ampicillin/ml

xiii

CHAPTER ONE: LITERATURE REVIEW
Introduction

Strawberry black root rot (BRR) is a disease complex that is widespread around
the world wherever strawberries have been planted for multiple years (Watanabe et al. ,
1977; D’Ercole et al., 1989). Initial symptoms of BRR include brown lesions on the
feeder and structural roots of the strawberry plant and blackening of the root cortex while
the stele remains white, initially (Maas, 1998). Root lesions range from 0.5 to 5 cm.
Feeder roots disintegrate due to infection. Infected roots darken and die, eventually
becoming completely black in severe cases. Infected plants produce smaller leaves,
fewer main and lateral roots, have slower growth, and reduced runner production
(Hancock 8! al., 2001). Severely infected plants wilt at the onset of dry weather. The
disease can be spread via infected nursery stock, movement of infested soil, or infected
plant debris (Maas, 1998; Strong and Strong, 1927; Hildebrand, 1934). Also known as
strawberry decline, many organisms and abiotic factors have been implicated in the cause
of the disease; however, Rhizoctoniafragariae Husain & McKeen, Pythium spp., and the
root lesion nematode Pratylenchus penetrans (Cobb) Filipjev and Shuurmans Stekhoven
are generally considered the primary pathogens (D’Ercole et al., 1989; Wing et al., 1995;
Maas, 1998).

In Michigan, the matted-row system is used to grow strawberries, where plants
are typically planted at wide spacing in spring so that runners ﬁll in between the plants.
Harvest occurs the year after planting and continues for 3 to 4 years. The fruit ripens for
3 to 5 weeks in May to mid-June (Pritts and Handley; 1998). In Michigan ‘U-Pick’

operations, continual strawberry production or short rotations are common due to the few

suitable locations for these enterprises. The shorter a rotation is between strawberry
plantings, the higher the potential economic return to the grower. However, continual
cropping allows pathogen establishment and build-up to deleterious levels. In this
system, fumigation is often employed to help control soil-bome pathogens and weeds
before planting (Perry and Ramsdell, 1994; Rosskopf er al. , 2005).

A common and effective fumigant used by strawberry growers is methyl bromide.
In 1992, the Montreal Protocol established methyl bromide as an ozone-depleting
substance and instituted its ban by January 1, 2005 (Anonymous, 1998; Rosskopf et al.,
2005). Methyl bromide was predominantly used as a soil fumigant, but also in the
disinfestation of durable and perishable commodities, and structures. Besides being
detrimental to the environment, concerns for operator safety, residues in food, effects on
soil biodiversity, and pollution of surface and ground water all inﬂuenced the decision to
ban methyl bromide use (Anonymous, 1998; Rosskopf et al., 2005). Methyl bromide is
emitted from many sources, some being natural, but it is estimated that 30% of emissions
come from soil fumigation (Rosskopf et al., 2005).

Currently, control of BRR consists of using crop rotation, cover crops, good
aeration and drainage, and fumigation (Maas, 1998; Martin and Hancock, 1983; Perry
and Ramsdell, 1994). Some chemicals, such as Telone (1,3-dichloropropene) and
chloropicrin have shown success as alternative fumigants. Vapam (metam sodium) and
Basamid (dazomet) have shown comparable results in some countries to methyl bromide
as well (Anonymous, 1998). Chemical control can cost between $700-$2000 depending
on chemical and application method (Anonymous, 1998; Rosskopf et al., 2005). With

one of the most effective fumigants being banned, and no speciﬁc crop rotation that has

shown consistent, effective control, growers are left with vague recommendations.
Prevention is key; planting disease-free stock in fertile, well-drained sandy loam is the
best way to avoid this disease (Perry and Ramsdell, 1994).

One problem when trying to develop a management strategy for BRR stems from
the variability of the disease. Rhizoctoniafragariae does not always have to be present to
get BRR symptoms (Wing at al. , 1994; Wing et al., 1995). While studies have
implicated P. penetrans, the disease can occur with low nematode populations or even in
their absence (Wing at al., 1994; Wing et al., 1995). It seems that a combination of
abiotic and biotic factors predispose strawberry plants to invasion by perhaps otherwise
saprophytic or weakly parasitic fungi that then cause severe damage to the root system
(Wing et al. , 1994; Wing et al. , 1995).

With the primary control measure due to be eliminated, it becomes necessary to
ﬁnd alternative reduce-risk chemicals, biological, and cultural alternatives. These
alternatives should not be expected to work as effectively alone as methyl bromide did.
The best control still resides in integrated management practices. Thus the objectives of
this research are: 1) Evaluate reduced-risk fungicides and biological products for the
control of BRR at the time of planting, 2) Evaluate various crop rotations in combination
with biological control products at the time of planting, 3) Assess integration of host
plant resistance with chemical and cultural controls to develop a “best practices”
approach, in order to make a recommendation to growers and 4) Evaluate reduced-risk

fungicides in already established strawberry ﬁelds.

Strawberry Production

The cultivated strawberry, F ragaria x ananassa Duchesne, is thought to be a
cross between F ragaria virginiana Duch. and Fragaria chiloensis Linn. (Galleta, 1990;
Hancock, 1999; Pritts and Handley, I998). The strawberry plant, a member of the rose
family, is a perennial that consists of a crown from which leaves, stolons, branch crowns,
ﬂower clusters, and adventitious roots grow (Hancock, 1999; Pritts and Handley, 1998).
Axillary buds produced at the base of each leaf may become a stolon or branch crown
depending on the environment, with long days and warm temperatures encouraging
runners, while cooler short days favor branch crowns (Hancock, 1999; Pritts and
Handley, 1998). The stolons have two nodes, the ﬁrst of which may become another
runner or remain dormant while the second becomes a daughter plant. A healthy plant
can develop 10-15 runners a year (Hancock, 1999; Pritts and Handley, 1998). ‘Allstar’ is
currently a widely grown variety in the Eastern, Mid-Atlantic, and Midwestern United
States. It is an early variety with large to medium sized, light colored fruit developed in
Maryland by the United States Department of Agriculture; and it also has resistance to
Verticillium wilt (Hancock, 1999).

Flower buds are initiated in one season for the following season. Initiation occurs
after certain day length and temperature requirements are met, which is often variety
speciﬁc. The inﬂorescence has a primary ﬂower, two secondary, four tertiary, and
possibly eight quartemary ﬂowers (Pritts and Handley, 1998; Hancock, 1999). In the
Northeast, plants are often short-day or June-bearing, meaning that ﬂower bud initiation
occurs during September and October when the days are shortening and cooler. Some

varieties are not sensitive to day length, and are called day neutral; these initiate ﬂower

buds between 4.4-29.4°C, and still others have a weak day length response (Pritts and
Handley, 1998; Hancock, 1999). Flowering in day neutral varieties occurs about six
weeks after bud initiation which is continuous from late spring through fall. The plants
are self—fertile, but fruit size is greatly improved with pollinators. The fruit develops
from the ﬂower receptacle (Pritts and Handley, 1998; Hancock, 1999).

The fruit is composed of numerous ovaries, each with seeds referred to as achenes
(Pritts and Handley, 1998; Hancock, 1999). Ripening depends on the pre-harvest
environment and the cultivar, with pectinmethylesterases and cellulases thought to be the
most important enzymes involved in strawberry softening; anthocyanins cause the
characteristic reddening (Hancock, 1999). In Michigan, three to four harvests are
possible, but plant yield and berry weight decrease through the season as the tertiary and
quartemary ﬂowers develop(Pritts and Handley, 1998; Hancock, 1999).

Strawberries have two types of roots, perennial (primary) and lateral (feeder,
secondary) (Pritts and Handley, 1998; Hancock, 1999). Primary roots arise from the
crown chieﬂy in late summer and fall while lateral roots originate in the pericycle, push
through the cortex of the primaries and are the main source of absorption. Root growth
occurs primarily during non-fruiting and vegetative dormancy periods (Pritts and
Handley, 1998; Hancock, 1999). Good aeration is important as low soil oxygen and
water-logging favors root damaging fungi and death of rootlets depending on the duration
of standing water (Galleta, 1990; Hancock, 1999). The lateral roots live 1 to 2 years
while primary roots may live 2 to 3 years. The largest root concentration is in the top 15
cm of the soil with each plant usually maintaining about 20 to 30 primary roots with an

average length of 10t015 cm (Pritts and Handley, 1998; Hancock, 1999). Dorrnancy is

caused by 4 to 6 weeks of short-day periods and is broken after sufﬁcient chilling at -1 to
10°C (Hancock, 1999).

Commercially, strawberries are primarily propagated by digging runners in late
fall and early spring, then storing them at 0°C until spring planting. Runner tips are also
used for fall planting in the annual plasticulture system used in California and Florida
where most of the strawberries in the United States are produced. In the annual
plasticulture system, the plants are set at a high density on raised beds covered with black
polyethylene plastic in late summer after the day length decreases. The plants produce
large crowns during fall and they fruit in spring (Hancock, 1999). In polyethylene-
mulched production systems, the use of pre-plant soil fumigants is essential to control
soil pathogens, weeds, and nematodes (Locascio, 2005; and Rosskopf et al., 2005).

The United States produces approximately 20% of the world’s supply of
strawberries (Hancock, 1999). The strawberry crop is the highest valued berry crop,
accounting for about two-thirds of all berry revenues since the 1980’s in the United States
(Pollack and Perez, 2005). The United States is a net exporter of fresh strawberries,
primarily to Canada, but it is a primary importer of frozen strawberries from Mexico
(Cook, 2002). In 2006, the United States’ strawberry crop was valued at $1.5 billion and
harvested from 53,280 acres. Califomia’s crop was valued at $1.2 billion and harvested
from 35,800 acres. Florida had a strawberry crop valued at $239 million in 2006.
Michigan’s crop was valued at $6.3 million and harvested from 950 acres (NASS, 2007).
California produces strawberries from January through October, and Florida’s harvest

covers the winter months. (NASS, 2007).

California is able to have such high yields because of the annual, plasticulture
system that utilizes soil fumigation to control pests and improved region-speciﬁc
varieties that can produce for 6 months instead of 4 weeks (Pritts and Handley, 1998;
Hancock, 1999; Cook, 2002).

The plasticulture system is not suitable for northern regions because of the risk of
spring frosts and the shorter growth period. In these regions, the matted row system is
used. This system requires that the ﬂowers are removed the ﬁrst year. In this system the
strawberry is grown as a perennial for 3 to 4 years. Harvest occurs for 3 to 5 weeks in
May to mid-June (Pritts and Handley; 1998). In Michigan, the strawberry season starts in
early June in the Lower Peninsula and ends in late July in the Upper Peninsula. Berrien,
Leelanau and Van Buren are Michigan's largest strawberry-producing counties (Long,
2002). Most of the Michigan crop is produced on “U-Pick” operations. In 2006,
Michigan had 200 strawberry farms totaling 850 acres (MDA, 2007).

In both of these systems, fumigation is employed to help control soil-bome
pathogens and weeds. Roots in fumi gated soils have deeper penetration with more root
branching, and have lateral roots that live longer. Lateral roots are continuously replaced
as they die so the plant can continue to get nutrients from the same soil area (Galletta,
1990).

Strawberry growers in the United States use chemicals to control a variety of
diseases including: gray mold (Botrytis cinerea), anthracnose (Colletotrichum spp.),
powdery mildew (Podosphaera aphanis), leather rot (Phylophthora cactorum), angular
leaf spot (Xanthomonas fragariae), red stele (Phytophthorafragariae var. fragariae),

common leaf spot (Mycosphaerellafragariae), phomopsis leaf blight (Phomopsis

obscurans), leaf scorch (Diplocarpon earlianum) and BRR. In 2002, the ten primary
strawberry-producing states purchased and applied a total of 864,000 pounds of
fungicides, at a value of $12.09 million. A loss of 1.1 billion pounds of production with a
value of $707 million was predicted if fungicides had not been used (Gianessi and
Reigner, 2005). In 2001 , it was estimated that in Georgia, methyl bromide fumigation
accounted for the largest single expenditure for disease control in strawberries. Root rots,
especially BRR, were estimated to have reduced crop value by 3%, with total costs,
including damage and control expenses, amounting to $255,000 (Williams-Woodward,
2001). It has been estimated that yields can be reduced by 50% if BRR is not controlled,
and control with fumigation can cost $1000 per acre (Long, 2002; EPA, 2006). Yuen et
al. (1991) found that soil fumigation with methyl bromide and chloropicrin (MBC)
reduced the severity of BRR of strawberries in California. Root density was increased by
19-61% over the non-fumigated control, while harvests from fumi gated plots were 24-

29% greater than untreated controls.

Black Root Rot Complex

First reported by Zeller in 1932, BR is characterized by reddish brown lesions
on the feeder and structural roots of the strawberry plant, which darken to black with age
and result in the death of the root (Zeller, 1932). Many organisms and abiotic factors
have been implicated in the cause of the disease; however, Rhizoctoniafragariae Husain
& McKeen, Pythium spp., and the nematode Pratylenchus penetrans (Cobb) F ilipjev and
Shuurmans Stekhoven are generally accepted as the primary pathogens (D’Ercole et al. ,

1989; Wing et al., 1995; Maas, 1998). The role of R. solam’ Ktihn in the disease is

unclear, as much work done prior to Husain and McKeen’s discovery of R. fragariae
does not identify, or may have misidentiﬁed, the species of Rhizoctom'a (Husain and
McKeen 1963a; Parmeter et al., 1967). Rhizoctoniafragariae has been more frequently
isolated than R. solani in several studies, but both have been found on strawberry roots
(Wilhelm et al., 1972; D’Erocole et al., 1989; Martin, 1988; Watanabe et al., 1977).
Hildebrand (1934) found Gliocladium, F usarium, Pythium, Hainesia,
C ylindrocladium, Coniothyrium, Rhizoctonia, Helmimhosporium, Asterocystis spp., and
members of the Plasmodiophoraceae associated with BRR, while Nelson ( I 957)
demonstrated the pathogenicity of [driella lunata P.E. Nelson & S. Wilh. on strawberry
in California. 1driella lunata was also recovered during root isolations from strawberry
in Italy (D’Ercole et al., 1989). Katznelson and Richardson (1948) found C ylindrocarpon
associated with ‘Premier’ strawberries. Yuen et al. (1991) isolated C ylindrocarpon
destructans, Pythium ultimum Trow, and Pythium irregulare most frequently from
diseased strawberry plants in California. Damage caused by R. ﬁagariae and other
primary pathogens may allow secondary fungi to invade the strawberry roots (Husain and
McKeen, 1963b). Weak parasites and saprophytes are more likely to be found in samples
collected in late spring and early summer, while samples taken in the late fall and early
winter have a better chance of containing R. fragariae (Husain and McKeen, 1963a).
Infection is most severe in moderately wet soils and when environmental
conditions are not conducive to plant growth. D’Ercole et a1. (1989) found that the
appearance of decline 10 to 15 days after planting was caused by agronomic problems,
while plants declining 50 to 60 days aﬁer planting harbored pathogens such as

Rhizoctom'a, Verticillium, Pythium, ldriella, F usarium and C ylindrocarpon.

 

 

Rhizoctonia spp.

While Rhizoctom'a spp. are an immense group of fungi that are grouped
largely by their lack of distinctive taxonomic features, with teleomorphic states in both
the basidiomycetes and ascomycetes, the three groups associated with plant diseases
includes R. solam', the binucleate species, and the isolates with a Waitea teleomorph
(V ilgalys and Cubeta, 1994).

Rhizoctom'a spp. have a highly variable growth rate, and some isolates may
produce spores. Under certain conditions, some species produce sclerotia-like tufts
consisting of short, broad cells that function as chlamydospores. The hyphae display
characteristic right-angle branching with dolipore septa and a moniliform resting cell
(Maas, 1998). The branches are slightly constricted and cross walls are present near the
junction. Infrequently, the perfect stage, Thanatephorus cucumeris (A.B. Frank) Donk, is
formed in the multinucleate species (R. solani), or Ceratobasidium spp. in the binucleate
species (Ogoshi and Ui, 1983; Burpee et al., 1980).

Rhizoctom'a solam' is a signiﬁcant pathogen of many crops. Rhizoctonia solam'
can cause rot in the strawberry crown and affects roots near the crown at 2 to 18°C with
crown infection favored at 18 to 32°C (Maas 1998). This and other Rhizoctonia species
are “collective” species consisting of several more or less unrelated strains. Strains are
distinguishable from each other by their relative ability to form anastomoses. Ogoshi and
Ui (1983) developed anastomosis groups (AG) for Japanese isolates, while Burpee et al.
(1980) developed groups for North America. Ogoshi (1985) then compared the two
different groups and found that Burpee’s seven groups corresponded to several groups in

the Ogoshi system. Ogoshi’s anastomosis groups AG-A, AG-G, and AG-I have often

10

been implicated in BRR of strawberries (Martin, 1988). The fungus overwinters usually
as mycelium or sclerotia in soil, infected perennial plants, propagation material, or even
seed depending on the host. It is present in most soils, and once established, impossible
to eliminate.

Rhizoctoniafragariae is represented in groups AG-A, AG-G, and AG-I. Isolation
frequency and virulence vary between and within each group and by site (Burpee et al. ,
1980; Martin, 1988; Mass, 1998; Martin, 2000). Martin (2000) found that isolates within
AG-I were particularily virulent and that AG groups differed between locations, and even
within a single location depending on the time of year. Each of these AG’s have different
host ranges that should be considered before incorporating certain crops into a rotation
prior to strawberry (Martin, 1988).

Husain and McKeen (1963a) ﬁrst implicated R. fragariae in BRR, and reported
that it was primarily isolated from roots during cold periods. Disease seems to be
conditional on temperature, with R. fragariae germinating most rapidly in exudates on
strawberry roots grown at 5-10° C (Husain and McKeen, 1963b).

In some cases, Rhizoctoniafragariae has also been found to stimulate plant
growth. Scott et al. (2003) found that R. fragariae may be tolerated by strawberries
during cool weather due to more favorable conditions for root growth over fungal growth.
However, as temperatures increase the plant begins to show symptoms of infection that
had occurred earlier in the season (Scott et al., 2003). In greenhouse trials using the
variety ‘Redgauntlet’, R. ﬁagariae was not as aggressive as R. solani (Molot and
Ferriere, 1989). Ribeiro and Black (1971) found that R. fragariae could actually

stimulate plant growth depending on environmental and nutritional conditions. This may

11

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'.ka.I-'..{'?:‘.‘I" “If" ~ ..i“,., -

     

mgr-

. . M 2» . . .. . ' . < . .
Apr...“ ‘ 'I‘T'K’IQWFW‘I 330:5 ‘.:..~“'gv.,,.....t.r,;,u;.3,0,.
f . . . , 14.. . u... _. ‘

explain why some Rhizoctonia spp. have been isolated from apparently healthy plants.

Another consideration is the stage of the infection process.

Pythium spp.

Pythium species belong to the class Oomycetes and are primarily known for
causing damping off in seeds and seedlings. The damage caused by Pythium spp. is
proportional to the amount of soil moisture present with it being greatest near the
saturation point (Hendrix and Campbell, 1973). These pathogens are pandemic.

Pythium produces a white, fast-growing mycelium that gives rise to sporangia,
which then form a vesicle, containing 100 or more zoospores. When the zoospores are
released, they swarm about and then encyst (Hendrix and Campbell, 1973). Zoospores
are attracted to the roots by exudates, with soil moisture inﬂuencing the distance over
which the exudates can stimulate the fungus (Hendrix and Campbell, 1973). Germination
of encysted zoospores occurs with the production of a germ tube, which usually directly
penetrates the host through the use of pectinolytic enzymes; the fungus then grows
between and through the cells, while proteolytic and sometimes cellulytic enzymes break
down protoplasts and cause complete collapse of invaded cells (Hendrix and Campbell,
1973)

The mycelium may give rise to oogonia, which, once fertilized, produce a thick
wall and become oospores that serve as the survival and resting stage on dead plant and
animal material as a saprophyte, or as a parasite on ﬁbrous roots. Stanghellini and
Hancock (1970) found that sporangia are important survival structures for some species

such as P. ultimum. Germination of oospores is similar to that of sporangia, but is

12

favored at temperatures above 18°C while temperatures between 10 to 18°C favor
zoospore germination (Hendrix and Campbell, 1973). Oospore germination is stimulated
by root exudates that contain sugars and amino acids (Hendrix and Campbell, 1973;
Nelson, 1990).

Pythium spp. rarely kill older plant hosts, but the plants develop root and stem
lesions and root rots; their growth is retarded, yields decrease, and they may wither and
die. Rootlets may be attacked at any point in the plant growth and the fungus can
proliferate quickly. Pythium diseases are favored by prolonged wetness and unfavorable
temperatures for the host, excess nitrogen, and growing the same crop for several years at
the same location (Hendrix and Campbell, 1973).

Watanabe et al. (1977) found that Pythium spp. played an important role in the
occurrence of strawberry stunt disease in the cool, wet conditions of Japan. Of the 58
fungal genera isolated from strawberry roots in Japan, Rhizoctom'a spp. accounted for
25.2%, followed by F usarium spp. at 19.6%, and Pythium spp. at 14.5%. Despite the
lower frequency of isolation, Watanabe et al. (1977) found that P. ultimum was a primary
pathogen causing BRR symptoms in strawberry at temperatures below 20°C. They
suggested that the wetness of the drained rice paddy ﬁelds in which strawberries were
grown in Japan may be a contributor to the severity of root rot caused by Pythium spp.

In Japan, Watanabe et al. (1977) isolated P. sylvaticum Hendrix & Campbell
more frequently than P. ultimum from strawberry roots. In the United States, P.
sylvaticum was the most isolated Pythium species from strawberry roots in Southern

Illinois, but P. irregulare, and P. perniciosum were also common (Nemec and Sanders,

l3

1970; Nemec, 1970). Wilhelm (1953) found P. ultimum to be one of the most common
fungi associated with strawberry roots in California and established its role as a pathogen.

Increasing moisture affects the pathogen, which multiplies and disperses best in
wet soils, but higher moisture may also decrease the ability of the host to defend itself
because oxygen availability and soil temperature are reduced (Hendrix and Campbell;
1973). Pythium ultimum has been found to survive at -18°C for 24 months and in air-
dried soil for 12 years (Hendrix and Campbell, 1973). Pythium spp. frequently occurs in
a complex with other fungi such as in peanut pod rot with F usarz'um spp. (Hendrix and

Campbell, 1973).

Nematodes

Nematodes in the genus Pratylenchus are commonly called root lesion
nematodes. Although the nematodes typically damage plant roots through the formation
of lesions, they are capable of damaging other underground shoot tissues such as those of
potatoes or peanuts. Many species have a wide host range, with some hosts able to
support multiple species (Mai and Mullin, 1960; Mai et al., 1977). Pratylenchus
penetrans is a migratory, endoparasitic root lesion nematode with nearly 400 known
hosts. It is considered the most important plant parasitic nematode in the northern United
States and Canada (Maas, 1998).

Pratylenchus penetrans has been shown to play a role in BRR. Hildebrand (1934)
originally suggested that BRR was caused by nematodes, while in the Netherlands
Klinkenberg (195 5) concluded that nematodes may be a primary cause, but that fungi

move in secondarily to the wound sites to create more disease symptoms. Raski (1956)

I4

provided evidence that P. penetrans was probably not the most important factor in
plantings showing BRR symptoms, while Goheen and Bailey (1955) found that BRR was
associated with small as well as large populations of nematodes. Goheen and Smith
(1956) then showed that nematode-infested soils contributed to typical BRR symptoms
and severe stunting of strawberries, but Chen and Rich (1962) found that fungi more
readily infected necrotic tissue, suggesting that the nematodes predisposed the host for
infection by surrounding soil fungi. Finally, Townshend (1963) demonstrated the
pathogenicity of Pratylenchus penetrans on strawberries, showing that the nematode
causes root necrosis and polyderrn formation beneath the damaged endodermis in the
stele.

The root lesion nematode can be identiﬁed by its ﬂat, rounded head, overlapping
esophagus, a stylet that is 14 tol9 pm long with a prominent basal bulb at the base of the
stylet (Mai and Mullin, 1960). The overlapping esophagus and head are the primary
means to make a positive identiﬁcation; other characteristics discussed by Mai and
Mullin (1960) include a body that is less than 1.0 mm long, phasmids that are 1/3 the
length of the tail or more behind the anus, and a blunt rounded tail. On the lateral ﬁeld,
four incisures can be found. When viewed under a microscope root lesion nematodes
often move slowly and gracefully (Mai and Mullen, 1960).

The root lesion nematode overwinters as eggs, juveniles, or adults in infected
roots (Dunn, 1972). Females lay a cluster of eggs in the cortex cells of the root or in the
soil. The ﬁrst-stage larva stays in the egg and molts into a second-stage larvae, which
emerges from the egg 9 to 25 days after egg deposition, depending on temperature, and

starts feeding on root parenchyma cells (Mamiya, 1971). All stages are vermiform, and

15

are morphologically similar except for reproductive structures. The nematode can infect
roots or other below-ground plant structure, but the third juvenile and females appear in
roots more often than males (Mai et al., 1977; Olthof, 1982). After hatching, the
nematode may move to new sites of infection, but all stages are usually found on the
same host (Dunn, 1972). The male-to-female ratio and life expectancy are inﬂuenced by
temperature, with higher temperatures shortening the life cycle. At 30°C, the life cycle
can be completed in 30 days but survival is better at 15°C. On average, the life cycle is
45 to 65 days (Mai et al., 1977; Kable and Mai, 1968).

Soil with a moisture tension (pF) around 1.8 to 2.5 maximizes root penetration,
while tensions above pF 5.06 result in the death of nematodes (Kable and Mai, 1968).
Soil texture plays a critical role in determining water tension, while organic soil
amendments can also inﬂuence populations of P. penetrans by changing soil structure
and water holding capacity (Miller et a1. , 1973).

Lee (2002) reported that the root lesion nematode ﬁnds it host most likely by
chemo-attraction or by sensing a potential gradient of ions. Root invasion occurs
preferentially 3-13 mm behind the root tip. A potential feeding site is created by rubbing
the epidermal cells with the lips and stylet, which is then thrust into the cell wall at rates
of up to 140 times per minute, starting at the comers of the cell and working along the
cell wall till it breaks (Mountain and Patrick, 1959; DiEdwardo, 1960; Freckman and
Chapman, 1972; Oyekan et al., 1972; Olthof, 1982; Kurppa and Vrain, 1985; Lee, 2002).
Penetration of the root is complete within 6-12 hours, and the mid-cortex is reached by
18-24 hours after inoculation. The endodermis acts as a barrier to invasion, except in

some plants after prolonged feeding (Mountain and Patrick, 1959; DiEdwardo, 1960;

16

Freckman and Chapman, 1972; Oyekan et al., 1972; Olthof, 1982; Kurppa and Vrain,
1985)

Feeding can last for hours after a short salivation period, but feeding and
migration are interrupted by periods of rest that can last for hours (Lee, 2002). The
nematode moves through the cortical cells (Mai et al. , 1977). The lesions appear mainly
on the younger feeder roots but may appear anywhere along the roots. Affected cortex
cells in the lesions collapse and the lesion area appears constricted. Cell death is often
delayed until after the nematode leaves, with nematode numbers often higher in diseased
tissue than in dead tissue (Lee, 2002).

Pathogenicity of P. penetrans is inﬂuenced by host resistance due to phenolics
produced in the roots or anatomical differences among plant species. Some hosts can
support large numbers of nematodes without showing injury. Other hosts may be
penetrated, but only a few cells around the infection site die where as others, such as
alfalfa, have numerous cells that die along the pathway of the nematode (Mai et al. , 1977;
Lee, 2002). Secondary fungi and bacteria usually invade the lesions and contribute to the
discoloration and rotting.

Strawberry plants infected with P. penetrans appear stunted, have increased
drought sensitivity, fewer runners, and shorter, more erect petioles (Maas, 1998).
Pralylenchus penetrans alone or in combination with Rhizoctoniafragariae was found to
reduce strawberry yield over time (LaMondia, 1999). Stunted plants with BRR have
harbored R. fragariae and P. penetrans while symptomless plants nearby were infected
only with R. ﬁagariae. The two pathogens act additively and not synergistically

(LaMondia and Martin, 1989). Pratylenchus penetrans can directly damage plants and

17

may lower the host’s natural resistance to fungi making the combined damage of the
nematode and fungi worse than either alone (Powell, 1971). The nematode was found to
interact with R. ﬁ'agariae among several other pathogens, allowing the associated
diseases to develop more quickly (Szczygiel and Proﬁc—Alwansiak, 1989; Powell, 1971).
The interaction was more pronounced in sterilized than in unsterilized soil, probably due
to decreased antagonism by any other organisms naturally present. Szczygiel and Proﬁc-
Alwansiak (1989) found that P. penetrans, Meloidogyne hapla Chitwood 1949, and

Longidorus elongatus (de Man, 1876) Thome & Swanger 1936 could all interact with R.

] xv-

fragariae to the detriment of the strawberry plant. .
Other nematodes associated with strawberry roots include Longidorus elongatus,

X iphinema americanum, and Meloidogyne spp. (Brown et al., 1993). Chapman (1956)

found Xiphinema, Pralylenchus, and Tylenchorhynchus spp., all of which he felt should

be considered potential pathogens in Kentucky. Any nematode which damages the root

offers a potential entry point for fungi.

Other Factors Inﬂuencing Black Root Rot

Drought, winter injury, excessive fertilizer application, and excessive soil
moisture are all detrimental to the strawberry plant and can cause symptoms similar to
BRR (Perry and Ramsdell, 1994; Miller, 1948). Wing et al. (1995) discussed that no
single factor accounted for the majority of the observed variation in root health, and that
BRR may be caused by different factors in different ﬁelds. They also suggested that
several interacting factors are necessary. Poor root health was associated with soil

compaction and high soil clay and silt content. Wing et al. (1995) also found that raised

18

beds (10-20 cm) were associated with better root health, while those under 10 cm had
poor root health. The age of the current planting, strawberry production on the site
within the previous 5 years, and cumulative period of strawberry production were all
signiﬁcantly associated with poor root health (Wing et al. , 1995). Higher rates of the
herbicide terbacil were associated with poor health, possibly due to the stress imposed on
plants, predisposing them to infection by pathogens. Recently, however, Mervosh and
LaMondia (2004) found that rates of terbacil four times the maximum recommended
dosage did not result in an increased occurrence of BRR or reduced yield. Use of the
fungicide metalaxyl was associated with good root health (Wing et al. , 1995).
Fumigation had a negative correlation with root health, possibly due to the ‘boomerang’
effect where fumigated areas are more quickly re-colonized by pathogens than by
beneﬁcial antagonists, or it could be that areas with a history of disease were more likely

to have been fumigated (Wing et al. , 1995).

Black Root Rot in Michigan

Over three years, seven samples suspected of BRR from various locations across
the state of Michigan, were processed. Over all these locations, F usarium spp. were
commonly isolated (Glass, unpublished). While the cause of the decline at two of these
sites could not be adequately determined, Rhizoctom'a spp., and at a separate site Pythium
spp., were each suspected to be the cause of decline. P. penetrans was determined to be
the cause at another location, while L. elongatus was causing the decline on a different

farm (Glass, unpublished).

19

Through personal communications with extension staff and cooperators, the use
of fumigation, particularily methyl bromide seems to be limited. While a nursery in
Michigan fumigates for production of their nursery stock, each grower has a preferred
method that works at their location and in their management scheme (Bardenhagen and

DeLange, personal communication).

Current Control

Currently, recommendations for control of BRR consist of using crop rotation,
cover crops, good aeration and drainage, and fumigation (Maas, 1998; Martin and
Hancock, 1983; Perry and Ramsdell, 1994). If an existing planting develops BRR, a new
site should be chosen, or the old plants plowed under and the soil cultivated for several
months followed by fumigation and planting of healthy strawberries in the spring (Perry
and Ramsdell, 1994). Prevention is key; planting disease—free stock in fertile, well-
drained sandy loam is the best way to avoid this disease (Perry and Ramsdell, 1994).

Incorporation of organic matter can encourage beneﬁcial organisms that are
antagonistic to pathogens (Perry and Ramsdell, 1994, Hoitink et al., 1997). Also, good
cultural practices to prevent drought stress and winter injury, and a 3-to 5-year rotation
between strawberry plantings helps prevent disease (where fumigation is not possible)
(Perry and Ramsdell, 1994). LaMondia (2004) evaluated 21 commercial strawberry
varieties in naturally infested BRR soil for up to 3 years after planting, and found that
loss in plant vigor and increased plant mortality occurred during harvest, especially under
conditions of environmental stress. The best performing cultivars were ‘Earliglow’ (early

' season), ‘Cavendish’ and ‘Lester’ (early-midseason), ‘Primetime’ (mid-season), and

20

‘Idea’ and ‘Latestar’ (late—season). LaMondia (2004) found that evaluations should be
done over many years due to the complexity, and often speciﬁcity, of the disease within a
ﬁeld. In addition, out of 20 strawberry genotypes evaluated over 2 years, ‘Cavendish,’
‘Bounty,’ and ‘Cabot’ were all found to perform well on soil naturally infested with
Rhizoctoniafragariae, Pythium spp., and P. penetrans in Michigan (Particka and

Hancock, 2005).

Methyl Bromide Fumigation

Fumigation with methyl bromide and chloropicrin was reported by Wilhelm et al.
(1963) to control Verticillium wilt of strawberry. Since that time it has been used as a
pre-plant eradicant of weeds, nematodes, and soil-bome pathogens. They showed that
soil productivity was limited not just by nutrients, but could be enhanced by fumigation
that eliminates pathogens (Wilhelm, 1965, 1984). The effectiveness of methyl bromide
in root rot disease control minimized the need for developing strawberry varieties with
resistance to root disease.

In 1992, the Montreal Protocol established methyl bromide as an ozone-depleting
substance and banned its use by January 1, 2005 in developed countries, or January 1,
2015 in developing countries (Anonymous, 1998; Rosskopf et al., 2005). When methyl
bromide is applied, it reacts in the soil to leave bromide ions, various methylated products
and carbon dioxide. The remainder of the gas escapes into the atmosphere (WMO,
2003). Reactions involving bromide are thought to contribute to 50% of the loss of the

ozone over Antarctica annually (Rosskopf et al. , 2005).

21

 

In 1996, global usage of methyl bromide for fumigation was 66,750 tonnes with
76% of that being used to fumigate soil within the United States (Anonymous, 1998).
Methyl bromide was widely used due to its penetrative nature and effectiveness over a
broad range of temperatures. Furthermore it did not disrupt farming practices due to the
quick efﬁcacy and ability to air rapidly after application (Rosskopf et al., 2005). Methyl
bromide was of particular use as a pre-plant fumigant where a broad range of soil pests
limited economic production and where land was limited, forcing continuous same-crop
production. It was of particular value to the vegetable, fruit, ornamental, tobacco, and
nursery industries (Rosskopf er al. , 2005). Until recently, methyl bromide was widely
used in the strawberry industry as a soil fumigant in production ﬁelds, but also in
nurseries to guarantee disease-free transplants. Florida alone accounted for 36% of pre-
plant methyl bromide use in 1997, with strawberry accounting for 9% of the total use in
that state (Rosskopf et al. , 2005).

In 1998, the Methyl Bromide Technical Options Committee (MBTOC), met to
develop feasible alternatives to methyl bromide. Although there are critical use
exemptions for areas and industries that could not operate without methyl bromide, there
has been a re-examination of existing fumigants. These materials are limited in their
applicability as fumigants, as they can be highly variable in efﬁcacy both by year and
location. In Florida, the best available alternative for strawberry consists of Telone +
35% chloropicrin, applied in-bed at 331 liters per treated hectare, 3-5 weeks before
transplanting. Fumigant application is supplemented by an herbicide tank mix of Goal
(oxyﬂuorfen) 0.56 kg/ha plus napropamide 4.5 kg/ha. A minimum 30—day interval is

required for Goal between applications and before transplanting (Rosskopf er al., 2005).

22

There are many chemicals still under regulatory scrutiny including compounds from
plants or processed plant by-products (Rosskopf et al. , 2005).

Each region will have to ﬁnd alternatives that best ﬁt the production schemes and
pests for that area. Soil type, climate, social, economic, regulatory, and political
situations all play into the success of a particular alternative. Factors limiting the
acceptance of alternatives include local availability, registration status, costs, labor, and
efﬁcacy of control (Anonymous, 1998). Methyl bromide will continue to be used in
limited land areas where replant is essential and for pest-free propagation material, but
the methyl bromide use for these is small (Rosskopf er al. , 2005). Although there is no
single replacement for methyl bromide, further research promises to develop an
integrated approach that will be safer for the environment and society. The future of
chemicals as a replacement for methyl bromide is unknown, and a movement towards a

more sustainable system is essential. One of the alternatives is biological control.

Biological Control

The rhizosphere is a dynamic environment. The mucilage excreted by the roots
supports both beneﬁcial and potentially harmful organisms seeking to colonize the
growing root. Mycorrhizal fungi colonize the root cortex, forming arbuscules, vesicles,
and extramatrical hyphae or ensheath short roots helping the plant to acquire nutrients,
especially iron (Buyer and Sikora, 1991). Other microbial interactions involve antibiosis
against other microbes and induction of plant resistance mechanisms. The beneﬁcial
microbes are antagonistic to plant pathogens by competition for food, essential elements,

and space (Whipps, 2001; Parke, 1991). Beneﬁcial microbes include bacteria in the

23

genera Streptomyces, Pseudomonas, and Bacillus, and fungi in several genera, with
potentially the most important being Trichoderma. These microbes enhance plant growth
by suppressing major pathogens, increasing nutrient availability, decreasing chemical
toxicity levels around the plant, or a combination of these (Whipps, 2001; Parke, 1991).
Some of the microbes parasitize pathogenic fungi by producing chitinases or antibiotics.
For instance, Streptomyces hygroscopicus var. geldonus produces a toxin called
geldanomycin which can inhibit R. solani (Chet et al., 1991; Fravel and Keinath, 1991).
Temperature, soil moisture, soil texture, pH, varietal differences and overall health of the
host, and growth rate of the microbe all inﬂuence the potential success of a biocontrol
organism (Parke 1991).

One biocontrol organism that has been used in the past is T. harzianum Rifai.
Chet and Henis (1983) reported 70% control of R. solani with 150 g (dry wt) of their T.
harzianum preparation per square meter in a broadcast application in carnation. Control
was enhanced further by establishing carnations in peat moss with 15% by volume of the
T. harzianum preparation prior to planting in the infested ﬁeld. Chet and Henis (1983)
also report that under ﬁeld conditions, seed treatment with T. hamatum (Bonord.) Bainier
reduced cotton damping-off caused by R. solani by 60%, and bare patches 23 days later
by 39%. It also increased density of plants by 14%. An integrated approach with soil
solarization or PCNB (pentachloronitrobenzene) improved control further. The use of
fumigation in conjunction with Trichoderma helped prevent the re-establishment of R.
solani and S. rolﬁrii in a peanut ﬁeld. The Trichoderma prolonged control over methyl
bromide alone (Chet and Henis, 1983). Chet and Henis (1983) detail the ﬁndings that

Trichoderma is antagonistic through parasitism, and competition for nutrients for

24

germinating sclerotia of R. solani and S. rolﬂrii . Trichoderma harzianum can use the R.
solani cell wall as a sole carbon source. Isolates of T. harzianum differed in the levels of
hydrolytic enzymes produced when mycelia of S. rolfsii, R. solani, and Pythium
aphanidermatum (Edson) Fitzp. were attacked in the soil. This correlated with the level
of control of each pathogen, suggesting that a speciﬁc Trichoderma species may be
required to control a speciﬁc pathogen (Chet and Henis, 1983).

Different groups within Trichoderma virens (J .H. Mill., Giddens & A.A. Foster)
Arx produce different antibiotics that differ in their control of Pythium ultimum versus
Rhizoctonia solani (Whipps, 2001). D’Ercole et al. (1989) found a reduction of post-
transplant blight incidence from 24.5% in the control to 11.5% in the treatment with T.
harzianum as a liquid dip on ‘Gorella’ strawberries. Studies found that T-22
(Trichoderma harzianum) could increase nitrogen fertilizer efﬁciency in corn (Harman,
2000). It causes plants to be more robust and have more extensive root systems by
suppressing disease as well as stimulating plant metabolism (Harman, 2000). A
combination of a binucleate Rhizoctonia spp. and Gliocladium virens (J .H. Mill., Giddens
& A.A. Foster) Arx provided control of Rhizoctonia blight on tall fescue in laboratory
assays decreasing percent blighted plants to 14 to 26%, compared to 30-36% in the
control (Yuen et al. , 1994).

Walker and Morey (1999) found that in pot and ﬁeld experiments, commercial
microbial nematicidal products were not as effective as conventional control measures in
controlling the nematodes T ylenchulus semipenetrans Cobb, ParatrichodOrus lobatus
Colbran, and the root rot fungi Pythium ultimum and Phytophthora nicotianae var.

parasitica (Dastur.) Waterhouse in citrus. They did ﬁnd that Actizyme [Bacillus subtilis

25

(Ehrenberg) Cohn.] stimulated citrus growth, as did the herbicide oryzalin, but effective
control was not obtained by biological measures, and some of the products harmed the

plant.

Cultural Control

Besides biological controls, other non-chemical measures have been tested.
Katznelson and Richardson (1948) found that treatment of soil infested with strawberry
BRR with dried blood, acetic acid, or steam sterilization all resulted in the reduction of
the disease, while oat straw appeared to increase the severity of the disease. In California,
chloride salts decreased Pythium ’s ability to colonize the soil resulting in a chance for
antagonists to become established (Martin and Hancock, 1983).

The build up of BRR pathogens over time has led to the recommendation of crop
rotation (Maas, 1998; Martin and Hancock, 1983; Perry and Ramsdell, 1994; LaMondia
et al., 2002). In Michigan ‘U-Pick’ operations, continual strawberry production is
common due to the few suitable locations for these enterprises and other economic
pressures. Continual cropping allows pathogen establishment and inoculum build-up. In
potato, wheat, barley, and com, the more frequently a crop is in a rotation, the higher the
decrease in yield of that crop (Schippers et al., 1987). Hildebrand and West (1941) found
that strawberries grown after a soybean series in greenhouse experiments approached the
same level of disease as sterilized soil. ‘Saia’ oats (A vena strigosa Ard.) has had some
success in controlling P. penetrans and R. fragariae in a pot trial in the greenhouse
(Townshend, 1989). Elmer and LaMondia (1999) found that an application of

ammonium sulfate with ‘Saia’ oats or sorgho-sudangrass reduced P. penetrans

26

populations in strawberry roots, and that a rotation with ‘Garry’ oats and ammonium
sulfate reduced root colonization by R. fragariae in a microplot study using ‘Honeoye’
strawberries. Brassica species such as oilseed radish, mustard, and canola have also
received attention due to the formation of isothiocyanates that have fungicidal and
nematicidal properties (Ettlinger and Kjaer, 1968; Snapp and Mutch, 2003).

Compost offers another means of control. Hoitink and Fahy (1986) discuss the
use of organic material for disease control, such as the incorporation of arnmoniated
Douglas ﬁr bark into soil which provided control for red stele in strawberry during the
ﬁrst two years of the planting, and the use of composted hardwood bark to suppress
several species of nematodes including P. penetrans. Plant pathogens are removed from
composted materials by three mechanisms discussed by Hoitink and Fahy (1986): (1) the
high temperature achieved during the composting procedure, (2) release of lethal
chemicals during the process, and (3) microbial antagonism. Once the pathogens have
been eliminated, successful disease suppression is affected by the ﬁnal particle size,
nitrogen, cellulose, lignin, and soluble salts content, as well as pH, any inhibitory
compounds released by the compost, and the population of beneﬁcial microbial
populations (Hoitink et al. , 1997). The microorganisms provide control through the same
concepts as biocontrol products such as competition, antibiosis, hyperparasitism, and the
induction of systemic acquired resistance (Hoitink et al. , 1997). The success of the
compost depends on the raw product from which it is derived and how it is handled. The
variability in compost stability is one obstacle that must be overcome. For instance,
control of R. solani by Trichoderma is better in mature compost as compared to fresher

organic matter because R. solani is a more ﬁt saprophyte under those conditions (Hoitink

27

et al., 1997). At the same time, excessively mature compost does not support as diverse a
microbial population, allowing pathogens to cause disease (Hoitink et al. , 1997).

The recommendations for controlling BRR have not changed much over the past
seventy years. The central difﬁculty is that BRR is a disease complex and the pathogens
vary from site to site. Studies have implicated P. penetrans, but the disease can occur
with low populations of this nematode, or without them at all. Rhizoctoniafragariae
does not always have to be present to get BRR symptoms either. It seems that a
combination of abiotic and biotic factors predispose strawberry plants to invasion by
perhaps otherwise saprophytic or weakly parasitic fungi that then cause severe economic
loss. With the primary control measure being eliminated, it becomes necessary to ﬁnd

alternative environmentally safe chemicals, biological alternatives, or cultural controls.

28

LITERATURE CITED

Anonymous. 1998. United Nations Environmental Programme (UNEP) Methyl Bromide
Technical Options Committee (MBTOC). 1998 Assessment of the Alternatives to
Methyl Bromide. United Nations Environmental Programme, Nairobi: 374 pp.

Brown, D. J. F, Dalmasso, A., and Trudgill, D. L. 1993. Plant Parasitic Nematodes in

Temperate Agriculture. Nematode Pests of Soft Fruits and Vines. Evans, K., D. L.
Trudgill, and J. M. Webster, Eds. CAB, International.

Burpee, L. L., Sanders, P. L., and Cole Jr., H. 1980. Anastomosis groups among isolates
of Ceratobasidium cornigerum and related fungi. Mycologia 72: 689-701.

Buyer, J. S. and Sikora, L. J. 1991. Rhizosphere interactions and siderophores. Pages

263-269 In The Rhizosphere and Plant Growth. D. L. Keister and P. B. Cregan, Eds.
Kluwer Academic Publishers, Dordrecht, The Netherlands.

Chapman, R. A. 1956. Plant parasitic nematodes associated with strawberries in
Kentucky. Plant Dis. Reporter 40: 179-1 83.

Chen, T., and Rich, A. E. 1962. The role of Pratylenchus penetrans in the development
of strawberry black root rot. Plant Disease Reporter. 46:839-843.

Chet, I., and Henis, Y. 1983. Trichoderma as a biocontrol agent against soilborne root
pathogens. In Ecology and Management of Soilbome Plant Pathogens. C. A. Parker, A.
D. Rovira, K. J. Moore, and P. T. W. Wong, Eds. American Phytopathological Society.
St. Paul. 110-112.

Chet, 1., Ordentlich, A., Shapira, R., and Oppenheim, A. 1991. Mechanisms of
biocontrol of soil-bome plant pathogens by Rhizobacteria. Pages 229-336 In The
Rhizosphere and Plant Growth. D. L. Keister and P. B. Cregan, Eds. Kluwer Academic
Publishers, Dordrecht, The Netherlands.

Cook, R. 2002. Strawberry Production in the United States 1990-2000. Department of
Agriculture Resources and Resource Economics.

D’Ercole, N., Nipoti, P., and Manzali, D. 1989. Research on the root rot complex of
strawberry plants. Acta Horticulturae 265:497-501.

DiEdwardo. 1960. Time-lapse studies of movement, feeding and hatching of
Pratylenchus penetrans. Phytopathology 50:570-571.

Dunn, R. A. 1972. Importance of depth in soil, presence of host roots, and role of eggs
as compared to vermiform stages in overwintering of Pratylenchus penetrans at Ithaca,
New York. Journal of Nematology 4:221-222.

29

Elmer, W. H., and LaMondia, J. A. 1999. Inﬂuence of ammonium sulfate and rotation
crops on strawberry black root rot. Plant Dis. 83:119-123.

EPA (Environmental Protection Agency). 2006. Metam sodium as an alternative to
methyl bromide for fruit and vegetable production. US. Environmental Protection
Agency: Ozone Depletion Rules and Regulations.

Ettlinger, M. G., and Kjaer, A. 1968. Sulfur compounds in plants. Pages 59-144 In
Recent advances in phytochemistry. T. J. Mabry, ed. New York: Appleton-Century-
Crofts.

Fravel, D. R., and Keinath, A. P. 1991. Biocontrol of soilbome plant pathogens with
fungi. Pages 237-243 In The Rhizosphere and Plant Growth. D. L. Keister and P. B.
Cregan, Eds. Kluwer Academic Publishers, Dordrecht, The Netherlands.

Freckman, D. W., and Chapman, R. A. 1972. Infection of red clover seedlings by

Heterodera trifolii (Goffart) and Pratylenchus penetrans (Cobb.) Journal of Nematology
4:23-28.

Galletta, G. J., and Himelrick, D. G. 1990. Pages 83-156 In Small Fruit Crop
Management. Prentice Hall, New Jersey.

Gianessi, L., and Reigner, N. 2005. The Value of Fungicides in US. Strawberry
Production: Preliminary Findings. CropLife Foundation: Crop Protection Research
Institute.

Goheen, A. C., and Bailey, J. S. 1955. Meadow nematodes in strawberry plantings in
Massachusetts. Plant Disease Reporter 39:879-880.

Goheen, A. C., and Smith, J. B. 1956. Effects of inoculation of strawberry roots with
meadow nematodes, Pratylenchus penetrans. Plant Disease Reporter 40: 146-149.

Hancock, J. F., Callow, P. W., Serce, S., and Schilder, A. C. 2001. Relative performance
of strawberry cultivars and native hybrids on fumigated and nonfumigated soil in
Michigan. HortScience 36: 136-138.

Hancock, J. F. 1999. Strawberries. Crop Production Science in Horticulture. CABI
Publishing, New York.

Harman, G. E. 2000. Myths and dogmas of biocontrol: changes in perceptions derived
from research on Trichoderma harzianum T-22. Plant Disease 84:377-392.

Hendrix Jr., F. F., and Campbell, W. A. 1973. Pythz’ums as plant pathogens. Ann. Rev.
Phytopathol. 11: 77-98.

30

Hildebrand, A. A. 1934. Recent observations on strawberry root rot in the Niagara
peninsula. Can. J. Research, 1 1:18-31.

Hildebrand, A. A., and West, P. M. 1941. Strawberry root rot in relation to

microbiologiclal changes induced in root rot soil by the incorporation of certain cover
crops. Can. J. Research Sec. C 19:183—198.

Hoitink, H. A. J ., and Fahy, P. C. 1986. Basis for the control of soilbome plant
pathogens with composts. Ann. Rev. Phytopathol. 24:93-114.

Hoitink, H. A. J., Stone, A. G., and Han, D. Y. 1997. Suppression of plant diseases by
composts. HortScience 32:184-187.

Husain, S. S., and McKeen, W.E. 1963a. Rhizoctoniafragariae sp. nov. in relation to
strawberry degeneration in southwestern Ontario. Phytopathology 53:532-540.

Husain, S. S., and McKeen, W.E. 1963b. Interactions between strawberry roots and
Rhizoctoniafragariae. Phytopathology 53 :54 1 -545.

Kable, P. F ., and Mai, W. F. 1968. Inﬂuence of soil moisture on Pratylenchus
penetrans. Nematologica 14: 101-122.

Katznelson, H., and Richardson, L.T. 1948. Rhizosphere studies and associated
microbiological phenomena in relation to strawberry root rot. Scientiﬁc Agriculture
28:293-307.

Klinkenberg, C. H. 1955. Nematode diseases of strawberries in the Netherlands. Plant
Dis. Reporter 9:603-606.

Kurppa, S., and Vrain, T. C. 1985. Penetration and feeding behavior of Pratylenchus
penetrans in strawberry roots. Revue Nematology 8:273-276.

LaMondia, J. A. 2004. Field performance of twenty-one strawberry cultivars in a black
root rot infested site. Journal of the American Pomological Soc. 58:226-232.

LaMondia, J. A., Elmer, W. H., Mervosh, T. L., and Cowles, R. S. 2002. Integrated
management of strawberry pests by rotation and intercropping. Crop Protection 21 :83 7-
846.

LaMondia, J. A. 1999. Effects of Pratylenchus penetrans and Rhizoctoniafragariae on
vigor and yield of strawberry. Journal of Nematology 31 :418-423.

LaMondia, J. A., and Martin, S. B. 1989. The inﬂuence of Pratylenchus penetrans and

temperature on black root rot of strawberry by binucleate Rhizoctonia spp. Plant Disease
73:107-110.

31

Lee, D. L. 2002. The Biology of Nematodes. University of Leeds, United Kingdom.
Taylor and Francis, London.

Locascio, S. J. 2005. Alternatives to methyl bromide fumigation for polyethylene-
mulched strawberries. University of Florida.

Long, S. 2002. MSU Researchers seek multicropping strawberry variety. Project
Greeen. Michigan State University.

Maas, J. L. 1998. Compendium of Strawberry Diseases. 2nd Ed. American
Phytopathological Society.

Mai, W. F., and Mullin, P. G.. 1960. Plant-Parasitic Nematodes: A Pictorial Key to
Genera. 5’h Ed. Cornell University Press. Ithaca, New York.

Mai, W. F ., Bloom, J. R., and Chen, T. A. 1977. Biology and Ecology of the Plant-
Parasitic Nematode Pratylenchus penetrans. Pennsylvania State University. Bulletin
815.

Marniya, Y. 1971. Effect of temperature on the life cycle of Pratylenchus penetrans on
Cryptomeria seedlings and observations on its reproduction. Nematologica 17:82-89.

Martin, F. N. 2000. Rhizoctonia spp. recovered from strawberry roots in central coastal
California. Phytopathology 90:345-353.

Martin, F. N., and Hancock, J. G. 1983. Chemical factors in soils suppressive to
Pythium ultimum. Pages 113-1 16 In Ecology and Management of Soilbome Plant
Pathogens. C.A. Parker, A.D. Rovira, K.J. Moore, and P.T.W. Wong, Eds. American
Phytopathological Society. St. Paul.

Martin, S. B. 1988. Identiﬁcation, isolation frequency, and pathogenicity of anastomosis
groups of binucleate Rhizoctonia spp. from strawberry roots. Phytopathology 78:379-
384.

MDA (Michigan Department of Agriculture). 2007. Michigan Fruit Inventory 2006-
2007. United States Department of Agriculture.

Mervosh, T. L., and LaMondia, J. A. 2004. Strawberry black root rot and berry yield are
not affected by terbacil herbicide. HortScience 39: 1339-1342.

Miller, P. M., Sands, D. C., and Rich, S. 1973. Effect of industrial mycelial residues,
wood ﬁber wastes, and chitin on plant-parasitic nematodes and some soilbome diseases.

Plant Disease Reporter 57:43 8-442.

Miller, P. W. 1948. Relation of desiccation to the development of black root rot of
strawberries. Plant Dis. Reporter 32:315-316.

32

Molot, P. M., and F erriere, H. 1989. Susceptibility of strawberry cultivars to
Rhizoctonia solani and R. fragariae as inﬂuenced by inoculation technique, seasonal
variations and physiological condition of the plants. Acta Horticulturae 265:535-538.

Mountain, W. B., Sands, D. C, and Rich, S. 1959. The peach replant problem in Ontario.
VII. The pathogenicity of Pratylenchus penetrans (Cobb, 1917) Filip. and Stek., 1941.
Canadian Journal of Botany 37:459-470.

NASS (National Agricultural Statistics Service). 2007. 2006 Strawberry Data. United
States Department of Agriculture.

Nelson, E. B. 1990. Exudate molecules initiating fungal responses to seeds and roots.
Plant and Soil 129:61-73.

Nelson, P. E. 1957. Pathogenicity of Idriella lunata on strawberry. Phytopathology
47:438-443.

Nemec, S. 1970. Pythium sylvaticum pathogenic on strawberry roots. Plant Dis. Rep.
54:416-418.

Nemec, S., and Sanders, H. 1970. Pythium species associated with strawberry root
necrosis in southern Illinois. Plant Disease Reporter 54:49-51.

Ogoshi, A. 1985. Anastomosis and intraspeciﬁc groups of Rhizoctonia solani and
binucleate Rhizoctonia. F itopatol. Brasileira 10: 371-390.

Ogoshi, A., and Ui, T. 1983. Anastomosis groups of Rhizoctonia solani and binucleate
Rhizoctonia. Pages 57—58 In Ecology and Management of Soilbome Plant Pathogens.
C. A. Parker, A. D. Rovira, K. J. Moore, and P. T. W. Wong, Eds. American
Phytopathological Society. St. Paul.

Olthof, Th. H. A. 1982. Effect of age of alfalfa root on penetration of Pratylenchus
penetrans. Journal of Nematology 14:100-105.

Oyekan, P. 0., Blake, C. D., and Mitchell, J. E. 1972. Histopathology of pea roots
axenically infected by Pratylenchus penetrans. Journal of Nematology 4:32-35.

Parke, J. L. 1991. Root colonization by indigenous and introduced microorganisms.
Pages 33-42 In The Rhizosphere and Plant Growth. D. L. Keister and P. B. Cregan, Eds.
Kluwer Academic Publishers, Dordrecht, The Netherlands.

Parmeter Jr., J. R., Whitney, H. S., and Platt, W. D. 1967. Afﬁnities of some

Rhizoctonia species that resemble mycelium of Thanatephorus cucumeris.
Phytopathology 57: 218-223.

33

Particka, C. A., and Hancock, J. F. 2005. Field evaluation of strawberry genotypes for
tolerance to black root rot on fumigated and nonfumigated soil. J. Amer. Hort. Sci.
130:688-693.

Perry, 8., and Ramsdell, D. 1994. Strawberry diseases in Michigan. Ag Facts, Michigan
State University Extension Bulletin E-1728: 1-4.

Pollack, S., and Perez, A. 2005. Fruit and Tree Nuts Outlook. Economic Research
Service. USDA. FTS-315.

Powell, N. T. 1971. Interactions between nematodes and fungi in disease complexes.
Annual Review of Phytopathology 9:253-274.

Pritts, M., and Handley, D. Eds. 1998. Strawberry Production Guide: For the Northeast,
Midwest, and Eastern Canada. Northeast Regional Agricultural Engineering Service
Cooperative Extension. Ithaca, New York.

Raski, D. J. 1956. Meadow Pratylenchus penetrans tested on strawberries grown in
black root rot soil. Plant Disease Reporter 40:690-693.

Ribeiro, O. K., and Black, L. L. 1971. Rhizoctioniaﬁagariae: a mycorrhizal and
pathogenic fungus of strawberry plants. 55:599-603.

Rosskopf, E. N., Chellemi, D. O., Kokalis-Burelle, N., and Church, G. T. 2005.
Alternatives to methyl bromide: A Florida perspective. Plant Management Network 27
Oct.

Schippers, B., Bakker, A. W., and Bakker, P. A. H. M. 1987. Interactions of deleterious
and beneﬁcial rhizosphere microorganisms and the effect of cropping practices. Ann.
Rev. Phytopahtology 25:339-358.

Scott, R., Pritts, M., and Kelly, M. J. 2003. Effects of Rhizoctonia/fragariae infection on
growth and productivity of strawberry plants grown under different temperature regimes.

Advances in Strawberry Research 22: 26-33.

Snapp, S. S., and Mutch, D. R. 2003. Cover crop choices for Michigan vegetables.
Michigan State University Extension Bulletin E 2896: 1-6.

Stanghellini, M. E., and Hancock, J. G. 1970. The sporangium of Pythium ultimum as a
survival structure in soil. Phytopathology 61 :157-164.

Strong, F. C., and Strong, M. C. 1927. Investigations on the black root of strawberries.
Phytopathology 21 :1041-1059.

Szczygiel, A., and Proﬁc-Alwasiak, H. 1989. Studies on the interaction between
nematodes and fungi in infecting strawberry plants. Acta Horticulturae 2651561 -565.

34

Townshend, J. L. 1963. The pathogenicity of Pratylenchus penetrans to strawberry.
Can. J. of Plant Science 43:75-78.

Townshend, J. L. 1989. Population densities of four species of root-lesion nematodes
(Pratylenchus) in the cat cultivars, Saia and OAC Woodstock. Can. J. Plant Science
69:903-905.

Vilgalys, R., and Cubeta, M. A. 1994. Molecular systematics and population biology of
Rhizoctonia. Annual Rev. Phytopathol. 32:135-155.

Walker, G. E., and Morey, B. G. 1999. Effects of chemicals and microbial antagonists

on nematodes and fungal pathogens of citrus roots. Australian Journal of Exper. Ag. 39:
629-637.

Watanabe, T., Hashimoto, K., and Sato, M. 1977. Pythium species associated with
strawberry roots in Japan, and their role in the strawberry stunt disease. Phytopathology

67: 1324-1332.

Whipps, J. M. 2001. Microbial interactions and biocontrol in the rhizosphere. Journal of
Experimental Botany 52:487-51 1.

Wilhelm, S. 1953. Pythium ultimum, an injurious component of the rhizosphere in
certain soils. Phytopathology 43:590.

Wilhelm, S. 1963. The control of Verticillium wilt of strawberry and of weeds by
preplant fumigation with chloropicrin and chloropicrin-methyl bromide mixtures. 16th
Inter. Hort. Congr. 3 (1962): 263-264.

Wilhelm, S. 1965. Pythium ultimum and the soil fumigation growth response.
Phytopathology 55: 1016-1020.

Wilhelm, S., Nelson, P. E., Thomans H. E., and Johnson, H. 1972. Pathology of
strawberry root rot caused by Ceratobasidium species. Phytopathology 62:700-705.

Williams-Woodward, J. L. 2001. Georgia Plant Disease Loss Estimate: Strawberry. The
University of Georgia College of Agriculture and Environmental Sciences Cooperative
Extension Service.

Wing, K. B., Pritts, M. P., and Wilcox, W. F. 1994. Strawberry black root rot: A Review.
Advances in Strawberry Research. 13:11-19.

Wing, K. B, Pritts, M. P., and Wilcox, W. F. 1995. Biotic, edaphic, and cultural factors
associated with strawberry black root rot in New York. HortScience 30:86-90.

35

WMO (World Meteorological Organization). 2003. Scientiﬁc Assessment of Ozone
Depletion: 2002, Global Ozone Research and Monitoring Project -Report No. 47, 498 pp.
Geneva.

Yuen, G. Y., Craig, M. L., and Giesler, L. J. 1994. Biological control of Rhizoctonia
solani on tall fescue using fungal antagonists. Plant Disease 78:118-123.

Yuen, G. Y., Schroth, M. N, Weinhold, A. R., and Hancock, J. G. 1991. Effects of soil
fumigation with methyl bromide and chloropicrin on root health and yield of strawberry.
Plant Disease 75:416-420.

Zeller, S. M. 1932. A strawberry disease caused by Rhizoctonia. Oregon Agricultural
Experiment Station Bulletin 295:1-22.

36

CHAPTER TWO: BIOCONTROL AND REDUCED-RISK FUNGICIDE
EFFICACY TRIAL
Introduction

Strawberry black root rot (BRR) is a disease complex that is widespread around
the world wherever strawberries have been planted for multiple years (Watanabe et al. ,
1977; D’Ercole et al., 1989). Infected plants produce smaller leaves, fewer main and
lateral roots, have slower growth, and reduced runner production (Maas, 1998; Hancock
et al., 2001). Severely infected plants wilt at the onset of dry weather. The disease can
be spread via infected nursery stock, movement of infested soil, or infected plant debris
(Maas, 1998; Strong and Strong, 1927; Hildebrand, 1934). Also known as strawberry
decline, many organisms and abiotic factors have been implicated in the cause of the
disease; however, Rhizoctoniaﬁagariae Husain & McKeen, Pythium spp., and the root
lesion nematode Pratylenchus penetrans (Cobb) F ilipjev and Shuurmans Stekhoven are
generally considered the primary pathogens (D’Ercole er al., 1989; Wing et al., 1994;
Maas, 1998).

In Michigan, the matted-row system is used to grow strawberries, where plants
are typically planted at wide spacing in spring so that runners ﬁll in between the plants.
In Michigan ‘U-Pick’ operations, continual strawberry production or short rotations are
common due to the few suitable locations for these enterprises. The shorter a rotation is
between strawberry plantings, the higher the potential economic return to the grower.
However, continual cropping allows pathogen establishment and build-up to deleterious

levels. In this system, fumigation is often employed to help control soil-bome pathogens

37

and weeds before planting. A common and effective fumigant used by strawberry
growers is methyl bromide. In 1992, the Montreal Protocol established methyl bromide
as an ozone-depleting substance and instituted its ban by January 1, 2005 (Anonymous,
1998; Rosskopf et al., 2005).

The primary problem when trying to develop a management strategy for BRR is
that the causative organisms of the disease are variable. Rhizoctoniafragariae does not
always have to be present to get BRR symptoms (Wing et al., 1994; Wing et al., 1995).
While studies have implicated P. penetrans, the disease can occur with low nematode
populations or even in their absence (Wing et al., 1994; Wing et al. , 1995). A
combination of abiotic and biotic factors may predispose strawberry plants to invasion by
perhaps otherwise saprophytic or weakly parasitic fungi that then cause severe damage to
the root system (Wing et al., 1994; Wing et al. , 1995). With the primary control measure
due to be eliminated, it becomes necessary to ﬁnd alternative reduce-risk chemicals,
biological, and cultural alternatives.

An alternative to methyl bromide are reduced-risk fungicides and biocontrol
products which could be applied at planting. This would require the least change in a
production scheme that is heavily reliant on pre-plant fumigation.

The mucilage excreted by the roots supports both beneﬁcial and potentially
harmful organisms seeking to colonize the growing root. The beneﬁcial microbes are
antagonistic to pathogens by competition for food, essential elements, and space
(Whipps, 2001; Parke, 1991). These microbes enhance plant growth by suppressing
major pathogens, increasing nutrient availability, decreasing toxicity levels around the

plant, or a combination of these (Whipps, 2001; Parke, 1991). Some of the microbes

38

parasitize pathogenic fungi by producing chitinases or antibiotics. For instance,
Streptomyces hygroscopicus var. geldonus produces a toxin called geldanomycin which
can inhibit R. solani (Chet et al., 1991; Fravel and Keinath, 1991). Trichoderma is
antagonistic through parasitism, and competition for nutrients for germinating sclerotia of
R. solani and S. rolfsii . Trichoderma harzianum can use the R. solani cell wall as a sole
carbon source (Chet and Henis, 1983).

One biocontrol organism that has been used in the past is T. harzianum Rifai.
Chet and Henis (1983) reported 70% control of R. solani with 150 g (dry wt) per square
meter in a broadcast application in carnation. Chet and Henis (1983) also report that
under ﬁeld conditions, seed treatment with T. hamatum (Bonord.) Bainier reduced cotton
damping-off caused by R. solani by 60%, and bare patches 23 days later by 39%. It also
increased density of plants by 14%. Trichoderma virens (J .H. Mill., Giddens & A.A.
Foster) Arx produce different antibiotics that differ in their control of Pythium ultimum
versus Rhizoctonia solani (Whipps, 2001). D’Ercole et al. (1989) found a reduction of
post-transplant blight incidence from 24.5% in the control to 11.5% in the treatment with
T. harzianum as a liquid clip on ‘Gorella’ strawberries.

This experiment was conducted to evaluate biocontrol agents and reduced-risk
fungicides applied at planting, or through the establishment season, as possible
alternatives to methyl bromide for strawberry production, and to enable the selection of
promising products for further assessment in an integrated approach to managing BRR
(Chapter 4).

All the products tested were fungicides, except for Ditera, a biological nematicide.

Some products, such as Abound, claim to control root rot caused by Rhizoctonia spp., but

39

have not been tested in Michigan. Some of the chemicals, like Ridomil Gold EC, are
used in strawberry production, but not necessarily in this application method or timing.
Other products are not labeled for strawberries, but claim to control similar diseases on
other hosts such as brown patch and large patch caused by Rhizoctonia spp. on turf
(Endorse). There are also many biological products that make claims of disease control

or enhancement of plant growth based on Streptomyces or Trichoderma spp.

Materials and Methods
Field Establishment and Measurements

On 16 June 2005, 15 treatments, replicated four times, were established in 3.05 x
3.05 m plots in a randomized complete block design to establish efﬁcacy against BRR
(table 1). The ﬁeld was a sandy-loam located at the Michigan State University
Horticulture Farm (East Lansing, MI) with a 4-year history of continuous strawberry
production and BRR. Each plot consisted of four rows of strawberries planted 61 cm
apart with six plants set 45.7 cm apart. There were 45.7 cm of cultivated buffers around
each plot. Plots used as positive controls received fumigation with 448 kg/ha of methyl
bromide and chloropicrin (methyl bromide 66%, Chlor-o-pic 33%) 14 d before planting.
Plots receiving no treatment served as negative controls. Napropamide (Devrinol) was
applied over the whole planting 10 d before planting for weed control. Susceptible
cultivar ‘Allstar’ transplants were obtained from Krohne Plant Farms (Hartford, MI).
These were pulled out of storage three days before planting due to severe etiolation and
placed in lugs with Baccto High Porosity Professional Planting Mix (Michigan Peat

Company, Houston, TX) to ensure the plants were healthy for transplanting.

40

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41

C/G was applied two days before planting to avoid phytotoxicity that had been
observed in previous experiments and to allow any precipitation to further distribute it in
the soil. Rainfall totaled 68 mm over the two days after planting. All strawberry
transplants were planted the same day with the rest of the drenches applied as 473 ml per
planting hole and the plant placed in the hole immediately. T-lO was grown on potato
dextrose agar (PDA) for 7-10 d until sufﬁcient spore production was achieved for making
a spore suspension the day of planting. The drench application procedure was done to
decrease the chance of contamination between treatments, increase consistency of
volume applied, and get each product around the roots. Since many of the products are
biocontrol agents, getting them in the root zone was essential to assist in potential
colonization, and consequently, disease control.

Roots receiving the dip treatments were treated with product mixed in 3.79 liters
of water in buckets. Planting was followed by 25 mm of irrigation. Standard cultural
practices were implemented including a spring and fall fertilization of 19-19-19 at the
rate of 32 kg/ha and weekly hand weeding. In August, 57.7 kg/ha of nitrogen was
applied as urea. Flowers were removed the ﬁrst year and runners were racked back into
the rows to establish a perennial bed 50 cm wide. Irrigation was applied as needed.

A count was taken of the mother plants, within the two center rows, two weeks
after planting to determine loss due to planting and establish a base number of plants used
for data collection. The mother plant count difference was calculated using the starting
count from 2005 through the fall count in 2005. Ditera, although originally applied at 2.8
kg/ha,was re-applied each month for the ﬁrst season at a rate of 5.6 kg/ha as a 473-ml

drench around each plant.

42

Each application of Ditera was followed by at least 6.4 mm of irrigation except
for the last treatment in September which was not irrigated because the irrigation system
was being repaired. On 13 September 2005, 100 g of soil surrounding the plant dug for
root samples were taken from each replication of the control, fumigated, and Ditera plots
to assess nematode populations. All samples were taken from the outside rows of the
treatments so that interior plants were kept for future data collection. Plant vigor and bed
fill ratings were obtained in 13 September 2005 and were based on a 1 to 5 scale (table
2). The total number of crowns and number of remaining mother plants were counted on
18 November 2005 from the middle two rows. Total crown counts include daughter and
mother crowns. Crown counts and erect petiole biomass were calculated for an area of
38.1 cm x 61 cm for a single row. Approximately 7 cm of straw was placed over the

plants in November.

43

Table 2. Plant vigor and bed ﬁll ratings used in the strawberry biocontrol and fungicide
products applied at planting efﬁcacy trial in East Lansing, MI during 2005-2007.

 

PlantVigor Characteristics

 

5

4

3

2

1

Bed Fill
5

4

3

Plants in excellent health, vigorous growth, superior runnering
33% plants diseased or stunted

50% plants diseased or stunted

Majority of plants diseased or stunted

Plants very stunted, diseased

Characteristics
Runners healthy and establishing well, full bed

Fewer runner plants than in 5
Thinning evident, runner size and establishment good
Few runners, mother plants obvious

Few to no runners establishing, mother plants dead

 

44

The straw was removed on 9 April 2006. Three harvests were taken a week apart
starting on 8 June 2006. The middle meter of each of the interior two rows was
harvested. Ripe berries were picked regardless of condition or disease. Yield was
standardized to one meter of a single row by dividing collected data by two. All plots
were subsequently clean picked to ensure plant health for the following year. Bed ﬁll and
plant vigor ratings were taken on 4 August 2006. In September, lime and 19-19-19
fertilizer were applied at the rates of 271 .5 kg/ha and 254.5 kg/ha, respectively. Plants
were dug for fungal assays on 9 October 2006, and then the rows were roto-tilled by
treatment to prevent cross contamination. All samples for fungal isolations were taken
from the outside rows of the treatments so that interior plants were kept for data
collection. Plants were covered with approximately 7 cm of straw in November 2006 .

On 21 April 2007 the straw was removed. Berries were counted as they were
harvested in June. There were two harvests 8 d apart. The same interior meters and
procedures were used as in 2006. Bed ﬁll, plant vigor, and fresh erect petiole and leaf
biomass were collected on 19 July 2007. A wooden frame measuring 38.1 cm x 61 cm
was centered over each of the center rows and all leaves were collected within this area.

The collected material was subsequently dried for 48 h at 80°C and weighed.

Root and Soil Analysis

The plants and soil collected in 2005 were submitted to the Michigan State
University Diagnostic Lab for nematode analysis. Roots were taken from the untreated
and fumigated treatments and put on water agar for fungal analysis. A total of ten root

pieces (ﬁve root pieces per plate) were analyzed in addition to ﬁve root pieces from

45

plants showing root tip discoloration after obtaining whole plant fresh weights. Root
pieces with lesions were selected. Isolated fungi were sub-cultured one week later after
growing at room temperature. They were subsequently identiﬁed to genus using classic
taxonomic procedures and growth characteristics on media (Barnett and Hunter, 1998;
Domsch et al., 1980; Barron, 1968). Those fungi that could not be identiﬁed in this
manner were subsequently subjected to DNA sequencing of the internal transcribed
spacer (ITS) regions by Dr. Mursel Catal (Sambrook et al. 1989; Altschul et al., 1990).

On 9 and 10 October 2006 mother plants were again taken from the outside rows
of each treatment for a total of 120 mother plants. These were selected as representatives
of the entire plot. These plants were dug for fresh and dry biomass, percent root necrosis,
and a visual root quality rating (on a scale of 1-5: 1= <20% of root mass is secondary
roots, 2= 20-40% secondary roots, 3= 40-60% secondary roots, 4= 60-80% secondary
roots, 5= >80% secondary roots). Dead material and daughter plants were removed, the
roots washed under running cold water, and then the plants were air dried before the fresh
weight was taken. Approximately 200 g of soil was collected from the Ditera, untreated,
and fumigated plots for nematode analysis done by the Michigan State University
diagnostic lab. Nematode presence was determined using the modiﬁed Jenkins technique
and a modiﬁed root extraction process (Jenkins, 1964; Bird, 1971).

The foliage and roots were separated by cutting the crown in half above the
uppermost adventitious roots. After rating and taking the fresh weights, the foliage and
roots were dried at 80°C for 48 h and weighed again.

Additional 1 year old plants were also dug for fungal isolations from roots to

evaluate general plant health. After washing as above, root pieces were surface sterilized

46

in 20% bleach for 2 min followed by two rinses in sterile deionized water for 1 min.
Root pieces were dried on sterile paper towels. Ten 0.64-cm pieces were placed onto
water agar (5 pieces per plate) with 80 pieces/treatment. Isolated fungi were sub-cultured
over the next 7-10 (I, being incubated on the laboratory bench at room temperature, and
identiﬁed to genus as described previously. In 2007, despite the treatments Plantshield,
Endorse, and the untreated control having biomass measurements and crown count not
being taken from the two center rows, but one center and one outside row, these data
were not removed and no signiﬁcant differences were observed. The Endorse and
Mycostop plots in block 2 were improperly harvested by not harvesting the same interior
meter as the previous year on the ﬁrst harvest date, no signiﬁcant differences are evident.
The frequency of fungi recovered was calculated from total fungi that grew. Total roots
showing no growth was calculated from total root number.

On 11 September 2007, two mother plants were dug from the middle rows of each
treatment plot to obtain a root quality rating, percent root necrosis, and fresh and dry
weights as previously described. An additional mother plant was dug for fungal analysis
from the same treatments assayed in 2006. The same procedure was used as previously
described except that there were only 10 root pieces per treatment. A mother plant and
200 g of soil were also removed from the untreated, fumigated, and Ditera plots for

nematode analysis.

Data Analysis

All statistical analyses were performed using the ANOVA and mean separation

functions (Fisher’s protected least signiﬁcant difference test p=0.05) of the StatGraphics

47

Plus 4.1 (StatPoint Inc., VA) statistical computer program. Initially a variance check was
performed; all data that did not pass the variance check were subsequently transformed.
When analyzing the data over multiple years, it was necessary to utilize SAS 9.1 (SAS

Institute Inc., Cary, NC.) using repeated measurement ANOVA in Proc GLM.

Results and Discussion

Year effect was signiﬁcant in the repeated measurements ANOVA of the ﬁeld
data (p<0.0001). There was not a block x treatment interaction (p=0.3033).

During establishment in 2005, the fumigated and control treatments did not differ
from one another signiﬁcantly for any parameter, except total crown number where
fumigated plants had more crowns than the untreated (table 3). Ditera, Abound, ProPhyt
and ProPhyt + Abound treated plants tended to have the highest vigor, best bed ﬁll, and
most crowns, but were not signiﬁcantly superior to those untreated. C/G was the only
treatment causing a signiﬁcant reduction in mother crowns from the untreated plants in
2005. The reason for this difference is that C/G can be phytotoxic to strawberries if not
properly applied. Plants receiving Ridomil Gold EC also appeared stunted, but were not
signiﬁcantly different than the untreated controls. Wing et al. (1995) discusses that
Ridomil is typically associated with healthier plants, but it is only effective in BRR sites
with oomycetes. Ridomil Gold EC is typically applied to older strawberry plantings, so
the age of the plants and the method used to apply it may not have been optimal for

strawberry growth. This toxicity was also observed by Louws et al. (2004).

48

Table 3. Effects of different biocontrol and fungicide products applied at planting on
plant vigor and bed ﬁll of strawberry cv. Allstar in a black root rot infested soil in East
Lansing, MI, in 2005 and 2006.2

x Plant vigor scale of l to 5 where 5 = Plants in excellent health, vigorous growth,
superior runnering, 4 = 33% plants diseased or stunted, 3 = 50% plants diseased
or stunted, 2 = Majority of plants diseased or stunted, and l = Plants very stunted,
diseased.

y Bed ﬁll scale of 1 to 5 where 5 = Runners healthy and establishing well, full bed, 4 =
Fewer runner plants than in 5, 3 = Thinning evident, runner size andestablishment
good, 2 = Few runners, mother plants obvious, and 1 = Few to no runners
establishing, mother plants.

2 Values in columns followed by differing letters are signiﬁcantly different according to
Fisher’s protected least signiﬁcant difference test p =0.05. Pairwise comparisons
performed with n=4.

49

 

2005

 

 

 

 

 

 

Bed fill Plant vigor Difference

rating rating Total crown in mother Fruit
Treatment (l-SJy (1'5)x number/m crown number yield/m (kg)
Fumigated 4.50 d 4.75 NS 12.31 c 0.00 c -
Abound 4.00 cd 4.25 11.67 be -0.25 bc -
Actinovate 3.75 cd 3.75 10.38 be -0.50 bc -
C/G 1.25 a 3.25 6.46 a -5.75 a -
Ditera 4.00 cd 4.50 10.36 be 025 be -
Endorse 3.50 cd 3.75 9.70 be -1.50 b -
Mycostop 4.00 cd 3.75 9.02 ab -0.25 bc -
Plantshield 2.75 bc 3.50 8.84 ab -0.25 bc -
Polyversum 3.25 bcd 3.50 10.04 be -1.00 bc -
ProPhyt 4.00 cd 4.50 11.59 be 0.00 c -
ProPhyt+Abound 3.50 cd 4.50 11.06 bc 0.00 c -
ProPhyt+T-10 3.50 cd 4.25 9.77 be -0.50 bc -
Ridomil Gold EC 2.00 ab 2.75 8.84 ab -1.00 bc -
T-10 3.00 be 3.50 9.70 be -1.25 bc -
Untreated 3.25 bcd 3.75 8.92 ab -1.00 bc -
ANOVA
Effect Df Signiﬁcance (p)
Treatment 14 0.0035 0.0546 0.0305 0.0000 -
Block 3 0.7981 0.5958 0.0019 0.7605 -
Residual 42
Total (Con) 59

2006

Fumigated 5.00 a 5.00 a 13.79 NS - 1.31 abc
Abound 4.25 ab 4.50 ab 12.41 - 1.67 a
Actinovate 3.75 be 3.75 bcde 12.39 - 1.11abc
C/G 2.75 c 3.25 def 11.36 - 0.44 d
Ditera 4.00 ab 4.00 bcd 12.77 - 1.31abc
Endorse 2.75 c 3.50 cde 13.50 - 1.19 abc
Mycostop 3.50 be 3.50 cde 12.80 - 1.16 abc
Plantshield 3.50 be 3.50 cde 10.89 - 0.96 bcd
Polyversum 3.25 be 3.00 ef 10.95 - 1.10 abc
ProPhyt 4.25 ab 4.25 abc 11.91 - 1.31 abc
ProPhyt+Abound 3.75 be 3.50 cde 11.91 — 1.29 abc
ProPhyt+T-10 4.00 ab 4.00 bcd. 10.93 - 1.25 abc
RidomilGold EC 2.75 c 2.50 f 10.36 - 0.76 cd
T-lO 3.75 be 3.50 cde 9.85 - 1.09 bc
Untreated 3.50 be 3.75 bcde 11.12 - 1.36 ab
ANOVA
Effect Df gniﬁcance (p)
Treatment 1 4 0.0074 0.0003 0.0652 - 0.0505
Block 3 0.6471 0.1363 0.5244 - 0.2621
Residual 42
Total (Con) 59

 

50

In 2006, the negative impact of C/G and Ridomil Gold EC on bed ﬁll and plant
vigor was still apparent. Abound, Ditera, ProPhyt, and ProPhyt + T-10 all tended to
increase bed ﬁll and plant vigor ratings over the untreated plants, but the differences were
not signiﬁcant over the untreated control (table 3). Although total crown number was not
signiﬁcantly different among treatments, Ditera, Endorse, and Abound tended to have
numerically more crowns (table 3). Abound, ProPhyt, and ProPhyt + Abound did not
have the highest averages for total crown number in 2006, which may be a result of
already having well established beds from 2005, meaning that the beds did not have as
many new additional crowns. Although the fumigated and untreated control treatments
signiﬁcantly differed for plant vigor and bed ﬁll, the lack of yield differences may be a
result of the fumigated strawberries putting on more vegetative growth, making them
bigger and more vigorous, but not necessarily produce more fruit (table 3).

Although there was a lack of signiﬁcant differences amongst the majority of the
treatments, and even between the treatments and the untreated control, there were three
treatments that consistently tended to have averages closer to the fumigated control for
each parameter in 2005 and 2006. These treatments were ProPhyt, Abound, and Ditera.
When examining just the biocontrol products over 2005 and 2006, Actinovate also tended
to have higher plant vigor and bed ﬁll than other biocontrol treatments.

Treatments subjected to fungal assays were selected because they showed initial
promising results and were being considered for evaluation in the Integrated Management
Experiment (Chapter 4). These included: untreated control, fumigated control, ProPhyt +

Abound, Abound, ProPhyt, Actinovate, and Plantshield.

51

The treatments were not signiﬁcantly different for a single parameter in 2007.
However, Abound, Ditera, ProPhyt, and ProPhyt + Abound-treated plants continued to
show averages approaching those of the plants in the fumigated control (table 4). Plants
receiving the Actinovate and T-lO dip also had averages approaching those of plants in
the fumigated plots for ﬁll, vigor, total berries, fresh biomass, and yield. The untreated
plants had the lowest average bed ﬁll and plant vigor rating, and dry biomass weights,
while the fumigated control had the highest averages. The signiﬁcance of the blocks in
the plant vigor rating data may be due to some of the Ridomil Gold EC treated plants
recovering more ﬁilly than others due to possible differences in local soil fertility and
microbiology. In the third season (2007), treatment effects, especially from those
treatments that appeared detrimental initially, had disappeared.

Despite the set backs the strawberry plants suffered from the C/G and Ridomil
Gold EC treatments, the ability of strawberry plants to compensate for the death of plants
is apparent from the 2007 yield data (table 4). The plants had also been able to outgrow
any of the initial toxicity. Due to the lack of a general decline being observed and the
increasing risk of not being able to conﬁdently identify all surviving mother crowns in

each treatment, a total surviving mother crown count was not attempted in 2007.

52

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53

For the two years that yield data were collected, Abound treated plants had the
highest average, but plants in the two control treatments did not differ signiﬁcantly from
one another. This lack of difference between years is maybe due to a late spring frost in
2007 that reduced fruit set. This was also a hotter, drier year resulting in lower yields.

Over the three years that the ﬁll rating was obtained, plants treated with Ditera or
Abound were the closest to the plants in the fumigated control, but none of the treatments
resulted in signiﬁcantly fuller beds than the untreated control (table 5). Ditera, Abound,
ProPhyt, and ProPhyt + T-lO treated plants also had the same plant vigor as the
fumigated control, but treatment effects were not signiﬁcant. ProPhyt and Abound
treated beds tended to have the same number of crowns as beds in the fumi gated control,
but there were no signiﬁcant differences among treatments. Average total crowns are
expected to increase each year in healthy strawberry beds, this is particularly true in beds
that may have started out thin due to phytotoxic treatments. This compensation would

also be reﬂected in bed ﬁll.

54

Table 5. Effects of different biocontrol and fungicide products applied at planting on
plant vigor, bed ﬁll, and yield of strawberry cv. Allstar in a black root rot infested soil in
East Lansing, MI, in 2005-2007.x

 

 

 

All three years 2006-2007

Bed ﬁll rating Plant vigor rating Total crown 1‘0“"
Treatment (1-5)z (1-5)y number/m2 yield/m (kg)
Fumigated 4.83 a 4.92 a 16.03 NS 0.96 NS
Abound 4.33 ab 4.50 ab 16.02 1.10
Actinovate 4.17 abc 4.08 bcd 15.01 0.87
C/G 2.67 e 3.67 cde 1 1.19 0.44
Ditera 4.33 ab 4.42 ab 14.88 0.96
Endorse 3.50 cd 3.92 bcde 14.15 0.82
Mycostop 4.l7 abc 4.00 bcd 14.44 0.86
Plantshield 3.75 bcd 3.92 bcde 13.74 0.78
Polyversum 3.50 cd 3.58 de 13.58 0.77
ProPhyt 4.25 abc 4.50 ab 15.58 0.94
ProPhyt + Abound 4.08 abc 4.25 be 15.28 0.92
ProPhyt + T-10 4.17 abc 4.33 ab 14.65 0.91
Ridomil Gold EC 3.08 de 3.33 e 12.61 0.64
T-10 3.92 be 3.92 bcde 14.18 0.90
Untreated 3.58 bcd 3.92 bcde 13.30 1.01
ANOVA
Effects Df Signiﬁcance (p)
Treatment 14 <0.0001 <0.0001 0.8729 0.4867
Block 3 0.9227 0.0052 0.9763 0.6857
Residual 162

Total (Corn) 179

x Results of pairwise comparison of averages performed on n=15, for block, n=4.

Values in columns followed by differing letters are signiﬁcantly different according to
Fisher’s protected least signiﬁcant difference test p=0.05.

y Plant vigor rating 5 = Runners healthy and establishing well, full bed, 4 = Fewer runner
plants than in 5, 3 = Thinning evident, runner size and establishment good, 2 = Few
runners, mother plants obvious, and 1 = Few to no runners establishing, mother plants.

2 Bed ﬁll rating 5 = Plants in excellent health, vigorous growth, superior runnering, 4 =
33% plants diseased or stunted, 3 = 50% plants diseased or stunted, 2 = Majority of
plants diseased or stunted, and 1 = Plants very stunted, diseased.

 

55

When considering individual plant parameters in 2006, average fresh foliage
weight and fresh root weight there were not signiﬁcantly different among treatments,
although the plants from the fumigated treatments were signiﬁcantly larger than all other
treatments (table 6). A similar pattern was found for fresh and dry total weights. There
was no difference between the plants from the untreated control for any weight parameter
except for Plantshield which had lower whole plant weights. Plants receiving the
ProPhyt + Abound, ProPhyt + T-lO, Ditera, Mycostop, and Polyversum treatments
tended to have root quality ratings similar to those of the plants in the fumigated
treatment. Plants treated with Polyversum, Mycostop, and ProPhyt + T-10 had the lowest
percent root necrosis next to plants from the fumigated plots, although they were not

signiﬁcantly different than the untreated control.

56

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57

In 2007 the effect of treatment was not signiﬁcant for fresh foliage, dry foliage, or
total dry weights, according to the ANOVA. Although no treatment caused plants to
signiﬁcantly differ from the untreated control plant weights, Ditera, ProPhyt + Abound,
ProPhyt + T-lO, and T-lO had average weights that approached the same fresh and dry
foliar weights as the fumigated treatment (table 7). The plants within the two controls did
differ from one another in fresh root, dry root, and total fresh weights, and root necrosis.
Although plants within the treatments Actinovate, Mycostop, ProPhyt + T-lO, and T-IO
alone approached the same average necrosis as plants within the fumigated control, they
did not differ from the untreated. Actinovate and T-lO having plants with better root

quality than the untreated plants.

58

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In 2006 and 2007, the fumigated treatment had signiﬁcantly smaller P. penetrans
populations than the untreated or Ditera-treated plants (table 8). This difference was also
evident for total parasitic nematodes present. In 2007 a higher average number of
Criconemella spp. were observed in the Ditera treatment when compared to the controls.
Using the risk rating table for parasitic nematodes on strawberries in Michigan (Appendix
C), the field did not exceed a risk rating of two after 2005.

For the root isolations, ProPhyt + Abound had the lowest recovery of Rhizoctonia in
2006. With 2007 being a hotter, drier year, recovery overall was lower compared to
2006. Pythium was recovered at low rates indicating the primary fungus present in the
BR complex was Rhizoctonia. The exact strain of Rhizoctonia was not determined, so
the inﬂuences of temperature and other strain specific characteristics observed by Martin
(2000, 1988) can not be discussed. C ylindrocarpon spp. was also recovered (table 9). It
is not surprising that Rhizoctonia was recovered from plants in the fumigated treatment as
the plots were surrounded by non-fumigated buffers, allowing a fast-growing fungus, like
Rhizoctonia, to quickly re-establish, as has been noted by other authors (Wing et al.,

1994). There may have also been pathogens present on the planting stock.

60

Table 8. Root (1 g) and soil (100 cm3) nematode samples taken from efﬁcacy trial of
different biocontrol and fungicide products applied at planting on individual plant
parameters of strawberry cv. Allstar in a black root rot infested soil in East Lansing, MI,
in 2005, 2006, and 2007.x

 

Pratylenchus penetrans

 

Treatment 2005 20062! 2007
Fumigated - 1.8 a 0.0
Untreated 11.5 19.5 b 19.5
Ditera 27.5 12.8 b 12.8
ANOVA

Effects Signiﬁcance(p)

Treatment 0.3 805 0.0227 0. 1 171
Block 0.6129 0.5519 0.8872

C riconemel/a spp.

 

Treatment 2005 2006 20072
Fumigated - 0.0 0.0 a
Untreated 5.0 18.0 0.5 a
Ditera 8.5 13.8 8.0 b
ANOVA

Effects Signiﬁcance (p)

Treatment 0.59 1 9 0.2708 0.0003
Block 0.4594 0.5279 0.1318

Total Plant Parasitic Nematodes Presenty

 

Treatment 2005 20061 2007
Fumigated - 1.8 a 0.0
Untreated 19.5 39.0 b 22.0
Ditera 43.0 31.5 b 39.5
ANOVA

Effects Df p value Df p value Df p value
Treatment 1 0.2167 2 0.0100 2 0.0615
Block 3 0.4573 3 0.7808 3 0.2672
Residual 3 6 6

Total (Corn) 7 11 1 1

 

w Appendix C has risk ratings for parasitic nematodes on strawberries in Michigan.

" Values in columns followed by differing letters are signiﬁcantly different according to
Fisher’s protected least signiﬁcant difference test p =0.05. Pairwise comparisons
performed with n=4.

y Total nematode averages includes: Pratylenchus penetrans, Trichodorus spp.,
Criconemella spp., Meloidogyne spp., Longidorus elongatus, and Paratylenchus spp.

2 Statistical separations were performed on log(x+l) transformation.

61

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62

With initial differences due to chemical toxicity, either due to unsuitable
application or the products being detrimental to strawberry plants, it became evident that
strawberry plants can compensate for initial damage, if properly grown. Although this
evaluation only covered three years, BRR is a progressive disease, with disease
symptoms taking varying amount of times to become evident depending on location.

Over the duration of the study, it did not seem that BRR was a signiﬁcant problem
at this location; however, the pathogens implicated in this disease complex were present.
While others have had success in inhibiting Rhizoctonia blight on tall fescue using
biocontrols, the chemicals ProPhyt and Abound showed the most promising trends when
compared to the untreated control (Yuen, 1994). The trends observed for ProPhyt and
Abound may be the result of these dips controlling any pathogens still on the crown from
the nursery. In addition, the ProPhyt may have provided some initial nutritional beneﬁts
that assisted in initial establishment. Since this location had low levels of nematodes, and
the strawberries were optimally maintained, BRR was not a severe problem. Thus it is
evident that carefully cultivated strawberries, even in the presence of pathogens, will be
productive, especially if they are established well initially. Developing a threshold for
Rhizoctoniafragariae, like that which exists for P. penetrans (Appendix C), would be

useful when developing a recommendation for a speciﬁc BRR site.

63

LITERATURE CITED

Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. 1990. Basic local
alignment search tool. J. Mol. Biol. 215:403-410.

Anonymous. 1998. United Nations Environmental Programme (UNEP) Methyl Bromide
Technical Options Committee (MBTOC). 1998 Assessment of the Alternatives to
Methyl Bromide. United Nations Environmental Programme, Nairobi: 374 pp.

Barnett, H. L., and Hunter, B. B. 1998. Illustrated Genera of Imperfect Fungi. 4‘h ed.
APS Press. St. Paul, Minnesota.

Barron, G. L. 1968. The Genera of Hyphomycetes from Soil. Williams and Wilkins
Company, Baltimore.

Bird, G. W. 1971. Inﬂuence of incubation solution on the rate of recovery of
Pratylenchus brachyurus from cotton roots. J. of Nematology 32378-3 85.

Chet, I., and Henis, Y. 1983. Trichoderma as a biocontrol agent against soilbome root
pathogens. Pages 110-112 In Ecology and Management of Soilbome Plant Pathogens.
C. A. Parker, A. D. Rovira, K. J. Moore, and P. T. W. Wong, Eds. American
Phytopathological Society. St. Paul. 1 10-1 12.

Chet, 1., Ordentlich, A., Shapira, R., and Oppenheim, A. 1991. Mechanisms of
biocontrol of soil-borne plant pathogens by Rhizobacteria. Pages 229-336 In The
Rhizosphere and Plant Growth. D. L. Keister and P. B. Cregan, Eds. Kluwer Academic
Publishers, Dordrecht, The Netherlands.

D’Ercole, N., Nipoti, P., and Manzali, D. 1989. Research on the root rot complex of
strawberry plants. Acta Horticulturae 265:497-501.

Domsch, K. H., Gams, W., and Anderson, T-H. 1980. Compendium of Soil Fungi.
Academic Press Inc., New York.

Fravel, D. R. and Keinath, A. P. 1991. Biocontrol of soilbome plant pathogens with
fungi. Pages 237-243 In The Rhizosphere and Plant Growth. D. L. Keister and P. B,
Cregan, Eds. Kluwer Academic Publishers, Dordrecht, The Netherlands.

Hancock, J. F., Callow, P. W., Serce, S., and Schilder, A. C. 2001. Relative performance
of strawberry cultivars and native hybrids on fumigated and nonfumigated soil in
Michigan. HortScience 36:136-138.

Hildebrand, A. A. 1934. Recent observations on strawberry root rot in the Niagara
peninsula. Can. J. Research, 1 1218-31.

64

Jenkins, W. R. 1964. A rapid centrifugal—ﬂotation technique for extracting nematodes
from soil. Plant Dis. Reporter 48:692

Louws, F. J ., Driver, J. G., and Leandro, L. 2004. Evaluation of fungicide pre-plant dip
and spray applications to manage Phytophthora crown rot on strawberry plugs.
American Phytopathological Society. F ungicide and Nematicide Tests, 60:SMFO41

Maas, J. L. 1998. Compendium of Strawberry Diseases. 2nd Ed. American
Phytopathological Society. St. Paul, MN.

Martin, S. B. 1988. Identification, isolation frequency, and pathogenicity of anastomosis
groups of binucleate Rhizoctonia spp. from strawberry roots. Phytopathology 78:379-
384.

Martin, F. N. 2000. Rhizoctonia spp. recovered from strawberry roots in central coastal
California. Phytopathology 90:345-353.

Parke, J. L. 1991. Root colonization by indigenous and introduced microorganisms.
Pages 33-42 In The Rhizosphere and Plant Growth. D. L. Keister and P. B. Cregan, Eds.
Kluwer Academic Publishers, Dordrecht, The Netherlands.

Rosskopf, E. N., Chellemi, D. O., Kokalis-Burelle, N., and Church, G. T. 2005.
Alternatives to methyl bromide: A Florida perspective. Plant Management Network 27
Oct.

Sambrook, J ., Fritsch, E. F ., and Maniatis, T. 1989. Molecular Cloning: A Laboratory
Manual, 2"d Edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

Strong, F. C., and Strong, M. C. 1927. Investigations on the black root rot of
strawberries. Phytopathology 21 : 1041-1059.

Watanabe, T., Hashimoto, K., Sato, M. 1977. Pythium species associated with
strawberry roots in Japan, and their role in the strawberry stunt disease. Phytopathology
67:1324-1332.

Whipps, J. M. 2001. Microbial interactions and biocontrol in the rhizosphere. Journal of
Experimental Botany 52:487-51 1.

Wing, K. B., Pritts, M. P., and Wilcox, W. F. 1994. Strawberry black root rot: A Review.
Advances in Strawberry Research. 13:11-19.

Wing, K. B, Pritts, M. P., and Wilcox, W. F. 1995. Biotic, edaphic, and cultural factors
associated with strawberry black root rot in New York. HortScience 30:86-90.

Yuen, G. Y., Craig, M. L., and Giesler, L. J. 1994. Biological control of Rhizoctonia
solani on tall fescue using fungal antagonists. Plant Disease 78:118-123.

65

CHAPTER THREE: GREENHOUSE BIOCONTROL AND REDUCED RISK
FUNGICIDE EFFICACY TRIAL
Introduction

Strawberry black root rot (BRR) is a disease complex that is widespread around
the world wherever strawberries have been planted for multiple years (Watanabe et al. ,
1977; D’Ercole et al., 1989). Infected plants produce smaller leaves, fewer main and
lateral roots, have slower growth, and reduced runner production (Maas, 1998; Hancock
et al., 2001). The disease can be spread via infected nursery stock, movement of infested
soil, or infected plant debris (Maas, 1998; Strong and Strong, 1927; Hildebrand, 1934).
Also known as strawberry decline, many organisms and abiotic factors have been
implicated in the cause of the disease; however, Rhizoctoniafragariae Husain &
McKeen, Pythium spp., and the root lesion nematode Pratylenchus penetrans (Cobb)
Filipjev and Shuurmans Stekhoven are generally considered the primary pathogens
(D’Ercole et al., 1989; Wing etal., 1994; Wing et al., 1995; Maas, 1998).

In Michigan ‘U-Pick’ operations, continual strawberry production or short
rotations are common due to the few suitable locations for these enterprises. Continual
cropping allows pathogen establishment and build-up to deleterious levels. In this
system, fumigation is often employed to help control soil-bome pathogens and weeds
before planting. In 1992, the Montreal Protocol established methyl bromide as an ozone-
depleting substance and instituted its ban by January 1, 2005 (Anonymous, 1998;
Rosskopf et al. , 2005).

The primary problem when trying to develop a management strategy for BRR is

that the disease expression is variable. Rhizoctoniafragariae does not always have to be

66

present to get BRR symptoms (Wing et al., 1994; Wing et al., 1995). A combination of
abiotic and biotic factors may predispose strawberry plants to invasion by perhaps
otherwise saprophytic or weakly parasitic fungi that then cause severe damage to the root
system (Wing et al., 1994; Wing 61 al., 1995). With the primary control measure due to
be eliminated, it becomes necessary to ﬁnd alternative reduce-risk chemicals, biological,
and cultural alternatives. Applying reduced-risk fungicides and biocontrol products at
planting would require the least change in a production scheme that is heavily reliant on
pre-plant fumigation.

The mucilage excreted by the roots supports both beneﬁcial and potentially
harmful organisms seeking to colonize the growing root. The beneﬁcial microbes are
antagonistic to pathogens by competition for food, essential elements, and space (Parke,
1991). These microbes enhance plant growth by suppressing major pathogens, increasing
nutrient availability, decreasing toxicity levels around the plant, or a combination of these
(Parke, 1991). Some of the microbes parasitize pathogenic fungi by producing
chitinases or antibiotics. For instance, Streptomyces hygroscopicus var. geldonus
produces a toxin called geldanomycin which can inhibit R. solani (Chet et al., 1991;
Fravel and Keinath, 1991). Trichoderma is antagonistic through parasitism, and
competition for nutrients for germinating sclerotia of R. solani and S. rolfsii .
Trichoderma harzianum can use the R. solani cell wall as a sole carbon source (Chet and
Henis, 1983). D’Ercole et al. (1989) found a reduction of post-transplant blight incidence
from 24.5% in the control to 11.5% in the treatment with T. harzianum as a liquid dip on

‘Gorella’ strawberries.

67

These experiments were performed to support ﬁeld trials (Chapter 2). The
greenhouse provided a more controlled environment to evaluate the effects on the two
pathogens separately and in a co-inoculation experiment. In addition, experiments were
conducted to establish an inoculation protocol that will consistently cause disease
(Appendix A and B) in evaluating biological and reduced risk fungicide efﬁcacy against
Rhizoctonia spp. and Pythium spp. as other work on strawberry resulted in disease
development, but the amount of inoculum was not as quantitative (Olatinwo and Schilder,
2002)

A product evaluation trial was conducted in 2005 to test fungicides targeting R.
fragariae. Products were initially chosen for their reduced-risk characteristics and
potential efﬁcacy against R. ﬁagariae and Pythium spp. Some of the products (Ridomil
Gold EC, ProPhyt, Abound, Plantshield) were already labeled for use on strawberry.
Several of the products are biocontrol products that offer a unique control mechanism,
but need further evaluation for efﬁcacy (Actinovate, Mycostop, Plantshield, Trichoderma
rossicum, Polyversum, Ditera). From the 2005 trial and ﬁeld experiments (Chapter 2),
the ﬁve most promising products were chosen for further assessment. These were
evaluated in 2006 against R. ﬁagariae and P. ultimum var. ultimum, independently, and
co—inoculated. Efﬁcacy was tested against R. fragariae again in 2007.

All the products tested in this study were fungicides, except for BioNem and
Ditera, which are biological nematicides. Some products, such as Abound, claim to
control root rot caused by Rhizoctonia spp., but have not been tested in Michigan. Some
of the chemicals, like Ridomil Gold EC, are used in strawberry production, but not

necessarily in this application method or timing. Other products are not labeled for

68

strawberries, but claim to control similar diseases on other hosts such as brown patch and
large patch caused by Rhizoctonia spp. on turf (Endorse). There are also many biological
products that make claims of disease control or enhancement of plant growth using

Streptomyces or Trichoderma spp.

Materials and Methods
Plant Material

‘Allstar’ strawberry plants were obtained from Krohne Plant Farms, Inc.
(Hartford, MI). These plants were potted in Baccto High Porosity Professional Planting
Mix (Michigan Peat Company, Houston, TX) soil in 15 x 15 cm pots. Daughter plants
were propagated in August and September 2005 into 10 x 10 cm black plastic pots ﬁlled
with 2 NS-grade sand which was autoclaved in a metal bin at 121°C for at least 16 h
prior to planting. The plants were kept outside on a bench between greenhouses at
Michigan State University. Once established in the sand, they were stored in a coldroom
at 4°C with a light bank set for ll-h days until January 24, when they were brought to the
greenhouse to acclimate. Plants were selected for uniformity and root health when the
experiment was setup.

Due to the possibility of contamination when establishing the daughter plants
outside and with overhead hand watering, a modiﬁcation was made for subsequent
experiments. During July through September 2006, the same mother plants from 2005
were kept under a polyethylene tunnel outside and used to propagate daughter plants into
52 x 64 x 6.4-cm aluminum pans ﬁlled with 2 NS-grade sand. The sand was autoclaved

in the pans for 4 h prior to plant establishment. Drip nozzles were used to prevent splash

69

contamination from the mother plants. Once the daughter plants were established in the
sand, the pans were moved into the greenhouse where they were placed under grow lights
set at l2-h day length.

In July of 2007, daughter plants were established from the same mother plants as
had been previously used into autoclaved, 2 NS-grade sand that had been placed in pans
in the greenhouse. These plants were carefully hand watered to prevent contamination.

In 2006 and 2007, the planting stock and soil were examined for any fungal

presence prior to planting.

Pathogen Evaluation in Planting Stock

In 2006, prior to the single pathogen inoculation experiment, ten 6-mm long, root
pieces were collected from each of four randomly selected daughter plants. The root
pieces were surface disinfested for 2 min in 20% bleach solution, followed by two l-min
rinses in sterile distilled water. The root pieces were dried on sterile paper towels, and
placed aseptically on water agar. No fungal growth was observed. This procedure was
repeated prior to the co-inoculation experiment, using three randomly selected daughter
plants. When fungal grth was observed, fungi were sub-cultured, and identiﬁed based
on morphology. In September 2007, ﬁve daughter plants were assessed using the

procedure previously described.

Inoculum Preparation

All cultures used in the experiments were obtained from Dr. Annemiek

Schilder’s laboratory at Michigan State University, East Lansing, MI. Cultures were

70

maintained on potato dextrose agar amended with ampicillin (50 ug/ml) (PDAamp)
throughout the duration of the experiments. Isolates were from strawberry roots from
samples taken in Michigan.

For the experiment in 2005, R. fragariae (Rhfr0303 8) was grown on PDAamp.
Three to four plugs were then placed on 240 g of oat bran in a 500-ml beaker with 143 m1
of sterile deionized water. Beakers with oat bran were autoclaved for 30 min. After 1 wk
of growth at 25°C the bran was stirred with a sterile rod; after the second week the
colonized bran was air dried in a laminar ﬂow hood for 2 d. The bran was broken into
small pieces (<2.5 cm) prior to air drying to aid in the drying process and to ensure even
inoculation. The bran was then stored covered on a laboratory bench overnight.

The same procedure was utilized in October 2006 except that ﬂasks were used
instead of beakers. The bran was inoculated using two to three 6—mm mycelial plugs
from cultures of either P. ultimum var. ultimum (Pyth03008) or R. ﬁ'agariae (Rhfr0303 8)
after the bran had cooled. The bran was dried in a laminar flow hood for a total of 26.5 h
over a period of 3 d. When the hood was not in operation, the bran was stored, covered,
on a laboratory bench. The bran was stirred and broken up the morning of each day the
bran was in the hood to help it dry thoroughly. Inoculum for the co-inoculation
experiment was started on 8 November 2006. The same isolates and procedure were
used as previously described.

In September 2007, only R. fragariae (Rhfr03038) was grown on bran as
previously described. After one week of growth at 25°C, the bran was stirred with a
sterile rod; after the second week the bran was air dried in a laminar ﬂow hood for 36 hr

over 4 d. The bran was stored covered on a laboratory bench overnight. The bran was

71

mixed the morning of each day to aid in drying. The bran was ground with a mini prep
plus food processor (Cuisinart, Stamford, CT) before drying. A sieve was used to ensure
that the bran was less than 0.6 cm in size. The procedure was as described by Martin

(2000) except that the bran was not passed through nested sieves.

Pathogen Evaluation in Soil Prior to Experiment

For the 2006 efﬁcacy trial, the sandy-sandy loam greenhouse soil mix was
evaluated and found to contain F usarium spp., Penicillium spp., Pericom'a spp., making
autoclaving the soil necessary. After autoclaving for 4 hr, 5 g of soil was taken from a
single pot in each of the four autoclaved batches of soil, and was prepared by mixing the
soil with 125 ml of sterile distilled water in sterile 125-ml ﬂasks for a 1:25 dilution. One
milliliter of this dilution was taken to make a 1:500 dilution. Each dilution was plated
twice onto water agar amended with ampicillin (50 ug/ml), streptomycin (20 ug/ml), and
gentomycin (1 ug/ml) for a total of four plates per batch of autoclaved soil. A sterile bent
glass rod was used to spread the soil dilution over the plate evenly, and the plates were
evaluated 5 d later. No pathogens were recovered. On 30 November 2006, soil dilution
plating was done as before from batches of soil autoclaved for the co-inoculation
experiment. One milliliter of a 1:25 dilution was plated onto a water agar plate for each
of the three autoclaved soil batches. In 2007, using a 1:25 dilution on PDA and water

agar, the autoclaved soil was assessed for fungi as previously described.

72

Inoculation Procedures

On 2 February 2005, 13 x l3-cm diameter standard clay pots were ﬁlled with the
greenhouse mixed sandy loam to mimic ﬁeld conditions such as drainage and conditions
conducive to disease development. The soil was autoclaved within theipots for 4 h. The
following day the contents of each pot were placed into sterile aluminum pans and
incorporated with 0.75% wth inoculum. This mixture was then returned to the pots.
No bran and autoclaved bran without Rhizoctonia served as controls. Each pot contained
approximately 1 kg of oven-dry soil. For treatments to be applied as a drench, it was
determined that 250 ml of each treatment would wet the soil, but not cause excess run off
out of the bottom of the pot.

On 8 November 2006, clay pots were prepared as previously described for the
2005 trial. Pots were prepared for the co-inoculation experiment 21 (1 later. The day
following autoclaving, the pots were individually inoculated with 1.5% wt/wt for the
single pathogen trials (Rhizoctonia and Pythium). Each organism was inoculated at
0.75% wt/wt per pot for the co-inoculation (total—=1 .5% wt/wt). The single fungus control
treatments within the co-inoculation experiment received 0.75% wt/wt of either
Rhizoctonia or Pythium inoculum. The pots were prepared with their respective
pathogens as previously described. Autoclaved bran served as a control. In September
2007, pots were inoculated with 3% WM of non-inoculated bran or bran with R.

fragariae using the same procedure as previously described (table 10).

73

Table] 0. Potted plant experiments conducted, inoculum level used, pathogens tested
against, and number of blocks used in greenhouse trials evaluating efﬁcacy of strawberry
biocontrol and reduced-risk fungicide applied at planting in East Lansing, MI.

 

 

Year of Inoculum Level (v/v) Pathogen Used in Number of Replicates

Experiment Experiment“
2005 0.75% R. fragariae 4
2006 1.5% R. fragariae 8
2006 1.5% P. ultimum var. ultimum 6

. R. fragariae and
2006 0.75% of each organism . . 7

P. ultimum var. ultimum

2007 3.0% R. fragariae 9

 

*Isolates used were Pyth03008 and Rhfr03038.

74

Treatments

In 2005, the day after autoclaving, all pots were inoculated and lightly watered to
rehydrate the soil as it was not watered prior to autoclaving and became too dry to plant
and apply treatments. The treatments were applied at the label rate using the method as
described in table 11. There were four replications per treatment.

The plants in the dip treatments and untreated controls were watered after planting
with 50 ml of water per pot. Pots were arranged in a randomized complete block design.
Each pot was placed in an individual 8-cm deep plastic tray to prevent cross
contamination and to prevent contact with the greenhouse bench. All plants were potted
and treatments applied the same day, with the exception of the C/G treatments, in which
the plants were potted one day after the treatments were applied. The C/G was allowed
to maintain contact with the soil and inoculum for 24 h. The C/G treated pots were then
drenched with 300 ml of water to wash out the C/G, since previous studies had found it to
be phytotoxic (Sabaratnam and Schilder, unpublished data; Chapter 2). Strawberry plants
were then planted in the pots when the water had disappeared from the soil surface. The
T-lO (Trichoderma rossicum) spore suspension was prepared the morning of the
application in sterile deionized water, using sporulating cultures that had been growing
on PDAamp for 23 d. This culture (Trsp02024), originally isolated from strawberries, is
available from Dr. Annemiek Schilder’s laboratory, Michigan State University, East
Lansing, MI. The spore concentration was selected due to information available on
commercial products utilizing Trichoderma spp. Plants were hand watered using a plastic

beaker as needed. Any ﬂowers or runners that formed were removed.

75

Table 11. Treatments, manufacturer rates, active ingredient and application method used
in a greenhouse trial evaluating the efﬁcacy of biocontrol and reduced-risk fungicide
applied at planting to strawberry cv. Allstar for control of black root rot pathogens in East

 

 

Lansing, MI from 2005.
Application
Product Label Rate Active Ingredient Method
Abound 8 ﬂ 02/100 gal Azoxystrobin 5 min dip
Actinovate 1 tsp/ gal Streptomyces lydicus 250 ml drench
BioNem 135 lbs/acre Bacillus ﬁrmus 250 ml drench
00 0.2% v/v Fatty acid 250 ml drench
C/G 0.5% v/v Fatty acid 250 ml drench
Ditera 2.5 lbs/acre Myrothecium verrucaria 250 ml drench
Endorse ll lbs/acre Polyoxin D zinc salt 250 ml drench
Mycostop 2 g/ 100 ft2 Streptomyces griseoviridis 250 ml drench
Plantshield 2.5 lbs/5 gal Trichoderma harzianum 250 ml drench
Polyversum 0.1 g/m2 Pythium oligandrum 250 ml drench
ProPhyt 2 pt/ 100 gal Potassium phosphite 30 min dip
ProPhyt + Abound Rates as above Potassium phosphite + azoxystrobin 15 min dip
ProPhyt + T-IO"‘ Rates as above gotasswm phosphite + 5 min dip
rtchoderma rossrcum

Ridomil Gold EC 1 pt/acre Mefenoxam 250 m1 drench
T-lO“ 1.3 8x I 07 spores/ml Trichoderma rossicum 5 min dip

 

*Dosage determined in laboratory.

76

Table 12. Treatments, manufacturer rates, and application method, used in a greenhouse
trial evaluating biocontrol and reduced-risk fungicide applied at planting in strawberry
cv. Allstar inoculated with black root rot pathogens in East Lansing, MI in 2006 and
2007.

 

Treatment Label Rate Application Method
T-IO" 1.75x107 spores/ml 5 min dip

ProPhyt + Abound 2 pt/100 gal + 8 ﬂ 02/100 gal 15 min dip
Actinovate 1 tsp/ gal Drench

Plantshield 2.5 lbs/5 gal Drench

Ditera 2.5 lbs/acre Drench

 

*Dosage determined in the lab.

77

In the 2006 experiments strawberries were planted and treatments applied the day
following inoculation with Rhizoctonia and Pythium on 1 December. Treatments were
applied, according to the label, either as a 250-ml drench per pot or as pre-plant root dip
(table 12). Plants receiving the fungicide dips were placed into plastic buckets containing
3.79 L of water for the appropriate amount of time. Plants receiving the T-10 dip were
placed in a 500-ml beaker containing the spore suspension.

Untreated control plants and those plants receiving dip treatments received an
additional 50 m1 of water to ensure good establishment. Drenches were applied post-
planting. Plants were grown under 400 W grow lights (P.L. Light Systems, Hortilux
Shreder Group, The Netherlands) set at 12-h day length and were hand watered using a
plastic beaker as needed to prevent splash between treatments. The greenhouse
temperature was kept between 21-27°C. Pots were arranged in a randomized complete
block design replicated eight times for the Rhizoctonia only experiment, six times for the
Pythium only experiment, and seven times for the combined inoculation experiment
(table 1 1). The co-inoculation experiment pots were arranged in a randomized complete
block design with seven replications per treatment. The T-10 suspension of 1.26 x 107
conidia/ml was prepared the morning of application in sterile deionized water when all
treatments, except ProPhyt +Abound, were applied. ProPhyt + Abound was not applied
until 4 wk after other treatments in the co-inoculation experiment, using fresh inoculum
and following the procedures previously described. This delay was due to a mistake
during the initial set—up. Each pot was individually placed on a plastic tray to prevent

contact with the greenhouse bench. Imidacloprid (Marathon) was applied 1 1 December

78

2007 and 8 February 2007 to control fungal gnats. Flowers and runners were removed if
they developed.

On 26 September 2007 the trial was repeated. The T-10 suspension 1.77x107
conidia/ml was prepared the morning of the application. Approximately 14 d after
planting, replicate 3 of the T-10 treatment was replanted and treated due to initial
planting error. Imidacloprid (Marathon) was applied on 17 October. Flower and runners
were removed as they developed. All treatments were applied as previously described
(table 12). Pots were placed in a randomized complete block design upon inverted 8-cm

deep plastic trays. Each treatment was replicated 9 times.

Evaluation Procedure

In 2005, all plants were evaluated 6 weeks after planting. Percent root necrosis
was visually estimated and root quality rated using scale of 1-5 (1= <20% of root mass is
secondary roots, 2= 20-40% of root mass is secondary roots, 3= 40-60% of root mass is
secondary roots, 4= 60-80% secondary roots, 5: >80% of root mass is secondary roots.)
(Appendix A, Figure 1). Fresh weight of the foliage and roots were obtained separately
by splitting the crown horizontally just above the uppermost adventitious roots. Plant
parts were then dried at 80°C for 48 h and weighed. Total fresh and dry weights were
obtained by adding the foliage and root weights together. For fungal isolations, fresh
roots were then taken from all plants and kept in a separate Petri plate containing a piece
of moist Watman ﬁlter paper for each treatment/inoculation combination. The root

pieces were stored at 4°C until processing the next day. Soil was reserved from each pot

79

in each treatment and kept at 4°C for evaluation of fungal contamination using Czapek’s
media.

The 2006 experiments were evaluated 68 d after planting. Plants were washed
under running water and then placed in trays with water to keep roots moist to evaluate
root quality as previously described. Percent root necrosis was also visually estimated.
The biomass measurements were obtained as previously described. One plant of each
treatment was taken for root plating on water agar to assess pathogen recovery rate. Soil
was taken randomly from different pots to enumerate colony forming units (CFUs) of
Rhizoctonia. Roots and soil were stored at 4°C until processing.

In December 2007, 72 d after planting, plants were processed as previously
described. For each treatment, the roots of the eighth replicate were set aside for root
isolations after taking the fresh weight. These were plated the next day. CFUs per unit

soil were not determined in 2007.

Pathogen Recovery

The day following fresh biomass assessment, in 2005, 10 root pieces with lesions
per treatment were selected and plated on water agar (ﬁve 6-mm pieces per plate). Each
piece was taken from a different lesion margin and surface sterilized in a 20% bleach
solution for 2 min, rinsed in sterile water twice for l min, and dried on sterile paper
towels. Hyphae from the root pieces were subcultured onto PDA and identiﬁed based on
morphological characteristics after plate colonization. The frequency of recovery was
calculated by dividing the number of root pieces that yielded Rhizoctonia sp. by the total

number of fungi that grew from all root pieces.

80

The same day, 0.5 g of soil was taken from the soil from each treatment. The soil
was dried for 72 h in a fume hood, diluted in 100 ml sterile water in sterile ﬂasks, and
then spread on Czapek’s media plates with a sterile bent glass rod. This procedure served
to evaluate the extent of any possible fungal contamination.

In 2006, at the time of fresh weight measurements, roots from the last replication
of each treatment in the single pathogen trials were set aside. Roots from the ﬁrst
replication of each treatment in the co-inoculation experiment were set aside, after taking
the fresh weight. Two or three days later, ﬁve 6-mm root pieces with lesions were placed
on each of two water agar plates, for a total of 10 root pieces per plant, using the
technique described previously. The roots in the ProPhyt +Abound treatment were
evaluated in the same manner after the same number of days as the other treatments.
Fungi growing from the root pieces were subcultured onto PDA identiﬁed based on
morphological characteristics (Barnett and Hunter, 1998; Domsch et al. , 1980; Barron,
1968). Frequency of recovery of Rhizoctonia was calculated as previously deﬁned. The
same isolation procedure as previously described was used in 2007 using roots from the

plants in the eighth replicate of each treatment.

Recovery of Rhizoctonia from Soil

For the 2006 greenhouse experiments, 30-g soil samples were taken from random
pots within the inoculated and non-inoculated control treatments within the Rhizoctonia-
only evaluation trial. The non-inoculated control, Rhizoctonia-only control, and the
Rhizoctonia + Pythium control were evaluated from the co-inoculation experiment. The

soil was plated 2 d after experiment evaluation using a pellet soil sampler described by

81

Henis et al. (1978) after spreading the soil in a sterile Petri dish bottom. The soil was
plated onto semi-selective media for Rhizoctonia spp., with three plates per treatment for
a total of 45 pellets (Gutierrez et al. , 1997). Four sets of pellets were taken, with one set
randomly put aside to obtain the weight for calculation of CFUs per gram of soil. Plates
were evaluated once mycelium became evident to the unaided eye. CFUs were
calculated by taking the average weight of pellets set aside for all treatments evaluated,

and then multiplying by the counts for each treatment.

Data Analysis

For each year that entire root systems were sacriﬁced for fungal isolations, the
replicate utilized was removed from the analysis for both dry root weight and total dry
weight data.

All data were analyzed, after performing a variance check, using ANOVA and
means separated by Fisher’s protected least signiﬁcant difference test (p=0.05) in
StatGraphics Plus 4.1 (StatPoint Inc., VA). In the interaction analysis, the following

effects were analyzed: treatment (T), inoculation (1), Block (B), I x T, and error.

Results and Discussion

2005
Based on the inoculation procedure evaluation (Appendix A) the 0.75% bran

inoculum rate was chosen. The soil was autoclaved within the clay pots to ensure that
both the soil and the pots were pathogen free and to allow more control over the process

than was obtained in an earlier trial (Appendix A).

82

In 2006, the fungi found (Epicoccum sp. and Periconia sp.) were isolated from the
roots of the strawberries sampled prior to the setup of the experiment. In 2007 the fungi
found (Alternaria sp., Cladosporium sp., Phoma sp. and yeast), all are considered
saprophytic. When assessing the soil prior to experiment setup in 2006, no growth was
observed on any of the plates 5 d later. In 2007 a Penicillium sp. and a Zygomycete were
found, but these were thought to be contaminants on the plates while they were on the
bench in the laboratory.

Inoculated control weight averages for each variable tended to be lower than the
non-inoculated control averages. Plants treated with ProPhyt + Abound had signiﬁcantly
higher average weights and better root quality than the control plants (table 13). C/G
0.5% and Polyversum may have damaged the plants and allowed Rhizoctonia sp. to cause
more necrosis in inoculated pots, since these two treatments consistently produced the
lowest means in all variables. A combination of ProPhyt and Abound resulted in plants
with greater fresh total weight than the untreated control.

Although Ridomil Gold EC is labeled for control of oomycetes in strawberries,
this product is usually applied to older plants, so the young transplants may have been
adversely affected. In addition, the method used may have resulted in a higher rate than
is recommended on strawberry. Also, Rhizoctonia is a basidiomycete, so control was not
expected. Control was also not expected from the two biocontrol nematicides, BioNem
and Ditera. Plants treated with Ditera showed promising growth, however, it did not

differ from the control for any parameter (table 13).

83

Table13. Effects of different biocontrol and fungicide products on plant biomass
and root health of strawberry cv. Allstar in a greenhouse study in soil with and
without inoculation with Rhizoctoniaﬁagariae in East Lansing, MI, in 2005."

V Values in columns followed by differing letters are signiﬁcantly different
according to Fisher’s protected least significant difference test at p=0.05. Pairwise
comparisons of treatments performed with n=8. For inoculum presence, n=64.

w Scale of 1-5 (1= <20% secondary roots, 2= 20-40% secondary roots, 3: 40-60%
secondary roots, 4: 60—80% secondary roots, 5: >80% secondary roots).

x Statistical analysis performed after log(x+1) transformation.

y Statistical analysis performed after sqrt(x) transformation.

2 Statistical analysis performed after sqrt(x+l) transformation.

84

 

 

 

 

 

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85

Pathogen Recovery

Rhizoctonia was recovered from all treatments applied to inoculated soil, but not
from the non-inoculated control. The lowest frequency was found in C/G 0.2% and
Abound, at 55.6% and 80%, respectively. In addition to the inoculated pots, Rhizoctonia
was also isolated from the non-inoculated Abound, C/G 0.2%, C/G 0.5%, Ditera,
ProPhyt+T-10, and Plantshield treatments, indicating there was some of contamination.
A zygomycete, Penicillium spp., and F usarium spp. were found in all treatments, using
Czapek’s media, except C/G 0.2%, C/G 0.2% inoculated with Rhizoctonia sp., and the
control treatment without bran.

Fungal gnats may explain some contamination that became evident after plating
the root lesions. There was also some doubt as to the success that Rhizoctonia sp. was
kept out of the transplants in 2005. This problem was remedied the following year. The
contamination by a zygomycete, Penicillium spp., and F usarium spp. may be a result of
contaminated bran used in the controls since there was some problem with contamination
in the laboratory. Although the contaminated bran was disposed of, the contamination
may only have become evident after planting due to the moisture from watering.
However, they may have come from outside sources once the plants were in the
greenhouse.

The fungicide combination of ProPhyt + Abound seems to offer a measure of
control. The lower rate of recovery of Rhizoctonia sp. from root lesions in the Abound-
treated inoculated pots indicates that Abound may prevent root infection by Rhizoctonia.
ProPhyt may be providing extra potassium to the plant boosting the plants ability to cope

with disease and possibly by inducing plant defenses. This is made more likely since

86

ProPhyt is labeled for control of Phytophthora, an oomycete. ProPhyt + Abound, T-10,
Actinovate, Ditera, and Plantshield were chosen for further evaluation due to the
tendency of producing higher average weights and higher root quality and performance in
the ﬁeld (Chapter 2). Actinovate was also chosen due to its selection for evaluation in
the Integrated Management Trial (Chapter 4) and low root necrosis.

It was decided that the experiment should be able to produce reproducible results
in approximately two months time. To achieve a deﬁnitive assessment of these products,

it was also necessary to increase the number of replications.

2006

The check for pathogens in the planting stock and the assessment of the
autoclaved soil yielded no pathogenic ﬁangi. The condition of plants (disease free) and
methods (sterile soil) was important for the accurate assessment of pathogen and
treatment effects. The incorporation of inoculum simulated infested ﬁeld conditions with
Rhizoctonia, Pythium, and a co-inoculation of the two, something a grower may face after

continuous strawberries in the perennial systems used in Michigan.

Rhizoctonia

For the Rhizoctonia inoculation, results of the ANOVA showed that dry root
weight differences were not signiﬁcant amongst treatments. As seen in table 14, ProPhyt
+ Abound and T-IO treated plants had the the highest average weights for all response

variables.

87

All treatments applied to inoculated pots yielded 100% recovery of Rhizoctonia.
Rhizoctonia was also isolated from the Ditera, Actinovate, and Plantshield untreated
control treatments, although at lower frequencies (table 15). Pythium spp. were isolated
from the Ditera control, and Cylindrocarpon spp. were isolated from the Actinovate
control. Other fungi include Alternaria spp., Myrothecium spp., and Phoma spp. The soil
evaluation demonstrated that Rhizoctonia was establishing. No fungi were evident from
the non-inoculated controls. The Rhizoctonia-inoculated control had 17.55 CFU/ g of
Rhizoctonia. For the co-inoculation experiment the combined inoculation control had
16.62 CFU/g Rhizoctonia, the Rhizoctonia only control had 16.62 CFU/g Rhizoctonia,
and the untreated bran control had 0.37 CFU/ g Rhizoctonia.

There were problems with fungal gnats which may supply a reason for the

recovery of Rhizoctonia sp. from non-inoculated pots.

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9O

Pythium

For the Pythium inoculation, ANOVA results showed that no treatment had a
signiﬁcant effect on any of the variables, but plants in pots inoculated with Pythium had
signiﬁcantly lower foliage weights and more necrosis (table 16). For fresh foliage weight,
the plants treated with T-l 0 had the highest average fresh foliar weight, followed by
ProPhyt +Abound. Plantshield treated plants tended to have higher average dry root and
total dry plant weight than those treated with ProPhyt + Abound.

Pythium was only recovered from the Ditera control plants and Actinovate
applied to Pythium-inoculated soil. Rhizoctonia sp. was isolated from Ditera, Actinovate,
Plantshield control plants, and Actinovate-treated plants in Pythium inoculated pots.
Trichoderma spp. were recovered from ProPhyt + Abound, Plantshield, and T-l 0
controls, but only from the T—10 applied to inoculated soil were Trichoderma spp.

recovered (table 17).

91

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93

C o-inoculation

In the co-inoculation study, the results of ANOVA showed that treatment did not
signiﬁcantly explain differences for any parameter except fresh root weights and percent
root necrosis. ProPhyt + Abound treated plants had the highest fresh root weight, but did
not differ signiﬁcantly from the control (table 18). Inoculation with the pathogens did
signiﬁcantly reduce fresh and dry foliage, total plant fresh and dry weights, and root
quality.

F usarium spp. were a common contaminant when isolations were conducted after
the experiment. Pythium sp. was not recovered from any treatment. Rhizoctonia sp. was
recovered from every treatment except the ProPhyt + Abound, Ditera, and T-l 0
treatments applied to non-inoculated soil (table 19). Trichoderma sp. was recovered from
ProPhyt + Abound and T-lO treatment controls. Rhizoctonia colony forming units were
obtained only from inoculated and non-inoculated pots that served as controls. Both the
co-inoculation and Rhizoctonia only control had 13.53 CFUs of Rhizoctonia/ g of soil
while the un-inoculated control had 0.9 CFUs/g of soil.

Overall, plants in inoculated pots were smaller with poorer root quality than those
in the non-inoculated pots. The ﬁnding that the non-inoculated control had 0.9 C FUs of
Rhizoctonia/g of soil indicates cross contamination by Rhizoctonia sp. among the
treatments, especially in the co-inoculation experiment, but since all three of the
experiments in 2006 overlapped for several weeks, this potential source of error may

have been present for the other evaluations as well.

94

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96

All three experiments were conducted in the same greenhouse at overlapping
times, so fungal gnats could have contributed to cross contamination amongst all
treatments. Although fungal gnat control was implemented, the gnats were not
preemptively controlled. The possibility of the planting stock harboring pathogens before
planting seems unlikely as the plants were evaluated prior to the start of the experiments
and did not show evidence of infection. A signiﬁcant problem in 2006 was lack of
proper care for the plants, particularly in the co-inoculation experiment. The plants
probably went through a severe wet and dry cycle due to improper watering. However,
this source of error was consistent amongst all treatments, therefore the data were
considered valid.

Due to the condition of the plants, the experiments were evaluated earlier than had
been; if the experiment were conducted longer, signiﬁcant differences may have become
evident. The conditions seem to have inhibited Pythium sp., which prefers cool, moist
soil conditions instead of warm, dry soil. The presence of Rhizoctonia in the co-
inoculation and the establishment of contaminant fungi, such as F usarium spp. in all the
experiments, could have outcompeted Pythium since it is a weak pathogen (Hendrix and
Campbell, 1973). This would explain the low recovery rates, particularly in the co-
inoculation evaluation. The isolation of F usarium spp. and Cylindrocarpon spp., from
the control pots may explain the poor health of treated plants, since both have been
implicated in BRR (Hildebrand, 1934; Hildebrand and West, 1941). The possibility of
infected planting stock still exists, as evidenced by Rhizoctonia sp. found in root lesions

of non-Rhizoctonia inoculated pots, but not in the CFUs of the soil in the treatments in

97

the Rhizoctonia evaluation. However, contamination may have been at such a low level
it could not be detected with the soil isolation procedure.

Since the bran was not ground as recommended by Martin (2000) in the 2005 and
2006 experiments, there may have been a problem with the speed with which the bran
broke down and the release of inhibitory compounds. The inoculum was mixed wt/wt, so
again the treatments were exposed to the same conditions; however, even distribution of
the inoculum may also explain the variability observed. The bran is important to have as
a carrier and initial nutrient source until the pathogen can establish, but perhaps other
carriers, and the quantity used should be evaluated. Despite these concerns, ProPhyt +
Abound showed potential as a reduced risk fungicide alternative to minimize damage
caused by black root rot. T-lO and Plantshield also have potential as more
environmentally benign alternatives to control black root rot as other Trichoderma spp.
have shown (Chet and Henis, 1983).
2007

After the inoculum optimization experiment (Appendix B), the inoculum level
was increased for this experiment. As with all the greenhouse experiments, artiﬁcially
severe infestations were created. In this experiment a distinct separation was observed
between plants in the Rhizoctonia-inoculated pots and those receiving the same quantity
of non-inoculated bran, which served as control checks for each treatment. Pots with
Rhizoctonia actually had higher average weights and better root quality. This split may
be a result of Rhizoctonia actively breaking down the bran, while the bran in the un-

inoculated pots hindered plant growth. The lack of mechanical or nematode damage

98

made this greenhouse experiment, like the others, possibly less severe than if damage had
been present.

Although not signiﬁcantly different from the inoculated control plants,
Plantshield-treated plants had the highest average weights in the presence of Rhizoctonia
for all parameters except fresh and dry root weights (table 20). ProPhyt + Abound-
treated plants had the lowest percent root necrosis.

Alternaria, F usarium, and a zygomycete were found to be colonizing the surface
of the soil within a few days after planting. These were probably establishing from
surrounding greenhouses. Rhizoctonia was isolated from every inoculated treatment, but
only from the Plantshield control, indicating much of the cross contamination issues had
been resolved. Trichoderma was isolated from the T-10 and Plantshield controls only
(table 21).

Although this inoculation technique worked for Martin (2000), there were several
differences from their original protocol. Instead of using ‘Selva’, the variety ‘Allstar’
was used. Also, Martin conducted his experiments in a growth chamber, which is a much
more controlled environment than the greenhouse. Their nested sieving of the bran is
also a consideration. Their soil was also sieved field soil that was pasteurized at 82°C for
2 h. Although the soil used in these experiments was autoclaved for considerably longer,
table 22 provides evidence that the autoclaved soil does not hinder the plants. It was also
apparent, during 2005, that non-inoculated bran (0.75% wt/wt) did not signiﬁcantly
reduce plant weights compared to inoculated bran. In 2007, the non-inoculated bran
reduced plant weights and root quality compared to the inoculated bran, probably because

Rhizoctonia was breaking down the bran in inoculated pots.

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101

Table 22. Effects of oat bran as an inoculum carrier on plant weights and root quality in
a greenhouse study using cv. Allstar in a greenhouse study using autoclaved soil
inoculated with and without Rhizoctoniafragariae in East Lansing, MI in 2005 and
2007.y

 

 

 

 

 

 

 

Total Total

Fresh Fresh Dry fresh dry Root

f0|iage root Dry root plant plant quality

weight weight foliage weight weight weight rating
Bran g) (g)2 weight (g) (g) (g)‘ (9’ (1-5)‘

2005 (0.75% wat)
Inoculated 3.25 NS 1.48 a 0.93 NS 0.40 a 4.73 a 1.33 NS 2.50 a
Non-inoculated 4.18 2.88 b 1.40 0.53 a 7.05 a 1.93 2.00 a
None 7.13 9.70 c 2.18 1.68 b 16.83 b 3.85 4.50 b
ANOVA
Effects Df Signiﬁcance (p)
Inoculation 2 0.0813 0.0016 0.1 103 0.0165 0.0187 0.0798 0.0020
Block 3 0.3556 0.0320 0.6019 0.2514 0.1423 0.5131 0.0701
Residual 6
Total (Corr.)
1 1
2007 (3.0% wt/wt)

Inoculated 8.36 b 4.53 a 2.68 b 1.14 a 12.89 b 3.70 b 3.44 b
Non-inoculated 3.75 a 3.29 a 1.25 a 0.84 a 7.05 a 2.00 a 1.78 3
None 12.94 c 9.57 b 4.06 c 2.43 b 22.51 c 6.22 c 5.00 c
ANOVA
Effects Df Signiﬁcance (p)
Inoculation 2 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000
Block 8 0.0811 0.0129 0.1973 0.1956 0.0154 0.0594 0.0689
Residual 16
Total (Corn)

26

" Scale of 1-5 (1= <20% secondary roots, 2: 20-40% secondary roots, 3= 40-60%
secondary roots, 4= 60-80% secondary roots, 5= >80% secondary roots).

y Values in columns followed by differing letters are signiﬁcantly different according to
Fisher’s protected least signiﬁcant difference test at p=0.05. Pairwise comparisons
performed on n=4 for 2006. Pairwise comparisons performed on n=9 for 2007.

2 Statistical analysis was performed after log(x) transformation.

102

Despite the continual modiﬁcation of the greenhouse trials, it is clear that ProPhyt
+ Abound offers a likely alternative to fumigation in an integrated management scheme
for BRR. The success of this product combination in the ﬁeld is probably due to the
removal of any pathogens that may be on the roots from the nursery, despite their best
efforts to prevent this from occurring. In the greenhouse it is harder to determine why
this speciﬁc treatment results in larger, healthier plants since the planting stock was
checked before each trial. However, the Abound may provide a layer of protection that
allows these plants to get a ‘jump start’ over their counterparts receiving other treatments,
especially with a little extra nutrition from the ProPhyt. Plantshield, T—l 0, and Ditera
have shown continued promise as well. As Harman (2000) discusses, T. harzianum T-22
does not always cause visual improvement of plants, but it can improve root development
of omamentals and protect tomato against Fusarium crown and root rot in the
greenhouse. He further discusses that control may be possible with one application at the
beginning of the growing season; however, T-22 can be overwhelmed by high disease
pressure and must be used as a preventative. In the BRR study conducted, disease
pressure was artiﬁcially high, this may explain why Plantshield and T-10 did not perform
consistently better than control plants.

All the products need further evaluation, and a quantitative, consistent system to
evaluate these products in the greenhouse remains to be determined. It would be
interesting to examine the efﬁcacy of integrating biologicals with fungicdes as Elmer and
McGovern (2004) studied in cyclamen and Harman (2000) suggests for T. harzianum T-

22, particularly in conjunction with fumigation.

103

LITERATURE CITED

Anonymous. 1998. United Nations Environmental Programme (UNEP) Methyl Bromide
Technical Options Committee (MBTOC). 1998 Assessment of the Alternatives to
Methyl Bromide. United Nations Environmental Programme, Nairobi: 374 pp.

Barnett, H. L., and Hunter, B. B. 1998. Illustrated Genera of Imperfect Fungi. 4‘h ed.
APS Press. St. Paul, Minnesota.

Barron, G. L. 1968. The Genera of Hyphomycetes from Soil. Williams and Wilkins
Company, Baltimore.

Chet, I., and Henis, Y. 1983. Trichoderma as a biocontrol agent against soilbome root
pathogens. Pages 110-112 In Ecology and Management of Soilbome Plant Pathogens.
C. A. Parker, A. D. Rovira, K. J. Moore, and P. T. W. Wong, Eds. American
Phytopathological Society. St. Paul.

Chet, I., Ordentlich, A., Shapira, R., and Oppenheim, A. 1991. Mechanisms of
biocontrol of soil-borne plant pathogens by Rhizobacteria. Pages 229-336 In The
Rhizosphere and Plant Growth. D. L. Keister and P. B. Cregan, Eds. Kluwer Academic
Publishers, Dordrecht, The Netherlands.

D’Ercole, N., Nipoti, P. and Manzali, D. 1989. Research on the root rot complex of
strawberry plants. Acta Horticulturae 265:497-501.

Domsch, K. H., Gams W., and Anderson, T-H. 1980. Compendium of Soil Fungi.
Academic Press Inc., New York.

Elmer, W. H., and McGovern R. J. 2004. Efﬁcacy of integrating biologicals with

fungicides for the suppression of Fusarium wilt of cyclamen. Crop Protection 23:909-
914.

Fravel, D. R., and Keinath, A. P. 1991. Biocontrol of soilbome plant pathogens with

fungi. Pages 237-243 In The Rhizosphere and Plant Growth. D. L. Keister and P. B.
Cregan, Eds. Kluwer Academic Publishers, Dordrecht, The Netherlands.

Gutierrez, W. A., Shew, H. D., and Melton, T. A. 1997. Sources of inoculum and
management for Rhizoctonia solani damping-off on tobacco transplants under
greenhouse conditions. Plant Dis. 81 :604-606.

Hancock, J. F., Callow, P. W., Serce, S., and Schilder, A. C. 2001. Relative performance
of strawberry cultivars and native hybrids on fumigated and nonfumigated soil in
Michigan. HortScience 36:136-138.

Harman, G. E. 2000. Myths and dogmas of biocontrol: changes in perceptions derived
from research on Trichoderma harzianum T-22. Plant Disease 84:377-392.

104

Hendrix Jr., F. F., and Campbell, W. A. 1973. Pythiums as plant pathogens. Ann. Rev.
Phytopathol. 11: 77-98.

Henis, Y., Ghaffar, A., Baker, R., Gillespie, S. L. 1978. A new pellet soil—sampler and
its use for the study of population dynamics of Rhizoctonia solani in soil.
Phytopathology 68: 371 -376.

Hildebrand, A. A. 1934. Recent observations on strawberry root rot in the Niagara
peninsula. Can. J. Research, Sec. C. 11:18-31.

Hildebrand, A. A., and West, P. M. 1941. Strawberry root rot in relation to
microbiologiclal changes induced in root rot soil by the incorporation of certain cover
crops. Can. J. Research Sec. C 19:183-198.

Maas, J. L. 1998. Compendium of Strawberry Diseases. 2nd Ed. American
Phytopathological Society.

Martin, F. N. 2000. Rhizoctonia spp. recovered from strawberry roots in central coastal
California. Phytopathology 902345-353.

Olatinwo, R. 0., and Schilder, A. M. C. 2002. Transplant root clips with biocontrol
agents reduce strawberry black rot. Phytopathology 92:861.

Parke, J. L. 1991. Root colonization by indigenous and introduced microorganisms.
Pages 33-42 In The Rhizosphere and Plant Growth. D. L. Keister and P. B. Cregan, Eds.
Kluwer Academic Publishers, Dordrecht, The Netherlands.

Rosskopf, E. N., Chellemi, D. 0., Kokalis-Burelle, N., and Church, G. T. 2005.
Alternatives to methyl bromide: A Florida perspective. Plant Management Network. 27
Oct.

Strong, F. C., and Strong, M. C. 1927. Investigations on the black root of strawberries.
Phytopathology 21 : 1041 -1059.

Watanabe, T., Hashimoto, K., Sato, M. 1977. Pythium species associated with

strawberry roots in Japan, and their role in the strawberry stunt disease. Phytopathology
67:1324-1332.

Wing, K. B., Pritts, M. P., and Wilcox, W. F. 1994. Strawberry BRR: A Review.
Advances in Strawberry Research. 13:11-19.

Wing, K. B, Pritts, M. P., and Wilcox, W. F. 1995. Biotic, edaphic, and cultural factors
associated with strawberry BRR in New York. HortScience 30:86-90.

105

CHAPTER FOUR: EVALUATION OF AN INTEGRATED APPROACH FOR
THE MANAGEMENT OF BLACK ROOT ROT

Introduction

Strawberry BRR is a disease complex that is widespread in the United States and
around the globe from Japan to Europe, wherever strawberries have been planted for
multiple years (Watanabe et al., 1977; D’Ercole et al., 1989). Characterized by 0.5-5 cm
reddish brown lesions on the feeder and structural roots of the strawberry plant,
eventually all root tissue becomes involved, as the stele, vascular cylinder, and cortical
tissue become infected (Maas, 1998). As feeder roots disintegrate at the point of
infection, shoots become stunted and wilt at the onset of dry weather. Roots darken with
age resulting in the death of the root. The roots may become completely black in severe
cases, with a corky texture. Infected younger plants will produce smaller leaves, fewer
main and lateral roots, have slower growth, and reduced runner production (Maas, 1998).

The disease can be spread through infected nursery stock, movement of infested
soil, or infected plant debris (Maas, 1998; Strong and Strong, 1927; Hildebrand, 1934).
Also known as strawberry decline, many organisms and abiotic factors have been
implicated in the cause of the disease. Wing et al. (1995) discussed that no single factor
explained a substantial part of the observed variation in root health, and that BRR may be
caused by different factors in different ﬁelds or that several interacting factors are
necessary. Poor root health was associated with soil compaction and high soil clay and
silt content. The implication is that speciﬁc pathogens are not known to consistently
cause the disease; however, Rhizoctoniaﬁagariae Husain & McKeen, Pythium spp., and

the nematode Pratylenchus penetrans (Cobb) Filipj ev and Shuurmans Stekhoven are

106

generally accepted as the primary pathogens (D’Ercole et al., 1989; Wing et al. , 1995;
Maas, 1998).

Currently, control of black root rot consists of using crop rotation, cover crops,
good aeration and drainage, and fumigation (Maas, 1998; Martin and Hancock, 1983;
Perry and Ramsdell, 1994). Recommendations for alternative control measures include
planting resistant varieties, crop rotations, and incorporating organic matter. Prevention
is key; planting disease-free stock in fertile, well-drained sandy loam is the best way to
avoid this disease (Perry and Ramsdell, 1994). Also, good cultural practices to prevent
drought stress and winter injury, and a 3 to 5-year rotation between strawberry plantings
helps prevent disease (Perry and Ramsdell, 1994).

The strawberry cultivar ‘Cavendish’ has been found to perform well on naturally
BRR infested soil (LaMondia, 2004; Particka and Hancock, 2005). In Michigan ‘U-Pick’
operations, continual strawberry production is common due to the few suitable locations
for these enterprises and other economic pressures. Continual cropping allows pathogen
establishment and inoculum build-up to deleterious levels. Hildebrand and West (1941)
found that strawberries grown after a soybean series in greenhouse experiments
approached the same level of disease as sterilized soil. ‘Saia’ oats (A vena strigosa) has
had some success in controlling P. penetrans and R. fragariae in greenhouse pots
(Townshend, 1989). Elmer and LaMondia (1999) found that an application of
ammonium sulfate with ‘Saia’ oats or sorgho-sudangrass reduced P. penetrans
populations in strawberry roots, and that only a rotation with ‘Garry’ oats and ammonium
sulfate reduced root colonization by R. fragariae. Brassica species such as oilseed

radish, mustard, and canola have also received attention due to the formation of

107

isothiocyanates that have fungicidal and broad spectrum nematicidal properties (Ettlinger
and Kjaer, 1968; Snapp and Mutch, 2003). Based on ﬁndings in New York the use of
sweet corn, rye, and mustard offer economic return, addition of organic matter,
respectively, and a natural fumigation that can be easily incorporated into strawberry
production operations.

Compost, already widely used in the nursery industry, offers another means of
control. Hoitink and F ahy (1986) discuss several diseases controlled by organic matter
such as the incorporation of ammoniated Douglas ﬁr bark into soil which provided
control for red stele in strawberry during the ﬁrst two years of the planting and the use of
composted hardwood bark to suppress several species of nematodes including P.
penetrans. Using compost contained in a mesh tube to create a raised bed, Millner
(2006) found that ‘Allstar’ and ‘Chandler’ strawberries grown in 100% compost had 16-
32 times higher yield than from non-compost rows.

Considering the complexity of BRR, a multi-faceted approach incorporating
chemical, host resistance, and cultural management to determine the best control strategy
of BRR was taken to enable a recommendation for growers. This included a drench of
Actinovate (Streptomyces lydicus), a pre-plant fungicide dip of ProPhyt (potassium
phosphite) + Abound (azoxystrobin), and compost. The susceptible variety ‘Allstar’ and
the tolerant variety ‘Cavendish’ were planted within these treatments. These sub-
treatments were each evaluated within a crop rotation, fumigation, and a continuous

strawberry main treatment.

108

Materials and Methods

In September 2004 at the Michigan State University Horticulture Farm, an
experiment was established on a sandy loam soil with a four year history of strawberry
production and BRR. In May 2005, 40.2 x 3.7 m strips of rye or fallow ground were
worked up to a depth of 15 cm starting with the fallow ground and ﬁnishing on the areas
planted with rye. Devrinol 50 DF (napropamide) was applied 10 (1 prior to planting at
8.96 kg/ha. Strawberry transplants (Krohne Plant Farms, Hartford, M1) were planted in
areas that were previously fallow. Plants were irrigated with 2.5 cm of water following
planting in 17 June 2005. Ten days later, sweet corn seed (Zea mays var. rugosa
‘J ackpot’) (Roger Seed, Boise, Idaho) treated with Captan, Thiram, and carboxin
(Vitavax) was planted in areas previously planted to rye in rows 76.2 cm apart using a
Mini Nibex (Markaryd, Sweden) so that the seeds were planted 10 cm apart. The
rye/corn/mustard rotation plot measured 10.1 x 3.7 m and was replicated four times. In
June 2005, 31.9 kg/ha of nitrogen was broadcast over the entire planting. In August, 57.7
kg/ha of nitrogen was applied to the entire planting. The com was harvested and stalks
removed in September 2005, with the stubble left to be incorporated. After incorporating
the corn stubble, brown mustard [Brassicajuncea (L.)] was planted 18 d after the corn
harvest by hand broadcasting at 30.8 kg/ha. Lime and 19-19-19 fertilizer were applied at
the rate of 36.8 kg /ha as well. Straw was placed over the strawberries in November.

On 9 April 2006, the ﬁeld was rototilled starting with the rye/corn/mustard
rotation plots and then the continuous strawberry plots. Using a randomized complete
block design, 10.2 x 3.7-m plots were chosen to receive fumigation with Telone C-35

(1,3-dichloropropene + chloropicrin) at the rate of 39.2 kg/ha knifed in with shanks

109

placed at 20.3 cm spacing three days after being cultivated. Prior to planting on 25 May
2006, the area to be planted was fertilized with 19-19-19 at the rate of 44.6 kg/ha and
then cultivated in the order of fumi gated, rotation, and continuous in preparation for
planting 2 (I later. Using a randomized complete block design, the large plots were split
into subplots which were further divided by variety. Each subplot consisted of two rows
of ten strawberry plants, planted 45.7 cm apart in rows 83.8 cm apart. The plants within
the subplots then received either: I) a 15-min root dip of Prophyt (potassium phosphite)
at the label rate of 0.95 L/3.79 L and Abound (azoxystrobin) at the label rate of 236.6
ml/3.79 L, 2) 473 m1 drench per transplant hole of Actinovate (Streptomyces Iydicus) at
the label rate of 1.10 g/3.79 L, 3) planting in compost contained within a mesh
polyethylene tube, or 4) no treatment. Each sub-plot was further divided into two
varieties, ‘Cavendish,’ a tolerant variety, and ‘Allstar’ a susceptible control (table 23).
There were a total of ﬁve plants in each of the two rows of each variety within each sub-
sub-plot. There was 66.0 cm between varieties and 81.3 cm between subtreatments.
Daughter plants were trained to form 50 x 270 cm beds. There were 4 blocks for each
treatment.

Planting was followed by 1.27 cm of irrigation. The compost used was mature
leaf-yard trimmings (MulchPIus, LaPorte, IN) blown into 20-cm diameter polyethylene
netting tubes (Filtrexx, Grafton, OH) using a long ﬂexible hose the length of the ﬁeld. A
drip irrigation system, 15 mil T-tape (Barry Hill Irrigation, Buffalo Junction, VA), with
emitters spaced 20.3 cm apart and an emitter ﬂow rate of 25.4 L/min -305 linear m (6.7
gal/min-IOOO linear ft.) of row, was placed under the netting on the surface of the

compost. The socks were planted 15 (I later after thoroughly soaking the compost.

110

Table 23. Main treatments, sub-treatments, and strawberry varieties used in the
integrated alternatives experiment conducted in a black root rot infested ﬁeld in East
Lansing, MI during 2006-2007.

 

 

Main plots Sub-plots Sub-sub-plots
Compost Cavendish or Allstar
Actinovate Cavendish or Allstar

Continuous strawberry
Untreated Cavendish or Allstar
ProPhyt+Abound Cavendish or Allstar
Compost Cavendish or Allstar
Actinovate Cavendish or Allstar

Fumigated continuous strawberry
Untreated Cavendish or Allstar
ProPhyt+Abound Cavendish or Allstar
Compost Cavendish or Allstar
Actinovate Cavendish or Allstar

Rye/Sweet Corn/ Mustard

Prior to strawberry Untreated Cavendish or Allstar
ProPhyt+Abound Cavendish or Allstar

 

111

During June and July 2006 ﬂowers were removed. During the summer, runners
were raked into the row centers to form matted-row beds. All weed control was done by
hand and irrigation was applied as needed. On 8 August 2006, mother crown counts, bed
ﬁll, and plant vigor ratings were collected from all treatments except the compost socks.
Crown counts were obtained by using a frame measuring 38.1 cm x 61 cm which was
centered over the row. Ratings were obtained visually using table 24. Fifteen days later,
to allow for the same length of period from planting, the data were collected from the
compost socks. On 23 and 25 August, total crown counts were collected from all
treatments except the compost socks, which were again collected 15 (I later. Total crown
number includes original parent crowns. In September 2006, lime and 19-19-19 fertilizer
were applied at the rates of 271.5 kg/ha and 254.5 kg/ha respectively. Soil was collected
for nematode analysis in May and September from each untreated strawberry sub-plot
within each main treatment, regardless of cultivar by digging a plant and 200 g of the
surrounding soil. Three ‘Allstar’ plants were dug within each sub-treatment within the
ﬁrst replication for fungal assays in November before the straw was put down. These
were sent to the Milner laboratory (USDA-ARS-BARC-Sustainable Agricultural Systems
and Food Safety Labs, 10300 Baltimore Ave., Bldg. 001, Rm 140, Beltsville, MD USA
20705-2350) for a root necrosis rating and fungal assays. In addition, root lesions were
also assessed on a scale from 1-4 on roots 2-3 mm in diameter where l=0-25°/o of 2-3
mm roots w/lesions, no feeder roots black/brown; 2=25-49% of 2-3 mm roots w/lesions,
a few feeder roots black/brown; 3=50-74% of roots with lesions, several clusters of
feeder roots black/brown;4= >75% of roots with lesions or most feeder roots

black/brown.

3" ‘“ Table 24. Bed ﬁll and plant vigor ratings used in the integrated management practices
experiment conducted in a black root rot infested ﬁeld in East Lansing, MI during 2006-
2007 using strawberry cvs. Allstar and Cavendish.

Plant Vigor Characteristics

 

 

Li..- 5 Plants in excellent health, vigorous growth, superior runnering
Mi 4 33% plants diseased or stunted
his 3 50% plants diseased or stunted
3553: 2 Majority of plants diseased or stunted
rt: 1 Plants very stunted, diseased
1 Bed Fill Characteristics
5 Runners healthy and establishing well. full bed
i :- 4 Fewer runner plants than in 5
ii i: 3 Thinning evident, runner size and establishment good
i: 2 Few runners, mother plants obvious
up: 1 Few to no runners establishing, mother plants dead

 

like

11: if

113

On 4 April 2007, straw was removed. During June 2007 there were three harvests
every 7 d obtained from the center meter of each sub-subplot. Berries were also counted
at the time of harvest. Iron phosphate (Slug Magic) was applied (4.8 g/mz) to prevent
slug damage. It was noted that the compost socks seemed to speed up ripening, possibly
due to the black fabric retaining heat. As no fungicides were sprayed to control fruit rots,
berries were picked regardless of disease or damage. At the third harvest all remaining
berries were picked regardless of size, maturity, or condition.

The erect petioles and leaves, along with total and parent crown counts were taken
using frames measuring 38.1 cm x 61 cm. These frames were centered over the middle
of the row. Erect petioles and leaves were clipped at the crown and the fresh weight
obtained on 31 July 2007. They were subsequently dried for 48 h at 80°C to obtain dry
weights. On 27 July, 3 ‘Cavendish’ plants from all subplots, and 3 ‘Allstar’ plants from
only the untreated strawberry subplots were dug from replications 2 and 4 for fungal
assays from the rotation, fumigation, and continuous strawberry main treatments. Before
the fungal assays, the root necrosis was evaluated on a scale, provided by the Milner
laboratory, of 1-5 where I = mostly black, dark brown, no ﬁnely branched roots, and a
single crown; 2 = same as 1, except one or two ﬁnely branched roots present; 3 = half of
all roots black/dark brown and unbranched, and l or 2 crown branches; 4 = more white,
ﬁne, branched roots present than black/brown unbranched roots, and two crown
branches; and 5 = white, ﬁne, and multi-branched roots and crown.

One plant, with surrounding soil, was removed from each variety within the
untreated subplot of each block of each main treatment. Nematode presence was

determined using the modiﬁed Jenkins technique and a modiﬁed root extraction process

114

(Jenkins, 1964; Bird, 1971). The counts of nematodes in the soil were compiled across
the varieties to be similar to the process used in 2006, as no roots were collected during
that year and soil was collected from each untreated sub-plot without regard to variety.
The focus was on the main treatments as none of the sub-plots were expected to affect
nematode populations within the ﬁeld soil.

Fungal assays were conducted in the Milner laboratory at the USDA facilities in
Maryland. For fungal assays, when possible, adventitious roots with obvious lesions with
distinct diseased-to-healthy transition zones were used with a note made if diseased roots
were unavailable. Healthy roots were not plated. Roots were prepared for isolation by
removing adhering soil by rinsing roots in cold tap water, surface-disinfesting for 2 min
in 0.5% NaOCl, and ﬁnally rinsing three times in sterile deionized water. After blotting
them dry, 8 segments (approximately 5 to 10 mm in length) were collected from a
composite of the roots of all the plants. The eight root segments are plated on water agar
containing 100 ppm streptomycin and 30 ppm vancomycin or 50 ppm penicillin. Plates
were incubated at 18-22°C in the dark for 2 wks and checked daily for fungal growth.
Isolated fungi were subcultured on 0.50-strength PDA and incubated at 18-22°C and
identiﬁed to genus using classic taxonomic procedures and growth characteristics on

media.

Field Inoculum Level Assessment

The effects of the main plots on colony forming units (CFUs) of Rhizoctonia in

the soil was of interest to determine if there was a reduction in propagule quantity.

115

Besides looking at infections on plants, the desire to know the disease level in the ﬁeld

spurred the assessment of inoculum level.

Soil Collection

Soil was collected on 10 June 2006, 19 July 2006, 17 August 2006, and 20
September 2006 using a soil probe to gather a composite sample across varieties at a
depth of 15.2 to 20.3 cm from all untreated subplots. Twelve composite samples were
taken back to the lab, one for each of the 12 plots, and 30 g was weighed out in the
bottom of a sterile petri dish and spread evenly over the bottom. Soil was taken from the
entire area, regardless of the variety planted. Soil was stored at 4°C until processing

within 1 to 2 d.

Soil Inoculum Evaluation

Soil (30 g) was spread on a sterile Petri dish bottom and a pellet soil-sampler, as
described by Henis er al. 1978, was used to plate 15 soil cores onto two semi-selective
media plates, for the ﬁrst soil sample (30 cores/sample), and then three plates for all
subsequent samples (45 cores/sample) (Gutierrez et al., 2001; Henis et al, 1978). The
pellet soil-sampler was rinsed in distilled water, followed by alcohol, and then ﬂamed
between each sample. It was kept at a constant depth and pressed ﬁrmly into the soil
with the bottom of the petri dish used to level off the pellet tubes. Plant debris and
pebbles were avoided. For each sample an additional set of soil cores were randomly set
aside to obtain weight for calculation of CFUs. All the additional sets from each

sampling time were dried for 24 h at 105°C. Colony assessments were made after 2 to 3

116

d of growth. Rhizoctonia inoculum levels were determined by morphological
identiﬁcation upon plate colonization. The procedure was repeated in 2007 with samples
taken the following dates: 11 May, 9 June, 9 July, 10 August, and 10 September.
Data Analysis

All statistical analyses was performed using the ANOVA and mean separation
(Fisher’s protected least signiﬁcant difference test p=0.05) functions of the StatGraphics
Plus 4.1 (StatPoint Inc., VA) statistical computer program after checking for equal
variance. In the interaction analysis, model variance components were estimated due to
main treatment (M), sub-treatment (S), variety (V), Block (B), M x S, M x V, S x V, M x
S x V, and error. All data obtained in the ﬁeld were collected from both rows, and then
calculated for one square meter centered over one row. All yield data were calculated for
the center meter of one row. When analyzing the data over multiple years, it was
necessary to utilize SAS 9.1 (SAS Institute Inc., Cary, NC.) using repeated measurement
ANOVA in proc glm.

For the inoculum level assessment, analysis was conducted with SAS 9.1 (SAS
Institute Inc., Cary, NC.) using proc glm. 1n the interaction analysis, model variance
components were estimated due to main treatment (M), time (T), Block (B). M x T, M x

B, T x B, and error.

Results and Discussion
Year and the year x main treatment interaction were signiﬁcant in the repeated

measurements ANOVA of the ﬁeld data (p<0.0001 and p=0.0003, respectively). The

year x block interaction was also signiﬁcant (p=0.0305) while year x sub-treatment was

117

not signiﬁcant (p=0.0527). Although block was signiﬁcant for the plant vigor rating
(p=0.0113) and total crown number (p=0.0198) over the two years, there was not a block
x main treatment interaction for total crown number. The plant vigor rating had a
signiﬁcant block x main treatment interaction (p=<0.0001). However, with a signiﬁcant
year x main treatment interaction, each year could only be examined separately. The
block x subtreatment was not signiﬁcant for any parameter.

The parameters measured in the current study were shown to differ between
plants grown on fumigated and non-ﬁrmigated ground (Hancock et al., 2001). In 2006,
the plants in the fumigated main treatment all had signiﬁcantly greater plant vigor, bed
ﬁll, and more total crowns than the plants in either the rotation or continuous main
treatments. The plants within the fumigated main treatment continued to have
signiﬁcantly higher bed ﬁll and plant vigor ratings in 2007. The plants in the sub-plots of
ProPhyt + Abound and compost tended to have higher vigor, but the plants in the
compost socks had the poorest bed ﬁll because the runners could not establish directly
into the socks; instead the runners established alongside the socks into the infested
ground. While ProPhyt + Abound tended to have the best ﬁll, the plants receiving this
dip were not signiﬁcantly different from the control (table 25).

None of the plants in any of the sub-treatments differed signiﬁcantly from the
plants within the untreated sub-plot in 2007 for bed ﬁll or plant vigor. The fumigated
main treatment plants continued to be superior to those plants within the other main
treatments in regards to total crown number (table 25). Although not statistically
different from the untreated sub-plot, the plants treated with ProPhyt + Abound tended to

have more total crowns. Differences between the varieties became evident when

118

examining the fruit data with Cavendish being more productive and having larger fruit
than Allstar. Individual berry size was greatest from plants in the compost socks. There
was no difference in berry size from plants in the untreated continuous or fumigated
continuous main treatments (table 25). Plants in the fumigated main treatment had the
highest total berries and greatest yield. Although not statistically different from the
control, plants treated with ProPhyt + Abound numerically had the greatest number of
berries and higher yields. The signiﬁcance observed in blocks was probably due to
differences in soil fertility and microbial populations caused by previous studies. The
plants placed in the compost socks and ProPhyt + Abound tended to have greater fresh
biomass than the control. There was some interaction between the varieties and the main
or subplots, but there was not a consistent interaction. There was no interaction between

main and subtreatments.

119

Table 25. Effects of cultural and chemical treatments applied at planting to strawberry cv.
Allstar and Cavendish on plant vigor, bed ﬁll, biomass, and yield in a black root rot
infested soil in East Lansing, MI, in 2006 and 2007.u

" Values in columns followed by different letters are signiﬁcantly different according to
Fisher’s protected least signiﬁcant difference test at p=0.05. Pairwise comparisons
performed for main treatments, n=32, sub-treatments, n=24, variety, n=48, and block,
n=24.

V Did not pass variance check.

w Scale 1 to 5 with 5 = Runners healthy and establishing well, full bed, 4 = Fewer runner
plants than in 5, 3 = Thinning evident, runner size and establishment good,

2 = Few runners, mother plants obvious, and 1 = Few to no runners establishing,
mother plants.

" Scale 1 to 5 with 5 = Plants in excellent health, vigorous growth, superior runnering,

4 = 33% plants diseased or stunted, 3 = 50% plants diseased or stunted, 2 = Majority
of plants diseased or stunted, and l = Plants very stunted, diseased.

y Statistical analysis performed after sqrt(x) transformation.

2 Statistical analysis performed after log(x) transformation.

120

 

 

 

 

 

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121

Table 26. Root lesion and general plant health ratings of strawberry cv. Allstar and
Cavendish grown under in different cultural and chemical treatments in a black root rot
infested soil in East Lansing, MI, in 2006 and 2007.

 

 

Main 2006 Root

Treatment Sub-Treatment Cultivar Health Rating 2007 Plant Health Ratingy_
Rotation Strawberry Allstar 3.67 -
Rotation ProPhyt+Abound Cavendish - 3
Rotation Strawberry Cavendish - 3
Rotation Compost Cavendish - 5
Rotation Actinovate Cavendish - 2
Fumigated Strawberry Allstar 2.33 -
Fumigated ProPhyt+Abound Cavendish - 4.5
Fumigated Strawberry Cavendish - 5
Fumigated Compost Cavendish - 5
Fumigated Actinovate Cavendish - 4.5
Continuous Strawberry Allstar l .67 -
Continuous ProPhyt+Abound Cavendish - 2.5
Continuous Strawberry Cavendish - 3 .5
Continuous Strawberry Allstar - 2
Continuous Compost Cavendish - 5
Continuous Actinovate Cavendish - 4.5

 

x All averages calculated from n=3.
3’ Plant health rating on scale of 1-5 for necrosis where l = mostly black, dark brown, no
ﬁnely branched roots, and single crown; 2 = same as 1, except 1 or 2 ﬁnely-branched
roots present; 3 = half of all roots are black/dark brown and unbranched, and l or 2

crown branches; 4 = more white, ﬁne, branched roots present than black/brown
unbranched roots, and 2 crown branches; and 5 = white, ﬁne, and multi-branched roots

and crown.

2 Root health rating on 2-3 mm roots from 1-4 where 1=0-25% of roots with lesions, no
feeder roots black/brown; 2=25-49% of roots with lesions, a few feeder roots
black/brown; 3=50-74% of roots with lesions, several clusters of feeder roots
black/brown;4= >75% of roots with lesions or most feeder roots black/brown.

122

In 2006, nematode sampling revealed that the fumigation main treatment had the
lowest total plant parasitic nematode counts. There were no signiﬁcant differences
between season sampled or blocks (table 27). In 2007 the detection of plant parasitic
nematodes was improved with root sampling. While the fumigated main treatment had
the highest counts, it was not signiﬁcantly different from the continuous strawberry main
treatment. Over the two years, the total number of parasitic nematodes in the soil was
higher in 2007 than 2006. This was also evident for total P. penetrans in the soil,
indicating a build up over time, but there were no signiﬁcant differences observed
amongst the main treatments. Some initial sampling done from plants in the compost
within the ﬁrst block of each main treatment revealed the presence of T richodorus spp.,

Criconemella spp., Meloidogyne spp., Pratylenchus penetrans, Longidorus elongates.

123

Table 27. Plant parasitic nematode populations in 100 cm3 soil or 1 g roots from roots of
strawberry cv. Allstar and Cavendish grown under different cultural and chemical
treatments in a black root rot infested soil in East Lansing, MI, in 2006 and 2007.m

 

 

 

 

2006 2007
Total Plant Total Plant Total Plant
Parasitic Parasitic Parasitic
Nematodes in Nematodes in Nematodes in
Main Treatment soilz“ soily m rootsm
Fumigated 0.50 a |07.00 NS 56.00 NS
Continuous strawberry 3.50 b 54.38 2 I .75
Rotation 8.25 b 101.25 44.88
ANOVA

Effects Df Signiﬁcance(p)
Main (M) 1 0.0017 0.7714 0.9211
Season (S) 1 0.1174 - -
Variety 2 - 0.8292 0.942]
Block (B) 3 0.8966 0.6547 0.728]
Residual l7
Total (Corr.) 23

 

' See Appendix C for risk ratings for parasitic nematodes on strawberries in Michigan.
5 Total nematode averages includes:, Trichodorus spp., Criconemella spp., Meloidogyne
spp., Pratylenchus penetrans, and Longidorus elongatus.

‘ Total nematodes include: Meloidogyne spp. and Pratylenchus penetrans.

“ Values followed by differing letters are signiﬁcantly different according to Fisher’s
protected least signiﬁcant difference test p=0.05.

V For pairwise comparisons, main treatment, n=8, for variety, n=12, and for block, n=6.

w For pairwise comparisons, main treatment, n=6, for variety, n=9, and for block, n=6.

x For pairwise comparisons, main treatment, n=8, for season, n=12, and for block, n=6.

y Statistical separation performed after sqrt(x+l) transformation.

7‘ Statistical separation performed after log(x+1) transformation.

124

Table 28. Fungal genera isolated from roots of strawberry cv. Allstar and Cavendish
grown under different cultural and chemical treatments in a black root rot infested soil in
East Lansing, MI, in 2006 and 20073“

 

2006 2007

 

ProPhyt
+

Genera present Control Control Compost Abound Actinovate Control

 

Allstar Cavendish Allstar

 

Lesion Lesions
free present Lesions present

 

 

Fumigated

 

Alternaria spp. - - - X X X -
Chaetomium spp. X X X - - - -
Com'olhyrium spp. - - X
C oniothyrium-like - X -
C ylindrocarpon spp. —
F usarium spp. -
Mucor spp. X
Penicillium spp. X
Pestalolia spp. - - - X
Phoma spp. - - - —
Pyrenochaeta spp. - - - -
Pythium spp. X X - -
Rhizoctonia spp. - - X X
Robillarda spp. - - - -
Trichoderma spp. X X X X -

.xx.
.xx.
><

XXIXX' i><><I

X XXI'XX'

x I

 

Rotation

 

Alternaria spp. -
C ylindrocarpon spp. -
F usarium spp.
Mucor spp.
Penicillium spp.
Phoma spp.
Pythium spp.
Rhizoctonia spp.
Robillarda spp.
Trichoderma spp.

“xx-x.
x.><><)<><)<u
I><|i><><><
I IX
IXX-IXXI
I .xx.

x I

 

Continuous

 

X

X
X

Alternaria spp. -
Chaelomium spp. -
Coniothyrium spp. -
Coniothyrium-like X
Cylindrocarpon spp. X
Doratomyces spp. -
F usarium spp. X
Mucor spp. X
Phoma spp. -
Pyrenochaeta spp. -
Rhizoctonia spp. - -
Robillarda spp. - X
Trichoderma spp. X X - - _

x I

x I

x I
x I
x I
x I
X I

.xx..
><><><l

><><><I
.x.
x.
.x.

I><I
IXXI
)(I

x I
x I
x I

 

x I

 

* Table obtained from Milner laboratory. X indicates genera most frequently recovered.

125

Inoculum Level Assesment

In 2006 time and time x block were not signiﬁcant in the ANOVA, but time x
treatment and the block main effect were signiﬁcant (p=0.0037 and 0.0188, respectively).
Treatment main effect was not signiﬁcant (p=0.2363). There was no block effect in
2007, but treatment and the time x treatment interaction were signiﬁcant (p<0.0001 and
0.0028, respectively).

At the beginning of 2006 the fumigation and rotation main treatments had
signiﬁcantly fewer CFUs of Rhizoctonia/g soil than the untreated continuous main
treatment; however, this difference was lost by the end of the ﬁrst growing season, and in
fact a reversal was seen (table 29).

In 2007, a general trend was observed where the fumigated main treatment had
signiﬁcantly fewer propagules than the other main treatments, which did not signiﬁcantly
differ (table 30). Again, at the end of the season the effect of the treatments blurred,
possibly indicating that September is an important time for BR and strawberry growth.
This fluctuation over the season, possibly inﬂuenced by temperature has been observed

by others (Martin, 1988; Scott et al., 2003).

126

Table 29. Effect of fumigation and rotation crops on soil inoculum concentration of
Rhizoctonia in a strawberry black root rot infested ﬁeld in East Lansing, MI in 2006*

 

 

 

 

CFU Rhizoctonia/g soil
Treatment June July August September
Continuous strawberry 2.38 a 2.25 1.08 1.00 b
Fumigated 0.75 b 1.00 1.67 2.92 a
Rotation 0.13 b 1.50 1.08 1.00 b
ANOVA
Effect Df Siiniﬁcance (p)
Treatment 2 0.0003 0.2166 0.4957 0.0010
Block 3 0.5295 0.3214 0.1276 0.0163
Error 30

 

* Values followed by differing letters are signiﬁcantly different according to Fisher’s
protected least signiﬁcant difference test p=0.05. Pairwise comparisons performed
with n=12, except for June where n=8.

127

Table 30. Effect of fumigation and rotation crops on soil inoculum concentration of
Rhizoctonia in a strawberry black root rot infested ﬁeld in East Lansing, MI in 2007.*

CPU Rhizoctonia/g soil

 

 

 

Treatment May June July August September
Continuous strawberry 13.83 a 14.42 a 14.58 a 15.00 a 14.08 NS
Fumigated 9.08 b 12.42 b 13.00 b 13.58 b 12.83
Rotation 10.25 b 13.33 b 14.58 a 14.50 c 13.92
ANOVA

Effect Df Signiﬁcance (p)

Treatment 2 0.0015 0.0007 0.0055 <0.0001 0.0739
Block 3 0.3250 0.2638 0.3546 0.9557 0.3128
Error 30

 

* Values followed by differing letters are signiﬁcantly different according to Fisher’s
protected least signiﬁcant difference test p=0.05. Pairwise comparisons performed

with n=12

128

It is well known that fumigation results in larger, more productive strawberry
plants (Wilhelm, 1965; Hancock et al., 2001). While the rotation and untreated
continuous strawberry main treatments did not signiﬁcantly differ for most parameters
over the two growing seasons, the rotation treatment did tend to produce more total
crowns and better bed ﬁll. The plants within the rotation main treatment were
signiﬁcantly more vigorous than the untreated continuous plants over the two years.

The utilization of ProPhyt + Abound, although not signiﬁcantly different from
untreated plants, deserves further evaluation as a potential, more environmentally benign,
alternative because of work in other trials (Chapter 1 and 2) and it tended to produce beds
with better ﬁll initially, indicating repeated measures like those in Chapter 6, may
provide acceptable control. In addition, a ProPhyt + Abound dip provides one of the
easiest alternatives to implement. This dip may work well in a weakly infested soil by
initially protecting the plant as it establishes in the ﬁeld. Another way is that the
fungicides may help decrease any residual organisms coming in on the planting stock
from the nursery. This could be determined by a thorough survey of nursery stock.

While the compost socks show potential as well, there are some downsides. The
socks do not readily degrade, so eventually, there will be a disposal cost. In addition, the
roots do eventually grow out of the sock into the infested soil, and all the daughter plants
must establish in this soil too. Fruit production is dependent on the initial mother plants,
not so much on the establishment of a perennial bed. It is worthwhile considering a
larger row spacing to aid in weed control and equipment maneuvering. Also, winter

injury or drought stress may be greater since the socks are lying above ground.

129

If BRR is approached as a complex of several independent factors, tailor
recommendations should be made for a given situation. These factors include: 1) poor
cultural practices, 2) fungi, 3) nematodes or grubs, 4) some combination of the previously
listed causes, and 5) a strawberry planting that has reached its maximum lifespan.
Rhizoctoniaﬁagariae is ubiquitous, particularly in strawberry ﬁelds. However, the
literature fails to describe all factors that may have been involved in any particular
instance of strawberry decline. This contributes to the confusion of the actual cause, as it
seems BRR encompasses any strawberry decline situation in which Rhizcotom'a, along
with several other fungi, is isolated from the strawberry roots and the cause can not be
contributed to something else. This does not mean that it is the cause, but that the correct
questions and information are not being asked or exchanged. In other words, the fungi
only complicate a problem caused by something else.

The nurseries need to be held accountable for their crowns, growers need to be
encouraged to do regular sampling, and the people evaluating the samples need to get
thorough background information of the ﬁeld. Only with more thorough information can
the vagueness and inconsistency of this disease complex become elucidated and

understood.

130

LITERATURE CITED

Bird, G. W. 1971. Inﬂuence of incubation solution on the rate of recovery of
Pratylenchus brachyurus from cotton roots. J. of Nematology 3:378-3 85.

D’Ercole, N., Nipoti, P., and Manzali, D. 1989. Research on the root rot complex of
strawberry plants. Acta Horticulturae 265:497-501.

Elmer, W. H., and LaMondia, J. A. 1999. Inﬂuence of ammonium sulfate and rotation
crops on strawberry BRR. Plant Dis. 83:119-123.

Ettlinger, M. (3., and Kjaer, A. 1968. Sulfur compounds in plants. Pages 59-144 In
Recent advances in phytochemistry. T. J. Mabry, Ed. New York: Appleton-Century-
Crofts.

Gutierrez, W. A., Shew, H. D., and Melton, T. A. 1997. Sources of inoculum and
management for Rhizoctonia solani damping-off on tobacco transplants under
greenhouse conditions. Plant Dis. 81:604-606.

Hancock, J. F., Callow, P. W., Serce, S., and Schilder, AC. 2001. Relative performance
of strawberry cultivars and native hybrids on fumigated and nonfumigated soil in
Michigan. HortScience 36:136-138.

Henis, Y., Ghaffar, A., Baker, R., Gillespie, S. L. 1978. A new pellet soil-sampler and
its use for the study of population dynamics of Rhizoctonia solani in soil.
Phytopathology 68: 371-376.

Hildebrand, A. A. 1934. Recent observations on strawberry root rot in the Niagara
peninsula. Can. J. Research, 1 1:18-31.

Hildebrand, A. A., and West, P. M. 1941. Strawberry root rot in relation to
microbiologiclal changes induced in root rot soil by the incorporation of certain cover
crops. Can. J. Research 19 6:183-198.

Hoitink, H. A. J ., F ahy, P. C. 1986. Basis for the control of soilbome plant pathogens
with composts. Ann. Rev. Phytopathol. 24:93-114.

Jenkins, W. R. 1964. A rapid centrifugal-ﬂotation technique for extracting nematodes
from soil. Plant Dis. Reporter 48:692

LaMondia, J. A. 2004. Field performance of twenty-one strawberry cultivars in a black
root rot infested site. Journal of the American Pomological Soc. 58:226-232.

Maas, J. L. 1998. Compendium of Strawberry Diseases. 2nd Ed. American
Phytopathological Society.

131

Martin, F. N., and Hancock, J. G. 1983. Chemical factors in soils suppressive to
Pythium ultimum. Pages 113-116 In Ecology and Management of Soilbome Plant
Pathogens. C. A. Parker, A. D. Rovira, K. J. Moore, and P. T. W. Wong, Eds. American
Phytopathological Society. St. Paul.

Martin, S. B. 1988. Identiﬁcation, isolation frequency, and pathogenicity of anastomosis
groups of binucleate Rhizoctonia spp. from strawberry roots. Phytopathology 78:379-
384.

Millner, P. D. 2006. Control of strawberry black root rot with compost socks. Biocycle
47:1212, 56-58.

Particka, C. A., and Hancock, J. F. 2005. Field evaluation of strawberry genotypes for
tolerance to black root rot on ﬁlmigated and nonfumigated soil. J. Amer. Hort. Sci.
130:688-693.

Perry, 8., and Ramsdell, D. 1994. Strawberry diseases in Michigan. Ag Facts, Michigan
State University Extension Bulletin E-172821-4.

Scott, R., Pritts, M., and Kelly, M. J. 2003. Effects of Rhizoctoniaﬁagariae infection on
growth and productivity of strawberry plants grown under different temperature regimes.
Advances in Strawberry Research 22: 26-33.

Snapp, S. S., and Mutch, D. R. 2003. Cover crop choices for Michigan vegetables.
Michigan State University Extension Bulletin E 2896: 1-6.

Strong, RC, and Strong, M. C. 1927. Investigations on the black root of strawberries.
Phytopathology 21 : 1041 -1059.

Townshend, J. L. 1989. Population densities of four species of root-lesion nematodes
(Pratylenchus) in the oat cultivars, Saia and OAC Woodstock. Can. J. Plant Sci. 69:903-
905.

Watanabe, T., Hashimoto, K., Sato, M. 1977. Pythium species associated with
strawberry roots in Japan, and their role in the strawberry stunt disease. Phytopathology
67:1324-1332.

Wilhelm, S. 1965. Pythium ultimum and the soil fumigation growth response.
Phytopathology 55:1016-1020.

Wing, K. B, Pritts, M. P., and Wilcox, W. F. 1995. Biotic, edaphic, and cultural factors
associated with strawberry BRR in New York. HortScience 30:86-90.

132

CHAPTER FIVE: ESTABLISHMENT OF BIOCONTROLS IN ROTATIONS
Introduction

Currently, recommendations for control of BRR consist of using crop rotation,
cover cr0ps, good aeration and drainage, and fumigation (Maas, 1998; Martin and
Hancock, 1983; Perry and Ramsdell, 1994). If an existing planting develops BRR, a new
site should be chosen, or the old plants plowed under and the soil cultivated for several
months followed by fumigation and planting of healthy strawberries in the spring (Perry
and Ramsdell, 1994). Incorporation of organic matter can encourage beneﬁcial
organisms that are antagonistic to pathogens (Perry and Ramsdell, 1994, Hoitink, 1997).

Crop rotation is known to encourage a more diverse, healthier soil microorganism
community (Snapp and Mutch, 2003; LaMondia et al., 2002). Continual cropping allows
pathogen establishment and inoculum build-up to deleterious levels. In potato, wheat,
barley, and com, the more frequently a crop is in a rotation, the higher the decrease in
yield of that crop (Schippers etal., 1987). Despite this knowledge, growers often have
land constraints in their strawberry U-pick operations, and rotation cr0ps do not generally
have the economic value of strawberries. Thus there is an interest in using the shortest
rotation between strawberry plantings possible. Hildebrand and West (1941) found that
strawberries grown after a soybean series in greenhouse experiments approached the
same level of disease as sterilized soil. ‘Saia’ oats (Avena strigosa Ard.) has had some
success in controlling P. penetrans and R. fragariae in a pot trial in the greenhouse
(Townshend, 1989). Elmer and LaMondia (1999) found that an application of
ammonium sulfate with ‘Saia’ oats or sorgho-sudangrass reduced P. penetrans

populations in strawberry roots, and that only a rotation with ‘Garry’ oats and ammonium

133

sulfate reduced root colonization by R. fragariae in a micrOplot study using ‘Honeoye’
strawberries. Brassica species such as oilseed radish, mustard, and canola have also
received attention due to the formation of isothiocyanates that have fungicidal and
nematicidal properties (Ettlinger and Kjaer, 1968; Snapp and Mutch, 2003).

In addition, the rhizosphere is a dynamic environment. The mucilage excreted by
the roots supports both beneﬁcial and potentially harmful organisms seeking to colonize
the growing root. The beneﬁcial microbes are antagonistic to pathogens by competition
for food, essential elements, and space (Whipps, 2001; Parke, 1991 ). These microbes
enhance plant growth by suppressing major pathogens, increasing nutrient availability,
decreasing toxicity levels around the plant, or a combination of these (Whipps, 2001;
Parke, 1991). Some of the microbes parasitize pathogenic fungi by producing chitinases
or antibiotics. For instance, Streptomyces hygroscopicus var. geldonus produces a toxin
called geldanomycin which can inhibit R. solani (Chet et al. , 1991; Fravel and Keinath,
1991). One biocontrol organism that has been used in the past is T. harzianum Rifai.
Chet and Henis (1983) reported 70% control of R. solani with 150 g (dry wt) per square
meter in a broadcast application in carnation. Control was signiﬁcantly better than that
achieved in the ﬁeld by establishing carnations in peat moss with 15% by volume of the
T. harzianum preparation. Chet and Henis (1983) also report that under ﬁeld conditions,
seed treatment with T. hamatum (Bonord.) Bainier reduced cotton damping-off caused by
R. solani by 60%, and bare patches 23 days later by 39%. It also increased density of
plants by 14%. Trichoderma harzianum can use the R. solani cell wall as a sole carbon
source. D’Ercole et al. (1989) found a reduction of post-transplant blight incidence from

24.5% in the control to 11.5% in the treatment with T. harzianum as a liquid dip on

134

‘Gorella’ strawberries. Studies found that T-22 (Trichoderma harzianum) could increase
nitrogen fertilizer efﬁciency in corn (Harman, 2000). It causes plants to be more robust
and have more extensive root systems by suppressing disease as well as stimulating plant
metabolism (Harman, 2000).

There are several biological control products emerging on the market, many of
which claim to control root pathogens, but have not been labeled for strawberries. These
products have also not been proven to be reproducibly successful in a ﬁeld situation. This
experiment was conducted to evaluate whether suppressive conditions can be created
prior to strawberry planting by utilizing different crop rotations in conjunction with
applications of Plantshield (Trichoderma harzianum) or Mycostop (Streptomyces
griseoviridis). Rotation crops were chosen by their being mentioned in either product’s
label, their ability to grow in Michigan, their potential to provide economic return
(summer crop), and their potential to naturally fumigate the soil (kale) or build up organic

matter to support a diverse microbe population.

Materials and Methods

Plots (1.8 x 1.8 m) were established in three 40.2 x 3.7 m blocks in a sandy loam
soil at the Michigan State University Horticulture Farm, East Lansing, MI, which had
been previously planted to wheat in September 2004. This ﬁeld had a four-year history
of strawberry production and a history of BRR. A randomized complete block design
was used. There were 61.0 cm between each plot and 91.4 cm buffer strips on the North
and South sides. The wheat was plowed under in May 2005. The area around each of the

blocks was kept in a winter rye and buckwheat rotation to control weeds and reduce

135

contamination when walking in the ﬁeld. The 61 .0-cm buffers between plots were kept
fallow and weed free.

On 16 June 2005, the continuous strawberries were planted 10 d after an
application of Devrinol 50 DF (napropamide) to control weed emergence. The summer
rotation crops of squash and sweet corn were planted 21 June 2005. Plantshield and
Mycostop were applied to each rotation crop at the label rate of 2.27 kg/ha and 1.13
kg/ha, respectively, on 12 July 2005 after crop emergence with a hand pump sprayer in
0.33 L of water per plot aimed at the base of the plants followed by 0.6 cm irrigation the
next day. Rotation crops not receiving either of these products served as a control. In
June 2005, 32 kg/ha of 19-19-19 was applied. In August, 57.7 kg/ha of urea was applied.
C ucurbita moschata ‘Pilgrim’ seeds (Horrocks Farm Market, Lansing, M1) were planted
in two rows at a rate of 18 seeds per 1.8 m row and thinned to a population of 6 plants per
meter row. Zea mays var. rugosa 'Checkered Choice' (W. Atlee Burpee & Co.,
Warminster, PA) was planted in three 1.8 m rows, 18 seeds/row, and thinned to a
population of 8 plants per meter of row.

In September 2005 the above ground parts of the sweet corn and squash plants
were removed from the ﬁeld after harvest so that the plots could be cultivated and re-
planted. Prior to planting with the winter rotation crops, the ﬁeld was prepared with
standard cultural practices including the addition of lime and 19-19-19 fertilizer applied
at the rate of 36.8 kg /ha. The seed bed was prepared for each plot in the order of
untreated, Mycostop, and Plantshield with the rototiller washed thoroughly between
different product treatments using a pressure washer. The products were applied at the

same rate as that for the summer crops but only in 500 ml of water per plot prior to

136

planting on 27 September 2005. The crops were hand broadcast at the following rates:
rye (Secale cereale)-100.8 kg/ha, buckwheat (F agopyrum sp.)-80.6 kg/ha, white clover
(Trifolium repens)-5.6 kg/ha, and hairy vetch (Vicia villosa Roth.)—67.2 kg/ha. Kale
(Brassica oleracea) was planted in two rows at 11.1 kg/ha (table 31). The kale was
thinned to a population of 7 plants per meter row. All seed was obtained from Michigan
State Seed Solutions (Grand Ledge, MI). Watering and manual weed control were done
as needed. Straw was placed over the strawberries in 29 November 2005 at a depth of 7
cm.

The straw was removed on 9 April 2006 and the area to be planted was fertilized
with 19-19-19 at the rate of 44.6 kg/ha in May. The plots were rototilled in a sequence to
prevent cross contamination between the Plantshield and Mycostop treatments. Two
rows of ﬁve strawberry plants were set in each plot. The rows were 83.8 cm apart, 15.2
cm from the edge of the plot and the plants were 30.5 cm apart within the rows. Initial
mother plant counts were taken a week after planting. During the summer of 2006,
ﬂowers were removed and runners were manipulated to form beds measuring 45 x 160
cm. On 8 August total crown count was taken along with visual bed ﬁll and plant vigor
ratings (table 32). In September, lime and 19-19-19 fertilizer were applied at the rates of
271.5 kg/ha and 254.5 kg/ha respectively. Soil was collected for nematode analysis on
22 May and 22 September. Straw was placed over the rows in 13 November at a depth of

7 cm and removed on 21 April 2007.

I37

Table 31. Rotations utilized in establishing biocontrols in rotations in a black root rot
infested ﬁeld in East Lansing, MI prior to planting strawberry cv. Allstar from 2005-
2007.

 

 

Summer 2005 Fall 2005 Summer 2006

Strawberry Strawberry Strawberry (Fumi gated)
Strawberry Strawberry Strawberry (Non-Fumigated)
Squash Rye Strawberry (Non-Fumigated)
Squash Hairy Vetch Strawberry (Non-Fumigated)
Sweet Corn Buckwheat Strawberry (Non-Fumigated)
Sweet Corn Kale Strawberry (Non-Fumigated)
Sweet Corn Clover Strawberry (Non-Fumigated)

 

* Each rotation received Mycostop, Plantshield, or No treatment.

138

Table 32. Bed ﬁll and plant vigor rating used in establishing biocontrols in rotations in a
black root rot infested ﬁeld in East Lansing, MI prior to planting strawberry cv. Allstar

from 2005-2007.

 

 

Plant Vigor Characteristics

5 Plants in excellent health, vigorous growth, superior runnering
4 33% plants diseased or stunted

3 50% plants diseased or stunted

2 Majority of plants diseased or stunted

1 Plants very stunted, diseased

Bed Fill Characteristics

5 Runners healthy and establishing well. full bed

4 Fewer runner plants than in 5

3 Thinning evident, runner size and establishment good
2 Few runners, mother plants obvious

Few to no runners establishing, mother plants dead

 

139

Starting 12 June 2007, yield data were collected including total berry number and
weight of berries from the interior meter of both rows. Fruit were harvested every 7 d for
3 wk. Iron phosphate (Slug Magic) was applied to prevent slug damage. As no
fungicides were sprayed to control fruit rots, berries were picked regardless of disease or
damage. At the third harvest all remaining berries were picked regardless of size,
maturity, or condition.

On 31 July, erect petioles and leaves were clipped at the crown and the fresh
weight obtained. The erect petioles and leaves, along with total crown counts were taken
using frames measuring 38.1 x 61.0 cm centered over the middle of each row. They were
subsequently dried for 48 h at 80°C to obtain dry weights. Plant vigor and bed ﬁll ratings
were visually taken on 19 July (table 32). Plants and 200 g of surrounding soil were
collected on 6 August for nematode analysis. These analyses were preformed by Fred
Warner in the Michigan State University Diagnostic services. Nematode presence was
determined using the modiﬁed Jenkins technique and a modiﬁed root extraction process
(Jenkins, 1964; Bird, 1971).

An additional plant was also dug from rotation combinations that appeared to
have fuller beds and healthier plants. This resulted in a total of 3 plants per rotation
treatment, both with and without biocontrols. Fresh biomass was obtained along with a
visual assessment of root quality (a qualitative scale of 1-5: 1 1= <20% of root mass is
secondary roots, 2= 20-40% secondary roots, 3= 40-60% secondary roots, 4= 60-80%
secondary roots, 5= >80% secondary roots) and percent necrosis. The roots from these
plants were used for isolations. The crown was cut in half above the uppermost

adventitious roots. The roots were washed under cold water and visually rated. After the

140

roots were surface sterilized in 20% bleach solution followed by two 1 min rinses in
sterile distilled water, ﬁve, 6-mm root pieces with lesions were dried on sterile paper
towels and placed on two separate petri plates with PDA amended with 100 til/ml of
streptomycin sulfate and penicillin-G sodium salt (10 root pieces/plant). Isolated fungi
were subcultured one week later after growing at room temperature. They were
subsequently identiﬁed to genus using classic taxonomic procedures and growth
characteristics on media (Barnett and Hunter, 1998; Domsch et al., 1980; Barron, 1968).
Those fungi that could not be identiﬁed in this manner were subsequently subjected to
DNA sequencing of the internal transcribed spacer (ITS) region by Timothy Miles

(Sambrook et al., 1989; Altschul et al., 1990).

Data Analysis

All biomass and crown count data obtained in the ﬁeld were collected
from both rows using frames previously described, and then calculated for one row.
Total crown number includes original parent crowns. Frequency of fungi recovered was
calculated based on total number of fungi that grew. Percentage of lesions with no fungal
growth was calculated from total lesions plated. All yield data were calculated for the
center meter of one row.

Initially, the statistical analysis was performed on the rotation combinations with
respective biocontrol products as one ‘management treatment’ because the fumigated and
continuous controls were not done with either product and the products were not
evaluated alone, resulting in an inability to study all interactions fully. In the interaction
analysis, model variance components were estimated due to type of rotation (R), product

(P), Block (B), R x P, and error after removing the fumigated and continuous strawberry

141

controls. Analysis was performed with the ANOVA and means separation (Fisher’s
protected least signiﬁcant difference test p=0.05) procedures in StatGraphics Plus 4.1
(StatPoint Inc., VA) using after performing a check for equal variance. When analyzing
the data over multiple years, it was necessary to utilize SAS 9.1 (SAS Institute Inc., Cary,

NC.) using repeated measurement ANOVA in proc glm.

Results and Discussion

In the repeated measures ANOVA the year and year x block effects were both
signiﬁcant (p<0.0001), but the year x rotation interaction was not signiﬁcant (p=0.6990).
It was of interest to look at the rotation and product combinations within each year as
well as over both years. Block was signiﬁcant for every parameter and the year x block
interaction was signiﬁcant for bed ﬁll and plant vigor (p=<0.0001 and 0.0009,
respectively), but not for total crown number (p=0.9402)

For the analysis of the interactions between the rotations and products, the year
and year x block interaction were again signiﬁcant (p<0.0001) while the year x treatment
interaction was not signiﬁcant (p=0.3733). The interaction year x product x rotation was
also not signiﬁcant (p=0.9849). Although block was signiﬁcant for each parameter, there
was not a block x treatment interaction for any of the measurements. Again, it was of
interest to look at the rotations and products within each year.

No rotation/product combination was signiﬁcantly different in the number of
beginning mother plants at the start of the experiment (table 33 and 36). However,

signiﬁcant differences were evident between blocks. The differences derive from

142

planting error due to poor crown placement and deaths. This began the differences
between the replicates that became more apparent over the next season.

Replanting, once it became evident that mother plants were not surviving so early
in the study, was not done for several reasons. There was a concern for differences in
timing among the treatments replanted versus those that were not. Also, when working
with crop rotations, the changes in fungal populations may not always be beneﬁcial for a
given crop (i.e. Verticillium wilt after solanaceous crops) (Pritts and Handley, 1998).
Although not expected, the crop rotations may have encouraged the build-up of
pathogens that contributed to early decline of the strawberries. In addition, each rotation
was planted to strawberries by a given pair of people randomly, so the risk of consistent
errors was small.

Despite these conditions, analysis did show some promising results. The rotation
combinations were not a signiﬁcant source of variance in the ANOVA for the bed ﬁll
rating or plant vigor rating (table 33), although the untreated continuous strawberry
consistently had the lowest average for every parameter obtained from the strawberry
beds (table 33). Strawberry plants planted after the squash/rye with Plantshield or
Mycostop were signiﬁcantly more vigorous than the untreated control, approaching the
same plant vigor as those plants in the fumigated control. In addition, these same rotation
combinations tended to produce better bed ﬁll than the untreated control. For total crown
number, strawberry plants in any of the squash/rye rotations, with or without products
applied, squash/hairy vetch and squash/hairy vetch with Mycostop all had more crowns

than the untreated control strawberries (table 33).

143

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144

In 2007, treatment only accounted for signiﬁcant variance in the ANOVA of total
berry number. However, strawberries planted after squash/rye and sweet com/kale with
Plantshield had bed ﬁll ratings approaching that of strawberries in the fumigated plots
(table 34). Plants planted after squash/rye, with or without either product, all produced
total crown numbers approaching that of strawberry plants in the fumigated plots. For
total berry number, strawberries planted after squash/hairy vetch, squash/hairy vetch with
Mycostop, and squash/rye with Mycostop produced more berries than the continuous
strawberries. The continuous plot strawberries did not produce as many berries, but they
were larger, causing there to be no difference between the controls for individual berry
weight (table 34).

Plant vigor rating, bed ﬁll rating, and total crown count were taken both years.
When considering the variables over 2006 and 2007 together, the strawberries planted
after the squash/rye rotations tended to have the same plant vigor and total crown
numbers as the fumigated plots (table 35). The difference in years not surprising as more
crowns should be evident the second year in bed establishment, especially when
considering the poor initial bed conditions in some blocks. Over time, bed ﬁll and plant
vigor should be expected to improve with healthy plants, particularly in this situation
where the beds had a rough-establishment year, as the strawberry plants compensate and

become more established.

145

Table 34. Effects of creating disease suppressive conditions using different rotations and
biocontrol products on plant vigor, bed ﬁll and total crown number of strawberry cv.
Allstar in a black root rot infested soil in East Lansing, MI, in 2007.

w Values in columns followed by differing letters are signiﬁcantly different according to
Fisher’s protected least signiﬁcant difference test at p=0.05. Pairwise comparisons
performed with n=3.

x Plant vigor scale of l to 5 where 5 = Plants in excellent health, vigorous growth,
superior runnering, 4 = 33% plants diseased orstunted, 3 = 50% plants diseased
or stunted, 2 = Majority of plants diseased or stunted, and l = Plants very stunted,
diseased.

y Bed ﬁll scale of l to 5 where 5 = Runners healthy and establishing well, full bed,

4 = Fewer runner plants than in 5, 3 = Thinning evident, runner size and
establishment good, 2 = Few runners, mother plants obvious, and l = Few to no
runners establishing, mother plants.

Z Statistical analysis performed after log(x+1) transformation.

146

 

 

 

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148

Comparing interactions between the products and the rotations, revealed that
rotations had signiﬁcant effects in 2006, but not 2007. Strawberries that followed the
squash/rye or squash hairy vetch rotation tended to have better plant vigor, bed ﬁll, and
total crown number in 2006 than any of the other rotations (table 36). The success of
these rotations could be due to these being the only rotations that had two well
established crops prior to the strawberries. The other fall rotation crops did not establish
well do to time of planting. Table 36 also indicates that the effects of rotation crops may
be lost aﬁer the ﬁrst year. Plants in plots receiving Plantshield had better bed ﬁll than
either Mycostop or nothing at all. Plantshield tends towards causing plants to have more
mass and be healthier (table 36). Rotations and products were not signiﬁcant for any
parameter measured over the two years (table 37).

Due to the large number of rotation and product combinations, the most
promising were chosen from data collected on the beds. These rotations (squash/rye and
squash/hairy vetch) were subjected to more intensive sampling for nematodes and fungal
isolations. These rotations still supported strawberry growth that tended to be better than

the untreated continuous control and were most like the growth in the fumigated plots.

149

Table 36. Effects of creating disease suppressive conditions using different rotations and
biocontrol products on plant vigor, bed ﬁll, total crown number, biomass and yield of
strawberry cv. Allstar in a black root rot infested soil in East Lansing, MI, in 2006 and
2007.w

w Values in columns followed by differing letters are signiﬁcantly different according to
Fisher’s protected least signiﬁcant differences test p=0.05. Pairwise comparisons
performed with n=9.

" Plant vigor scale of 1 to 5 where 5 = Plants in excellent health, vigorous growth,
superior runnering, 4 = 33% plants diseased or stunted, 3 = 50% plants diseased or
stunted, 2 = Majority of plants diseased or stunted, and l = Plants very stunted,
diseased.

y Bed ﬁll scale of 1 to 5 where 5 = Runners healthy and establishing well, full bed, 4 =
Fewer runner plants than in 5, 3 = Thinning evident, runner size and establishment
good, 2 = Few runners, mother plants obvious, and l = Few to no runners establishing,
mother plants.

2 Statistical separations performed after log(x) transformation.

150

 

 

 

 

 

 

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151

Table 37. Effects of creating disease suppressive conditions using different rotations and
biocontrol products on plant vigor, bed ﬁll, and total crown number of strawberry cv.
Allstar in a black root rot infested soil in East Lansig, MI, in 2006-2007.x

 

 

 

Fall Plant vigor Bed ﬁll Total crown
Summer rotation rotation rating (l-S)y rating (l-S)z number/m2
Squash Hairy Vetch 3.89 NS 3.6] NS 9.33 NS
Squash Rye 4.39 3.89 l 1.87
Sweet Corn Buckwheat 3.89 3.22 8.19
Sweet Corn Clover 3 .44 2.94 8.24
Sweet Corn Kale 3.72 3.56 8.84
Product
None 3.97 NS 3.33 NS 8.86 NS
Plantshield 4.07 3.67 10.00
Mycostop 3.57 3.33 9.03
ANOVA
Effects Df Signiﬁcance (p)
Rotation (R) 4 01 I34 0.0532 0.2589
Product (D) 2 0.1552 0.3289 0.6923
Block (B) 2 <0.0001 <0.0001 <0.0001
R x D 8 0.2250 0.8763 0.9542
Residual 73
Total (Corr.) 39

 

x Values in columns followed by differing letters are signiﬁcantly different according to
Fisher’s protected least signiﬁcant differences test p=0.05. Pairwise comparisons
of averages n=18. For product, n=30.

y Plant vigor scale of l to 5 where 5 = Plants in excellent health, vigorous growth,
superior runnering, 4 = 33% plants diseased or stunted, 3 = 50% plants diseased or
stunted, 2 = Majority of plants diseased or stunted, and l = Plants very stunted,
diseased.

7‘ Bed ﬁll scale of l to 5 where 5 = Runners healthy and establishing well, full bed, 4 =
Fewer runner plants than in 5, 3 = Thinning evident, runner size and establishment
good, 2 = Few runners, mother plants obvious, and l = Few to no runners establishing,
mother plants.

152

Again, rotations did not signiﬁcantly account for variance for any individual plant
parameter. For individual mother plant measurements, there were no signiﬁcant
differences amongst the rotations (table 38). The plants from the squash/hairy vetch
rotations had averages closest to the plants from the fumigated control over all
parameters. The rotations containing a fall component of kale, buckwheat, or clover were
essentially a rotation of squash or sweet corn followed by a fallow ﬁeld because none of
the crops established to any degree before being killed by frost. This explains the lack of
differences evident in many of the variables between these rotations.

Although populations did not differ among the treatments, it became apparent that
nematode populations did build up over time. There were no signiﬁcant differences
between total parasitic nematode population in any of the rotations for either year (table
39).

There was particular interest in the root lesion nematode (P. penetrans) due to its
implication in BRR. Sweet corn/buckwheat, sweet com/clover, and sweet com/kale all
tended to have populations that were similar to the fumigated plot while the other
rotations had more root lesion nematodes than the fumigated control (table 39). This is
probably due to the fallow condition that followed the buckwheat, clover, and kale since
these failed to establish and did not provide a conducive environment for survival. The
needle nematode, Longidorus elongatus, is also damaging to strawberry roots, so, due to

its presence, it was also analyzed.

153

Table 38. Effects of creating suppressive soils using different rotations and biocontrol
products on individual plant biomass and root health of strawberry cv. Allstar in a black
root rot infested soil in East Lansing, MI, in 2007.*

 

 

 

Fresh Fresh Total Root
Summer Fall foliage root wt. fresh wt. quality Necrosis
rotation rotation Product wt. (g) (g) (g) rating (“/o)
Fumigated - - 24.42 NS 15.29 NS 39.71 NS 3.33 NS 30.00 NS
Hairy
Squash Vetch - 26.78 18.78 45.57 3.00 46.67
Hairy
Squash Vetch Plantshield 17.29 12.95 30.24 2.00 40.00
Hairy
Squash Vetch Mycostop 22.14 16.26 3 l .35 2.00 60.00
Squash Rye - 19.50 17.57 37.07 2.33 66.67
Squash Rye Plantshield l 1.60 8.75 20.35 1.33 63.33
Squash Rye Mycostop 22.14 14.65 36.79 2.00 70.00
Continuous - - 9.12 15.85 24.97 2.33 73.33
ANOVA
Effects Df Signiﬁcance (p)
Rotation (R) 7 0.7914 0.8711 0.8560 0.8561 0.0892
Block (B) 2 0.5212 0.2769 0.4275 0.1492 0.0072
Residual 14
Total (Corr.) 23

 

* Values followed by differing letters are signiﬁcantly different according to Fisher’s
protected least signiﬁcant difference test p=0.05. Pairwise comparisons performed
with n=3.

154

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The overriding principle from this study is that strawberries, managed properly
from the beginning of the planting, can handle limited disease pressure. The major issue
throughout this study is the quality of the initial planting and possible desiccation of the
crowns because they were not planted deep enough. This cultural factor may have
weakened the plants to become more susceptible to the ﬁJngal and parasitic nematode
populations. The development a decline often has origins in the cultural practices of the
grower. This study provides strong evidence that, although the BRR pathogens are
present, properly cultivated strawberries can still be productive and that rotations can
make a difference, but one year of rotations is not sufﬁcient. In addition, suppressive
soils are difﬁcult to develop. Multiple applications of biocontrol products should be
considered to replenish the populations and increase the chances of establishment for the
beneﬁcial organisms.

Further evidence that this disease complex is highly variable is apparent when
considering that the fumigated continuous strawberry control had a high average of
pathogenic fungi isolated from it, yet this treatment tended to have the highest averages
over most of the bed parameters. These plants may have had an early establishment
advantage. Rhizoctonia is a fast growing fungus that re-populates fumigated soil quickly
(table 40), especially in this study where the small plots were surrounded by non-
fumigated soil. This ability makes it more likely to be the only fungus in the soil to
populate the roots of plants in a fumigated area, while plants in the non-fumigated soil
have many different genera of fungi and other microbes that may exclude the pathogens.

Although, the untreated continuous strawberries had Rhizoctonia and parasitic nematodes

157

present, along with other fungi that have been implicated in strawberry decline such as
C ylindrocarpon spp.

Overall, a crop rotation prior to establishing strawberries is a good practice.
Others have found that short rotations are not sufﬁcient, in studies with ‘Honeoye’
strawberries, LaMondia et al. (2002) did not ﬁnd that rotation crop affected pathogen
recovery from roots of 2-year old strawberry crowns. LaMondia (1999) found that there
were no differences. in nematode populations after one year of strawberry growth,
regardless of the previous crop. A one year crop rotation can not be expected to compete
with fumigation, but it can provide economic beneﬁts, allow healthy strawberry bed
establishment, and be environmentally conscious. Crops that establish well, such as rye
and hairy vetch, and grow for a longer time in the climate of Michigan, can enhance the
soil conditions for a following strawberry crop. While there is a strong trend for the
beneﬁts of Plantshield, more work should be done, perhaps with multiple applications, to
discover if this product really does limit BRR pathogens. This study also made it
apparent more nematodes or fungi does not mean a decline in strawberry production if
the plants become well established at planting and are kept healthy with proper cultural

practices that meet the needs of the plant.

158

LITERATURE CITED

Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. 1990. Basic local
alignment search tool. J. Mol. Biol. 215:403-410.

Barnett, H. L., and Hunter, B. B. 1998. Illustrated Genera of Imperfect Fungi. 4‘h ed.
APS Press. St. Paul, Minnesota.

Barron, G. L. 1968. The Genera of Hyphomycetes from Soil. Williams and Wilkins
Company, Baltimore.

Bird, G. W. 1971. Inﬂuence of incubation solution on the rate of recovery of
Pralylenchus brachyurus from cotton roots. J. of Nematology 3:378-3 85.

Chet, I., and Henis, Y. 1983. T richoderma as a biocontrol agent against soilbome root
pathogens. Pages 110-112 In Ecology and Management of Soilbome Plant Pathogens.
C. A. Parker, A. D. Rovira, K. J. Moore, and P. T. W. Wong, Eds. American
Phytopathological Society. St. Paul.

Chet, 1., Ordentlich, A., Shapira, R., and Oppenheim, A. 1991. Mechanisms of
biocontrol of soil-borne plant pathogens by Rhizobacteria. Pages 229-336 In The
Rhizosphere and Plant Growth. D. L. Keister and P. B. Cregan, Eds. Kluwer Academic
Publishers, Dordrecht, The Netherlands.

D’Ercole, N., Nipoti, P. and Manzali, D. 1989. Research on the root rot complex of
strawberry plants. Acta Horticulturae 265:497-501.

Domsch, K. H., Gams., W., and Anderson, T-H. 1980. Compendium of Soil Fungi.
Academic Press Inc., New York.

Elmer, W. H., and LaMondia, J. A. 1999. Inﬂuence of ammonium sulfate and rotation
crops on strawberry BRR. Plant Dis. 83:119-123.

Ettlinger, M. G., and Kjaer, A. 1968. Sulfur compounds in plants. Pages 59-144 In
Recent advances in phytochemistry. T. J. Mabry, Ed. New York: Appleton-Century-
Crofts.

Fravel, D. R., and Keinath, A. P. 1991. Biocontrol of soilbome plant pathogens with
ftmgi. Pages 237-243 In The Rhizosphere and Plant Growth. D. L. Keister and P. B.
Cregan, Eds. Kluwer Academic Publishers, Dordrecht, The Netherlands.

Harman, G. E. 2000. Myths and dogmas of biocontrol: changes in perceptions derived
from research on Trichoderma harzianum T-22. Plant Disease 842377-392.

159

Hildebrand, A. A., and West, P. M. 1941. Strawberry root rot in relation to

microbiologiclal changes induced in root rot soil by the incorporation of certain cover
crops. Can. J. Research 19:183-198.

Hoitink, H. A. J ., Stone, A. G. and Han, D. Y. 1997. Suppression of plant diseases by
composts. HortScience 32:184-187.

Jenkins, W. R. 1964. A rapid centrifugal-ﬂotation technique for extracting nematodes
from soil. Plant Dis. Reporter 48:692

LaMondia, J. A. 1999. Inﬂuence of rotation crops on strawberry pathogens:
Pratylenchus penetrans, Meloidogyne hapla, and Rhizoctoniafragariae. Supp. Journal
of Nematology, 31 :4S.

LaMondia, J. A., Elmer, W. H., Mervosh, T. L., and Cowles, R. S. 2002. Integrated
management of strawberry pests by rotation and intercropping. Crop Protection 21 :83 7-
846.

Maas, J. L. 1998. Compendium of Strawberry Diseases. 2nd Ed. American
Phytopathological Society.

Martin, F. N., and Hancock, J. G. 1983. Chemical factors in soils suppressive to
Pythium ultimum. Pages 113-116 In Ecology and Management of Soilbome Plant
Pathogens. C. A. Parker, A. D. Rovira, K. J. Moore, and P. T. W. Wong, Eds. American
Phytopathological Society. St. Paul.

Parke, J. L. 1991. Root colonization by indigenous and introduced microorganisms.
Pages 33-42 In The Rhizosphere and Plant Growth. D. L. Keister and P. B. Cregan, Eds.
Kluwer Academic Publishers, Dordrecht, The Netherlands.

Perry, S. and Ramsdell, D. 1994. Strawberry diseases in Michigan. Ag Facts, Michigan
State University Extension Bulletin E-1728zl-4.

Pritts, M., and Handley, D. Eds. 1998. Strawberry Production Guide: For the Northeast,
Midwest, and Eastern Canada. Northeast Regional Agricultural Engineering Service
Cooperative Extension. Ithaca, New York.

Sambrook, J ., Fritsch, E. F ., and Maniatis, T. 1989. Molecular Cloning: A Laboratory
Manual, 2"d Edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

Schippers, B., Bakker, A. W., and Bakker, P. A. H. M. 1987. Interactions of deleterious
and beneﬁcial rhizosphere microorganisms and the effect of cropping practices. Ann. Re.

Phytopahtoogy 251339-358.

Snapp, S. S., and Mutch, D. R. 2003. Cover crop choices for Michigan vegetables.
Michigan State University Extension Bulletin E 2896:1-6.

160

Townshend, J. L. 1989. Population densities of four species of root-lesion nematodes

(Pratylenchus) in the oat cultivars, Saia and OAC Woodstock. Can. J. Plant Sci. 69:903-
905.

Whipps, J. M. 2001. Microbial interactions and biocontrol in the rhizosphere. Journal of
Experimental Botany 52:487-51 1.

161

CHAPTER SIX: SLOWING BLACK ROOT ROT IN DECLINING
ESTABLISHED STRAWBERRY FIELDS

Introduction

In Michigan, strawberries are typically produced in perennial, matted-row
plantings in U-Pick operations. Black root rot is a disease complex that plagues older
strawberry ﬁelds and results in diminished yields to the point where the planting becomes
economically unsustainable. A number of fungi and the root lesion nematode have been
implicated in this disease (Hildebrand, 1934; Wing et al., 1994; Maas, 1998). Black root
rot often develops in ﬁelds that have been replanted to strawberries.

The recommendation is frequently made to increase the length of time when the
ﬁeld is planted to crops other than strawberries, or to ﬁnd a new location for the
strawberries (Perry and Ramsdell, 1994). However, land constraints are frequently the
reason why strawberries cannot be grown in a different area on a given farm, and
cessation of strawberry production is not a viable choice. In such cases, prevention or
slowing of crop decline are extremely desirable. Testing of treatments that might prolong
the useful life of the planting would be of value.

While the product fungicide Abound (azoxystrobin) is labeled as a drench in
strawberries for control of Rhizoctonia root rot, this claim has not been tested in
Michigan. ProPhyt is one of the phosphorous acid fungicides, which are highly systemic.
Since this fungicide has good efﬁcacy against oomycete pathogens, it may have the
potential to control BRR-related decline. Furthermore, the potassium in this product may
have some nutritional value as well. The phosphorus component is not available to the

plant in the phosphite form. however.

162

While several products are labeled for use speciﬁcally against pathogenic
organisms that are part of the black root rot complex, growers are continually supplied
information on numerous products that are said to improve plant health, but that have
little scientiﬁc support showing efﬁcacy. In order to develop recommendations for
increasing the longevity and proﬁtability of perennial strawberry plantings, this research
investigated the effectiveness of some products to slow or reverse BRR decline
(fungicides and nutritional amendments). While used as a foliar fertilizer, Vigor-Cal-
Phos contains phosphites, like ProPhyt, and may provide essential micronutrients (table
42) that aide in plant defenses. In ‘Sweet Charlie’ strawberries, a ProPhyt 4L dip
treatment followed by a foliar application increased the percentage of healthy plants
compared to plants that were not treated after 24 days in a North Carolina study looking
to control Phytophthora cactorum (Louws et al., 2004). Symbex 4x is designed to
provide nutritional beneﬁts as well as encouraging beneﬁcial microbial populations. C/G
is a fatty acid compound that has shown general fungicidal properties on other crops in

previous studies (Schilder el al., 2003).

Materials and Methods

Two commercial production sites with BRR symptoms were selected for the
efﬁcacy trial (site O in Ottawa County and site L in Leelanau County). Each site had
seven different treatments in a randomized complete block design with four blocks (table
42). Each treatment plot consisted of three 3.05-m rows. All rows were treated with
their respective product, with only the center row used for data collection. The

treatments were applied as either a foliar spray or a drench (table 43). Sprays were

163

applied at a rate of 467.5 L/ha, every 2 wk, at 50 psi with a backpack sprayer. The control
remained untreated. All plots received the standard practices of the cooperator. The
drenches were applied at a rate of 18,700 L/ha using l9-L containers to mix the drenches
and pour them over the rows. C/G was applied at 0.05% v/v rate at the ﬁrst application
and subsequently at 0.2%. The growers were permitted to continue their standard cultural
practices over the duration of the project (table 41).

At the time of set-up, plants were removed from rows beside each replication, but
not from rows within the plots, to allow for an initial pathogen evaluation. Plants were
removed with 200 g of surrounding soil for analysis. Fungi were isolated from the roots
using process described below. Nematode presence in soil and roots was determined by
the Michigan State University Diagnostic Laboratory. Nematode presence was
determined using the modiﬁed Jenkins technique and a modiﬁed root extraction process
(Jenkins, 1964; Bird, 1971). An initial bed ﬁll rating was also taken. At both locations,
bed ﬁll was assessed on a l to 5 scale, with 5 = runners healthy and establishing well, full
bed, 4 = fewer runner plants than in 5, 3 = thinning evident, runner size andestablishment
good, 2 = few runners, mother plants obvious, and l = few to no runners establishing,
mother plants.

Plant volume was calculated from two radius measurements at right angles to
one another, and a plant height measurement. Five mother plants were chosen in the
center row for this measurement. Two plants were removed (June or July, depending on
the site) from each treatment for fresh and dry biomass as well as a root necrosis rating.
This rating was on a l to 5 scale with l = mostly black, dark brown, no ﬁnely branched

roots and a single crown; 2 = same as 1, except one or two ﬁnely-branched roots present;

164

3 = half of all roots black/dark brown and unbranched, and l or 2 crown branches; 4 =
more white, ﬁne, branched roots present than black/brown unbranched roots, and > 2
crown branches; and 5 = white, ﬁne, and multi-branched roots and crown. From each of
these dug plants, 1 g of fresh root tissue was removed after the root necrosis rating, but

before taking the fresh root weight, for fungal pathogen evaluation.

165

Table 41. Cooperator application rates and dates in the effort to slow black root rot in
infested established strawberry ﬁelds in Ottawa (cv. ‘Jewel’) and Leelanau (cv.
‘Northeaster’), Ml durinL2007.

 

Leelanau Co. Site

 

 

 

Active Time of
Product Ingredient Rate Application
Fungicides Captan Captan 1.8 qts/A 20 May
Elevate Fenhexamid 1, 5 lbs/A 20 May
Captan Captan 1.8 qts/A 2 Jun
Pristine Pyraclostrobin 20 ozs/A 2 Jun
and boscalld
Insecticides Thiodan 3 EC Endosulfan L3 3 qts/A 20 May
Thiodan 3 EC Endosulfan 1,3 3 qts/A 2 Jun
Fertilizers Urea - 601bs/A 21 Apr
Ottawa Co. Site
Fungicides Quadris Azoxystrobin [0 (WA 28 April
Captec 4L Captan 2 qt/A 28 April
Cabrio EG Pyraclostrobin lZoz/A 11 May
Switch Cyprodinil and 11 May
Fludioxonil lO oz/A
Elevate Fenhexamid 1,5 lbs/A 29 May
Nova Myclobutanil 2.5 oz/A 29 May
Nova Myclobutanil 2.5 oz/A 6 Aug
Nova Myclobutanil 2.5 oz/A 5 Sept
Herbicides Select (+oil l%) Clethodim 6 021A 17 May
2,4-D Amine, 2, 4-D l qt/A 2 Aug
Weedar 64 47%
Sinbar Terbacil I qt/A 29 Aug
2, 4-D 2, 4-D 1 qt/A 20 Oct
Insecticides Thiodan 50 W Endosulfan 2 lb/A 11 May
Lorsban 4E Chlorpyrifos 2 pt/A l 1 May
Advise (Admire) Imidacloprid 23oz/A 1 Aug
- Dimethoate 0, 5 pt/A 6 Aug
Fertilizers 12-12-12 - 200 lbs/A 12 July
Calcium nitrate - 100 lbs/A 20 April
Potassium nitrate - IOO lbs/A 20 Sept

 

166

Table 42. Active ingredients of products used in effort to slow black root rot in infested
established strawberry ﬁelds in Ottawa (cv. ‘Jewel’) and Leelanau (cv. ‘Northeaster’), MI

 

 

during 2007.
Product
Product Company Classiﬁcation Active Ingredients
Symbex 4x Ago K Fertilizer Microbial enzymes, Calcium carbonate,
Cobalt carbonate, Zinc carbonate
Vigor-Cal-Phos Agro K Fertilizer Calcium phosphite, Copper phosphite
ProPhyt Helena Fungicide Potassium phosphite
Abound Syngenta Fungicide Azoxystrobin
Abound + ProPhyt Syngenta and Helena Fungicide As above
C/G Summerdale, Inc. Fungicide Pelﬁonic acid

 

167

Table 43. Application method, rate, and schedule for products used in effort to slow black
root rot in infested established strawberry ﬁelds in Ottawa (cv. ‘Jewel’) and Leelanau (cv.
‘Northeaster’), MI during 2007.

 

Application Schedule

 

Product Applied As Applied Rate Site 0* Site L”
Symbex 4x Drench 2 qts/acre 1,5,9 1,3
Vigor-Cal-Phos Spray 3 qts/acre I,2,3,4,5,6,7,8,9 1,2,3
ProPhyt Spray 4 pts/acre I,2,3,4,5,6,7,8,9 1,2,3
Abound Drench 0.8 ﬂ. oz./1000 ft. row 1,5,9 1,3
Abound + ProPhyt Drench + Spray As above 1,5,9 + I,2,3,4,5,6,7,8,9 1,3 + 1,2,3
C/G Drench 0.2% v/v 1,5,9 1,3

 

* Dates of applications for Ottawa Co. site: 1:23 May 2007, 2=6 June 2007, 3=20 June
2007, 4=5 July 2007, 5=18 July 2007, 6=2 August 2007, 7=15 August 2007, 8=29
August 2007, and 9:8 September 2007

** Dates of applications for Leelanau Co. site: l=22 May 2007, 2=5 June 2007, and 3=l 9

June 2007.

168

Leelanau County Site

The trial was begun on 22 May 2007 in a ﬁeld of ‘Northeaster’ strawberries that
had been established in 2004 after a fall fumigation with Telone C35 at the rate of 25
GPA in Lake Leelanau, MI. The transplants were originally purchased from Nourse
Farms (South Deerﬁeld, MA). The matted row planting necessitated the use of an aerator
(Swisher AE-48, Warrensburg, M0) by hand to ensure that the drenches would reach the
root zone. The aerator was used at the ﬁrst application only because of the concern of
damaging fruit in subsequent applications. The soil was aerated to a depth of 5-8 cm.
Applications were made on the schedule given in table 43. The strawberries were
harvested on 21 June 2007 and again on 28 June 2007. A bed ﬁll rating and plant volume
measurement were also obtained at the second harvest date. In June, after the last
harvest, two plants, chosen from the ﬁve plants that were used for the plant volume
measurement, were dug from the central meter of the plot to assess fresh foliage, fresh
root, dry foliage, and dry root mass, and obtain a root necrosis rating. The trial at this
location was ended in early July because the ﬁeld was plowed up by the grower because

of poor yield.

Ottawa County Site

On 23 May 2007 a trial was established in a ﬁeld of ‘Jewel’ strawberries in
Hudsonville, MI that had been established in 2005 following a plow-down of sorghum
sudan grass. The transplants were purchased from Krohne Plant Farms, Inc. (Hartford,
MI). The cooperator utilized raised beds with drip tape to establish the strawberry rows

at this location, making aeration unfeasible. Applications were made according to the

169

schedule given in table 43, starting when the strawberry fruit was thumb-sized. Harvests
occurred on 13 June, 19 June, and again on 27 June 2007. The ﬁrst plant volume
measurement and a bed ﬁll rating were also obtained at the third harvest date. Bed ﬁll
ratings were also taken on 23 July and at the end of the experiment on 14 September
2007. A second plant volume measurement was taken 23 July. On 30 July 2007, two
representative plants were dug to assess fresh foliage, fresh root, dry foliage, and dry root
mass, and obtain a root necrosis rating. These plants were taken from the center row, but

not the central meter so as not to interfere with harvest data the second year of the study.

Yield Estimation
The interior meter of the center row was marked and all ripe berries were picked
at each harvest date. Berries were counted at the time of picking and weighed on

location. The berries were picked without regard to size or condition.

Plant Material Processing

From each of the dug plants, 1 g of fresh root tissue was removed after the root
necrosis rating, but before taking the fresh root weight, for fungal isolations. All plants
were stored at 4°C until processing. The ﬁrst plant of each treatment, from Leelanau Co.
site, was processed on 5 July 2007 after removing all ﬂower stalks, runners, and dead
leaves, in addition to knocking as much soil off of the plant as possible before the roots
were washed under cold running water. The rating was taken, and then fresh weights
were obtained by cutting the crown in half just above the uppermost adventitious roots.

The foliage and roots were then dried for 48 h at 80°C to obtain dry weights. The second

170

plant of each treatment was processed 10 June 2007 using the same procedure. The
process was repeated for the Ottawa Co. site, with the exception that all plants were
processed on the same day in August.

The roots were surface sterilized with 20% bleach solution for 2 min and then
rinsed for 1 min in two sequential sterile water baths. The roots were dried on sterile
paper towels, and ten lesions were selected to be placed on water agar, at ﬁve 6-mm root
pieces per plate. Plates were left on the laboratory bench and checked daily for growth
for 8 d. If growth was observed, fungi were sub-cultured onto potato dextrose agar and
subsequently identiﬁed morphologically upon plate colonization (Barnett and Hunter,
1998; Domsch et al., 1980; Barron, 1968). Those fungi that could not be identiﬁed in
this manner were subsequently identiﬁed by sequencing the DNA of the internal
transcribed spacer (ITS) regions by Timothy Miles (Sambrook et al., 1989; Altschul et
al., 1990).

Nematode samples were taken at the Ottawa site again at the end of the
experiment in September. Nematode presence was determined using the modiﬁed
Jenkins technique and a modiﬁed root extraction process by Fred Warner in the Michigan
State University Diagnostic Services laboratory (Jenkins, 1964; Bird, 1971). A nematode
sample was taken again in October to ensure that the populations had been properly
evaluated at the Ottawa site. Plants and surrounding soil dug for the September sampling
were removed from the outside rows so as not to interfere with future data collection.
Only a composite soil sample from the top 1.5-20 cm within the center row was evaluated

for nematodes in October.

171

Data Analysis

All statistical analyses was performed using the ANOVA and mean separation
functions (Fisher’s protected least signiﬁcant difference test at p=0.05) of the
StatGraphics (StatPoint Inc., VA) statistical computer program after checking for equality
of variance. When analyzing the bed ﬁll data over multiple sampling times, it was
necessary to utilize SAS 9.1 (SAS Institute Inc., Cary, NC.) using repeated measurement
ANOVA in proc glm. This procedure was also used for the analysis of plant volume data
at the site in Ottawa county.

Each site was analyzed separately due to differences in variety, age of planting,
the duration of the experiment at each location, the presence of nematodes at one site, and
the difference in production and management practices.

The frequency of fungi recovered was calculated based on the total number of
fungi that grew. The number of roots showing no growth was calculated based on the

total number of roots.

Rsults and Discussion

Both strawberry sites were in serious decline; in fact, the Leelanau Co. site was
taken out of production in 2007. The initial pathogen assessment revealed that the
Ottawa Co. site suffered from high levels of needle nematodes (Longidorus elongatus) as
well as several fungal root pathogens (F usarium and to a lesser extent Rhizoctonia, and
Cylindrocarpon), whereas the Leelanau Co. site had predominantly fungal pathogens

(mostly Rhizoctoniaﬁagariae) and no nematodes (tables 44 and 45).

172

Table 44. Preliminary fungal isolations from 40 strawberry root pieces from infested
established strawberry ﬁelds in Ottawa (cv. ‘Jewel’) and Leelanau (cv. ‘Northeaster’), M1
prior to study evaluatingproducts in an effort to slow black root rot during 2007.

°/o Root Pieces Colonized*

 

Genera Isolated Ottawa Co. Leelanau Co.
Rhizoctoniafragariae - 50.0
Pythium sp. 26.9 —
Fusarium sp. 50.0 4.6
Cylindrocarpon sp. 11.5 9.1
Other“ 1 1.5 36.4
Colonized root pieces 65.0 55.0

No colonization 40.0 45.0

 

* Multiple fungi would colonize the plate from a singe root piece.
** Other fungi included unknown fungi and Phoma spp.

173

Table 45. Preliminary nematode presence at the Ottawa site (cv. ‘J ewel’) prior to the

study to evaluate products in an effort to slow black root rot in infested established

strawberry ﬁelds during 2007 and the risk rating for needle nematode at this location.
Nematodes in root

 

 

Number of Nematodes in Soil (100 cc) tissue (1 g)
Pratylenchus Longidorus X iphinema C riconemella Trichodorus Pratylenchus
penetrans elongatus americanum spp. spp. penetrans
0 33 4 0 0 4
0 36 2 0 O 0
8 145 0 3 0 8
0 I40 0 0 0 0
4 45 0 0 l 3

 

* There were no parasitic nematodes found at site L. Samples obtained from plants
surrounding area to be used in the study.
** Risk ratings for parasitic nematodes on strawberries in Michigan available in
Appendix C.

174

At the Leelanau county site both time and time x block were signiﬁcant (p<0.001)
in the repeated measures ANOVA, but time x treatment was not (p=0.6361). While
block was signiﬁcant for the bed ﬁll rating, upon further analysis the block x treatment
interaction was not signiﬁcant (p=0.3670). It was of interest to look at the bed ﬁll rating
over the entire season.

At the Ottawa county site time, time x block, and time x treatment were
signiﬁcant in the repeated ANOVA (p=0.0001, <0.0001, and 0.0458 respectively). Block
(p<0.0001) and treatment (p=0.0458) were signiﬁcant in the bed ﬁll rating ANOVA. The
block x treatment interaction was signiﬁcant at this site (p<0.001). With the signiﬁcance
of the time x treatment interaction, it was not possible to look at the bed ﬁll rating over
the entire season.

There were no signiﬁcant differences among treatments for any of the bed ﬁll
ratings. Abound did tend to produce the highest average ratings at both sites. The ﬁnal
bed ﬁll rating at the Ottawa site was higher for beds receiving Abound or Abound +
ProPhyt, but did not signiﬁcantly differ from the untreated control. The block
signiﬁcance at the Ottawa site was driven by the block closest to the ﬁeld which seemed
to decline further, possibly due to poorer irrigation towards the edge of the ﬁeld. At the
site in Leelanau Co. the lack of differences between treatments for each bed ﬁll rating is
not surprising since the plants did not have an opportunity to set new daughter plants
during the experiment (table 46). However, at the Leelanau site, over the season, beds
receiving any treatment did tend to improve, with C/G improving the most when

compared to the untreated control.

175

ProPhyt, C/G, and Symbex 4x tended to produce higher yields in Leelanau, but
higher yields were observed in almost all treatments at both sites (table 47). There were
no signiﬁcant differences at either sites among the treatments for average berry weight or

total berries (table 47).

176

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177

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178

In Ottawa, use of the fungicides Abound, ProPhyt, and Abound + ProPhyt tended
to have higher plant volumes when compared to the untreated control. For the second
plant volume measurement in Ottawa Go, all treatments had higher average plant
volumes when compared to the control. In Leelanau all treatments but ProPhyt +
Abound tended to have a higher average plant volume than the untreated control (table
47).

The Abound treatment numerically had the highest average fresh foliage weight,
but was not signiﬁcantly different from any other treatment at both sites. Abound
provided the only difference from the untreated control at the Ottawa site for average
fresh root weight (table 48). There were no significant differences among the treatments
for dry foliage weight at either site. In Ottawa county the only significant improvement
in dry root weight was provided by Abound, when compared to the untreated control. At
the site in Ottawa Co. use of C/G, Abound, and ProPhyt resulted in numerically less root
necrosis. All treatments differed from the control in Leelanau Co., with Abound
producing roots with the least necrosis. Abound was the only signiﬁcantly improved
treatment from the untreated control for total fresh and dry plant weights in Ottawa.
There were no significant differences between treatments for either parameter in

Leelanau Co., but Abound and Abound + ProPhyt tended to be higher.

179

Table 48. Effects of fungicide or fertilizer treatments on plant weights and root necrosis
in cv. ‘Jewel’ or ‘Northeaster’ strawberries in a study to slow black root rot decline
conducted in Leelanau and Ottawa counties in Michigan in 2007."1

" Values in columns followed by differing letters are signiﬁcantly different according to
Fisher’s protected least signiﬁcant differences test p=0.05. Pairwise comparisons of
averages perfomed with n=6.

y Rating was on a 1 to 5 scale with 1 = mostly black, dark brown, no ﬁnely branched
roots and a single crown; 2 = same as 1, except 1 or 2 ﬁnely-branched roots present;

3 = half of all roots black/dark brown and unbranched, and 1 or 2 crown branches; 4 =
more white, ﬁne, branched roots present than black/brown unbranched roots, and > 2
crown branches; and 5 = white, ﬁne, and multi-branched roots and crown.

2 Statistical analysis was performed after log(x) transformation.

180

 

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182

Table 50. Average number of nematodes present in 100 cm3 of soil and l g of roots at
the Ottawa county site in a study evaluating products in an effort to slow black root rot in
Hudsonville, MI in September and October 2007.

 

 

 

 

September
Treatment Soil Root
Pratylenchus Longidorus C riconemella Pratylenchus
penetrans elongatus spp. penetrans
Untreated 2.75 7.00 l 1.50 8.50
C/G 2.50 6.25 3.25 2.50
October
Untreated 5.50 1.0 0.75 0.0

 

183

Except for Vigor-Cal—Phos and ProPhyt, the plants dug out at the Ottawa county
site had an overall lower recovery of Rhizoctonia than those plants dug from the site in
Leelanau Co. (table 49). Pythium sp. was predominately found in roots from Ottawa Co.
The lowest recovery of Rhizoctonia occurred from plants treated with Abound + ProPhyt
in Ottawa and Abound in Leelanau. Other fungi often associated with BRR were isolated
from both sites, including Cylindrocarpon and F usarium spp. As has been observed
before with BRR complex, F usarium was found at the Ottawa site where there were high
parasitic nematode populations. This may indicate that some F usarium spp. readily
colonize roots after nematodes damage them.

This experiment had a few limitations. The experiment should have been started
in April when the strawberries ﬁrst began to grow for the season. The lack of suitable
sites and time constraints prevented early set-up. An earlier application of products may
have targeted the ﬁrst root growth of the season. It also would have been easier to get the
drenches into the root zone as there would have been less foliage.

Two weeks after the ﬁrst application, it was noted that plants receiving drenches
had visually more vigorous growth and higher fruit quality than the untreated control.
The 2007 growing season was very dry. Some measurements, particularly those taken
after harvest, were inﬂuenced by the irrigation schedule. This schedule was determined
by the cooperator, and was not recorded. It was not possible to aerate the site in Ottawa
Co. like was done in Leelanau Co. because of the drip tape under the hills. Fortunately,
the soil was sandy enough to allow application of the drenches. An important

consideration is that the site in Leelanau Co. did not receive as many applications as the

184

site in Ottawa Co. over the season because the evaluation ended when the grower plowed
down the ﬁeld.

The difference in speciﬁc (fungi vs. nematodes) pathogens present between the
two sites may help explain differences in treatment effects between the sites. Nematodes
alone can be very damaging, particularly with the populations found at the Ottawa Co.
site (Chapman, 1956; Brown et al. , 1993). The potential for an interaction between the
nematodes and the fungi also exists (Powell, 1971). The performance of the fungicides
would be expected to make a greater impact at site the site in Leelanau Co. At the
Ottawa Co. site, the addition of a nematicide may have improved the control of BRR.
The data indicates that there was an improvement over the season at both sites for some
treatments because of the reduction of fungal pathogens that may have been
compounding the damage caused by the nematodes in Ottawa Co. (table 50).

Late spring to early summer applications of fungicides or foliar fertilizers
improved root health and yield in strawberry ﬁelds declining due to black root rot. A
drench of Abound was generally the most effective at improving root health, although
foliar sprays of ProPhyt also worked well. While bed ﬁll was not signiﬁcantly different
between the treatments, plant grth tended to be visually better in all treatments
compared to the untreated plants. C/G is in the process of being developed as a fungicide
and may be suitable for organic production.

Considering the success of the treatments incorporating Abound, it becomes
necessary to consider resistance management, otherwise the use of this fungicide will be
short-lived. It is always important to consider how growers will apply this on their own

farm. In this instance a grower could apply Abound through trickle irrigation like that

185

used at the site in Ottawa county. One or two applications per season may provide some
measure of control, if the application is timed appropriately. In addition, alternating
between ProPhyt and Abound may provide control while managing resistance. Other
growers could make use of a sweep cultivator to furrow along the rows and contain the
drenches applied with a sprayer. This experiment will continue at the site in Ottawa in
2008. Larger-scale trials on farms with products applied through the irrigation system

may be conducted in the future.

186

LITERATURE CITED

Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. 1990. Basic local
alignment search tool. J. Mol. Biol. 215:403-410.

Barnett, H. L., and Hunter, B. B. 1998. Illustrated Genera of Imperfect Fungi. 4th ed.
APS Press. St. Paul, Minnesota.

Barron, G. L. 1968. The Genera of Hyphomycetes from Soil. Williams and Wilkins
Company, Baltimore.

Bird, G. W. 1971. Inﬂuence of incubation solution on the rate of recovery of
Pratylenchus brachyurus from cotton roots. J. of Nematology 3:378-385.

Brown, D. J. F, Dalmasso, A., and Trudgill, D. L. 1993. Plant Parasitic Nematodes in
Temperate Agriculture. Nematode Pests of Soft Fruits and Vines. K. Evans, D. L.
Trudgill, and J. M. Webster, Eds.. CAB, International.

Chapman, R. A. 1956. Plant parasitic nematodes associated with strawberries in
Kentucky. Plant Dis. Reporter 40:179-183.

Domsch, K. H., Gams, W., and Anderson, T-H. 1980. Compendium of Soil Fungi.
Academic Press Inc., New York.

Hildebrand, A. A. 1934. Recent observations on strawberry root rot in the Niagara
peninsula. Can. J. Research, Sec. C. 11:18-31.

Jenkins, W. R. 1964. A rapid centrifugal-ﬂotation technique for extracting nematodes
from soil. Plant Dis. Reporter 48:692.

Louws, F. J ., Driver, J. G., and Leandro, L. 2004. Evaluation of fungicide pre-plant dip
and spray applications to manage Phytophthora crown rot on strawberry plugs.

American Phytopathological Society. Fungicide and Nematicide Tests, 60:SMF041

Maas, J. L. 1998. Compendium of Strawberry Diseases. 2nd Ed. American
Phytopathological Society.

Perry, S., and Ramsdell, D. 1994. Strawberry diseases in Michigan. Ag Facts, Michigan
State University Extension Bulletin E-1728: 1-4.

Powell, N. T. 1971. Interactions between nematodes and fungi in disease complexes.
Annual Review of Phytopathology 9: 253-274.

Sambrook, J ., Fritsch, E. F., and Maniatis, T. 1989. Molecular Cloning: A Laboratory
Manual, 2nd Edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

187

Schilder, A. M. C., Gillett, J. M., and Sysak, R. W. 2003. Evaluation of fungicides for
control of foliar diseases and black rot in grapes. American Phytopathological Society.
F ungicide and Nematicide Tests, 59:SMF027

Wing, K. B., Pritts, M. P., and Wilcox, W. F. 1994. Strawberry BRR: A Review.
Advances in Strawberry Research. 13:11-19.

188

CHAPTER SEVEN: EVALUATING RHIZOCTONIA VIRULENCE IN-VITRO

Introduction

While Rhizoctonia spp. are a vast group of fungi that are grouped largely by their
lack of distinctive taxonomic feateures, with teleomorphic states in both the
basidiomycetes and ascomycetes, the three groups associated with plant diseases
includes: R. solani, the binucleate species, and the isolates with a Waitea teleomorph
(Vilgalys and Cubeta, 1994).

Rhizoctonia spp. have a highly variable growth rate. Under certain conditions,
some species produce sclerotia-like tufts consisting of short, broad cells that function as
chlamydospores. The hyphae display characteristic right-angle branching with dolipore
septa and a moniliform resting cell (Maas, 1998). The branches are slightly constricted
and cross walls are present near the junction. Infrequently, the perfect stage,
Thanatephorus cucumeris (A.B. Frank) Donk, is formed in the multinucleate species, or
Ceratobasidium spp. in the binucleate species (Ogoshi and Ui, 1983; Burpee et al., 1980).

Ogoshi and Ui (1983) developed anastomosis groups (AG) for Japanese isolates,
while Burpee et al. (1980) developed groups for North America. Ogoshi (1985) then
compared the two different groups and found that Burpee’s seven groups corresponded to
several groups in the Ogoshi system. Ogoshi’s anastomosis groups AG-A, AG-G, and
AG-I have been implicated in BRR of strawberries (Martin, 1988). Rhizoctonia

fragariae is represented in groups AG-A, AG-G, and AG-I. Isolation frequency and
virulence vary between and within each group and by site (Husain and McKeen, 1963;
Burpee et al., 1980; Martin, 1988; Mass, 1998; Martin, 2000). Martin (2000) found that

isolates within AG-I were particularily virulent and that AG groups differed between

189

locations, and even within a single location depending on the time of year. Each of these
AG’s have different host ranges that should be considered before incorporating into a
rotation prior to strawberry (Martin, 1988)

Due to the variability observed in disease development in ﬁeld situations, and
even in more controlled greenhouse environments, an efﬁcient and expedient way to
evaluate isolate pathogenicity was sought to ensure an optimum isolate was used in future

trials. Therefore, an in-vitro virulence assay was evaluated.

Materials and Methods
Isolate Selection and Staining

Seven isolates were randomly chosen from the cultures maintained in Dr.
Annemiek Schilder’s laboratory at Michigan State University, East Lansing, MI. The
only prerequisite was that the isolates had been recovered from strawberry roots. Prior to
the evaluation, the isolates were sequenced for identiﬁcation by Dr. Mursel Catal,
Michigan State University, East Lansing, MI. They were also stained with acridine
orange to determine the nuclear complement with the assistance of Dr. Mursel Catal
(Sambrook et al., 1989; Altschul et al., 1990; Séndor et al., 2000). All isolates were
obtained from strawberry plants in Michigan, except hasp06-121, which was isolated
from strawberries in Oregon, and hasp06-115 which originally came from the Driscoll
Strawberry Institute in California. Both haspO6-121 and hasp06-1 15 were provided

by Dr. Gerry Adams, Michigan State University, East Lansing, MI (table 60).

190

Root Preparation

Daughter plants were propagated in sterile, 2 NS grade sand from ‘Allstar’
strawberry plants obtained from Krohne Plant Farms (Hartford, MI). Roots were surface
sterilized in 20% bleach solution for 5 min to eliminate any organisms that may inhibit
infection. The daughter plants were approximately 6 months old. The roots were rinsed
twice in sterile water for l min, and left in ﬁnal sterile water wash until ready for plating
on 100 x 15 mm petri plates containing water agar. These plates were prepared by
placing an agar plug of an isolate in the middle of the water agar plate. Then, after being
dried on a sterile paper towel, root pieces were placed between the agar plug and the side
of the petri dish. The plugs were surrounded by ﬁve 6-mm, secondary root pieces placed
25 mm from the plug. There were a total of 15 root pieces per isolate. The experiment
was repeated in May 2007 using the same procedure with the exception that the root
pieces came from three different daughter plants that were approximately 6 wk old. In
June 2007, the experiment was repeated again, using plants that were approximately 12

wk old.

Inoculum Preparation

A 6-mm agar plug from ten different Rhizoctoniafragariae isolates grown on
potato dextrose agar (PDA) for 5 d was taken and placed in the middle of separate water
agar plates. A sterile PDA agar plug served as a control. The plates were not sealed with

paraﬁlm.

191

Evaluation
After inoculation for 12 tol3 d at 25°C on a laboratory bench under ambient light,
each root piece was evaluated for necrosis on a scale of 1-4 (1-<25% necrosis, 2-25-50%

necrosis, 3—50-75% necrosis, 4->75% necrosis).

Analysis
All statistical analyses was preformed with a one way ANOVA and mean
separations (Fisher’s protected least signiﬁcant difference test at p=0.05) in StatGraphics

Plus 4.1 (StatPoint Inc., VA) after checking for equality of variance.

Results and Discussion

The water agar allowed the isolates to grow and establish on minimal nutrition.
After approximately 5 (1, each isolate had colonized the entire plate. With the low
nutrient level of water agar, the theory was that the fungus would penetrate the
strawberry root in order to sustain itself. This penetration would be evident as the tissue

was degraded and darkened, which did occur when compared to the control.

192

 

Table 60. Percent necrosis caused by Rhizoctoniaﬁagariae isolates from strawberries in
an in-vitro evaluation of virulence and anastomosis group at Michigan State University,

 

 

 

East Lansing, MI.x
Root Necrosis Rating (1-4) w
Over All
Anastomosis First Second Third Three
Isolate Groupy Experiment Experiment Experiment Experiments
1 Control - 1.00 NS 1.00 NS 1.00 NS 1.00 a
2 Rhfr03-038 AG-A 2.80 1.67 2.00 2.16 bc
3 RhfrO3-047 AG-A 2.47 1.40 2.53 2.13 bc
4 Rhﬁ03-057 AG-G 2.93 1.60 2.00 2.18 bc
5 Rhfr03-077 AG-l 2.53 1.53 - 2.15 bc
6 RhfrO3-083 AG-G 2.67 1.60 2.80 2.36 bc
7 RhfrO5-096 AG-A 2,30 1.60 2.80 2.40 bc
8 hasp05-103 AG-A 2,40 1.20 2.80 2.13 bc
9 hasp06-1157‘ - 2.67 1.93 2.87 2.49 c
10 hasp06-|2|z - 2.47 1.13 2.00 1.87 b
ANOVA
Effect Signiﬁcance (p)
Isolate 0.1248 0.0876 0.0816 0.0001
Experiment - - - 0.0000
Df
Between 9 9 8 -
groups
Within groups 20 20 18 -
Isolate - - - 9
Experiment - - - 2
Residual - - - 75
Total (Corr.) 29 29 26 86

 

w Roots pieces were evaluated after inoculation for 12 to 13 d at 25°C, on a laboratory
bench for necrosis on a scale of 1-4 (1 -<25% necrosis, 2-25-50% necrosis, 3-50-75%
necrosis, 4->75% necrosis).

" Values in columns followed by differing letters are signiﬁcantly different according to
Fisher’s protected least signiﬁcant difference test p=0.05. Pairwise comparisons

performed with n=15 for each experiment and n=45 over all three. For isolate

Rhfr03077, n=30 when analyzing over all three experiments.
y Anastomosis groups determined by Dr. Mursel Catal using ITS sequencing and nuclear
staining with acridine orange.
2 Isolates originally from Dr. Gerry Adams laboratory at Michigan State University, East
Lansing, MI. Anastomosis group not determined for these isolates.

193

All isolates sequenced and stained were identiﬁed as Rhizoctoniafragariae. In all
experiments, with respect to the amount of necrosis they caused on the root pieces, the
isolates were not signiﬁcantly different from one another or the control. Isolate Rhfr03-
077 was not evaluated a third time because there were not enough root pieces to allow a
full assessment of the isolate. Isolates 115 caused the most necrosis over the three
experiments (table 60).

Virulence did not differ amongst isolates, but only a small number were tested.
The only difference in procedure over the three experiments was the age of the plant.
The ﬁrst experiment utilized the oldest plants, followed by the third evaluation, and the
second evaluation had the youngest plant tissue. The results observed may be a result of
the older plants having the potential to receive more root damage as they were moved in
their pans in the greenhouses or damage received upon removal from the sand. Older
plants may also have more naturally occurring damage and openings in their roots that
allow penetration as well. This would be indicative to observations made in the ﬁeld

where older plantings are more likely to have this disease develop.

194

LITERATURE CITED

Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. .I. 1990. Basic local
alignment search tool. J. Mol. Biol. 215:403-410.

Burpee, L. L., Sanders, P. L., and Cole Jr., H. 1980. Anastomosis groups among isolates
of C eratobasidium cornigerum and related fungi. Mycologia 72:689-701.

Husain, S. S., and McKeen, W. E. 1963. Rhizoctoniaﬁ'agariae sp. nov. in relation to
strawberry degeneration in southwestern Ontario. Phytopathology 53:532-540.

Maas, J. L. 1998. Compendium of Strawberry Diseases. 2nd Ed. American
Phytopathological Society.

Martin, F. N. 2000. Rhizoctonia spp. recovered from strawberry roots in central coastal
California. Phytopathology 90:345-353.

Martin, S. B. 1988. Identiﬁcation, isolation frequency, and pathogenicity of anastomosis
groups of binucleate Rhizoctonia spp. from strawberry roots. Phytopathology 78:379-
384.

Ogoshi, A. 1985. Anastomosis and intraspeciﬁc groups of Rhizoctonia solani and
binucleate Rhizoctonia. Fitopatol. Brasileira 10:371-390.

Ogoshi, A., and Ui, T. 1983. Anastomosis groups of Rhizoctonia solani and binucleate
Rhizoctonia. Pages 57-58 In Ecology and Management of Soilborne Plant Pathogens.
C. A. Parker, A. D. Rovira, K. J. Moore, and P. T. W. Wong, Eds. American
Phytopathological Society. St. Paul.

Sambrook, J ., Fritsch, E. F., and Maniatis, T. 1989. Molecular Cloning: A Laboratory
Manual, 2nd Edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

Séndor, E., Karaffa, L., Paul, G. C., Pocsi, 1., Thomas, C. R., and Szentinnai, A. 2000.
Assessment of the metabolic activity of Acremonium chrysogenum using acridine

orange. Biotechnology Letters 22:693-697.

Vilgalys, R., and Cubeta, M. A. 1994. Molecular systematics and population biology of
Rhizoctonia. Annu. Rev. Phytopathol. 32:135-155.

195

APPENDIX A
INOCULATION PROCEDURE EVALUATION

Introduction

Previous experiments (Olatinwo and Schilder, 2002; Sabaratnarn and Schilder
unpublished data) had varying success in achieving BRR symptoms in greenhouse
experiments. The inherent variability in disease pressure made it difﬁcult to create BRR
artiﬁcially, and consistently, in the greenhouse for further study. Evaluation of past
techniques, and additional techniques (Martin, 2000), was undertaken to aid in future

efficacy trials.

Materials and Methods
Plant Material

One or two ‘Allstar’ strawberry plants were propagated from mother plants from
Krohne Plant Farms, Inc. (Hartford, MI). Plants were placed in 10 x IO-cm black plastic
pots ﬁlled with sterilized 2 NS-grade sand that had been autoclaved in a metal bin at
121°C for at least 16 h. The establishing daughter plants were kept in place on the sand
using paper clips so that they would not touch the table top. The plants were kept outside
on benches between greenhouses at Michigan State University, East Lansing, MI.
Strawberry plants used in the experiment were selected for uniformity in size and root

mass.

196

Inoculum Techniques and Preparation

Two inoculation techniques were evaluated, with the pathogens either grown on
bran or as a mycelial suspension. The bran inoculations were at the rates of 0.75% and
1.5% wat using the Rhizoctonia spp. (isolate Rhfr0303 8) and the Pythium sp. (isolate
Pyth03007), both available from Dr. Annemiek Schilder’s laboratory at Michigan State
University, East Lansing, MI. These isolates were originally obtained from strawberry
roots in Michigan. Both were chosen due to previous work showing that they were
virulent (Sabaratnam and Schilder, unpublished). These inoculation techniques were
done in both autoclaved and steamed sandy-sandy loam with both pathogens (table 61).
On 14 September 2005, previously steamed sandy—sandy loam soil was autoclaved at
121°C for at least 16 h. Sandy-sandy loam soil (steamed at least 45 min at 82°C) is
available from the Michigan State University greenhouse. Standard clay pots (13 x 13
cm) were used and placed on individual plastic trays. Each pot contained approximately

1 kg of oven dry soil.

197

Table 61. Inoculation method, soil treatment, and pathogen inoculated in greenhouse
experiment to develop an inoculation protocol for creating black root rot conditions in
greenhouse experiments using strawberry cv. Allstar in East Lansing, MI in 2005.

 

 

 

 

Inoculation Soil Condition Pathogen
0.75% bran" Autoclaved None
0.75% bran Autoclaved Pythium
0.75% bran Autoclaved Rhizoctonia
0.75% bran Steamed None
0.75% bran Steamed Pythium
0.75% bran Steamed Rhizoctonia
1.50% bran Autoclaved None
1.50% bran Autoclaved Pythium
1.50% bran Autoclaved Rhizoctonia
1.50% bran Steamed None
1.50% bran Steamed Pythium
1.50% bran Steamed Rhizoctonia
Mycelium Autoclaved None
Mycelium Autoclaved Pythium
Mycelium Autoclaved Rhizoctonia
Mycelium Steamed None
Mycelium Steamed Pythium
Mycelium Steamed Rhizoctonia

 

* Wt/wt of bran mixed with soil.

I98.

The bran inoculum was created by putting 3 to 4 PDA-Amp plugs of each isolate
into separate ﬂasks that contained 169 g oat bran/100 ml distilled water that had been
autoclaved for 30 min. The fungus was allowed to grow at 25°C for one week before
being mixed with a sterile rod. Growth continued at the same temperature for another
week, after which the bran was allowed to air dry in a laminar ﬂow hood. The procedure
was similar to the one used by Martin (2000) except that the bran was not ground and
passed through sieves. The drying process took several days, and the bran was stored
overnight on a laboratory bench. After drying, the bran was broken to a consistent size
(<2.5 cm). There were three pots per soil x inoculation x pathogen combination placed in
a completely randomized design. Each pathogen x soil combination was thoroughly
mixed with the soil (1 kg oven dried/pot) in separate 52 cm x 64 cm x 6.4 cm autoclaved
aluminum pans.

For the mycelium inoculation, each fungus was grown separately in sterile potato
dextrose broth using fungal agar plugs to inoculate the broth. The cultures were allowed
to grow for seven days and then passed through a sterile Buchner funnel to collect the
mycelium on sterile Watman ﬁlter paper. The mycelium of each fungus was pressed dry
with sterile paper towels and re-suspended in sterile deionized water, to create a 100
ml/pot suspension. The suspension was added, as the strawberries were planted, at the
rate of 20 ml/pot. Sterile water served as the control. All 54 plants were allowed to grow
for six weeks before evaluation. Watering was done by hand using a plastic beaker as

needed, to prevent cross contamination.

199

Evaluation

The plants were removed from their pots and their roots were washed under
running water. All three plants from each inoculation x soil x pathogen combination
were placed on a plastic tray with water to keep them moist. Figure 1 shows the
qualitative rating scale used to rate roots from 1-5: 1: <20% of root mass is secondary
roots, 2= 20-40% of root mass is secondary roots, 3= 40-60% of root mass is secondary
roots, 4= 60-80% secondary roots, 5= >80% of root mass is secondary roots. Percent of

the root surface that was necrotic was also visually assessed.

Pathogen Recovery

Five root pieces from the edges of ﬁve lesions, for each treatment, were taken for
plating on water agar. These were kept in petri dishes with moist Watman paper at 4°C
until processing. The root pieces were surface sterilized for 2 min in 20% bleach solution
and rinsed in two sterile water baths for l min each, dried on sterile paper towels and then
placed on water agar. Fungi growing from the root pieces were subcultured onto V8
media (163 ml V8 juice, 1.8 g CaCO3, 15 g agar/1 L) and subsequently identiﬁed using
morphological characteristics after plate colonization. Those fungi that could not be
identiﬁed in this manner were subsequently processed for DNA sequencing from the
internal transcribed spacer (ITS) region by Dr. Mursel Catal (Sambrook et al., 1989;

Altschul et al., 1990).

200

 

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201

Data Analysis

All statistical analyses were performed using the ANOVA and mean separation
procedure (Fisher’s protected least signiﬁcant difference test at p=0.05) in StatGraphics
Plus 4.1 (StatPoint Inc., VA) after initially checking for equal variance. In the analysis
the mycelial suspension procedure was removed because the lack of necrosis and
resulting excellent root quality, did not allow for a valid variance check. In the analysis,
model variance components were estimated due to bran level (B), soil treatment (S),

pathogen (P), B x S, B x P, S x P, B x S x P, and error.

Results and Discussion

It seemed that the autoclaving process had its own impact on the plant with
autoclaved soil having a low root quality rating and high percent necrosis (table 62). The
B x S and B x S x P interactions were always signiﬁcant. However, pathogen presence
was not different from the absence of a pathogen in root quality or percent necrosis (table
62). In addition, the bran inoculum may have been contaminated with bacteria which
may have altered the true inoculum density and could have had a negative impact on the
strawberry roots.

Treatments with mycelial suspension of Rhizoctonia, Pythium, or none had
average root quality ratings of 5, regardless of soil, except for Rhizoctonia in steamed soil
which had an average root quality rating of 4.33. In addition, the mycelial suspension of
Rhizoctonia had 5.0% and 3.7%, Pythium had 5.0% and 0.0%, and no pathogen had 6.7%

and 0.0% average root necrosis in autoclaved and steamed soil, respectively.

202

Table 62. Effects of inoculation, soil treatment, and pathogen on root health of cv. Allstar
strawberries in a greenhouse experiment to develp an protocol for creating black root rot
conditions in East Lansing, MI in 2005.x

Root quality rating (1-5)z

 

Root necrosis ("/o)y

 

Bran Quantity
0.75%
1.50%

Soil Condition
Autoclaved
Steamed

Pathogen
Rhizoctonia
Pythium
None

ANOVA
Effect

U
-h

3.83a
3.17 b

2.39 a
4.61 b

3.25 NS

3.83
3.42

15.83 NS
38.00

42.83 a
11.00 b

28.75 NS
25.08
26.92

Signiﬁcance (p)

 

Bran Quantity (B)
Soil Condition (S)
Pathogen (P)
Block

BxS

BxP

SxP

BxSxP

Residual

Total (Corr.)

N
N

35

0.045l
0.0000
0.3138
0.7569
0.0213
0.0754
0.6703
0.0335

0.0613
0.0000
0.2968
0.72] 1
0.0028
0.8883
0. 1602
0.0241

 

" Values in columns followed by differing letters are signiﬁcantly different according to
Fisher’s protected least signiﬁcant difference test at p=0.05. Pairwise comparisons
performed with n=3.
y Statistical analysis was performed after log(x) transformation.

2 Qualitative rating scale used to rate roots from 1-5: I= <20% of root mass is secondary
roots, 2= 20-40% of root mass is secondary roots, 3= 40-60% of root mass is secondary
roots, 4: 60-80% secondary roots, 5= >80% of root mass is secondary roots.

203

Rhizoctonia sp. was recovered from roots of plants in every inoculation technique
x soil combination that had Rhizoctonia as the inoculated pathogen. In addition,
Rhizoctonia was recovered from the roots of plants from the mycelium x steamed soil
combination in which only Pythium sp. had been inoculated. Pythium sp. was re-isolated
from treatments that were and were not inoculated with Pythium as shown in table 63.
F usarium spp. was found to be the most common contaminant, being isolated from roots
several of the inoculation techniques x soil combinations, regardless of whether the pots
were inoculated with Pythium sp., Rhizoctonia sp., or neither. Other fungi isolated from
root pieces included Aureobasidium spp., Arthrobotrys spp., C ephalosporium spp., and

Stachybotrys spp.

204

Table 63. Fungal genera recovered from roots of cv. Allstar strawberries in a greenhouse
experiment to develp a protocol for creating black root rot conditions in East Lansing, MI

 

 

 

 

in 2005.
Fungal Presence"
Inoculation Soil Pathogn Rhizoctonia Pythium Fusarium Other
0.75% autoclaved Control - - X X
0.75% autoclaved Pythium - X - X
0.75% autoclaved Rhizoctonia X - - -
0.75% steamed Control - X X -
0.75% steamed Pythium - X - -
0.75% steamed Rhizoctonia X - - -
1 .50% autoclaved Control - X X X
l .50% autoclaved Pythium - - X -
I .50% autoclaved Rhizoctonia X - - -
1 .50% steamed Control - - X -
I .50% steamed Pythium - X - -
1 .50% steamed Rhizoctonia - - -
Mycelium autoclaved Control - X X
Mycelium autoclaved Pythium - - - X
Mycelium autoclaved Rhizoctonia X - - -
Mycelium steamed Control - - X
Mycelium steamed Pythium X - - -
Mycelium steamed Rhizoctonia X - - -

 

* Plated root tissue from ﬁve lesions for each treatment.

205

The largest concern was that the Pythium sp., upon identiﬁcation with DNA
sequencing of the ITS regions, turned out to be Geomyces pannorum, completely
invalidating any conclusions that could be drawn from the Pythium inoculations and in
fact confounding the autoclaved versus steam soil conclusions. Geomyces is a ubiquitous
saprophyte. Another isolate Pyth03-008, available from Dr. Annemiek Schilder‘s
laboratory, Michigan State University, East Lansing, MI, was identiﬁed as Pythium
ultimum var. ultimum. From this experiment, it was determined that it was sufﬁcient to
autoclave soil for less time, in smaller batches and that bran inoculum needs to be
evaluated more thoroughly. The autoclave processed used in this experiment may have
inhibited nutrient availability and released toxic compounds into the soil due to the length

of time.

206

LITERATURE CITED

Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. .1. 1990. Basic local
alignment search tool. .1. Mol. Biol. 215:403-410.

Martin, RN. 2000. Rhizoctonia spp. recovered from strawberry roots in central coastal
California. Phytopathology 90:345-353.

Olatinwo, R. 0., and Schilder, A. M. C. 2002. Transplant root dips with biocontrol
agents reduce strawberry black rot. Phytopathology 92:861.

Sambrook, J., Fritsch, E. F., and Maniatis, T. 1989. Molecular Cloning: A Laboratory
Manual, 2nd Edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

207

APPENDIX B
INOCULATION OPTIMIZATION
Introduction
After the inoculation procedure evaluation experiment, further assessment was
needed. Again, a previous inoculation procedure was re-evaluated (Olatinwo and
Schilder, 2002) and improvements made upon new techniques that had been used in the
previous experiment. The purpose of this experiment was to further evaluate and ﬁnd the
optimum bran quantity that would cause disease. Weed blocking fabric was used to

examine the prevention moss on the soil surface of pots.

Materials and Methods

Plant Material

During March and May 2007, daughter plants were propagated from ‘Allstar’
mother plants (Krohne Plant Farms, Inc., Hartford, MI) into 52 x 64 x 6.4-cm aluminum
pans ﬁlled with 2 NS-grade sand kept in a greenhouse at Michigan State University, East
Lansing, MI. The pans and sand were autoclaved for 4 h at 15 psi. The mother plants

were forced to produce runners in the greenhouse under lights set at a 12 h day length.

Pathogen Evaluation in Planting Stock
Prior to the experiment, ﬁve, 6-mm long root pieces (10 pieces/plant) of three
randomly selected daughter plants were placed on water agar. Root pieces were selected

for possible lesion formation. Root pieces were sterilized for 2 min in 20% bleach

208

solution, followed by two l-min rinses in sterile deionized water. The root pieces were
dried on sterile paper towels before placing on the media. Evaluation was performed

after plate colonization. No fungal growth was observed.

Inoculum Preparation

The Rhizoctoniafragariae inoculum was grown from 2 plugs of PDA-Amp agar
placed on autoclaved oat bran in ﬂasks with 0.6 ml/g of sterile distilled water. After one
week of growth at 25°C the bran was stirred with a sterile rod; after the second week the
bran was air dried in a laminar ﬂow hood for two days. The bran was stored covered on a
laboratory bench overnight. The bran was mixed the morning of each day to aide in
drying. The bran was ground with a mini prep plus food processor (Cuisinart, Stamford,
CT) after drying for 22 h, except for the unground bran treatment which was simply
broken to an approximate consistent size. A 6.30 mm sieve was used to ensure that the
bran was less than 0.6 cm in size. The procedure was used by Martin except that the bran
was not passed through nested sieves (Martin, 2000). Each pot held approximately 1 kg

ofsoﬂ.

Initial Pathogen Evaluation in Soil

Five grams of soil taken from a single pot in each of the four autoclaved batches
of soil was mixed with 125 in] sterile deionized water in sterile 125 ml ﬂasks to create a
I :25 dilution. One milliliter of this dilution was plated onto semi-selective media (water
agar, 100 mg/L streptomycin sulfate and penicillin-G sodium salt, and 800 ul/L sodium

hydroxide) or PDA for a total of two plates per batch of autoclaved soil. A sterile bent

209

glass rod was used to spread the soil dilution over the plate evenly. and the plates were

evaluated 4 (I later. No fungal growth was observed.

Inoculation

On 16 May 2007, 13 x l3-cm standard clay pots containing the greenhouse mixed
sandy-sandy loam were autoclaved for 4 h. The following day each pot was individually
mixed with the necessary wt/wt inoculum in sterile aluminum pans. Each bran quantity
was evaluated with and without Rhizoctonia. Control pots had autoclaved bran. The
PDA plate inoculation consisted of three, 15-d-old Rhizoctonia cultures grown on 20 ml
PDA plates. The cultures were started from 6-mm discs from a culture kept on PDAamp.
These plates were cut into quadrants and placed in half ﬁlled pots. The pots were ﬁlled
and strawberries planted. Control pots had only sterile PDA media placed in the pots.
Both the control and Rhizoctonia plates were kept unwrapped on a laboratory bench at
25°C. Weed blocking fabric (IS-year commercial landscaping fabric) was placed around
plants in certain treatments, as indicated in table64. This material was cut to cover the
entire surface of each pot with a hole cut in the middle to allow for planting and was
evaluated for its effect on the experiment. Treatments were replicated ﬁve times with
each replication consisting of one pot placed on an individual inverted plastic tray.
Flowers and runners were removed if they developed. Watering was done carefully to

prevent cross contamination.

210

Evaluation

All plants were evaluated 8 wk after planting. Roots were visually rated using a
qualitative scale of l-5 (Scale of 1-5: l= <20% of root mass is secondary roots, 2= 20-
40% of root mass is secondary roots, 3= 40-60% of root mass is secondary roots, 4= 60-
80% secondary roots, 5= >80% of root mass is secondary roots (Appendix A, Fig.1).
Fresh weight of the foliage and roots were obtained separately by splitting the crown
horizontally just above the uppermost adventitious roots. These were then dried in a
gravity convection oven set at 80°C for 48 h and dry weights were obtained. Total fresh

and dry weights were obtained by adding the foliage and root weights together.

Data Analysis
All data were analyzed using the ANOVA and mean seperations (Fisher’s
protected least signiﬁcant difference test at p=0.05) in .StatGraphics Plus 4.1 (StatPoint

Inc., VA) after checking for equality of variance.

Results and Discussion

The unground bran treatments served as a comparison since unground bran had
been used in the 2005 and 2006 greenhouse efﬁcacy experiments (chapter 2). In all other
treatments of this trial utilizing the bran carrier, the bran was ground.

The treatments having 1.50% bran with and without fabric allowed the evaluation
of the surface cover to see if that assisted in disease development. The grinding did not
make a signiﬁcant difference in plant weights when compared to the unground bran
treatments (table 64). The fabric did not inﬂuence disease development when

considering the parameters measured. It did become clear from this trial that 3% bran

211

could provide a signiﬁcant difference between plants inoculated with and without
Rhizoctonia. Only with 3% bran was there a difference between the plants placed in pots
inoculated with Rhizoctonia versus plants placed in non-inoculated pots that had only the
bran. In addition, plants in the non-inoculated pots did not differ from plants in pots that
contained no bran (table 64). This was consistent for every parameter measured. The
PDA plates made a difference between plants in inoculated and noninoculated pots.
Perhaps if the experiment had been run longer the PDA plates would also work.

The grinding procedure was adopted for the 2007 greenhouse efﬁcacy
experiment. Grinding does create a more consistent inoculum and more propagules to
infest the pot. In addition, it appears that bran can have a negative inﬂuence on
strawberry grth if it gets too high as evidenced by the 5.00% bran inoculation level.
Consideration should be made in evaluating additional carriers and amounts to maximize

the inoculation protocol.

212

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213

APPENDIX C

RISK RATINGS FOR MAJOR PARASITIC NEMATODES OF STRAWBERRIES
IN MICHIGAN

Table 65. Risk ratings of major parasitic nematodes found on strawberries grown in
Michigan. (Data furnished by Fred Warner, Michigan State University Diagnostic
Services)

 

Samples collected in fall prior to a strawberry crop (100 cc soil)
Pralylenchus Meloidogvne Longia’orus X iphinema C riconemel/a Trichodorus

 

Risk rating penetrans spp. elongatus americanum spp. spp.
0 0 0 0 0 0 0

1 1-5 1-5 1 1-5 1-20 l-5

2 6-15 6-20 2-5 6-15 21-50 6-15
3 16-30 21-50 6-15 16-30 51-100 16-30
4 31-50 51-100 16-30 31-50 101-250 31-50
5 >50 >100 >30 >50 >250 >50

 

Samples collected in the fall prior to a strawberry crop (1.0 g root tissue and 100 cc soil)
Pralylenchus Meloidogyne Longidorus X iphmema Criconemella Trichodorus

 

Risk rating penetrans spp. elongatus americanum spp. spp.
0 0 0 0 O 0 O

1 l-lO 1-20 1 1-5 1—20 1-5

2 11-25 21-50 2-5 6-15 21-50 6-15
3 26-50 51-100 6-15 16-30 51-100 16—30
4 51-100 101-200 16-30 31-50 101-250 31-50
5 >100 >200 >30 >50 >250 >50

 

Samyles collected in a strawberry crop (1.0 g root tissue and 100 cc soil)
Pralylenchus Meloidogyne Longidorus X iphinema Criconemella Trichodorus

 

Risk rating penetrans spp. elongatus americanum spp. spp.

0 O 0 O O 0 0

1 1-10 1-20 l-S 1-10 1-20 HO

2 11-25 21-50 6-15 11-25 21-50 11-25
3 26-50 51-100 l6-30 26-50 51-100 26-50
4 51-100 101-200 31-50 51-100 101-250 51-100
5 >100 >200 >50 > 100 >250 >100

 

* Risk Rating: 0=none, 1=1ow, 2=low-moderate, 3=moderate to high, 4=high, and
5=severe. A rating 23 would result in the recommendation of control measures.

214

   

 

 

 

 

 

 

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