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This is to certify that the

dissertation entitled

Influence of Biological Agents
on Asparagus Decline Syndrome

presented by

Marshall Kent Elson

has been accepted towards fulfillment
of the requirements for

Ph.D. degree in Horticulture

fl Major p m) ‘

 

 

Date “/18/9/‘5

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MSU Is An Affirmative Action/Equal Opportunity Institution
mena-p

 

INFLUENCE OF BIOLOGICAL AGENTS
ON ASPARAGUS DECLINE SYNDROIVIE

By

Marshall Kent Elson

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

DOCTOR OF PHILOSOPHY

Department of Horticulture

1993

ABSTRACT

INFLUENCE OF BIOLOGICAL AGENTS
ON ASPARAGUS DECLINE SYNDRONIE

BY
Marshall Kent Elson

Asparagus decline syndrome is caused by Fusan'um oxysporum f.sp. asparagi
(FOA) and F. moniliforme (FM). Effective methods for controlling Fusarium spp. in
asparagus are not available currently. Resistant varieties have not been found in
asparagus. Chemical treatments have failed to increase crown survival or yield of
asparagus.

Brassica spp. residues have been shown to control Fusarium diseases in other
crops. Seven brassicas were incorporated in the field and greenhouse. The brassicas
included ’Premier' kale (B. oleracea L. virdis), ’Rondo’ turnip (B. rapa L.), ’Global’
canola (B. napus L. annua), ’Glacier’ canola (B. napus L. biennis), yellow mustard
(B. nigra L.), ’Dwarf Essex’ oilseed rape (B. napus L. biennis), ’Humus’ oilseed rape
(B. napus L. biennis) and sorghum-sudangrass (Sorghum bicolor sudanensé) as the
control. Brassica residues had no effect on FOA or FM, but were phytotoxic to several
indicator crops. Chemical extraction of the brassica residues did not yield any Fusarium-
inhibitory compounds.

Several soil-bome microorganisms have been found inhibitory to Fusarium

diseases in other crops. A novel Streptomyces spp. (ME2-27-19A) was isolated from

 

asparagus field soil and found inhibitory to FOA and FM in vitro. Chemical extraction
of MEZ-27—19A yielded a fraction that was inhibitory. The inhibitory fraction was
purified by vacuum-liquid chromatography and C18 medium-pressure chromatography
and found inhibitory at 50 ug'ml“. ME2-27-19A extract produced variable control FOA
or FM in vitro, and was phytotoxic at 1000 ug'ml". In soil, ME2-27-19A extract
reduced the Fusarium population at 100 ug‘ml", but also reduced the asparagus shoot
length.

ME2—27-19A cells, applied as a root-dip in vitro, reduced the disease incidence
in asparagus. In soil, ME2-27-19A decreased the number of root lesions caused by
Fusarium spp., but also reduced the root length of the asparagus. ME2-27-19A cells
colonize the entire root system in sterilized soil, but not at sufficient concentration to
inhibit FOA and FM. Maltodextrin was a satisfactory carrier for ME2-27-19A and other
biological control agents. ME2-27—19A may compete with FOA and FM, but it is not

capable of controlling disease.

 

ACKNOWLEDGEMENTS

I would like to acknowledge the many people who have contributed to this
research. Generous thanks to Dr. John F. Kelly, my major professor, for his
encouragement and guidance throughout this project. Gratitude is extended to my
graduate committee for their time and efforts. Thanks to Dr. Muraleedharan G. Nair for
his enthusiasm and interest in my project, and direction of the actinomycetes work. My
graditude to Dr. Stanley K. Ries for helping me with statistics and allowing me to serve
as his teaching assistant in "Research Procedures for Plant Science" class. My
appreciation is extended to Dr. Christine T. Stephens for her willingness to share her
knowledge of plant pathology necessary for this interdisciplinary research project.
Thanks to Dr. Francis J. Pierce for his unique perspective and knowledge of soils and
cover—cropping systems. I would also like to acknowledge the efforts of Dr. Hugh C.
Price for initiating the brassica project and serving as my major professor for the first
two years.

My appreciation is extended to Dr. Bernard H. Zandstra for allowing me to use
the laboratory and greenhouse space after Dr. Price left and to Dr. Alvin J. M. Smucker

for use of the video imaging system.

 

 

Financial support was provided by the USDA Grant, titled "Asparagus Decline
Syndrome and Replant Problem in Michigan" and the Michigan Asparagus Growers
Association. Thanks to Max Kokx for providing the field site and John Baker for field
assistance in Oceana County.

I also wish to acknowledge the laboratory and field assistance of Phil Throop,
Shelia Linderman, Paul Pilos, Rhonda James, and Kathy Nelson. Special thanks to
Rebecca Baughan, Deborah Thorogood Williams, Anu Rangarajan, Amitabh Chandra and
Yu-Chen Chang for friendship, assistance and intellectual stimulation.

Finally, I acknowledge the love, encouragement and support of my parents who

have made this dissertation possible.

 

TABLE OF CONTENTS

List of Tables ......................................... xi

List of Appendices ...................................... xii

CHAPTER 1 - LITERATURE REVIEW

Introduction ...................................... l
Attempted Controls .................................. 5
Resistance ................................... 5
Chemicals ................................... 6
Salt ....................................... 6
Suppressive Soils ................................... 7
Introduced Suppression ............................... 8
Fusarium ................................... 9
Pseudomonas ................................. 10
Actinomycetes ................................ l 1
Colonization ................................. l3
Induced Suppression ................................. 15
Organic Amendments ............................ 17

vi

 

Brassica Residues .............................. 18
Brassica Compounds ............................ 20

Literature Cited .................................... 23

CHAPTER 2 - Influence of Brassica spp. residues

on Fusarium spp. in asparagus ............................... 35
Introduction ...................................... 36
Materials and Methods ............................... 37

Plants, Inocula and Media . . . . . . . . . . ............... 37
Selection in the Field ............................ 38
Soil Microflora ................................ 40
Phytotoxicity of Residues ......................... 41
Selection in the Greenhouse ........................ 42
Solarization in the Field .......................... 43
Extraction of Compounds ......................... 44
Conversion of Compounds ......................... 46
Results and Discussion ............................... 47
Selection in the Field ............................ 47
Soil Microflora ................................ 47
Phytotoxicity of Residues ......................... 49
Selection in the Greenhouse ........................ 51
Solarization in the Field .......................... 5 3
Extraction of Compounds ......................... 54

vii

 

Conversion of Compounds ......................... 54

Literature Cited .................................... 56

CHAPTER 3 - Influence of antifungal compounds from a soil-home

actinomycete on Fusarium spp. in asparagus .............. 58
Introduction ...................................... 59
Materials and Methods ............................... 59

Isolation and Culture ............................ 59

Extraction of cells .............................. 60

Antifungal/Antibacterial Bioassay ..................... 60

Agar Overlay ................................. 61

Purification .................................. 62

Minimum Inhibitory Concentration .................... 62

Separation by C18 Chromatography ................... 63

Insecticidal Assay .............................. 63

Phytotoxicity ................................. 64

In Vitro Assay with Asparagus and Fusarium ............. 64

Assay of ME2-27-19A Extract in Soil .................. 65
Results and Discussion ............................... 67

Agar Overlay ................................. 67

Purification .................................. 67

Minimum Inhibitory Concentration .................... 68

Separation by C18 Chromatography ................... 68

viii

Insecticidal Assay .............................. 69

Phytotoxicity ................................. 69
In Vitro Assay with Asparagus and Fusarium ............. 69
Assay of ME2-27-19A Extract in Soil .................. 72
Literature Cited .................................... 74

CHAPTER 4 - Influence of a novel soil-home actinomycete

on Fusarium spp. in asparagus ...................... 75
Introduction ...................................... 76
Materials and Methods ............................... 76

Microorganisms ............................... 76

Plants ..................................... 78

In Vitro Agar Assay ............................ 78

Greenhouse Assay .............................. 79

Video-Imaging ................................ 81

Root-Colonizing Ability .......................... 81

Maltodextrin ................................. 82
Results and Discussion ............................... 82

In Vitro Agar Assay ............................ 82

Greenhouse Assay .............................. 84

Video—Imaging ................................ 87

Root-Colonizing Ability .......................... 88

Maltodextrin ................................. 88

 

Literature Cited .................................... 90
Summary and Conclusions .................................. 92

Appendices ......................................... 94

LIST OF TABLES

Chapter 2:
Table 1. Influence of time on populations of microflora

from Spinks sandy loam field soil .................... 48
Table 2. Growth of asparagus roots (percentage of excelsior

control) 0, 2, 4, and 12 weeks after incorporation

of brassica residues ............................. 50
Chapter 3:
Table 1. Phytotoxicity of VLC fractions of ME2-27-19A crude

extract .................................... 70
Table 2. Effects of FOA and FM on asparagus seedlings

in vitro ..................................... 71
Table 3. Effects of ME2-27-19A crude extract on asparagus

seedlings inoculated with FOA or FM in

sterilized field soil .............................. 73
Chapter 4:
Table 1. Effects of FOA or FM on asparagus seedlings in vitro

after root-dip (dip) or surface-application (drench)

of ME2-27-19A ............................... 83
Table 2. Effects of ME2-27-19A supplemented with YMG on asparagus

inoculated with FOA or FM in vitro ................... 85
Table 3. Effects of MEZ-Z7-19A on asparagus inoculated with

FOA or FM in sterilized field soil in the greenhouse ......... 86
Table 4. Video imaging data from asparagus roots grown in

sterilized field soil with ME2-27-19A and FOA or FM ....... 89

xi

Appendix A

Appendix B

Appendix C

Appendix D

Appendix E

Appendix F

Appendix G

Appendix H

Appendix I

Appendix J

LIST OF APPENDICES

Effects of brassica cover crops on disease rating

and phytotoxicity ‘in the field ...................

Effects of brassica cover crops

on microflora counts in field soil .................

Growth as a percentage of excelsior control of
asparagus, cress, wheat, cucumber or cabbage roots
following incorporation of brassica residue

(2% of soil dry weight) .......................

Fusarium ratings and root growth of asparagus
seedlings following incorporation of
fresh or dry brassica residues (2% of soil dry weight)

in FOA- or FM-inoculated soil ..................

Effects of ’Humus’ cover crop and solarization on

95

96

97

disease rating and growth of asparagus in the field .......... 100

Effects of ME2—27-19A crude extract on asparagus

seedlings inoculated with FOA or FM in vitro ............. 101

Effects of benomyl on asparagus seedlings

inoculated with FOA or FM in sterilized field soil .......... 102

Effects of ME2-27-19A applied as a root-dip (dip) or
surface-application (drench) on asparagus inoculated

with FOA or FM in vitro ......................... 103

UV spectrum of Fusarium-inhibitory compound

from ME2-27-19A .............................. 105

1H-NMR spectrum of Fusarium-inhibitory

compound from ME2-27-19A in dé-DMSO ............... 106

xii

CHAPTERI

LITERATURE REVIEW

Introduction

Asparagus (Asparagus oflicinalis L.) is an important vegetable crop in the United
States. The total acreage in the United States increased from 86,675 in 1978 to 97,335
in 1987. Michigan acreage increased from 19,719 to 23,426 (Bureau of Census, 1978,
1987). The average price received at the wholesale level increased from $24.30/cwt in
1975 to a high of $59.00/cwt in 1988 (Michigan Agriculture Statistics Service, 1980,
1992). In 1991, Michigan produced 12,950 tons of ”snapped" asparagus with a value
of $14.1 million (Michigan Agriculture Statistics Service, 1992). Snapped asparagus is
hand picked by breaking the spears near the ground, giving less waste during packaging.

Michigan asparagus production averaged 1200 pounds per acre over the past
decade (Michigan Agriculture Statistics Service, 1992). However, yields of 3000 pounds
per acre are achieved in some fields (Hodupp, 1983), indicating that a majority of the
acreage is not producing at its full potential. Replanting these declining field is difficult
because 50% of young asparagus plants die within five years of planting and replanted
fields often yield only half of the expected production (Hodupp 1983).

Asparagus stands generally have a profitable life of 10 to 20 years (Tiedgens,

1924, 1926; Grogan and Kimble 1959). They reach peak production in the eighth year,

2

then spear size gradually decreases until the product is unmarketable (Grogan and
Kimble, 1959). In recent years, fields have been removed after six to eight years
because of declining yields (Takatori and Souther, 1978). This phenomenon of early
decline, known as Asparagus Decline Syndrome (ADS), threatens the profitability of the
affected fields (Grogan and Kimble, 1959). Additional symptoms of ADS are stunted,
yellow, wilted plants and blank spaces in the rows. It is caused by a complex of
organisms and allelopathic chemicals (Hartung, 1987).

Decline of asparagus was first reported as "Fusariosis" by Stone and Chapman
in 1908 (Cohen and Heald, 1941). Cook (1923) described a "Dwarf Asparagus" disease
resembling ADS. The plants were stunted and yellow, having vascular discoloration and
occurring in circular areas. Cohen and Heald (1941) were the first to attribute the
disorder to Fusarium oxysponun (Schlecht) emend Snyder and Hansen f.sp. asparagi
Cohen (FOA). Fusarium moniliforme (Seld.) emend Snyder and Hansen (FM) also is
recognized as a primary disease—causing organism (Johnson et al, 1979). Both are isolated
readily from asparagus seed and most cultivated soils (Correll et a1. , 1986; Elmer, 1989).
Inglis (1980) reported that up to 9.3% of the asparagus seed in Washington state was
infected with FOA and FM. Soil which had no history of asparagus production had low
populations of FOA and FM (Hartung et al., 1990).

Fusarium wilts are among the most common diseases of horticultural crops. They
affect asparagus, banana (Musa X paradisiana L.), cabbage (Brassica oleracea var.
capitata L.), celery (Apium graveolens L. var. dulce), cotton (Gossypium hirsutum L.),

cucumber (Cucumis sativus L.), flax (Linum usitatissum L.), muskmelon (Cucumis melo),

3

onion (Allium cepa L.), pea (Pisum sativum L.), sweet potato (Ipomea batatus Lam.),
tomato (Lycopersicwn esculentum L.), watermelon (Citrullus lanatus ('I‘humb.) Matsum.
& Nakai), aster (Aster dumosus L.), carnation (Dianthus caryophyllus L.),
Chrysanthemum (Chrysanthemum X morifloium Ramant.), gladiolus (Gladiolus X
hortulanus Bailey), and tulip (Tulipa gesnerana L.)(Louvet et a1. , 1981). Fusarium spp.
account for 80-90% of the total fungal populations in the rhizosphere of these crops
(Toussoun, 1975).

FOA causes vascular discoloration and reddish-brown, elliptical lesions on the
roots and crowns of asparagus (Grogan and Kimble, 1959). The lesions are associated
with the points of attachment of the lateral roots (Graham 1955). Fibrous roots may be
shrivelled or absent. Fusarium oxysporum strains are indistinguishable except by
pathogenicity tests (Correll et a1. 1986). The strains that are pathogenic on asparagus
also infect onion, gladiolus and celery (Elmer and Stephens, 1989) and cause stunting in
sorghum (Sorghum bicolor (L.) Moench) and sweet pepper (Capsicum annuum L.)
(Tu and Cheng, 1990). However, strains that are pathogenic on cotton, cucumber,
muskmelon, radish and sweet potato are not pathogenic on asparagus (Elmer and
Stephens, 1989). In addition, FOA can survive on other crops and wwds (Damicone et
al., 1988). Alfalfa (Medicago sativa L.), oats (Avena sativa L.), red clover (Tnfolium
pratense L.) and soybeans (Glycine max (L.) Merrill) tended to increase disease
incidence, whereas potatoes (Solanum tuberosum L.) maintained a low level of inoculum

(Graham 1955).

4

In asparagus, FM causes stem and crown rot which leads to yellowing, stunting
and wilting of the fern (Johnson et al. , 1979). FM is cross-pathogenic between corn
(Zea mays L.) and asparagus (Damicone et al., 1988), and spreads rapidly through
airborne conidia (Johnson et al., 1979). Damicone and Manning (1985) concluded, "It
is impossible to grow Fusarium-free asparagus, " since Fusarium was isolated from every
crown after just one growing season.

FOA and FM may invade at an early stage of plant growth and live as
saprophytes until a stress alters the host physiology, allowing the fungi to become
pathogenic (Nigh 1990). Injuries make asparagus more susceptible to Fusarium (Cohen
and Heald 1941; Cook 1923). Lacy (1979) found more Fusarium infection in hand-
wwded than in herbicide-treated asparagus.

Nigh (1990) suggested that FOA and FM might not be a problem if asparagus
could be maintained in an unstressed condition. Asparagus seedlings emerged best at
35 % of the water holding capacity (WHC) of the soil and emergence was lower at 10 and
80% WHC (Nigh, 1990). However, FOA showed no change in activity from 20 to 80%
WHC. Therefore, moisture stress predisposes the seedling to preemergence infection.
Cohen and Heald (1941) observed that Fusarium wilt was more abundant in low areas
and on steep, sandy slopes, where plants are likely to suffer from water stress.

Another factor of ADS is that asparagus produces several autotoxic compounds
including asparagusic, caffeic, ferulic and methylenedioxycinnamic acids (Hartung, 1987;
Hartung et al. , 1990). These autotoxins are heat-stable and water-soluble, and should

leach out of the soil (Hartung, 1987; Yang, 1982). However, asparagus debris still

 

5

contained some autotoxin eight months after the death of the plants (Yang, 1982). These
inhibitors have detrimental effects on certain microflora organisms including Pythium
(Hartung, 1983; Peirce and Colby, 1987). Caffeic and ferulic acid inhibit F. oxysporwn
f. sp. radicis-lycopersici (Kasenberg and Traquair, 1988). However, FOA, FM and some
soil bacteria are insensitive to the allelopathic substances (Hartung, 1987 ; Hazebroek et
al., 1989). Peirce and Colby (1987) determined that the filtrate from asparagus roots
predisposed asparagus seedlings to FOA infection and stimulated growth of FOA
chlamydospores. Caffeic and ferulic acids inhibited growth of asparagus seedlings and

appeared to damage root cells by erosion of the cell wall (Peirce and Miller, 1993).

Attempted Controls
Resistance. Numerous methods have been investigated in attempts to control Fusarium
wilt diseases in vegetables. Resistant varieties have been the most effective control of
Fusarium diseases in other vegetable crops (Mace et al., 1981). Although numerous
varieties of asparagus have been developed, resistance to FOA and FM has not been
found inA. ofiicinalis (Hartung, 1987). Stephens et a1. (1989) found ’Lucullus 234’ and
’Lucullus 328’ were the most tolerant cultivars. Sadowski and Knaflewski (1990) found
’Huchel’s L’, ST-3, and ’Danische Riesen’ were the most tolerant cultivars. However,
progeny of parents selected for Fusarium-resistance were among the most susceptible
when exposed to Fusarium (Bussell and Ellison, 1987). Asparagus densiflorus (Kuath)
Jessop ’Sprengeri’ and ’Myersii’ were resistant to FOA and FM, and offer potential for

resistance through recombinant breeding techniques (Stephens et al., 1989).

6

Resistance does not require elimination of Fusarium spp. from the plant roots.
In celery, the roots of the resistant cultivar were colonized with a population similar to
that of the susceptible cultivar, except that the Fusarium did not penetrate into the roots
(Elmer and Lacy, 1987). Resistance also can be overcome by pathogens. In cabbage,
monogenic type A resistance to Fusarium Yellows was overcome by Fusarium oxysporum
f.sp. conglutinans Race 5 , demonstrating the potential for the organism to mutate and the

need for additional controls (Ramirez-Villapudua et al. 1985).

Chemicals. Chemical treatments have not proven effective for control of Fusarium spp.
in asparagus. Fumigation with Vorlex (1 ,3-dichloropropene, 1,2-dichloropropane,
methyl isothiocyanate) gave variable results (Lacy 1979). Captafol drench after steaming
was effective but not practical for field situations (Komada, 1975). Crown-dip treatments
with benomyl [methyl l-(butylcarbamoyl)- 1H—benzimidazol-2-ylcarbamate] (300 ppm) and
captan [N-[(trichloromethyl)thio] -4-cyclohexene-1 ,2-dicarboximide]( 1 800 ppm) produced
no positive effect on survival or yield of asparagus (Lacy, 1979). Falloon and Fraser
(1991) observed some control with an acid formulation of thiabendazole, but it was too

expensive for field applications.

Salt. Liberal applications of rock salt (N aCl) once were recommended for production
of asparagus (Rudolph, 1921). Elmer (1992) found that asparagus seedlings grow best
when fertilized with NILNO, and NaCl. Treatments with 560-1120 kg-ha‘l of NaCl

suppressed FOA and increased asparagus dry weight, but were not herbicidal (Elmer

7

1992). The colony-forming units of FOA per centimeter of root was correlated with the
ratio of K“ to Cl“ ions. Therefore, the traditional practice of applying rock salt (2qt/yd2)
may have benefitted the asparagus plants as well as controlling weeds (Elmer, 1989).
Chloride ions were shown to inhibit FOA (Elmer 1992). Salt (NaCl) may
suppress Fusarium spp. through a fungistatic effect or by stimulating host resistance in
the asparagus (Elmer 1992). Chloride ions are associated with nitrate uptake and malate

synthesis, which may affect root exudation (Elmer 1992).

Suppressive Soils

Naturally occurring Fusarium-suppressive soils have been identified for over 100
years (Campbell, 1989). Disease-suppressive soils impede the development of specific
soil-bome diseases when susceptible plants are cultivated.

The classic example of naturally occurring suppression of F. oxysporum f. sp.
cubense in banana was studied by N .J . Volk (United Fruit Company, La Lima,
Honduras) and summarized by Stover (1962). Volk found that pH affected the
productive life of banana plantings (Wardlaw, 1972). Reinking and Manns (1933)
conducted extensive physical and chemical analyses of the suppressive soils, but did not
identify the Fusarium-suppressive e1ement(s). Since montmorillonite clay was found in
all of the suppressive soils, Reinking and Manns (1933) concluded that soil type affected
the survival of Fusarium spp. rather than pH. However, sandy loam soils also have been

found to be suppressive (Scher and Baker, 1980).

 

8

Other Fusariwn-suppressive soils also have been found that have a high clay
content (Walker and Snyder, 1933) or a high WHC (Cook and Schroth, 1965; Burke
1965). Stover (1962) suggested that the clay mineralogy maintained the soil moisture
content, stimulating antagonistic bacteria. Stover (195 3) studied the use of flooding for
control of Fusarium spp. in banana in Central America. All of the Fusarium species
tested were reduced by 50-85 % soil saturation. Six months of flood fallowing were
sufficient to eliminate F. oxysporum f. sp. cubense from the soil (Wardlaw, 1972).

Other researchers have found that soil pH influences germination and growth of
Fusarium spp. The optimum pH for germination of Fusarium oxysporum is 5-6 (Griffin,
1981). Jones et al. (1989) report that liming soils to pH 6.5-7.5 decreased Fusarium wilt
of tomato (F. oxysporum f. sp. lycopersicz). Raising the soil pH to 7.0 was as effective
as pasteurizing soil with a pH of 5.5 (Jones and Overman, 1971). Searseth (1945)
reported complete control of Panama disease of banana (F. oxysporum f. sp, cubense)
with heavy lime applications (12-15 tons/acre) and deep plowing (2 ft). Liming could
be effective in asparagus because asparagus tolerates pH up to 7.5. Alabouvette (1990)
claims that high pH does not directly affect Fusarium spp., rather it influences the
microbial interactions. Actinomycetes and bacteria are favored by high pH (Waksman,

1927), making them more competitive with Fusarium spp.

Introduced Suppression
Couteaudier et al. (1985) found that transferring 1 ppm of the Fusarium-

suppressive Chateaurenard soil established suppression in a conducive soil. Loss of the

 

9

suppression by autoclaving and selective growth of saprophytic Fusarium spp. in the
suppressive soil support the case for microbial competition (Smith and Snyder, 1972).
However, transferring suppressive soil to conducive soil is not a practical method of
introducing suppression. Therefore, a basic concept of introduced suppression is to
isolate antagonistic microorganisms from suppressive soils and inoculate them into
conducive soil. The information on introduction of organisms is complicated and

fragmented (Mutitu et al., 1991).

Fusarium. Several microorganisms have been isolated from Fusarium-suppressive
soils. It was observed that the population of non-pathogenic Fusarium spp. in the
suppressive Chateaurenard soil was ten times greater than in a similar conducive soil
(Alabouvette et al. , 1983). Several non-pathogenic fusaria were isolated and tested for
suppression of F. oxysporwn f. sp. melonis. Non-pathogenic Fusarium could compete
directly with the pathogen for nutrients or infection sites (Alabouvette, 1990). The ratio
of pathogenicznon—pathogenic Fusarium affects the amount of suppression observed
(Alabouvette et al. , 1983). Carbon and iron were the main nutrients for which
microorganisms competed; carbon seemed to be more important (Alabouvette, 1990).
Non-pathogenic isolates, which inhibited germination of F. oxysporum f. sp. lini, had a
high affinity for glucose (Couteaudier, 1992). The isolates with the best ability to inhibit
germination of pathogenic chlamydospores also reduced wilt symptoms (Alabouvette and
Couteaudier, 1992). Damicone and Manning (1982) found a non-pathogenic Fusarium

isolate that protected asparagus seedlings for 8 weeks.

10

Other researchers have suggested that inoculation with non-pathogenic Fusariwn
stimulates the defenses of the host, known as induced resistance (Kuc, 1990). Ogawa
and Komada (1983) found that dipping tomato seedlings in F. oxysporum f.sp. dianthi

protected the plants from F. oxysporum f.sp. lycopersici for several weeks.

Pseudomonas. Suppressiveness in soil generally is attributed to Pseudomonas spp.
(Alabouvette, 1990). Pseudomonas are gram-negative bacteria that produce siderophores
(Miller 1990). They comprise 0.5 % of the soil bacteria, and colonize the rhizosphere
more than the rhizoplane (Miller 1990). Cassini et al. (1985) found a Pseudomonas that
reduced Fusarium infection in asparagus. Tari and Anderson (1988) found that
Pseudomonas putida suppressed Fusarium oxysporum f. sp. cucumerinum in soil. It was
suspected that the organisms were competing for iron (Tari and Anderson 1988).
Siderophores are iron-binding organic molecules that inhibit fungi. The
siderophore produced by P. putida is pyoverdine (Paulitz and Loper, 1991). Sneh et al.
(1984) found a correlation between siderophore production and Fusarium chlamydospore
inhibition. Fusarium also was inhibited by the synthetic siderophore, ethylenediamine-
di[(o-hydroxyphenyl)acetic acid] (EDDHA) (Sneh et al. , 1984). The affinity for iron was
highest in EDDHA, slightly less for the plant and lowest for F. oxysporum (Campbell,
1989), giving the Pseudomonas and the plant a slight advantage. Pythium was inhibited
by both EDDHA and a Pseudomonas isolate (Becker and Cook, 1988; Paulitz and Loper,
1991). However, the inhibition was overcome by the addition of FeCl,, supporting the

. concept of competition for iron (Becker and Cook, 1988).

11

Siderophore production may not be necessary for biocontrol, since eight
siderophore-negative mutants did not inhibit Pythium, but did protect cucumber plants
from infection (Paulitz and Loper, 1991). Levy et al. (1989) isolated l-hydroxy-
phenazine as an antibiotic produced by Pseudomonas which inhibited F. oxysporum f .sp.
vasinfectum. A Pseudomonas spp. was isolated from the surface F. oxysporum f. sp. lini
hyphae in a Fusarium-suppressive soil (Scher and Baker, 1980). It was not clear whether

Pseudomonas could parasitize Fusarium.

Actinomycetes. Meniman et a1. (1974) found a Streptomyces griseus isolate that was
antagonistic to Rhizoctonia solani in barley (Hordeum vulgare L.), oats (Avena sativa
L.), wheat (Triticum aestivum L.), and carrot (Daucus carota var. sativa L.). It did not
control the root disease, but did give higher yields, suggesting that it produced a growth
regulator (Merriman et al. 1974). Sabaou and Bounaga (1987) found Nocardiopsis
dassonvillei and Streptomyces alni reduced populations of F. oxysporum f. sp. albedinis
in date-palm soil. Thus, actinomycetes offer some potential for control of Fusarium spp.

Actinomycetes are gram-positive, coccus and rod-coccus bacteria. Most are
saprophytes with resistant spores (Goodfellow and Williams, 1983) comprising 20% of
the soil bacteria, but only 4% of the rhizosphere bacteria (Nemec et al. 1989). Mayfield
et a1. (1972) found Streptomyces spp. in discreet, spherical sites, associated with dead
root fragments.

Streptomyces spp. produce some of the known antibiotics, such as cycloheximide

(Leach et al., 1947), tetracycline (Conover et al., 1953), nystatin (Hazen and Brown,

12

1951), and streptomycin (Schatz et al. , 1944). However, there has been much debate
as to whether antibiotics are produced in soil (Gottlieb, 1976). Inoculation of natural soil
with Streptomyces did not result in detectable amounts of antibiotics, but addition of
glucose and Streptomyces did yield some antibiotic compounds (Williams, 1982).
Williams (1982) suggested that the soil is a nutrient-limiting environment and antibiotics
are produced when nutrients are available. Nitrogen content of the organic material also
may limit antibiotic production in the soil (Isizawa and Araragi, 1976). Antibiotics still
have not been isolated from natural, unamended soil (Maplestone et al. , 1992).

Mayfield et al. (1972) found a Streptomyces spp. that colonized fungal hyphae,
but it was not clear if the actinomycete penetrated the cell walls. Sutherland and
Lockwood (1984) found that actinomycetes parasitized the oospores of Phytophthora
megasperma f. sp. glycine. Some actinomycetes use chitin as a carbon source, so it is
possible that they may parasitize fungi for the chitin in the cell walls.

In 1987, Tahvonen (1988) isolated a Streptomyces spp. from a light-colored
Sphagnum peat. It was found to control F. oxysporum f. sp. dianthi in camations
(Lahdenpera, 1987). It also reportedly inhibited Altemaria, Bonyis, Pythium,
Rhizoctonia, and Verticillum (Lahdenpera, 1987, 1990). The organism was formulated
by Kemira Oy into a powdered preparation called Mycostop (Lahdenpera, 1987).
Tahvonen (1988) claimed it was effective in peat, sand or clay. Although the yield of
treated plants increased by 20-35 % when using Mycostop, they rarely reached the yield

of healthy control plants (Lahdenpera, 1987).

13
Smith et al. (1990) found that S. griseus controlled FOA in asparagus. It

increased root dry weight when applied as a root dip. The antibiotic compound was
extracted and named Faerifungin (Nair et al. 1987). Faerifungin inhibited FOA at
25 ppm (Smith et al., 1990).

Other organisms capable of inhibiting Fusarium spp. were Arthrobacter (Barrow-
Broaddus and Kerr, 1981), Aspergillus, Penicillum, Dichoderma (Marois and Mitchell,
1981); Bacillus (Mutitu et al. , 1991). Trichoderma harzianum colonized the rhizosphere
of tomato and tobacco, reducing the Fusarium population from 8.8 x 10‘ to
6.3 x 102 cfu/g of soil (Sivan and Chet, 1989). However, the effect was nullified by the
presence of excessive root exudates. Trichoderma also increased the rate of seedling

emergence, suggesting that it produces plant growth substances (Windham et a1. , 1986).

Colonization. The ability of any microorganism to protect plant roots depends on
colonization of the root. Colonization has been studied more thoroughly for
Pseudomonas. Although Pseudomonas have flagella, and Weller (1983) found
colonization along the entire root system of wheat, other researchers have not found
evidence for active movement (Dijkstra et al., 1987; Howie et al., 1987). Percolating
water enhanced movement of the organism along the root (Chao et al. , 1986; Liddell and
Parke, 1989). Passive movement on the root tip was suggested by Dijkstra et al. (1987)
and Howie et a1. (1987) but Liddell and Parke (1989) found little evidence for such
movement. Tan et al. ( 1991) suggested that adsorption on the soil particles was the key

factor affecting movement of Pseudomonas in soil.

14

Colonization and consistency of the biological control are dependent on several
environmental conditions (Lemanceau and Alabouvette, 1991). Soils with a matric
potential of less than -.05kPa were too wet and greater than -4.0 kPa was too dry for
P. fluorescens (Howie et al. , 1987). Suppression was more effective at neutral to alkaline
pH (Park et al., 1988).

Colonization was also temperature-sensitive. P. aeruginosa and P. fluorescens
both survived temperatures from 4 to 42C. However, P. aeruginosa had a maximum cell
yield at 28C and a maximum siderophore yield at 19C. P. fluorescens had the maximum
cell yield at 19C and maximum siderophore yield at 12C (Seong et al., 1991). Weller
and Cook (1983) found that the Pseudomonas population declined to 2.8 x 103 cfu/ g over
winter, but increased 10-fold in spring and remained at that level. Miller et al. (1990)
interpreted the frequently observed decline in Pseudomonas as the inability of the plant
to support the organism in later development.

Rhizobacteria can be very specific in their interaction with the host plant and their
antibiosis (El-Meleigi, 1989; Huber et al., 1989). Weller and Cook (1983) found that
a combination of isolates was more effective than single isolates for control of Pythium
in wheat. Lemanceau and Alabouvette (1991) found that a particular strain may not have
the same effect on two different Fusarium diseases. Stotzky (1964) found that
Achromobacter inhibited F. oxysporum if inoculated three days before exposure to the
Fusarium, but not if the microorganisms were co-inoculated.

Weller (1983) found that Pseudomonas populations were higher on roots infected

with F. oxysporum. Nemec et al. (1989) also reported the Pseudomonas population was

15

higher with F. solani in citrus. Park et al. (1988) found that Pseudomonas and non-
pathogenic Fusarium controlled F. oxysporum in cucumber in combination, but were
ineffective alone. Lemanceau and Alabouvette (1991) found that a Pseudomonas isolate
plus non-pathogenic Fusarium controlled F. oxysporum f. sp. radicis-lycopersici in tomato
under greenhouse conditions. Park et al. ( 1988) suggest that Fusarium infection induces
increased root exudation, which leads to competition for iron by the microorganisms.
Organisms that are introduced into soil must compete for nutrients with the
organisms that are already established (Schroth and Hancock, 1982; Campbell 1989).
Therefore, biological controls often fail unless large quantities of inoculum are applied,
or the soil is partially sterilized. It has been economically unfeasible to apply
concentrated inoculum on a regular basis. Direct application to seed or roots was more
feasible (Schroth and Hancock, 1982). However, Zablotowicz et al. (1989) used an in-
furrow delivery system which required only one liter of Pseudomonas cell suspension per

hectare.

Induced Suppression
Suppression may be induced by germination and lysis of the pathogen propagules
or by promotion of native antagonists. Cook and Schroth (1965) found that F. solani
f. sp. phaseoli chlamydospores require exogenous sources of carbon and nitrogen for
germination. They hypothesized that reduction of the fungal propagules should be
feasible by adding the appropriate nutrients in the absence of a susceptible host.

Asparagine and ammonium-nitrogen were identified as the preferred sources (Cook and

l6

Schroth, 1965). Sivan and Chet (1989) found maximum germination of F. oxysporum
chlamydospores with 0.4 mg glucose and 0.08 mg of asparagine per gram of soil. The
success of this type of control depends on the inability of the organism to form daughter
chlamydospores. If daughter chlamydospores form, the inoculum density will increase
(Papavizas and ‘Lumsden, 1980).

A natural germination-lysis system was found in the coniferous forests of
California. These forest soils lacked Fusarium spp. propagules (Toussoun et al. , 1969).
F. oxysporum and F. solani could be isolated from seedlings grown in a nursery, but the
Fusarium populations rapidly declined in the forest. Aqueous extracts of conifer needles
(Pinus ponderosa Laws. , Abies concolor (Gord. & Glend.) Hoeps. , Sequoia sempervirens
(Lamb) Endl.) caused 96-98% germination of the chlamydospores, but the germ tubes
lysed before daughter chlamydospores could form. The germ tubes formed abnormally
small tips before lysis (Toussoun et al. , 1969). Additional studies revealed that water
extracts of conifer needles contained shikimic, malic, citric, quinic and phosphoric acids
(Muir et al., 1964). The germination of F. oxysporum f. sp. lini chlamydospores was
directly proportional to the concentration of the shilcimic and quinic acids (Hammerschlag
and Linderman, 1975). Hammerschlag and Linderman (1975) concluded that if the
germination-lysis reaction occurred repeatedly, Fusarium eventually would be eliminated
from the soil. The Fusarium isolated from the seedlings was associated with the grass
between the rows in the nursery, since Graminae species selectively encourage

Fusarium spp. (Toussoun, 1975).

l7

' Crop rotations have been effective for reducing disease in some crops. Fusarium
oxysporum f.sp. apii was reduced by rotating celery with onions (Elmer and Lacy, 1987).
Soluble residue extracts from the onions may have stimulated chlamydospore germination

in the absence of a suitable host (Elmer and Lacy, 1987).

Organic Amendments. Many organic amendments have been tested to induce
suppression of Fusarium spp. in soil. Mitchell and Alexander (1961) found that
application of 200 lbs/acre of chitin reduced the severity of F. solani f. sp. phaseoli and
F. oxysporum f. sp. conglutinans in radish. They suggested that chitin encouraged the
development of actinomycetes which degrade the cell walls of fungi. However, the
application rate was not economically feasible and was replaced by other materials in
later experiments. Adachi et al. (1987) revived the work on F. solani f. sp. phaseoli, but
found that the effects of chitin decreased after three cultivations.

Zakaria and Lockwood ( 1980) added barley, wheat, corn, soybean, sugarbeet
pulp, alfalfa meal, linseed meal, cottonseed meal, soybean meal and ground crab shells
to Fusarium-infested soil. They found that 2% linseed, cottonseed or soybean meal
reduced the populations of F. moniliforme and F. solani from 105 to 101-102 cfu/ g of soil
after 4 weeks in containers. Soybean meal was the most effective treatment, reducing
the Fusarium population at a concentration of 0.25% (w/w) over a range of WHC.
However, field experiments did not support these greenhouse results. In addition, linseed

and cottonseed meals were phytotoxic. They concluded that toxic, volatile substances

18
were produced during the decomposition of the oilseed meals. Ammonia was thought

to be the active component (Zakaria and Lockwood, 1980).

Tu and Cheng (1990) applied a complex mixture of bagasse (4.4%), rice husk
(8.4%), oyster shell powder (4.2%), urea (8.2%), Cal-IP04 (13.1%), potassium nitrate
(1.0%) and slag (60.5 %) and found that asparagus growth improved and the population
of saprophytic Fusarium increased. Inoculating soil with saprophytic Fusarium at
4.5 x 104 cfu/g reduced Fusarium infection from 24.45 to 12.16% .(Tu and Cheng,
1990).

Mutitu et al. (1991) found that farmyard manure and coffee hulls reduced
Fusarium in beans. They isolated a Bacillus spp. as the active organism, which suggests

that organic amendments control Fusarium spp. by modifying the soil microflora.

Brassica Residues. Ramirez-Villapudua and Munnecke (1988) added residues of
cabbage (B. oleracea var. capitata L.), kale (B. oleracea var. virdis L.), mustard
(B. nigra L.), cauliflower (B. oleracea var. batrytis L.), broccoli (B. oleracea var.
italica L.), collard (B. oleracea var. virdis L.), Brussels sprouts (B. oleracea var.
gemmifera L.), turnip (B. rapa L.), radish (Raphanus sativus L.), alfalfa hay
(Medicago sativa L.), wheat straw (Triticum aestivum L.), chicken manure, and steer
manure to Fusarium-infested soil. They found that 2% dry cabbage, kale, or mustard
reduced the population of Fusarium oxysponmr f. sp. conglutinans (FOC), whereas wheat
residue, chicken manure and steer manure increased the populations. A combination of

solar heating and cruciferous residues reduced the population and disease incidence to

19

near zero. Solarization also reduced populations of F. oxysporum f. sp. lycopersici in
tomato (Greenberger et al. 1987).

Additional research with dry cabbage residue indicated that survival of the FCC
propagules was directly proportional to the concentration of the amendment and the time
of exposure (Ramirez-Villapudua and Munnecke, 1988). In sealed jars, FOC survival
was reduced to zero after 15 days. However, in open jars, the population increased
sharply, then returned to near control levels, indicating the loss of volatile compounds.
Radial growth of the fungus was inhibited by the volatiles of cabbage after 24 h, but
resumed its original growth rate when transferred to fresh air. While the total culturable
fungi decreased with cruciferous residues, the total culturable bacteria increased 16-fold.
Ramirez-Villapudua and Munnecke (1988) suggested that volatiles stimulate germination
of FCC propagules but continued exposure makes them susceptible to attack by other
microflora.

The effects of cruciferous residues on fungi have been studied more thoroughly
in Aphanomyces euteiches root rot of pea (Pisum sativum). Papavizas and Lewis (1971)
incorporated 0.5% corn, Brussels sprouts, collards, soybeans, cress, turnip, mustard,
kohlrabi, kale, or cabbage into dry soil. They found that cruciferous and soybean
residues were effective against A. euteiches and did not reduce pea stand if added
3 weeks before planting. The beneficial effects may be due to the fumigation action of
volatile sulfur-containing compounds arising from microbial decomposition of the residue
(Papavizas and Lewis, 1971). Suppression of the Aphanomyces root rot lasted for

15 weeks (Papavizas 1966).

20

Lewis and Papavizas (1971) found that isothiocyanate (ITC) was extremely toxic
to Aphanomyces. Growth was prevented by 0.7 ppm, and the fungus did not recover
when transferred to fresh air (Lewis and Papavizas, 1971). Allyl-isothiocyanate (AITC)
and methyl-isothiocyanate (MITC) were the most effective forms. MITC is used in the
commercial fumigant Vorlex (1,3-dichloropropene + MITC). It also results from the
decomposition of dazomet [3,5-dimethyl-tetrahydro-1,3,5,2H-thiadiazine-2-thione] and
metham [sodium N-methyl-dithiocarbamate (anhydrous)]. Dazomet was effective against
Aphanomyces at 50-200 ppm, although lower concentrations were required in closed
containers (Papavizas and Lewis, 1971). ITC also is derived by enzymatic degradation

of glucosinolates found in the Brassica spp.

Brassica Compounds. Glucosinolates are a group of secondary compounds produced
by the Brassica spp. The glucosinolate-thioglucosidase system is a defining character of
the Capparales family (Chew, 1988; Larsen 1981). The thioglucosidase enzymes
(myrosinases) are produced by the plants, but are isolated from the glucosinolates in
idioblast cells (Davis 1988). When the plant is injured, the myrosinases interact with
glucosinolates and release isothiocyanate. Certain bacteria, fungi, mammals, and the
cabbage aphid have thioglucosidase enzymes (Davis 1988; Larsen 1981). Glucosinolates
are hydrophilic, non-volatile compounds that are stable at neutral pH (Larsen, 1981).
Isothiocyanates are volatile, highly reactive compounds with a strong smell and

taste (Larsen 1981). Isothiocyanates have been shown to reduce fungal growth. Allyl

21

isothiocyanate is a known inhibitor of protein synthesis, but is believed to dissipate too
quickly to affect Md germination and viability (Mason-Sedun 1986).

The morphology of the sexual structures and mycelium of A. euteiches were
affected adversely by ITC. Zoospore motility and formation stopped after 2-6 h of
exposure to ITC (Lewis and Papavizas, 1971). Hyphae exhibited abnormal protrusions,
and the oogonia were deformed (Papavizas and Lewis, 1971). Chan and Close (1987)
conclude that soil with a disease severity index of 50 could be reduced to a level of no
risk by one or two plantings of rape.

Brassicas are known to contain volatile compounds that are phytotoxic (Ramirez-
Villapudua, 1985). High concentrations of cabbage caused browning and stunting of pea
roots (Papavizas and Lewis, 1971). Ramirez-Villapudua and Munnecke (1988) found
that tomatoes quickly wilted and died in cabbage-amended soil, indicating a residual
allelopathic effect. The effects may last at least six months in the field (Papavizas and
Lewis, 1971).

The concentration of glucosinolates in the various Brassica species has been
investigated in relation to phytotoxicity to subsequent crops. B. campestris residue
inhibited wheat growth by 43-57% (Mason-Sedun 1986). Many of the Brassica spp.
have different quantities of glucosinolates depending on cultivar, seeding date, and
harvest date (Davis 1988). The toxicity range from lowest to highest is: B. oleracea <
B. campestris < B. napus < B. nigra < B. kaber (Mason-Sedun 1986). The conditions
that increase the phytotoxicity of brassica residues are high temperatures (> 30C), long

days, low nutrition, water stress, and autoclaving (Mason-Sedun 1986). The

22

glucosinolate concentration also depends on location within the plant. Glucosinolates are
10—fold higher in the seed meal than in leaves and stems of brassica plants (Davis 1988).

Most soils suppress disease or pathogens to some degree (Hornby, 1983).
Addition of organic amendments may improve the antagonistic qualities of these soils or
may cause germination and lysis of the pathogen propagules. The purpose of this
dissertation was to examine two methods for enhancing the natural suppressiveness and

reducing Asparagus Decline Syndrome.

23

LITERATURE CITED

Adachi, K., M. Kobayshi, and E. Takahashi. 1987. Effect of the application of lignin
and/ or chitin to soil inoculated with Fusarium oxysporum on the variation of soil
microflora and plant growth. Soil Sci. Plant Nutrition 33: 245-259.

Alabouvette, C. 1990. Biological control of Fusarium wilt pathogens in suppressive soils,
pp. 27-44. In: D. Hornby, (ed.). Biological Control of Soil-bome Plant
Pathogens. C A B International, Wallingford, Oxon, UK.

Alabouvette, C. and Y. Couteaudier. 1992. Biological control of Fusarium wilts with
nonpathogenic Fusaria, pp. 415-426. In: E. S. Tjamos, G. C. Papavizas and
R. J. Cook, (eds.). Biological Control of Plant Diseases: Progress and Challenges
for the Future. Plenum Press, New York.

Alabouvette, C., Y. Couteaudier, and J. Louvet. 1983. Soil suppressive to Fusarium
wilt: Mechanisms and management of suppressiveness, pp. 101-104. In: C. A.
Parker, A. D. Rovira, K. J. Moore and T. W. Wong, (eds.). Ecology and
Management of Soilbome Plant Pathogens: Proc. of Section 5 of the Fourth Intl.
Congress of Plant Pathology. APS Press, St. Paul.

Barrows-Broaddus, J. and T. J. Kerr. 1981. Inhibition of Fusarium monilzfonne var.
subglutinans, the causal agent of pine pitch canker, by the soil bacterium
Arthrobacter sp. Can. J. Microbiology 27: 20-27.

Becker, J. O. and R. J. Cook. 1988. Role of siderophores in suppression of Pythium
species and production of increased-growth response of wheat by fluorescent
pseudomonads. Phytopathology 78: 778-782.

Bureau of the Census. 1978 Census of Agriculture, Vol. 1: Summary and State Data.
Part 51, United States. United States Department of Commerce, Washington,
DC.

Bureau of the Census. 1987 Census Of Agriculture, Vol. 1: Summary and State Data.
Part 51, United States. United States Department of Commerce, Washington,
DC.

Burke, D. W. 1965. Fusarium root rot of beans and the behavior of the pathogen in
different soils. Phytopathology 55: 1122-1126.

24

Bussell, W. T., J. H. Ellison. 1987. Tolerance of first generation asparagus hybrids to
Fusarium spp. A preliminary study. New Zealand I. Expt. Agric. 15: 239-242.

Campbell, R. E. 1989. Chapter 5. Biocontrol of diseases of roots, pp. 112-159.
Biological Control of Microbial Plant Pathogens. Cambridge Univ. Press,
New York.

Cassini, R. C., S. El Medawar, and R. P. Cassini. 1985. A biological control technique
to prevent fusarium decline in the fields, pp. 228-237. In: E.C. Lougheed and
H. Tiessen, (eds.). Proceedings of the Sixth International Asparagus Symposium.
Univ. of Guelph, Guelph, Ont. Can.

Chan, M. K. and R. C. Close. 1987. Aphanomyces root rot of peas. 3. Control by the
use of cruciferous amendments. New Zealand I. of Agric. Res. 30: 225-233.

Chao, W. L., E. B. Nelson, G. E. Harman, and H. C. Hoch. 1986. Colonization of the

rhizosphere by biological control agents applied to seeds. Phytopathology 76:
60-65 .

Chet, I., A. Ordentlich, R. Shapira, and A. Oppenheim. 1990. Mechanisms of
biocontrol of soil-bome plant pathogens by Rhizobacteria. Plant Soil 129: 85-92.

Chew, F. S. 1988. Biological effects of glucosinolates, pp 155-181. In: Biologically
Active Natural Products. Amer. Chem. 800., Washington, DC.

Cohen, S. I. and F. D. Heald. 1941. A wilt and root rot of asparagus caused by
Fusarium oxyspomm Schlecht. Plant Disease Reporter 25: 503-509.

Conover, L. H., W. T. Moreland, A. R. English, C. R. Stephens, and F. J. Pilgrim.
1953. Terramycin. XI. Tetracycline. J. Amer. Chem. Soc. 75: 4622-4623.

Cook, M. T. 1923. Dwarf asparagus. Phytopathology. 13: 284.

Cook, R. J. 1981. Water relations in the biology of Fusarium, pp. 236-244. In:
P. E. Nelson, T. A. Toussoun and R. J. Cook, (eds.). Fusarium: Diseases,
Biology, and Taxonomy. Penn. State Univ. Press, University Park, PA.

Cook, R. J. and M. N. Schroth. 1965 . Carbon and nitrogen compounds and germination
of chlamydospores of Fusarium solani f. sp. phaseoli. Phytopathology 55:
254-256.

Correll, J. C., J. E. Puhalla, and R. W. Schneider. 1986. Identification of Fusarium
oxysporum f. sp. apii on the basis of colony size, virulence, and vegetative
compatibility. Phytopathology 76: 396-400.

25

Couteaudier, Y. 1992. Competition for carbon in soil and rhizosphere, a mechanism
involved in biological control of Fusarium wilts, pp. 99-104. In: E. S. Tjamos,
G. C. Papavizas, and R. J. Cook, (eds.). Biological Control of Plant Diseases:
Progress and Challenges for the Future. Plenum Press, New York.

Couteaudier, Y. , M. Letard, C. Alabouvette and J Louvet. 1985. Lutte biologique contre

la fusariose vasculaire de la tomate. Resultats en serre de production. Agronomie
5: 151-160.

Damicone, J. P. and W. J. Manning. 1985. Frequency and pathogenicity of Fusarium
spp. isolated from first-year asparagus grown from transplants. Plant Disease 69:
413-416.

Damicone, J. P., P. D. Vineis, and W. J. Manning. 1988. Cross-pathogenicity of
Fusarium moniliforme isolates from corn and asparagus. Plant Disease 72:
774-777.

Davis, J. B. 1988. Winter rapeseed (Brassica napus L.) with differential level of
glucosinolates evaluated as a green manure crop to suppress Aphanomyces root
rot of Peas (Pisum sativum L.). MS Thesis, Univ. Idaho, Moscow. 66p.

Dijkstra, A. F., G. H.N. Scholten, and J. A. vanVeen. 1987. Colonization of wheat
seedling (Triticum aestivum) roots by Pseudomonas fluorescens and Bacillus
subtilis. Biol. Fertil. Soils 4: 41-46.

El-Meleigi, M. A. 1989. Effect of soil Pseudomonas isolates applied to corn, sorghum
and wheat seeds on seedling growth and corn yield. Can. J. Plant Sci. 69:
101-108.

Elmer, W. H. 1989. Effects of chloride and nitrogen form on growth of asparagus
infected by Fusarium spp. Plant Disease 73: 736-740.

Elmer, W. H. 1992. Suppression of Fusarium crown and root rot of asparagus with
sodium chloride. Phytopathology 82: 97-104.

Elmer, W. H. and M. L. Lacy. 1987. Effects of crop residues and colonization of plant
tissues on propagule survival and soil populations of Fusarium oxysporum f.sp.
apii Race 2. Phytopathology 77: 381-387.

Elmer, W. H. and C. T. Stephens. 1989. Classification of Fusarium oxysporum f.sp.
asparagi into vegetatively compatible groups. Phytopathology 79: 88-93.

26

Engelhard, A. W. 1989. Historical highlights and prospects for the future, pp. 9-17. In:
A. W. Engelhard, (ed.). Soilbome Plant Pathogens: Management of Diseases
with Macro- and Microelements. APS Press, St. Paul.

Falloon, P. G. and H. A. Fraser. 1991. Control of establishment failures in asparagus
(Asparagus ofiicinalis L.) caused by Phytophthora rot. New Zealand I . Crop and
Hort. Sci. 19: 47-52.

Goodfellow, M. and S. T. Williams. 1983. Ecology of actinomycetes. Annu. Rev.
Microbiology 37: 189-216.

Gottlieb, D. 1976. The production and role of antibiotics in soil. J. Antibiotics 29: 987-
1000.

Graham, K. M. 1955. Seedling blight, and fusarial diease of asparagus. Can. J. Botany
33: 374-400.

Greenberger, A. , A. Yogev, and J. Katan. 1987. Induced suppressiveness in solarized
soils. Phytopathology 77: 1663-1667.

Griffin, G. J. 1981. Physiology of conidium and chlamydospore germination in
Fusarium, pp. 331-339. In: P. E. Nelson, T. A. Toussoun and R. J. Cook,
(eds.). Fusarium: Diseases, Biology, and Taxonomy. APS Press, St. Paul.

Grogan, R. G. and K. A. Kimble. 1959. The association of Fusarium wilt with the
asparagus decline and replant problem in California. Phytopathology 49: 122-125.

Hammerschlag, F. and R. G. Linderman. 1975. Effects of five acids that occur in pine

needles on Fusarium chlamydospore germination in nonsterile soil.
Phytopathology 65: 1 120-1124.

Hartung, A. C. 1983. Effects of allelopathic substances produced by asparagus on the
incidence and severity of Fusarium crown rot. MS Thesis, Michigan State Univ.,
East Lansing.

Hartung, A. C. 1987. Allelopathic potential of asparagus (Asparagus ofiicinalis L.).
PhD Diss., Michigan State Univ., East Lansing. 138p.

Hartung, A. C., M. G. Nair, and A. R. Putnam. 1990. Isolation and characterization of

phytotoxic compounds from asparagus (Asparagus ofiicinalis L.) roots.
J. Chem. Ecol. 16: 1707-1718.

27

Hartung, A. C. and C. T. Stephens. 1983. Effects of allelopathic substances produced
by asparagus on incidence and severity of asparagus decline due to Fusarium
crown rot. J. Chem. Ecology. 9: 1162-1174.

Hartung, A. C., A. R. Putnam, and C. T. Stephens. 1989. Inhibitory activity of
asparagus root tissue and extracts on asparagus seedlings. J. Amer. Soc. Hort.
Sci. 114: 144-148.

Hazebroek, J. P., S. A. Garrison, and T. Gianfagna. 1989. Allelopathic substances in
asparagus roots: Extraction, characterization, and biological activity. J. Amer.
Soc. Hort. Sci. 114: 152-158.

Hazen, E. L. and R. Brown. 1951. Fungicidin, an antibiotic produced by a soil
actinomycete. Proc. Soc. Expt. Biol. Med. 76:93-97.

Hodupp, R. M. 1983. Investigation of factors which contribute to asparagus (Asparagus
ofi'icinalis L.) decline in Michigan. MS Thesis, Michigan State Univ. , East
Lansing.

Homby, D. 1983. Suppressive soils. Annu. Rev. Phytopathology 21: 65-85.

Howie, W. J., R. J. Cook, and D. M. Weller. 1987. Effects of soil matric potential and
cell motility on wheat root colonization by fluorescent pseudomonads suppressive
to take-all. Phytopathology 77: 286-292.

Huber, D. M., H. El-Nasshar, L. W. Moore, D. E. Mathre, and J. E. Wagner. 1989.
Interaction between a peat carrier and bacterial seed treatments evaluated for

biological control of the take-all diseases of wheat (Triticum aestivum L.).
Biol. Fertil. Soils 8: 166-171.

Inglis, D. A. 1980. Contamination of Asparagus seed by Fusarium oxysporum f.sp.
asparagi and Fusarium monilifonne. Plant Disease 64: 74-76.

Isizawa, S. and M. Araragi. 1976. Composition of actinomycete population in soil,
pp. 97-107. In: T. Arai, (ed.). Actinomycetes: The Boundary Microorganisms.
Toppan Co. Ltd. Tokyo.

Johnson, S. A., J. K. Springer, and G. D. Lewis. 1979. Fusarium moniliforme as a
cause of stem and crown rot of asparagus and its association with asparagus
decline. Phytopathology 69: 778-780.

28

Jones, J. P., A. W. Engelhard, and S. S. Woltz. 1989. Management of Fusarium wilt
of vegetable and omamentals by macro- and microelement nutrition, pp. 18-32.
In: A. W. Engelhard, (ed.). Soilbome Plant Pathogens: Management of Diseases
with Macro- and Microelements. APS Press, St. Paul.

Jones, J. P. and A. J. Overman. 1971. Control of Fusarium wilt of tomato with lime and
soil fumigants. Phytopathology 61: 1415-1417.

Kasenberg, T. R. and J. A. Traquair. 1988. Effects of phenolics on growth of Fusarium
oxysporum f.sp. radicis-lycopersici in vitro. Can. J. Botany 66: 1174-1177.

Komada, H. 1975 . Development of a selective medium for quantitative isolation of
Fusarium oxysporum from natural soil. Rev. Plant Protection Res. 8: 114-124.

Kuc, J. 1990. Immunization for the control of plant disease, pp. 355-373. In:
D. Homby, (ed.). Biological Control of Soil-home Plant Pathogens.
C A B International, Wallingford, Oxon, UK.

Lacy, M. L. 1979. Effects of chemical on stand establishment and yields of asparagus.
Plant Disease Reporter 63: 612-616.

Lahdenpera, M. L. 1987. The control of Fusarium wilt on carnation with a Streptomyces
preparation. Acta Horticulturae 216: 85-92.

Lahdenpera, M. L., E. Simon, and J. Uoti. 1990. Mycostop - a novel biofungicide based
on Streptomyces bacteria. Biotic Interaction and Soil-home Diseases. Proceedings
from the First Conference of the European Foundation for Plant Pathology.

Larsen, P. O. 1981. Glucosinolates. The Biochemistry of Plants 7: 501-525.

Leach, B. E., J. H. Ford, and A. J. Whiffen. 1947. Actidione, an antibiotic from
Streptomyces griseus. J. Amer. Chem. Soc. 69: 474.

- Lemanceau, P. and C. Alabouvette. 1991. Biological control of fusarium diseases by

fluorescent Pseudomonas and non-pathogenic Fusarium. Crop Protection 10:
279-286.

Levy, E., Z. Eyal, S. Carmely, Y. Kashman, and I. Chet. 1989. Suppression of Septoria
tritici and Puccinia recondita of wheat by an antibiotic-producing fluorescent
pseudomonad. Plant Pathology 38: 564-570.

Lewis, J. A. and G. C. Papavizas. 1971.'Effect of sulfur-containing volatile compounds

and vapors from cabbage decomposition on Aphanomyces euteiches.
Phytopathology 61: 208-214.

29

Lewis, J. A. and G. C. Papavizas. 1970. Evolution of volatile sulfur-containing
compounds from decomposition of crucifers in soil. Soil Biol. Biochem. 2:
239-246.

Liddell, C. M. and J. L. Parke. 1989. Enhanced colonization of pea taproots by a
fluorescent pseudomonad biocontrol agent by water infiltration into soil.
Phytopathology 79: 1327-1332.

Louvet, J . , C. Alabouvette, and F. Rouxel. 1981. Microbiological suppressiveness of
soils to Fusarium wilts, pp. 261-275. In: P. E. Nelson, T. A. Toussoun and R.
J. Cook, (eds.). Fusarium: Diseases, Biology, and Taxonomy. Penn. State Univ.
Press, University Park, PA.

Mace, M. C., A. A. Belland, and C. H. Beckman. 1981. Fungal Wilt Disease of Plants.
Academic Press, New York.

Maplestone, R. A., M. J. Stone, and D. H. Williams. 1992. The evolutionary role of
secondary metabolites - a review. Gene 115: 151-157.

Marois, J. J. and D. J. Mitchell. 1981. Effects of fumigation and fungal antagonists on
the relationships of inoculum density to infection incidence and disease severity
in Fusarium crown rot of tomato. Phytopathology 71: 167-170.

Mason-Sedun, W. M. 1986. Differential phytotoxicity of residues from the genus
Brassica. PhD Diss., Univ. New England, Armidale, Australia. 154p.

Mayfield, C. 1., S. T. Williams, S. M. Ruddick, and H. L. Hatfield. 1972. Studies on
the ecology of actinomycetes in soil. IV. Observations on the form and growth -
of Streptomycetes in soil. Soil Biol. Biochem. 4: 79—91.

Merriman, P. R., R. D. Price, J. F. Kollmorgen, T. Piggott, and E. H. Ridge. 1974.
Effect of seed inoculation with Bacillus subtilis and Streptomyces griseus on the
growth of cereals and carrots. Aust. J. Agric. Res. 25: 219-226.

Michigan Agricultural Statistics Service. 1980. Michigan Agricultural Statistics.
Michigan Department of Agriculture, Lansing, MI.

Michigan Agricultural Statistics Service. 1992. Michigan Agricultural Statistics.
Michigan Department of Agriculture, Lansing, MI.

Miller, H. 1., E. Liljeroth, G. Henken, and J. A. VanVeen. 1990. Fluctuation in the
fluorescent pseudomonad and actinomycete population of rhizosphere and
rhizoplane during the growth of spring wheat. Can. J. Microbiology 36:
254-258.

30

Mitchell, R. and M. Alexander. 1961. Chitin and the biological control of Fusarium
diseases. Plant Disease Reporter 45: 487-490.

Muir, J. W., R. I. Morrison, C. J. Bown and J. Logan. 1964. The mobilization of iron
by aqueous extracts of plants. 1. Composition of the amino-acid and organic-acid
fractions of an aqueous extract of pine needles. J. Soil Sci. 15:220-225.

Mutitu, E. W., D. M. Mukunya, and S. O. Keya. 1991. Control of Fusarium yellows
of beans using bacterial antagonists. Discovery and Innovation 3: 83-86.

Nair, M. G., A. R. Putnam, S. K. Mishra, M. H. Mulks, W. H. Taft, J. E. Keller, and
J. R. Miller. 1989. Faeriefungin: A new broad-spectrum antibiotic from
Streptomyces griseus var. autotrophicus. J . Nat. Products 52: 797-809.

Nemec, S., R. M. Zablotowicz, and J. L. Chandler. 1989. Distribution of Fusarium spp.
and selected microflora in citrus soils and rhizospheres associated with healthy
and blight-diseased citrus in Florida. Phytophylactica 21: 141-146.

Nigh, E. L. Jr. 1990. Stress factors influencing Fusarium infection in asparagus. Acta
Horticulturae 271: 315-322.

Ogawa, K. and H. Komada. 1983. Biological control of Fusarium wilt of sweet potato
with cross-protection by nonpathogenic Fusarium oxysporum, pp.121-123. In:
C. A. Parker, A. D. Rovira, R. J. Moore and T. W. Wong, (eds.). Ecology and
Management of Soilbome Plant Pathogens: Proc. of Section 5 of the Fourth Intl.
Congress of Plant Pathology. APS Press, St. Paul, MN.

Papavizas, G. C. 1966. Suppression of Aphanomyces root rot of peas by cruciferous soil
amendments. Phytopathology 56: 1071-1075.

Papavizas, G. C. and J. A. Lewis. 1971. Effect of amendments and fungicides on
Aphanomyces root rot of peas. Phytopathology 61: 215-220.

Papavizas, G. C. and R. D. Lumsden. 1980. Biological control of soilborne fungal
propagules. Annu. Rev. Phytopathology 18: 389-413.

Park, C. S., T. C. Paulitz, and R. Baker. 1988. Biocontrol of Fusarium wilt of
cucumber resulting from interactions between Pseudomonas putida and
nonpathogenic isolates of Fusarium oxysporum. Phytopathology 78: 190-194.

Paulitz, T. C. and J. E. Loper. 1991. Lack of a role for fluorescent siderophore
production in the biological control of Pythium damping-off of cucumber by a
strain of Pseudomonas putida. Phytopathology 81: 930-935.

31

Peirce, L. C. and H. G. Miller. 1993. Asparagus emergence in Fusarium-treated and
sterile media following exposure of seeds or radicles to one or more cinnarnic
acids. J. Amer. Soc. Hort. Sci. 118: 23-28.

Peirce, L. C. and L. W. Colby. 1987. Interaction of asparagus root filtrate with
Fusarium oxysporum f.sp. asparagi. J. Amer. Soc. Hort. Sci. 112: 35-40.

Ramirez-Villapudua, J. 1985 . Identification of a new race of Fusarium oxysporum Schl.
f.sp. conglutinans (Wr.) Snyd. & Hans. from cabbage and its control by means
of solarization of soil amended with cabbage residues. PhD Diss. , Univ. Calif. ,
Davis. 125p.

Ramirez-Villapudua, J. and D. E. Munnecke. 1988. Effect of solar heating and soil
amendments of cruciferous residues on Fusarium oxysporwn f. sp. conglutinans
and other organisms. Phytopathology 78: 289-295.

Ramirez-Villupadua, J. and D. E. Munnecke. 1987. Control of cabbage yellows
(Fusarium oxysporum f. sp. conglutinans) by solar heating of field soils amended
dry cabbage residues. Plant Disease 71: 217-221.

Ramirez-Villupadua, J., R. M. Endo, P. Bosland, and P. H. Williams. 1985. A new
race of Fusarium oxysporum f. sp. conglutinans that attacks cabbage with Type A
resistance. Plant Disease 69: 612-613.

Reinking, O. A. and M. M. Manns. 1933. Parasitic and other fusaria counted in tropical
soil. Zeitschrift fur Parasitenkunde 6: 23-75.

Rudolph, W. 1921. Experiments with common rock salt: I. Effect on asparagus. Soil
Sci. 12:449-455.

Sabaou, N. and N. Bounaga. 1987. Actinomycetes parasites de champignons: etude des
especes, specificte de l’action parasitaire au genre Fusarium et antagonisme dans
le sol envers Fusarium oxysporum f. sp. albedinis (Killian et Maire) Gordon.
Can. J. Microbiology 33: 445-451.

Sadowski, C. and M. Knaflewski. 1990. Susceptibility of selected asparagus cultivars to
Fusarium spp. under field conditions. Acta Horticulturae 271: 343-351.

Schatz, A., E. Bugie, and S. A. Waksman. 1944. Streptomycin, a substance exhibiting
antibiotic activity against gram-positive and gram-negative bacteria. Proc. Soc.
Expt. Biol. Med. 55: 66-69.

Scher, F. M. and R. Baker. 1980. Mechanism of biological control in a
Fusarium-suppressive soil. Phytopathology 70: 412-417.

32

Seong, K. Y., M. Hofte, J. Boelens, and W. Verstraete. 1991. Growth, survival, and
root colonization of plant growth beneficial Pseudomonas fluorescens ANP15 and

Pseudomonas aeruginosa 7NSK2 at different temperatures. Soil Biol. Biochem.
23: 423-428.

Shafer, W. E. and S. A. Garrison. 1986. Allelopathic effects of soil incorporated
asparagus roots on lettuce, tomato, and asparagus seedling emergence.
HortScience 21: 82-84.

Sivan, A. and I. Chet. 1989. The possible role of competition between Trichodenna

harzianum and Fusarium oxysporum on rhizosphere colonization. Phytopathology
79: 198-203.

Smith, J. D., A. Putnam, and M. Nair. 1990. In vitro control of Fusarium diseases of
Asparagus ofiicinalis L. with a Streptomyces or its polyene antibiotic,
Faeriefungin. J. Agric. Food Chem. 38: 1729-1733.

Smith, S. N. and W. C. Snyder. 1972. Germination of Fusarium oxysponrm
chlamydospores in soils favorable and unfavorable to wilt establishment.
Phytopathology 62: 273-277.

Sneh, B., M. Dupler, Y. Elad, and R. Baker. 1984. Chlamydospore germination of
Fusarium oxysporum f. sp. cucumerium as affected by fluorescent and lytic
bacteria from a Fusarium-suppressive soil. Phytopathology 74: 1115-1124.

Stephens, C. T., R. M. DeVries, and K. C. Sink. 1989. Evaluation of Asparagus species

for resistance to Fusarium oxysporum f. sp. asparagi and F. moniliforme.
HortScience 24: 365-368. '

Stover, R. H. 1954. The effect of soil moisture on Fusarium species. Can. J. Botany 31:
693-697.

Stover, R. H. 1962. Fusarial wilt (Panama disease) of bananas and other Musa species.
Commonwealth Mycological Inst., Phytopathological Paper 4: 1-117.

Sutherland, E. D. and J. L. Lockwood. 1984. Hyperparasitism of oospores of some
Peronosporales by Actinoplanes missouriensis and Humicola fizscoatra and other
Actinomycetes and fungi. Can. J. Plant Pathology 6: 139-145.

Tahvonen, R. T. 1988. Microbial control of plant diseases with Streptomyces spp. EPPO
Bulletin 18: 55-59.

Takatori, F. and F. D. Souther. 1978. Asparagus Workshop Proceedings. Department
of Plant Sciences, Univ. Calif., Riverside. 100p.

33

Tan, Y., W. J. Bond, A. D. Rovira, P. G. Brisbane, and D. M. Griffin. 1991.
Movement through soil of a biological control agent, Pseudomonas fluorescens.
Soil Biol. Biochem. 23: 82-84.

Tari, P. H. and A. J. Anderson. 1988. Fusarium wilt suppression and agglutinability of
Pseudomonas putida. Applied Environ. Microbiology 54: 2037-2041.

Tiedjens, V. A. 1924. Some physiological aspects of Asparagus ofiicinalis. Proc. Amer.
Soc. Hort. Sci. 21: 129-140.

Tiedjens, V. A. 1926. Sone observations on root and crown bud formation in Asparagus
ofi'icinalis. Proc. Amer. Soc. Hort. Sci. 23: 189-195.

Toussoun, T. A. 1975. Fusarium-suppressive soils, pp. 145-151. In: G. W. Bruehl, (ed.)
Biology and Control of Soil-borne Plant Pathogens. APS Press, St. Paul.

Toussoun, T. A., W. Menzinger, and J. R. S. Smith. 1969. Role of conifer litter in

ecology of Fusarium: Stimulation of germination in soil. Phytopathology 59:
1396-1399.

Tu, C. C. and Y. H. Cheng. 1990. Recent advance in biological control of Fusarium
wilt of asparagus in Taiwan. Acta Horticulturae 271: 353-362.

Walker, I. C. and W. C. Snyder. 1933. Pea wilt and root rots. Wisconsin Agric. Expt.
Sta. Bulletin 424.

Waksman, S. A. 1927. Principles of Soil Microbiology. The Williams and Wilkins Co.,
Baltimore, MD.

Wardlaw, C. W. 1972. Panama disease (concluded), pp. 239-276. Banana Diseases,
including Plantains and Abaca, Second Edition, Longman Group Ltd., London.

Weller, D. M. 1983. Colonization of wheat roots by a fluorescent pseudomonad
suppessive to take-all. Phytopathology 73: 1548-1553.

Weller, D. M. and R. J. Cook. 1983. Suppression of Take-all of wheat by seed
treatments with fluorescent pseudomonads. Phytopathology 73: 463-469.

Williams, S. T. 1982. Are antibiotics produced in soil?. Pedobiologia 23: 427-435.

Windham, M. T., Y. Elad, and R. Baker. 1986. A mechanism for increased plant
growth induced by Trichoderma spp. Phytopathology 76: 518-521.

34

Yang, H. J. 1982. Autotoxicity of Asparagus oflicinalis L. J. Amer. Soc. Hort. Sci.
107: 860-862.

Zablotowicz, R. M., E. M. Tipping, F. M. Scher, M. Ijzerman, and J. M. Kloepper.
1991 . In-furrow spray as a delivery system for plant growth-promoting
rhizobacteria and other rhizosphere-competent bacteria. Can. J . Microbiology 37:
632-636.

Zakaria, M. A. and J. L. Lockwood. 1980. Reduction in Fusarium populations in soil
by oilseed amendments. Phytopathology 70: 240-243.

CHAPTER 2
INFLUEUNCE OF BRASSICA SPP. RESIDUES ON

FUSARI UM SPP. IN ASPARAGUS

Abstract

Brassica spp. residues have been shown to control Fusarium diseases in other
crops. Seven brassicas were incorporated in the field and greenhouse. The brassicas
included ’Premier’ kale (B. oleracea L. virdis), ’Rondo’ turnip (B. rapa L.), ’Global’
canola (B. napus L. annua), ’Glacier’ canola (B. napus L. biennis), yellow mustard
(B. nigra L.), ’Dwarf Essex’ oilseed rape (B. napus L. biennis), ’Humus’ oilseed rape
(B. napus L. biennis) and sorghum-sudangrass (Sorghum sudanense (Piper) Stapf.) as the
control. Brassica residues had no effect on disease incidence in asparagus or Fusarium
populations. Addition of plastic covers, which produced solarization, did not affect the
disease incidence. Brassica residues were phytotoxic to several indicator crops, including
asparagus. Chemical extraction of the brassica residues did not yield any Fusarium-

inhibitory compounds.

35

36

Introduction

Asparagus (Asparagus oflicinalis L.) is an important vegetable crop in Michigan.
In 1991, Michigan produced 12,950 tons of asparagus with a value of $14.1 million on
23,500 acres of land (Michigan Agricultural Statistics Service, 1992). Michigan
asparagus production has averaged 1200 pounds per acre over the past decade (Michigan
Agricultural Statistics Service, 1992). However, yields of 3000 pounds per acre are
achieved in some fields (Hodupp, 1983), indicating that a majority of the acreage is not
producing its full potential. Replanting these fields has limited benefit, because 50% of
young asparagus plants die within five years of planting and replanted fields often yield
only half of the expected production (Hodupp, 1983).

The phenomenon of declining yields has been termed Asparagus Decline
Syndrome (Grogan and Kimble, 1959). It is characterized by stunted, yellow, wilted
plants and blank spaces in the rows. The major causes are thought to be soil
microorganisms and allelopathic chemicals which adversely affect the growth of
asparagus plants (Hartung, 1987). The primary crown and root rot organisms are
Fusarium oxysporum f. sp. asparagi (FOA) and F. monilifonne (FM).

Methods for controlling Fusarium spp. in asparagus currently are not available.
Resistant varieties, which have proven effective for control of Fusarium spp. in other
vegetable crops (Mace et al., 1981) have not been found in asparagus (Stephens et al.,
1989). Chemical treatments with benomyl [methyl 1-(butylcarbamoyl)-lH-benzimidazol—
2-ylcarbamate] and captan [N-[(trichloromethyl)thio]-4-cyclohexene-1,2-dicarboximide]

produced no positive effects on crown survival or yields (Lacy, 1979).

 

37
Ramirez-Villapudua and Munnecke (1987) found that cabbage (Brassica oleracea

var. capitata), kale (B. oleracea var. virdis) or mustard (B. nigra)residue at 2 % of the
soil dry weight reduced the population of F. oxysporum f .sp. conglutinans in cabbage.
The addition of a plastic cover, which produced solar heating, reduced the Fusarium
populations to near zero.

Brassica spp. , such as cabbage, kale or mustard, contain glucosinolates which
degrade to isothiocyanate. The beneficial effects of brassica residues may be caused by
the fumigation action of these volatile, sulfur-containing compounds arising from
microbial decomposition of the residue (Papavizas and Lewis, 1971). Isothiocyanates are
extremely toxic to some fungi and are used in the commercial funrigant Vorlex
(1,3 dichloropropene + methylisothiocyanate). Many Brassica spp. contain higher
concentrations of glucosinolates than cabbage (Davis, 1989), and may be more effective
for controlling pathogens.

Brassicas also are phytotoxic. Ramirez-Villapudua and Munnecke (1987) found
that tomatoes quickly wilted and died when transplanted into cabbage-amended soil.
Mason-Sedun (1986) found that wheat yields were reduced 43-57% when following a
canola crop. The objectives of this study were to determine the effect of brassica

residues on FOA and FM; and to determine the duration of Brassica phytotoxicity.

Materials and Methods
Plants, inocula and media. The asparagus selection ’Syn 4-56’ (Nourse Farms Inc.,

South Deerfield, MA) was used in all experiments. Seeds were surface-disinfected with

 

38
0.5% NaOCl (20% bleach) for 20 min, followed by infusion with benomyl (2.5g) in

acetone (100ml) for 18-20 h (Damicone et al., 1981). Seeds were rinsed with acetone,
followed by sterile deionized water and then dried on a sterilized clay tile (Stephens and
Elmer, 1988). Seedlings were germinated on 0.6% water agar and grown in 96—ce11
(5 x 28 x 53 cm) plastic trays. Seedlings were fertilized once a week with 1000 ml/tray
of 200 ppm Peters 2ON-8.7P-16.6K fertilizer. Transplants used in the experiments were
washed to remove the. soil and inspected for absence of Fusarium symptoms.

Single-spore cultures of FOA (isolate FOASO) and FM (isolate F12) were
obtained from cultures stored in muck soil and grown on potato dextrose agar (PDA).
For in vitro studies, spores were washed from 14-day-old culture plates and diluted to
106 colony forming units per milliliter (cfu/ml). For soil studies, a 5-mm agar plug of
FOA or FM was inoculated into autoclaved fescue seed and grown for two weeks, with
mixing each day. The fescue seed then was dried and ground to pass a 2-mm sieve in
a Wiley mill.

The soil used in all experiments consisted of a Spinks loamy sand (Psammetitic
Hapludalf, sandy, mixed, mesic, pH 6.4, P 195 kg-ha", K 110 kg-ha", Ca 538 kg-ha“,
Mg 179 kg'ha", CEC 2 meq-100g“, bulk density 1.4-1.7 g'cm‘3, organic matter 2-4%)
obtained from Hart, Michigan. It was passed through a 2-mm sieve before use in the

greenhouse studies.

Selection in the Field. A 20-year-old asparagus field near Hart, MI was plowed out in

the summer of 1989 because of declining yields typical of Fusarium infection. The field

39
was prepared by adding 313.6 kg-ha" of 20N-4.4P-8.3K fertilizer and disking. Seven

brassicas and a control were planted in 9.1 x 18.3-m plots. The brassicas included
’Premier’ kale (B. oleracea L. virdis), ’Rondo’ turnip (B. rapa L.), ’Global’ canola
(B. napus L. annua), ’Glacier’ canola (B. napus L. biennis), yellow mustard
(B. nigra L.), ’Dwarf Essex’ oilseed rape (B. napus L. biennis), ’Humus’ oilseed rape
(B. napus L. biennis)(courtesy of Dr. Dick Auld, University of Idaho) and sorghum- -
sudangrass (Sorghum sudanense (Piper) Stapf.) as the control.

In September 1989, the number of plants and fresh weights were collected from
1 m2 of each plot. Soil samples (200 g) were collected randomly from the top 10 cm of
each plot to determine the microflora populations. The brassicas subsequently were

disked and plowed-down, and a rye (Secale cereale L.) cover crop was planted for the
1 winter.

In May 1990, the plots were divided in half. One half was used to determine
Fusarium infection and phytotoxicity, the other half was planted to the same brassica to
determine if a second year of cropping would increase disease suppression. Soil samples
from each plot were collected again on 3 May. On 19 May the rye was killed with
Ranger herbicide (glyphosate, 3378 ml-ha"). On 31 May 1990, 3 x 3-m indicator plots
were established containing ’Syn 4-56’ asparagus, ’Pik Red’ tomatoes (Lycopersicum
esculentum L.), ’Market Prize’cabbage (B. oleracea var. capitata L.), and ’Discover’
cucumbers (Cucumis sativa L.) for rating of the brassica phytotoxicity.

Three ’Syn '4-56’ asparagus seedlings were excavated from each plot on three

dates (3 July, 26 July, 20 August) to rate for Fusarium infection. The disease rating

 

40
scale was 1-5, where 1 = a healthy seedling, 2 = slight infection, 3 = moderate

infection, 4 = severe infection, and 5 = dead or dying seedling.

The other half of each plot was rototilled on 30 June and planted with the same
brassica which was grown in the previous year. Plant stands and fresh weights were
collected from 1 m2 of each plot in September 1990, before the brassicas were plowed
down. The experiment was terminated before the Spring of 1991 based on results from
other studies.

The field was returned to commercial asparagus production in the Spring of 1991.
In the Fall of 1993, stand counts from two rows in each plot were collected to evaluate
any residual effects of the brassicas on the asparagus.

The experimental design was a randomized complete block with five blocks. The
asparagus rating data were split over the three sampling times. The data were analyzed
by analysis of variance (ANOVA) using MSTAT-C (MSTAT-C, Michigan State
University, E. Lansing, MI). The brassica treatments were compared to the control

using non-orthogonal F-tests.

Soil Microflora. Soil samples collected from the field in Hart, MI were air-dried and
stored at 4C before assay. The samples were diluted serially and spread on plates
containing selective media to determine the microflora populations. Media included
Komada’s for Fusarium spp. (Komada, 1975), amended Czepek’s Rose Bengal for fungi
(Hartung, 1987), Tryptic Soy Agar for bacteria (Martin, 1975). Several media were

tested for selective isolation of actinomycetes: chitin agar (Hsu and Lockwood, 1975),

41
NZ-Amine (Hartung, 1987) and water agar (Tiedje and Dazzo, 1982). The final

procedure involved heat-shocking the sample for 2 min at 60C then plating on water agar
amended with cyclohexinride. Colonies were counted after two days for bacteria, four
days for fungi and Fusarium, and seven days for actinomycetes. FOA and FM were
identified by visible morphology.

The experimental design was a randomized complete block with five blocks and
two sampling times (Fall 1989, Spring 1990). The data were analyzed by ANOVA and

means were separated by LSD.

Phytotoxicity of Residues. Brassica residue (2 % of soil dry weight) was combined with
unsterilized Spinks loamy sand field soil (2500 g) in the greenhouse (21C day, 10C night)
to determine the duration of the phytotoxicity. Treatments included the seven brassicas
listed above plus sorghum-sudangrass and poplar excelsior (Populus alba L.) as controls.
All residues were dried at 60C and ground to pass a 2-mm sieve. The soil was placed
in 4—liter clay pots and watered twice a week to maintain soil moisture. Soil samples
(130 g) were collected in petri dishes at 0, 2, 4, and 12 weeks and 10 seeds of five
bioassay crops were germinated in the soil to determine phytotoxicity. The bioassay
crops included: ’Syn 4-56’ asparagus, ’Curly’ cress (Lepidium sativum L.), ’Yorkstar’
wheat (Triticum aestivum L.), ’Discover’ cucumber and ’Market Prize’ cabbage. The
asparagus seeds were pregerrninated on filter paper to assure viable seedlings. The

germination percentages were recorded and root lengths were measured with a ruler after

42
3 days for cucumber, 4 days for wheat, 6 days for cress, and 7 days for asparagus and

cabbage.

The experimental design was a randomized complete block with a split over time
and four blocks. The root length data were converted to a percentage of the excelsior
control for simplicity of interpretation and analyzed by AN OVA. The means were
separated by LSD. Asparagus root growth with Sudan and kale was compared to the

other brassica using non-orthogonal F-test.

Selection in the Greenhouse. In the Spring of 1990, brassica residue (2 % of soil dry
weight) was incorporated into pasteurized field soil in 500-ml pots to determine the
relative Fusarium-inhibition among the species tested. In January the seven brassicas
plus sorghum-sudangrass were seeded in the greenhouse. On 20 February, steam-
pasteurized Spinks loamy sand soil was inoculated with l g of fescue seed inoculum
containing FOA or FM and incubated for 5 weeks to allow the Fusarium to colonize the
soil. On 23 March, dry, ground brassica (12.5 g) or fresh, chopped brassica (2% on dry
weight basis) were incorporated and incubated for 5 weeks to allow dissipation of the
phytotoxic compounds. Poplar excelsior also was included as a control. On 4 May, a
five-week-old asparagus seedling was transplanted into each pot. The seedlings were
watered every other day with 100 m1 of deionized water and fertilized every 2 weeks
with 100 ml of 200 ppm 20N-8.7P-l6.6K fertilizer. After 10 weeks, the seedlings were
removed from the pots, rinsed and rated for Fusarium infection using the 1 to 5 scale.

The root lengths were estimated by the line-intersect method (Tennant, 1975).

43

The experimental design was a completely randomized design with nine brassica
treatments, three Fusarium species, fresh versus dry brassica and five replicates. The
data were analyzed by AN OVA and LSD. Relationships between the treatments were
examined by othogonal contrasts.

Soil samples (10 g) were collected from one replication on three dates (23 March,
4 May, 31 July) to determine if Fusarium was present at each stage of the experiment.
The samples were air-dried and stored at room temperature. At the end of the
experiment, the samples were diluted and plated on Komada’s medium (Komada, 1975).
The samples from a single Fusarium treatment for all brassica treatments were plated at

the same time to allow comparison.

Solarization in the Field. During the Summer of 1990 an old asparagus plot with a
history of Fusarium infection was removed at the Horticultural Teaching and Research
Center in East Lansing, MI. The old asparagus crown tissue was removed carefully to
reduce the Fusarium spp. inoculum. The plot was divided into twelve 4.5 x 5.4-m
sections. ’Humus’ oilseed rape, which had shown some control of Fusarium in other
studies, was planted (11.4 kg-ha“) in one-half of the sections on 17 September 1990.
The rape reached the five-leaf stage before cold weather halted its growth.

On 17 May 1991, the rape was 60-cm tall and flowering. Stand counts and fresh
weights were collected from 1 m2 of each plot. The rape was cut with a hand scythe and
disked into the soil. Selected plots were covered with 6-mil black plastic film to

determine if solarization would improve Fusarium inhibition. The soil temperature was

44

measured at 0, 1, 5, 10, and 15-cm depths on 28 June, which was a sunny, clear day
(29C air-temperature, 2 pm.). Soil samples (100 g) were collected randomly from the
top 10 cm of each plot before and after the plastic treatment to determine FOA and FM
populations. After 8 weeks, the plastic was removed and ’Syn 4-56’ asparagus mungs
were planted to test for disease. After 6 weeks more (5 September), three seedlings were
excavated from each plot and rated for Fusarium infection. Root lengths were estimated
by the line-intersect method (Tennant, 1975).

The experimental design was a two-factor randomized complete block with two
brassica treatments and two plastic-covered treatments and three blocks. The data were
analyzed by AN OVA and means were separated by LSD. The brassica treatments were
compared to bare soil and the covered treatments were compared to uncovered using

orthogonal contrasts.

Extraction of Compounds. Four procedures were utilized for extraction of Fusarium-

inhibitory compounds from brassicas.

Boiling Water. Brassica residue (10 g) was extracted with boiling water (300 ml) to
denature the myrosinase enzymes which cause conversion of glucosinolates to
isothiocyanates. The extracts were filtered through Whatman #1 filter paper and
lyophilized at 5C. Each extract was spotted at 125, 250, 500, 1000 pg in 10 ill water
on PDA plates spread evenly with either FOA or FM. The zone of inhibition was

measured after 4 days incubation at 27C.

45

Acetone-Ethyl Acetate. Frozen ’Humus’ rape or fresh mustard flowers and leaves were
extracted with acetone. The brassica tissue (200 g) was blended with acetone“ (1500 ml)
for 4 min. The extract was filtered through Whatman #1 filter paper and evaporated
under vacuum. The remaining solution was partitioned three times with ethyl acetate
(100 ml, 50 ml, 50 ml). The ethyl acetate fraction was dried under vacuum. The water
fraction was dried with excess methanol under vacuum. These extracts were spotted at
0, 250, 500 pg in 10 pl of methanol on PDA plates spread evenly with FOA or FM.

The zone of inhibition was measured after 4 days incubation at 27 C.

Sequential Extraction. Brassica seeds were extracted sequentially with hexane, ethyl
acetate, and methanol. Seeds contained the highest concentration of glucosinolates
(Davis, 1988) and glucosinolates (or the degradation product, isothiocyanate) were
inhibitory to Fusarium (Papavizas and Lewis, 1971). ’Global’ canola seeds (200 g) were
blended with hexane (1000 ml) for 4 min. The extract was filtered through Whatman
#1 filter paper and additional hexane (500 ml) was added to the seeds for 1 h. The
second extract was filtered through Whatman #1 filter paper and additional hexane (500
ml) was added to the seeds and allowed to soak for 24 h. The hexane was removed and
replaced with ethyl acetate (500 ml), which was removed after 1 and 24 h. The ethyl
acetate was replaced with methanol (500 ml), which was removed after 1 and 24 h. All
extracts were dried under vacuum and spotted at 0, 250, 500 ug in 10 ul of DMSO on
PDA plates spread evenly with FOA or FM. The zone of inhibition was measured after

4idays at 27c.

46
Soxhlet. Mustard residue (17.2 g) was extracted for 24 h with 250 ml of methanol in a

Soxhlet apparatus. Poplar excelsior was extracted as the control. The extracts were
dried and tested at 0 and 500 pg in 10 p1 of DMSO on PDA plates spread with FOA or

FM. The zone of inhibition was measured after 4 days at 27C.

Conversion of Compounds. Some naturally occurring compounds require microbial
conversion to become active (Chase, 1990). Since release of isothiocyanate from
glucosinolates in brassicas requires myrosinases (Davis, 1988), the inhibition of Fusarium
may require decomposition of glucosinolates by other soil microorganisms.

A culture of naturally occurring soil microorganisms was isolated from Spinks
loamy sand on PDA plates. A suspension of the microorganisms was made by adding
10 ml of saline solution to the plates and stirring with a glass rod.

’Humus’ canola residue (2 g) was added to 100 g of sterile, washed sand in 250-
ml Erlenmeyer flasks. Sterile water (10 ml) and the microorganism suspension (1 ml)
were added to each flask. The control flasks received 11 ml of sterile water. The flasks
were incubated at 27C. Samples were extracted at 0, 5, 10, 15 days of incubation with
methanol (50 ml x 3 times) and dried under vacuum. The extracts were spotted at 0,
125, 250, 500 pg in 25 pl of methanol on PDA plates spread evenly with FOA or FM.

The zone of inhibition was measured after 4 days at 27C.

47

Results and Discussion
Selection in the Field. Brassicas did not reduce the incidence of disease in asparagus
(Appendix A). However, the average rating of all of the brassica treatments combined
(2.1) was lower than the sudangrass control (2.3) at the 5% level.

The brassicas yielded from 1.9 (mustard) to 5.7 (turnip) kg-m’2 of fresh weight,
depending on type of crop (Appendix A). Horricks (1969) found that 7. 8 t-ha‘l of canola
residue reduced the dry weight and plant height of wheat, barley, and oats. However,
there were no differences among the toxicity ratings for cabbage, cucumber, and tomato
in this study, indicating that phytotoxic compounds were not left in the soil.

After three years, the asparagus field had many missing and stunted plants.
However, there were no differences among the stand counts that could be attributed to

the brassica treatments.

Soil Microflora. The brassica treatments generally did not affect the populations of FM,
total Fusarium or total fungi (Appendix B), so the data were combined by sampling time.
The decrease in FOA and PM from Fall 1989 to Spring 1990 suggests that Fusarium
declines over the winter, as one would expect (Fable 1).

Although the populations were "low" for all treatments (Stephens et al., 1989),
none were completely disease-free. For example, ’Humus’ reduced the FM population
to zero (Appendix B), but did not eliminate the disease because of the presence of FOA

(Appendix A).

48

Table 1. Influence of time on populations of microflora from Spinks loamy sand field

 

soil.
Total Total
Time FM FOA Fusgium Fungi
Fall 1989 141‘ 167‘ 3903 8547
Spring 1990 38 60 2945 9853

 

'F value significant at P g .05

49

’Humus’ rape, turnip and ’Dwarf Essex’ rape had the lowest combined
populations of FOA and FM. This information combined with the Fusarium ratings from
the Selection in the Field Study, above, suggested the use of ’Humus’ for subsequent
research.

No meaningful results are available for the bacteria and actinomycetes counts.
The actinomycetes were not plated on the same media, so no comparisons could be
made. The bacteria had extreme variability in- their counts, suggesting contamination
from external sources. Ramirez-Villapudua (1986) found a 16-fold increase in bacteria

following cabbage residue. This study can neither confirm nor challenge that result.

Phytotoxicity of Residues. The toxicity of the brassica residues on asparagus varied
over time. In general the brassicas inhibited root growth early, but this inhibition
dissipated by 12 weeks. Mustard was the least inhibitory at the beginning of the study.
After 12 weeks, ’Global’ canola and mustard stimulated root growth by 47 and 48%,
respectively, over the excelsior control. A interesting comparison was found between
sudan and kale, which were similar to the other brassica treatments for the first 4 weeks,
but continued to inhibit growth after 12 weeks (Table 2).

Curly cress was considered a sensitive indicator of phytotoxicity, which was
demonstrated by the amount of inhibition remaining after 12 weeks (Appendix C).
’Humus’ oilseed rape continued to inhibit root growth of cress to 44% of control after
12 weeks. ’Global’ canola and mustard stimulated root growth 45 and 65% ,

respectively, over the excelsior control.

50

Table 2. Growth of asparagus roots (percentage of the excelsior control) 0, 2, 4, and
12 weeks after incorporation of brassica residues.

T m 2 4 w 2 w
Sudan, Kale 30.6 68.2
mm 44.1 125.6‘

 

 

‘F values significant at P 5 .05

 

51

Wheat was less sensitive to the toxicity of brassica. All of the brassicas, except
’Global’ canola, inhibited wheat root growth after 2 weeks, but none inhibited after
12 weeks (Appendix C). ’Glacier’ canola was the least inhibitory (63 %) at the beginning
of the experiment. None of the treatments stimulated wheat root growth.

Cucumber was the least sensitive crop to brassica toxicity. Sudan, kale, turnip
and ’Global’ canola inhibited for just 2 weeks (Appendix C). None of the treatments
inhibited after 12 weeks. Mustard was the least inhibitory (58%) at the beginning of the
experiment. None of the treatments stimulated cucumber root growth.

Cabbage is sensitive to the compounds produced by other Brassica spp. The
inhibition was very strong at the beginning, but none remained after 12 weeks
(Appendix C). All of the brassicas inhibited cabbage root growth after 2 weeks, but not
after 4 weeks. Sudan inhibited root growth for less than 2 weeks.

Mason-Sedun (1987) found that canola inhibited the growth and yield of wheat.
Ramirez-Villapudua and Munnecke (1987) found that brassica residues improved the
growth of cabbage. This study demonstrates that the phytotoxic effects depend on the
subsequent crop and the degree of decomposition of the brassica residue. If conditions

are warm and water is not limiting, phytotoxic compounds will dissipate in 4 weeks.

Selection in the Greenhouse. The disease ratings in the greenhouse were highly
dependent on the treatments applied. Dry brassica alone increased the disease ratings of

asparagus seedlings an average of 66% over the excelsior control (Appendix D). The

52

dry brassica may have contained Fusarium spp. inoculum since it had been grown in an
old asparagus field.

Dry turnip, ’Global’ canola, ’Glacier’ canola, or ’Dwarf Essex’ rape residue
increased the disease ratings of FOA-inoculated plants by 25 % over the excelsior control.
All of the brassicas increased the disease ratings of FM-inoculated plants by an average
of 73% over the excelsior control. The disease rating with dry sudan was 63% higher
for FOA and 43 % higher for FM than the excelsior control.

Poplar excelsior reduced the development of FOA or FM infection by 35 % over
the no residue controls, giving disease ratings of 1.6 and 1.4, respectively. Stephens
et al. (1989) considered plants with a rating of 1.5 or less as disease-free. Excelsior may
limit disease development by limiting the nitrogen available to Fusarium spp. or by
improving the WHC of the soil, which will increase the population of microorganisms
that can compete with Fusarium spp. Brassica residues provide a much richer supply of
nutrients than the cellulose/lignan-carbon of excelsior.

The disease ratings for the fresh brassicas were compared with the no residue
controls, since fresh excelsior was not tested. Sudan, turnip, or mustard reduced the
disease rating of FOA-inoculated plants by 33%. The FM-inoculated plants were not
affected by any of the brassica treatments.

The root lengths also were highly dependent on the treatments applied. The
brassicas alone did not affect root growth of asparagus, except for dry mustard, which
increased by 116% over the excelsior control. ’Global’ canola, ’Glacier’ canola,

mustard, ’Dwarf Essex’ rape promoted the growth of FOA-inoculated plants an average

53

of 67% over the excelsior control. All of the brassicas promoted the root growth of FM-
inoculated plants an average of 150% over the excelsior control. Sudan with FOA did
not promote root growth, but sudan with FM promoted growth 88 % .

The root lengths from the fresh brassicas with FOA were not different than the
no residue control, but fresh sudan showed a 48 % increase in growth. Fresh ’Glacier’
canola with FM promoted root growth by 36% , whereas fresh ’Humus’ rape with FM
decreased growth by 59 % over the no residue control.

Dry ’Dwarf Essex’ rape residue with FM produced an average root length of
1396 cm, longer than any of the other brassicas. It also had one of the highest disease
ratings (2.8), suggesting that Fusarium infection was stimulating root growth to
compensate for the disease.

Dilution of the soil samples collected during the greenhouse experiment indicated
that Fusarium was present at each stage of the experiment (data not shown). Fusarium
was not present in the control pots until the asparagus seedlings were transplanted,
suggesting that it was transferred with the seedlings. FOA was the predominate organism

in the control pots.

Solarization in the Field. The disease ratings, root lengths and fresh weights of
seedlings grown in brassica-treated and solarized soil were not different than the control
(Appendix E). However, root length estimates for seedlings with brassica (461 cm) were

shorter than seedlings without brassica (631 cm), indicating a possible phytotoxic effect.

54

The toxicity should have dissipated from the uncovered plots during the plastic treatment;
however, the soil was dry so there was little decomposition.

Soil temperatures were measured under the plastic covers and compared with the
bare soil on 28 June 1991. The soil, temperature just below the plastic was 48C
compared with 39C for the bare soil. At the l-cm depth the temperature was 37C for
both covered and bare soil. At the 5- and 10-cm depths, the covered soil was 5C
warmer than the bare soil. At the 15-cm depth, both soils were the same as the ambient
temperature of 29C. Ramirez-Villapudua and Munnecke (1987) found reduced disease
in cabbage after the soil had been heated to 45C, which would suggest that only the
upper layers of soil are warm enough. However, no conclusions can be drawn from the
temperatures in this study, since they were sampled on a single date.

Extraction of Compounds. Compounds which inhibit Fusarium spp. were not isolated
by water or acetone extraction. Acetone extraction of mustard flowers yielded a fraction
that was slightly inhibitory at 500 pg-ml“. The hexane, ethyl acetate, and methanol
extracts of ’Global’ canola seeds did not inhibit FOA or FM. No Fusarium-inhibitory

compounds were extracted by Soxhlet extraction.

Conversion of Compounds. All treatments gave slight inhibition at 500 pg'ml'1 with
or without the microorganisms from soil extract. However, the zones of inhibition
quickly became covered with fungal growth. Methanol alone was inhibitory to FOA and

FM for a few days, therefore, DMSO was used and the inhibitory fractions were still

55

inhibitory. It was concluded that non-volatile Fusarium-inhibitory compounds were not
present in these extracts.

In conclusion, brassica residues may produce volatile compounds which are
inhibitory to FOA and FM. They cause a temporary suppression of Fusarium, which
would have limited benefit to a perennial crop such as asparagus. Brassica residues also
are toxic to certain crops, causing suppression of root growth for several weeks.

Therefore, growers using brassica cover crops should select subsequent crops carefully.

56
LITERATURE CITED

Chase, W.R. 1990. Response of cucumbers (Cucumis sativa L.), snap beans (Phaseolus
vulgaris L.) and wwds to cover crop residues and rye (Secale cereale L.)
allelochemicals. MS Thesis, Michigan State University, East Lansing, MI.

Damicone, J.P., D.R. Cooley, W.J. Manning. 1981. Benomyl in acetone eradicates
Fusarium moniliforme and F. oxysporwn from asparagus seed. Plant Disease
65:892-893.

Davis, LB. 1988. Winter rapeseed (Brassica napus L.) with differential levels of
glucosinolates evaluated as a green manure crop to suppress Aphanomyces root
rot of Peas (Pisum sativum L.). MS Thesis, University of Idaho, Moscow, ID.

Grogan, R.G. and K.A. Kimble. 1959. The association of Fusarium wilt with the
asparagus decline and replant problem in California. Phytopathology 49:122-125.

Hartung, A.C. 1987. Allelopathic potential of asparagus (Asparagus ofiicinalis L.).
PhD Diss., Michigan State University, East Lansing, MI.

Hodupp, RM. 1983. Investigation of factors which contribute to asparagus (Asparagus
ofiicinalis L.) decline in Michigan. MS Thesis, Michigan State University, East
Lansing, MI.

Horricks, J. 1969. Influence of rape residues on cereal production. Can. J. Plant Sci.
49:632-634.

Hsu, SC. and LL. Lockwood. 1975. Powdered chitin agar as a selective medium for
enumeration of Actinomycetes in water and soil. Applied Microbiology 29:422-
426.

Komada, H. 1975. Development of a selective medium for quantitative isolation of
Fusarium oxysporum from natural soils. Rev. Plant Prot. Res. 8:114—125.

Lacy, M.L. 1979. Effects of chemicals on stand establishment and yields of asparagus.
Plant Disease Reporter 63:612-616.

Mace, M.E., A.A. Belland and CH. Beckman. 1981. Fungal Wilt Diseases of Plants.
Academic Press, New York.

Michigan Agricultural Statistics Service. 1992. Michigan Agricultural Statistics.
Michigan Department of Agriculture, Lansing, MI

57

Martin, J.K. 1975. Comparison of agar media for counts of viable soil bacteria. Soil
Biol. Biochem. 7 :401-402.

Mason-Sedun, W.M.A. 1986. Differential phytotoxicity of residues from the genus
Brassica. PhD Diss., University of New England, Armidale, N .S.W., Australia.

Papavizas, G.C. and LA. Lewis. 1971. Effect of amendments and fungicides on
Aphanomyces root rot of peas. Phytopathology 61:215-220.

Ramirez-Villapudua, J. 1985. Identification of a new race of Fusarium oxysporum Schl.
f.sp. conglutinans (Wr.) Snyd. & Hans. from cabbage and its control by means
of solarization of soil amended with cabbage residues. PhD Diss. , Univ. Cal. ,
Davis, CA.

Ramirez-Villapudua, J. and DE. Munnecke. 1987. Control of cabbage yellows
(Fusarium oxysporum f. sp. conglutinans) by solar heating of field soil amended
with dry cabbage residues. Plant Disease 71:217-221.

Stephens, C.T., R.M. DeVries and KC. Sink. 1989. Evaluation of asparagus species for

resistance to Fusarium oxysporum f. sp. asparagi and F. moniliforme. HortScience
24:365-368.

Stephens, CT. and W.H. Elmer. 1988. An in vitro assay to evaluate sources of

resistance in Asparagus spp. to Fusarium crown and root rot. Plant Disease
72:334-337.

Stephens, C.T., S. Garrod and W.H. Elmer. 1985. A comparison of techniques for
evaluateion of Fusarium spp. resistance in asparagus, pp. 156-167. In:
E.C. Lougheed and H. Tiessen, eds. Proceeding of the Sixth International
Symposium on Asparagus. University of Guelph, Guelph, Canada.

Tennant, D. 1975. A test of a modified line intersect method of estimating root length.
J. Ecol. 63:995-1002.

Tiedje, J.M. and RB. Dazzo. 1982. Soil microbiology. p. 177-209. In: V.J. Kilmer, ed.
Handbook of Soil and Climate in Agriculture. CRC Press, Boca Raton, FL.

CHAPTER 3
INFLUENCE OF ANTIFUN GAL CONIPOUNDS FROM A SOIL-BORNE

ACTINOMYCETE ON FUSARI UM SPP. IN ASPARAGUS

Abstract

Asparagus Decline Syndrome is caused by Fusarium oxysporum f.sp. asparagi
(FOA) and F. moniliforme (FM). Several soil-home micrOorganisms have been found
inhibitory to Fusarium diseases in other crops. A novel Streptomyces spp. (ME2-27—19A)
was isolated from asparagus field soil and found inhibitory to Fusarium spp. in vitro.
Chemical extraction of ME2-27—19A yielded compound(s) that were inhibitory. The
inhibitory fraction was purified by vacuum-liquid chromatography and C18 medium-
pressure chromatography and found inhibitory at 50 pgrml". ME2-27-19A extract
produced variable control of FOA and FM in vitro, and was phytotoxic at 1000 pg-mll.
In soil, ME2-27-19A extract reduced the Fusarium population at 100 pg°ml", but also

reduce the asparagus shoot length.

58

59

Introduction

Fusarium crown and root rot of asparagus (Asparagus oflicinalis L.) are caused
by Fusarium oxysporum (Schlecht.) emend Snyd. & Hans. f.sp. asparagi Cohen (FOA)
and F. moniliforme (Sheld.) emend Snyd. & Hans. (FM). Effective methods of
controlling these diseases of asparagus are not currently available. Resistant varieties,
which have proven effective in other vegetable crops (Mace et al. , 1981) , have not been
found in asparagus (Stephens et al., 1989). Chemical treatments with benomyl [methyl
1-(buty1carbamoyl)-lH-benzimidazol-2-ylcarbamate] and captan [N- [(trichloromethyl)
thio]-4-cyclohexene-1,2-dicarboximide] produced no positive effects on crown survival
or yields over the controls (Lacy, 1979).

However, Smith et al. (1990) found that compounds produced by Streptomyces
griseus var. autotrophicus inhibited F. oxysporum f. sp. asparagi in vitro and in the
greenhouse. Kemira Oy, a European chemical company, has recently started marketing
a biofungicide named Mycostop. It is based on S. griseovirdis and claimed to be
effective against Fusarium spp., although it not registered in the United States. The
objective of this study was to determine if compounds extracted from a newly isolated

soil microorganism would control Fusarium spp. in asparagus.

Materials and Methods
Isolation and culture. Strain ME2-27-19A was one of eight actinomycetes isolated from
Spinks loamy sand soil (Psammetitic Hapludalf, sandy, mixed, mesic) collected from an

old asparagus field in Oceana County, MI. It was compared with six other actinomycetes

60
known to have fungicidal activity and it was selected as the most inhibitory to FOA and

FM. Biochemical tests were conducted using Biolog GN plates (Biolog, Inc. , Hayward,
CA). The microorganism was identified tentatively as a Streptomyces spp. by

Dr. S.K. Mishra (Microbiology laboratory, Krug International, Houston, TX).

Fermentation. Cultures of ME2-27- 19A were grown in 2-liter baffled-bottom
Erlenmeyer flasks, containing 400 ml of A-9 medium (peptone 5 g, glucose 10 g, "Brer
Rabbit green label " molasses 20 g in 1 liter of distilled water). The inoculated flasks

were placed on a rotary shaker at 150 rpm and 27C for 8 days.

Extraction of cells. Microbial cells from 6 liters of fermentation broth were centrifuged
(16300 x g) after 8 days of growth. The cells were filtered under vacuum to remove
excess medium and extracted by homogenizing in methanolzchloroform (MeOHzCHCl3,
1.25 liters, 4:1 v/v) for 10 min. The extract was filtered through a sintered glass funnel
under vacuum. The residue from the funnel was homogenized again with 1.25 liters of
MeOH:CHCl3 (4:1 v/v) and filtered. The filtrates were combined and dried under

vacuum at 40C.

Antifungal/Antibacterial Bioassay. Cultures of Fusarium oxysporum f. sp. asparagi
(MSU-FOA 50) and Fusarium moniliforme (MSU-FM F12) were grown on Potato
Dextrose Agar (PDA) for 14 days. Cultures of Aspergillus flavus and Candida albicans

were grown on YMG medium (yeast extract 4g, maltose 10g, glucose 4g, agar 12 g, in

61

1 liter of distilled water). Cultures of Streptococcus aureus, Staphylococcus epidermis,
and Escherichia coli were grown on Emmons medium (neopeptone 10 g, glucose 20 g,
agar 15 g in 1 liter of distilled water).

Cell or spore suspensions of the test organisms were made by adding 10 ml of
sterile saline solution to a culture dish and stirring with a glass rod. The cell
concentration was adjusted to 10‘5 colony forming units per milliliter (cfu/ ml) using a
hemacytometer. Bioassy plates were made by spreading evenly 100 p1 of the cell
suspension in petri dishes containing 20 m1 of the appropriate medium. Crude extract
of ME2-27-19A was bioassayed by placing 100 pg of extract dissolved in 10 pl of
DMSO (dimethyl sulfoxide) in the center of the bioassay plates and incubating at 27C for
4 days. A zone of inhibition characterized by absence of fungal growth was measured.

ME2-27-19A was also bioassayed by adding 500 or 1000 mg in 0.5 ml of DMSO
to 100 ml of warm (50C) PDA before pouring the medium into petri dishes. A plugs
(5mm) of FOA, FM, Alternaria solani, or Sclerotinia sclerotia were placed in the center

of each petri dish. Radial growth of the fungi was measured daily for 7 days.

Agar Overlay. Crude extract of ME2-27-19A was chromatographed on silica TLC (thin
layer chromatography) plates in CHC13:MeOH (2:1 v/v). The inhibitory bands were
identified on the TLC by pouring 10 ml of molten (50C) YMG agar (amended with 6 g
of agar in 1 liter of water) containing 105 cfu/ml of FOA, FM, Aspergillus or Candida
over the developed TLC plates. The plates were placed in a sterile moisture chamber

and incubated at 27C for 4 days.

 

62
Purification. The crude extract was purified by vacuum-liquid chromatography (VLC).

Crude extract (386 mg), dissolved in MeOH, was loaded on a silica gel column (100 g,
35-75 pm mesh) and eluted with CHC13:MeOH (1:1 v/v). Twenty fractions (30 ml each)
were collected and combined into three major fractions based on similar Rf values from
TLC. The silica was eluted with 100% MeOH to remove any remaining compound.
The fractions were bioassayed on PDA plates spread with FOA or FM. Zones of
inhibition were measured after 4 days at 27C.

A preliminary 1H-NMR spectrum of Fraction 2 was determined on a Varian

Gemini 500 MHz spectrometer in deuterated-DMSO (dg-DMSO).

Minimum Inhibitory Concentration. Fraction 2 from the VLC was bioassayed in test
tubes containing liquid Emmons medium to determine the minimum inhibitory
concentration. Fraction 2 (2 mg, major band at Rf .32) was dissolved in DMSO (200 p1)
and serially diluted in the same solvent. A 20 pl aliquot of each dilution was mixed with
1.96 ml of Emmons medium. The tubes were inoculated with 20 pl of 10’ cfu/ml
solution of FOA or FM. The final concentrations were 0, 20, 30, 40, 50, 100 pg-nrl‘1
of Fraction 2 in 2 ml of solution. The inoculated tubes were incubated on the shaker at
27C and the results were recorded after 4 days. The lowest concentration of compound
that totally inhibited growth of FOA or FM was recorded as the minimum inhibitory

concentration (MIC) for that species.

63
Separation by C18 chromatography. Separation on C18 TLC plates in MeOH:H20

(60:40 v/v) indicated that Fraction 2 contained three compounds. Additional crude
extract was purifiedby VLC. Fraction 2 was purified further by dissolving 0.4 g in 4 ml
MeOH and injecting it into a medium-pressure C18-column (Chemco Scientific Co. ,
Ltd., Osaka, Japan, 4 x 56 cm, 37-55 pm mesh). Forty fractions (10 ml each) were
eluted in MeOHzH20 (60:40 v/v, 25—30 psi, 2 rnl-nrin‘l flow rate) and combined into five
major fractions based on similar Rf from C18-TLC. These fractions were bioassayed on
PDA plates spread evenly with FOA or FM. Zones of inhibition were measured after
4 days at 27 C.

Fraction 3 from the medium-pressure column was further purified on C18
preparative-TLC plates (500 pm thickness) in CHCl3zMeOH (2:1 v/v). The ultra-violet
(UV) absorbance of the compound was determined from 800 to 200 nm on a Shimadru

(Kyoto, Japan) UV260 spectrometer.

Insecticidal assay. The insecticidal assay was conducted on fourth instar mosquito
larvae (Aedes aegypti). Mosquito larvae were hatched by placing eggs in 200 ml of
degassed, deionized water with a pinch of liver powder. After 2 weeks, five to seven
fourth instar larvae in 975 p1 of deionized water were placed in 4-ml test tubes.

ME2-27-19A crude extract (0, 125, 250, 500, or 1000 pg) in 25 pl of DMSO was placed
in the test tubes and left in the dark at room temperature. The numbers of dead larvae
were recorded at 2, 4, 6, 12, 24, 48 h. The experiment was repeated with 100 pg each

of the VLC and medium-pressure column fractions.

 

 

 

64
Phytotoxicity. The herbicidal assay was conducted on ’Syn 4-56’ asparagus, ’Curly’

cress (Lepidium sativum L.) and ’Campbell 1327’ tomato (Lycopersicum esculentum L.)
seedlings. The three fractions from VLC were dissolved in MeOH and placed on sterile
filter paper in petri dishes at 0, 500, and 1000 pg. The MeOH was allowed to evaporate
and 1 ml of sterile water was added to each petri dish. Ten mds of each bioassay crop
were placed in the petri dishes. The petri dishes were sealed with parafilm and incubated
at 27C. The root and shoot lengths of the swdlings were measured after 4 days for cress
and 14 days for asparagus and tomato.

The experimental design was a randomized complete block design with three
blocks. The data were analyzed by analysis of variance (AN OVA) using MSTAT-C
(MSTAT-C, Michigan State University, E. Iansing, MI). The means were separated by

LSD and relationship between treatments were analyzed by linear contrasts.

In Vitro Assay with Asparagus and Fusarium. The effects of ME2-27-19A extract on
FOA and FM in asparagus were determined by incorporating crude extract into 20 ml
of partially cooled (50C) Hoaglands agar (Hoagland and Amon, 1938). The final
concentration of the extract was 0, 100, 200, 1000 png1 in 20 m1 of agar. Asparagus
seedlings were surface-disinfected by acetone infusion (Damicone et al. , 1981) and
germinated on water agar. Germinated seedlings were transferred to the test tubes and
grown in a growth chamber under 16-h days with 15C night temperature and 25C day
temperature. After 3 weeks, as the seedlings were initiating lateral roots, 0.1 ml of

107 cfu/ml solution of FOA or FM was added. The seedlings were grown for an

65
additional 5 weeks and rated for Fusarium infection on a scale of 1-5 , where 1 = healthy

seedling, 2 = slighty infected, 3 = moderately infected, 4 = severely infected, 5 =
dead or dying seedling. The root and shoot dry weights also were measured.

The experimental design was a randomized complete block design with four
blocks. The data were analyzed by ANOVA using MSTAT-C. The means were
separated by LSD and the relationship between extract concentrations was analyzed by

linear contrasts.

Assay of ME2-27-19A Extract in Sterile Soil. ME2-27-19A extract was incorporated
into sterilized Spinks loamy sand soil to determine the effects of ME2-27-19A on
asparagus and FOA or FM in soil. Since purification required several steps and yielded
only a minute amount of pure compound, it was decided an active ingredient method
should be used. A known amount of crude extract (2g) was separated by VLC and
medium-pressure column as before. The active ingredient content was calculated from
the extraction procedure as follows: 2 g of crude extract separated by VLC yielded
0.8125 g of Fraction 2. Separation of Fraction 2 in the medium-pressure C18 column
yielded 0.0402 g of Fraction 3. Purification of Fraction 3 by C18 preparative-TLC
yielded 0.0260 g of pure compound, or 13 mg°g'l of crude extract.

Fusarium inoculum was prepared by adding a plug of FOA or FM to autoclaved
fescue seed (Festuca rubra L.). The fungi were grown for 3 weeks with mixing each

day. The seed was then air-dried and ground to pass a 2-mm sieve in a Wiley mill.

66
Asparagus swds were surface-disinfected with 0.5 % NaOCl (20% bleach) for

20 min, followed by infusion with benomyl (2.5 g) in acetone (100 ml) for 18 hr
(Damicone et al. , 1981). The seeds were rinsed with acetone, followed by sterile
deionized water and dried on a sterilized clay tile (Stephens and Elmer, 1988). The
seeds were germinated of 0.6% water agar.

The soil was Spinks loamy sand soil (Psammetitic Hapludalf, sandy, mixed,
mesic, pH 6.4, CEC 2 meq- 100 g", BD 1.4-1.7 gem", OM 2-4%) and was autoclaved
on three consecutive days. Crude extract was incorporated into dry soil to give 0, 100,
250, 500, 1000 pg a.i.Ig of soil. Benomy1(50% a.i., wettable powder) was incorporated
at the same active ingredient concentrations as the control. Fescue seed inoculum of
FOA or FM were added to give 106 cfu/ ml. Germinated asparagus seedlings were
transferred to the treated soil and 1 m1 of filter-sterilized, 100 ppm 20N-8.7P-16.6K
fertilizer solution was added. The seedlings were grown in the growth chamber for 10
weeks and rated for Fusarium infection on the 1-5 scale. The shoot lengths, number of
shoots, shoot dry weights and root dry weights also were measured.

The Fusarium population was counted by placing 10 g soil in 100 ml sterile saline
solution and diluting serially. The 10'2 dilutions were plated on Komada’s medium
(Komada 1975) and the number of colonies were counted after 4 days. Similarly,
bacterial counts were conducted by plating the 10" dilution on PDA.

The experimental design was a randomized complete block with five blocks.

Only four blocks were sampled for the microflora populations because of the time

 

 

67
required to sample each block. All data were analyzed by AN OVA and means were

separated by LSD. The extract concentrations were compared using orthogonal F-tests.

Results and Discussion

The wild type of ME2-27-19A produced 1 g~liter’1 of crude extract in A-9
medium after 8 days of fermentation. In the GN Biolog plates, ME2-27-19A used Tween
40, Tween 80, aD—glucose, methyl pyruvate, D-gluconate, D-glucuronate, and
asparagine as carbon sources. The crude extract inhibited growth of Aspergillus flavus,
Candida albicans, Fusarium oxysporum f. sp. asparagi, Fusarium moniliforme,
Gleosporium spp. , and Staphylococcus epidermidis at 100 p g-ml“. The inhibition of
FOA and FM was similar to that of nystatin, another microbially produced antibiotic

(Hazen and Brown, 1951).

Agar Overlay. Zones of inhibition remained over the bands at Rf .10, .65, and .68 on
the agar overlay TLC plates. All three bands fluoresced under 366 nm UV-light. The
band at Rf .10 was more stable when left exposed on silica TLC plates, and was selected

for further purification.

Purification. Fractions 2 and 3 from the VLC were inhibitory to FOA and FM in vitro.
The fraction that was eluted with 100% MeOH from the VLC column reacted with
ninhydrin, indicating an amino-group. This fraction had caused much difficulty in earlier

attempts to purify the compound because it would not remain soluble in MeOH or CHC13.

68
The preliminary 1H-NMR experiment with VLC Fraction 2 in deuterated-DMSO

revealed that it did not contain an aromatic moiety (Appendix J). The signal at 66.25
appeared as a multiplet suggested that it contained olifinic bonds. The group of signals
between 4.2-5 .1 ppm suggested the presence of sugar or aliphatic protons attached to
oxygenated carbon. The doublet at 65 .2 revealed the anomeric proton of a hexose
moiety. The multiplets from 3.0-4.0 ppm indicated the presence of aliphatic or amino
protons. Since the compound was not pure, additional spectral work will be carried out

later.

Minimum Inhibitory Concentration. The minimum inhibitory concentration of the
Fraction 2 from the VLC was 50 pg-mIl for FOA and FM. Smith et al (1990) found

the MIC of faeriefungin was 12.5 pg-ml'1 for FOA.

Separation by C18 chromatography. Fraction 3 from the C18 medium-pressure
column was the most inhibitory to FOA and FM. Further purification by C18
preparative-TLC was limited by the solubility of Fraction 3. It was soluble at 30 mg-ml'l
in MeOH, but insoluble in H20. On C18 preparative-TLC plates, it formed a precipitate
which prevented the solvent system from moving. It finally was separated by loading
just 21 mg on a 20 x 20 C18 plate (500 pm). The UV spectrum revealed absorption
maxima at 349, 331, and 316 nm (Appendix I) and were indicative of one or more

chromophores in the purified compound.

69
Insecticidal assay. The crude extract of ME2-27-19A killed 95 % of the mosquito larvae

at 125 pg-ml‘l after 12 h. However, the insecticidal fractions from the VLC were
relatively nonpolar and different from the fraction that inhibited Fusarium, as
demonstrated by the lack of insecticidal activity in the purified C18 fractions. Since
insecticidal compounds were not of direct interest in this project, the compounds were

not isolated.

Phytotoxicity. The seedling assay for toxicity showed no differences in the root
elongation of asparagus or tomato (Table 1). However, the root length of curly cress
was decreased 42% by VLC Fraction 3. The shoot length of asparagus was more than
doubled by 1000 pg-ml'l of VLC Fraction 3. The shoot length of tomato increased 21%
with 1000 pg-ml‘1 of VLC Fraction 1. Curly cress showed the least variation of these

indicator plants.

In Vitro Assay with Asparagus and Fusarium. Although asparagus seedlings showed
no inhibition from ME2-27-19A crude extract on filter paper, seedlings placed in
Hoagland’s agar containing 1000 pg-ml'1 of crude extract failed to grow. When these
seedlings were transferred to fresh Hoagland’s agar, they resumed normal growth.
Asparagus seedlings grown in Hoagland’s agar had higher disease ratings when
inoculated with FOA or FM than the control, as expected (Table 2). ME2-27-19A
extract had no effect, except for inhibiting the asparagus at 1000 pg-ml‘, so the data

were combined to compare the effects of Fusarium.

70
Table 1. Phytotoxicity from VLC fractions of ME2-27-19A crude extract.

 

 

 

 

 

 

 

Root Shoot
Concn. Length Length
Baptism (pg-ml") (cm) (cm) ASPARAGUS
1 0 2.7 0.73
500 1.9 0.99
1000 2.2 0.98
2 0 2.4 0.77
500 3.2 1.27
1000 3.4 1.23
3 0 1.4 0.69
500 2.5 0.95
1000 1.8 1.60
LSD,“ (interaction) ns ns
Root Shoot
Concn. Length Length
Fraction (pg-ml“) (cm) (cm) CURLY CRESS
1 0 1.9 1.6
500 1.5 1.4
. 1000 1.7 1.8
2 0 1.9 1.6
500 2.0 1.8
1000 1.8 1.7
3 0 1.9 1.6
500 1.4 1.6
1000 1.1 1.6
LSD_05 (interaction) 0.5 ns
Root Shoot
Concn. Length Length
Fr_a,gfign (pg-ml") (cm) (cm) TOMATO
l 0 1.2 3.3
500 1 2 3.4
1000 1.3 4.0
2 0 1.0 3.6
500 1.1 3.6
1000 0 9 3.6
3 0 1.0 2.9
500 1.2 3.2
1000 0.9 3.2
LSD”, (interaction) ns 0.6

zMeans represent the average of 3 replications.

71

Table 2. Effects of FOA and FM on asparagus mdlings in vitro.

 

 

 

Root Shoot
FUS Disease Dry Wt Dry Wt.
Ramgzy 1mg)”L (mg)'
0 1.3 5 .5 17.6
FOA 2.8 4.8 18.3
FM 3.5 4.8 18.6
LSD“ 1.3 ns ns

zMeans represent the average of 4 replications.

yFusarium infection rating based on the scale of 1-5, where
1 = healthy seedling, 2 = slightly infected, 3 = moderately infected,
4 = severely infected, 5 = dead seedling.

72
ME2-27-19A alone (200 pg-ml“) caused a browning effect on the asparagus roots

which increased the disease rating. FM-inoculated seedlings with 100 pg-mll of
ME2-27-19A extract had a disease rating of 4.3, while the FM-inoculated seedlings with

200 pg°ml'1 of extract had a rating of 3.7.

Assay of ME2-27-19A Extract in Sterile Soil. ME2-27-19A extract reduced the
population of FOA and FM at 1000 pg°g‘l in sterilized field soil (Table 3). It also
reduced the shoot length by 92% and the number of shoots by 82% at 1000 pg°ml‘l.
Bacterial populations were not affected by either ME2-27-19A or benomyl.

Benomyl reduced the FM population to zero at 100 pg-g", but required
1000 pg-g‘l to eliminate FOA (Appendix G). Benomyl increased the number of shoots
by 70%, suggesting that it removed apical dominance. Manning and Vardaro (1977)
found a similar increase in shoot number from benomyl treatments. Benomyl has not
been used for control of FOA and FM in asparagus because it produced no positive
effects on yield over the control (Lacy, 1979).

ME2-27-19A does not compare favorably with benomyl for control of FOA or
FM. While ME2-27—19A crude extract demonstrated some activity against FOA and
FM, the purified compound was not sufficiently active to warrant further testing in the
greenhouse or field. Although ME2-27-19A was not effective against FOA or FM, this
paper establishes a protocol which may be used for future attempts to control these

pathogens.

73

Table 3. Effects of ME2-27-19A crude extract on asparagus seedlings inoculated with
FOA or FM in sterilized field soil.

 

 

Fusarium Bacteria Number Shoot Shoot
Concn FUS Pop. Pop. of Shoots Length Rating
W-ml“) (log cfu) (log cfu) (cm)
19A 0 0 1.52 7.22 2.6"" 9.9"" ~ 1.6”
100 4.5 8.2 2.0 5.5 4.4
250 0.0 8.2 1.2 4.4 3.6
500 0.0 7.6 1.0 2.1 3.2
1000 0.0 7.6 0.6 0.7 3.4
0 FOA 5.9 7.6 1.6 4 8 5.0
19A 100 5.5 7.7 1.2 3.3 5.0
250 5.4 7.6 0.8 2.4 5.0
500 5.5 7.8 0.8 l 8 4.8
1000 1.5 7.3 0.4 1 0 4.2
0 FM 5 6 7.9 2.0 7.5 4.6
19A 100 5.7 7.4 1.2 3.2 5.0
250 4.1 7.3 1.0 2.4 5.0
500 2 8 7.7 0.4 1.1 4.8
1000 2 7 7.8 0.4 0.7 $6
LSD_05(interact.) 2.4 1.3 0.76 1.8 1.2

'Means represent the average of 4 replicates.
yMeans represent the average of 5 replicates.
"Shoot rating based on a scale of 1-5, where
= healthy seedling, 2 = slightly stunted, 3 = moderately stunted,
4 = severely stunted or diseased, 5 = dead seedling.
wLinear F-value significant at P S .01

74
LITERATURE CITED

Danricone, J.P., D.R. Cooley, W.J. Manning. 1981. Benomyl in acetone eradicates
Fusarium moniliforme and F. oxysporum from asparagus md. Plant Disease
65 :892-893.

Hazen, EL. and R. Brown. 1951. Fungicidin, an antibiotic produced by a soil
actinomycete. Proc. Soc. Expt. Biol. Med. 76:93-97.

Hoagland, DR. and DJ. Amon. 1938. The water-culture method for growing plants
without soil. Univ. Calif. Agric. Expt. Sta. Circular 347: 1-39.

Komada, H. 1975. Development of a selective medium for quantitative isolation of
Fusarium oxysporum from natural soils. Rev. Plant Prot. Res. 8:114-125.

Lacy, M.L. 1979. Effects of chemicals on stand establishment and yields of asparagus.
Plant Disease Reporter 63:612-616.

Mace, M.E., A.A. Belland and CH. Beckman. 1981.'Fungal Wilt Diseases of plants.
Academic Press, New York.

Manning, W.J. and PM. Vardaro. 1977. Soil fumigation and preplant fungicide crown
soaks: Effects on plant growth and Fusarium incidence in newly planted
asparagus. Plant Disease Reporter 61:355-357.

Smith, J ., A. Putnam and M. Nair. 1990. In vitro control of Fusarium diseases of
Asparagus ofiicinalis L. with a Streptomyces or its polyene antibiotic,
Faeriefungin. J. Agric. Food Chem. 38: 1729—1733.

Stephens, C.T., R.M. DeVries and KC. Sink. 1989. Evaluation of Asparagus species

for resistance to Fusarium oxysporum f. sp. asparagi and F. moniliforme.
HortScience 24:365-368.

Stephens, CT. and W.H. Elmer. 1988. An in vitro assay to evaluate sources of

resistance in Asparagus spp. to Fusarium crown and root rot. Plant Disease
72:334-337.

CHAPTER 4
INFLUENCE OF A NOVEL SOIL-BORNE ACTINOMYCETE

ON FUSARIUM DISEASES IN ASPARAGUS

Abstract

Asparagus decline syndrome is caused by Fusarium oxysponan f. sp. asparagi
(FOA) and F. moniliforme (FM). Several soil-home microorganisms have been found to
inhibit Fusarium diseases in other crops. A novel Streptomyces spp. (ME2-27-19A) was
isolated from asparagus field soil and found inhibitory to FOA and FM in
vitro. ME2-27-19A cells, applied as a root-dip in vitro, reduced the disease incidence
in asparagus. In soil, ME2-27-19A decreased the number of root lesions caused by
Fusariwn spp., but also reduced the root length of the asparagus. ME2-27-19A cells
colonize the entire root system in sterile system, but not at sufficient concentration to
inhibit FOA and FM. Maltodextrin was a satisfactory carrier for ME2-27-19A and other

biological control agents.

75

76
Introduction

Effective methods are not currently available for control of Fusarium oxysporum
f.sp. asparagi (FOA) and F. moniliforme (FM) in asparagus (Asparagus oflicinalis L.).
Resistant varieties, which have been effective in other vegetable crops (Mace et al. ,
1981), have not been found in asparagus (Stephens et al. , 1989). Crown-dip treatments
with benomyl [methyl 1-(butylcarbamoyl)- lH-benzimidazol-Z-ylcarbamate] and captan
[N-[(trichloromethyl)thio] -4-cyclohexene- 1 ,2-dicarboximide] produced no positive effects
on crown survival or yields of asparagus (Lacy, 1979) .

However, Cassini et al. (1985) employed an antagonistic microorganism,
Pseudomonas fluorescens, to reduce Fusarium oxysponim infection in asparagus. Smith
et al. (1990) found Streptomyces griseus var. autotrophicus was antagonistic to FOA
in vitro and limited disease severity in asparagus seedlings in the greenhouse. Kemira
Oy, a European chemical company, recently started marketing a biofungicide under the
name Mycostop (Iahdenpera et al. , 1990). It was based on Streptomyces griseovirdis
and claimed to be effective against Fusarium spp. The objective of this study was to

determine if a newly isolated actinomycete could control Fusarium spp. in asparagus.

Materials and Methods
Microorganisms. Cultures of the pathogens (MSU-FOA 50 and MSU-FM F12) were
grown on potato dextrose agar (PDA) for 14 days. Spore suspensions were made by

adding 10 ml of sterile saline solution to the petri dishes and stirring with a sterile glass

77

rod (Stephens et al., 1985). The spore concentration was adjusted to 10‘ colony forming
units per milliliter (cfu/ ml) using a hemacytometer.

Fescue-m inocula of FOA and FM were prepared by the method described by
Elmer (1987). Red fescue seed (Festuca rubra L.)(200 g) was combined with 100 ml
water and autoclaved on three consecutive days. The seed was inoculated with a plug
(5mm) of FOA or FM grown on PDA. The cultures were shaken daily for 3 weeks to I
assure uniform growth of the fungi. The colonized seed was air-dried and ground to pass
a 2-mm sieve in a Wiley mill.

The Fusarium-antagonistic actinomyces strain ME2-27-19A was isolated from
Spinks loamy sand soil (Psammetitic Hapludalf, sandy, mixed, mesic) collected from an
old asparagus field in Oceana County, MI. The organism was identified tentatively as
a Streptomyces spp. by Dr. S.K. Mishra (Microbiology Laboratory, Krug International,
Houston, TX). Preliminary tests showed a zone of inhibition when applied to PDA plates
spread with Fusarium inoculum.

ME2-27-19A was grown in 500-ml baffled-bottom flasks containing 100 ml of
A-9 medium (peptone 4 g, dextrose 10 g, "Brer Rabbit green label" molasses 20 g in
1 liter of distilled water). The flasks were incubated on a rotary shaker at 150 rpm and
27C for 8 days.

ME2-27-19A spore suspensions were prepared by adding 10 ml of sterile saline

to 8-day-old PDA plates and adjusting with a hemacytometer.

78
Plants. The asparagus cultivar Syn 4-56 (Nourse Farms Inc. , South Deerfield, MA) was

used in all experiments. Swds were surface-disinfected with 0.5 % NaOCl (20% bleach)
for 20 nrin, followed by infusion with benomyl (2.5 g) in acetone (100ml) for 18-20 h
(Damicone et al. , 1981). Seeds were rinsed with acetone, followed by sterile, deionized
water and dried on a sterilized clay tile (Stephens and Elmer, 1988). The seeds were
germinated on 0.6 % water agar. For in vitro experiments, germinated seedlings were
transferred to 20 ml of Hoagland’s nutrient agar (Hoagland and Amon, 1938; amended
with 6 g agar/liter). For greenhouse experiments, the seedlings were transferred to 96-

cell (5 x 28 x 53 cm) plastic trays containing sterilized Spinks loamy sand soil.

In Vitro Agar Assay. Two methods were. compared for introducing the antagonist:

root-dip or agar-surface inoculation. Smith et a1. (1990) used root dips to apply
S. griseus to asparagus seedlings. The root-dip treatments were achieved by. dipping
germinated seeds in saline suspensions of ME2-27-19A (0, 107, 109 cfu/ml) before
transferring them to Hoagland’s agar. Surface-inoculated treatments were achieved by
pipetting 0.1 ml of ME2-27-19A spore suspension (0, 107, 109 cfu/ml) into the test tubes
before transferring the seedlings. Both treatments were incubated 2 weeks before
inoculation with 0.1 ml of FOA or FM spore suspension (107 cfu/ml). The inoculated
seedlings were incubated for an additional 5 weeks with a 16-h photoperiod (25C day,
15C night). The seedlings were rated for disease infection on a scale of 1-5, where
l = healthy seedling, 2 = slightly infected, 3 = moderately infected, 4 = severely

infected, and 5 = dead seedling. The shoot and root dry weights were measured.

79
The experiment was repeated four times. In the last experiment, 0. 1 ml of YMG

medium (yeast extract 4 g, maltose 10 g, glucose 4 g, in 1 liter of distilled water) was
added to each test tube to give the antagonist additional nutrients.

The experimental design was a randomized complete block with five blocks. The
data were analyzed by analysis of variance (AN OVA) using MSTAT-C (MSTAT-C,
Michigan State University, E. Iansing, MI) and means were separated by LSD. The

results were examined by linear contrasts for the actinomycete treatment.

Greenhouse Assay. ME2-27-19A was applied to asparagus seedlings in the greenhouse
to determine its effects on FOA and FM in soil. Spinks loamy sand (Psammetitic
Hapludalf, sandy, mixed, mesic, pH 6.4, CEC 2 meq/ 100 g, BD 1.4-1.7 g-cm‘3,OM 2-
4%) was passed through a 2-mm sieve and autoclaved on three consecutive days. Dry
soil was inoculated with FOA or FM on fescue seed (2.5 g/500 ml soil) placed in 500-ml
clay pots and watered.

ME2-27-19A inoculum was prepared by centrifuging (16300 x g for 10 min)
900 ml of cell culture and resuspending the cells in 90 ml of fresh A-9 medium. The
cells were homogenized for 2 min to produce a uniform, thick liquid. Ten-week-old
asparagus seedlings were washed to remove the soil from their roots, inspected for
abscence of Fusarium symptoms, and sorted by weight. The seedlings were dipped in
the ME2-27-19A inoculum (0, 10", 108 cfu/ml) and immediately transplanted into the
soil. The seedlings were fertilized once a week with 100 ml of 200 ppm 20N-8.7P-

16.6K fertilizer.

80

After 10 weeks, the root-ball was sliced into 2-cm sections and samples were
collected to determine distribution of ME2-27-19A and Fusarium in the soil and in the
rhizosphere. For the soil, 10 g from each section was placed in 100 ml of saline. For
the roots, 1 g from each section was placed in 10 ml of sterile saline solution to
determine the rhizosphere micrOflora. The root samples were placed in an ultrasonic
water bath for 2 min to dislodge the rhizosphere organisms. The root and soil solutions
were diluted serially and plated on selective media. The Fusarium population was
counted on Komada’s medium (Komada, 1975). The actinomycete population was
counted on chitin agar (colloidal chitin 2g, agar 15 g, cycloheximide 45 mg in 1 liter of
distilled water). The colloidal chitin was prepared by the method of Lingappa and
Lockwood ( 1962). ME2-27-19A was counted after 2 days and FOA or FM were counted
after 4 days.

After plating the solutions, the number of Fusarium lesions were counted for each
root system and root lengths were estimated by the line-intersect method (Tennant,
1975). The number of lesions per meter of root was calculated from the number of
lesions and the root length. The disease incidence of the roots was rated on the 1-5 scale.
The root and shoot dry weights also were measured.

The experimental design was a two-factor randomized complete block with five
replicates. The greenhouse experiment was repeated with 20-week-old asparagus
seedlings. The data were analyzed by ANOVA and means were separated by LSD. The

treatments were analyzed for linear contrasts.

81

Video-Imaging. The root lengths of the plants from the second greenhouse experiment
were estimated by both line-intersect method and video-imaging (Smucker 1989). During
video-imaging, the root lengths are divided into groups based on root width. One
advantage for asparagus research is that fleshy (> 1mm) and fibrous (< 1mm) root
lengths may be determined without the labor of physically separating them.

The roots were washed and stored in a solution of 15 % methanol at 4C.
Malachite green (20 pg-ml“) was added to stain the roots 24 h before imaging. The
roots were spread evenly in a 45 x 60-cm glass tray and video-recorded with a robotic
camera system. The images were processed by the Root Image Processing Laboratory

(Michigan State University, E. Lansing) on a Viacom computer system.

Root-Colonizing Ability. The ability of ME2-27—19A to colonize the rhizosphere of
asparagus was determined by the method of Misaghi (1990). Germinated mds were
transferred to petri dishes containing water agar (Difco-Bacto agar 6 glitcr").

ME2-27-19A cell suspension (2 pl) was placed 2 mm below root tips. The roots were
inclined and allowed to grow through the cell suspension. The seedlings were observed
daily with a 100X microscope. In a second experiment, a plug of FOA or PM was place
at the edge of the petri dish to observe the interaction between ME2-27-19A, Fusarium

and asparagus.

82
Maltodextrin. Kemira 0y markets a powdered Streptomyces preparation under the name

Mycostop (Iahdenpera et al. , 1990). Preliminary work was conducted on formulation
of the antagonist to enhance colonization, viability and covenience of application.

A spore suspension of ME2-27-19A was made by adding 10 ml of sterile
maltodextrin solution (10%) to a PDA plate containing 8-day-old ME2-27-19A colonies
and stirring with a glass rod. The spore suspension was transferred to a sterile
scintillation vial and lyophilized at 4C and 20 mtorr. Samples were dissolved and plated

after 0, 5 and 12 months to determine viability.

Results and Discussion

In Vitro Agar Assay. The disease ratings for swdlings treated with FOA or FM were
always higher than the no-Fusarium control, as expected. In a preliminary experiment
the disease rating did not change, suggesting that the concentrations of antagonist (0, 107,
10‘, 10" cfu/ml) were too low (data not shown).

In the first experiment, the highest concentration of the actinomycete was
108 cfu/ ml. However, the disease ratings for the high actinomycete concentration were
not different than the antagonist-free control. Since there were no differences among the
actinomycete concentrations, the data were combined for presentation (Table 1).

In the second experiment, the disease rating and the root dry weight for the FOA-
inoculated seedlings improved as the concentration of surface-applied antagonist increased
(Appendix H). However, the disease rating still did not decrease to the level of the

Fusarium-free seedlings. The root and shoot dry weights were reduced 37 and 42%,

Table 1. Effects of FOA or FM on asparagus seedlings in vitro after root-dip (dip) or

83

surface-application (drench) of ME2-27-19A.

 

 

 

 

Drench Drench
Disease Disease Root Shoot
Fusarium Rating Rating Dry Wt. Dry Wt.
TimLspp (Dip)"y (Drench) (mg) (mg)
1 O 1.0 1.0 no data no data
FOA 2.4 2.4
PM 2.9 3.0
LSD 05 0.8 0.9
2 O 1.0 1.0 4.4 16.5
FOA 3.4 3.9 2.6 9.1
FM 3.0 35 2.9 9.9
LSD”, 1.1 1.2 1.7 7.6
3 0 1.0 4.2 19.1
FOA 3.7 3.4 13.5
FM 2.9 3.3 14.9
LSD_05 1.3 2.0 4.9

‘Means represent the average of four blocks and three actinomycete treatments
yDisease rating based on 1-5 scale, where l = healthy seedling, 2 = slightly infected,
3 = moderately infected, 4 = severely infected, 5 = dead seedling

84

respectively, by the Fusarium treatments. The antagonist alone decreased the dry weight
of asparagus shoots an average of 49% . The disease ratings from the root-dip treatments
were not different at any concentration, so the root-dip treatment was discontinued after
the second experiment.

In the third experiment, the disease rating of the FOA- and FM-inoculated
seedlings showed no difference over the range of antagonist concentrations (Appendix H)
and were combined for presentation (Table l). The root dry weight of the asparagus
seedlings decreased an average of 60% for FOA and 53% for PM as the antagonist
concentration increased. The antagonist alone decreased shoot dry weight an average of
40% (Appendix H).

Addition of YMG to the growth medium had no effect on the disease rating of the
Fusarium-treated plants (Table 2). However, the shoot dry weights for the FM-treated
seedlings were 64% higher at the high actinomycete concentration, and might have
improved even more if additional YMG had been added. On the other hand,
actinomycete populations are normally 104 cfu/ g in natural soil and the concentration of
the highest treatment was five-fold higher than a natural population. Therefore, any

beneficial effect would not likely be sustained for long.

Greenhouse Assay. In the first greenhouse study, the disease ratings for FOA- and FM-
inoculated plants were 100 and 187% higher, respectively, than the non-inoculated
control (Table 3). The disease rating for the FOA-treated plants increased 66% as the

concentration of ME2-27-19A increased. The shoot dry weights were 57% lower for

 

85
Table 2. Effects of ME2-27-19A supplemented with YMG on asparagus inoculated with

 

 

FOA or FM in vitro.
Root Shoot

ACT Disease Dry Wt. Dry Wt.
mpg BUS Rating2y (mg) (mg)
0 0 2.0 4.4 12.6
10‘ 1.8 3.1 13.0
10’ 2.3 3.3 16.4
0 FOA 2.5 3.4 9.3
10‘ 3.0 2.0 8.2
108 2.3 2.7 11.9
0 FM 3.5 2.9 7.0
106 3.8 2.2 10.1
H)“ 3.0 3.8 11.5
LSD,“ 1.0 2.2 4.4
(interaction)

‘Means represent the average of 4 replicates.

yFusarium disease rating based of the scale of 1-5 where
1 = healthy seedling, 2 = slightly infected, 3 = moderately infected
4 = severely infected, 5 = dead seedling.

 

86

Table 3. Effects of ME2-27-19A on asparagus inoculate with FOA and FM in sterilized
field soil in the greenhouse.

 

 

 

Root Shoot Root
ACT FUS Disease Lesions/ Dry Wt. Dry wt.
Tim n R m r (mg) (mg)
1 0 0 1.4 0.70 43.7 112.0
10‘ 1.4 0.68 43.0 102.7
10‘ 1.8 1.16 67.0 110.0
0 FOA 2.4 7.47 23.2 57.2
10‘5 2.8 9.93 19.9 41.2
108 4.0 22.62 22.7 51.8
0 FM 4.0 27.11 9.8 31.3
106 4.4 23.87 12.9 37.0
m8 4.4 22.01 13.8 33.9
LSD.05(interaction) 0.9 10.60 13 .9 24. 1
2 0 0 1.8 3.08 939 3380
10‘ 1.8 2.01 927 3532
108 l .6 2.29 893 3075
0 FOA 2.8 8.27 1000 2421
10" 3.0 9.45 680 2258
108 2.4 5.81 671 1669
0 FM 2.4 6.53 1131 3510
10‘ 2.2 6.21 671 1669
108 L4 6.54 888 2263
LSD,05(interaction) 0. 8 3. 76 ns 1 185

”Means represent the average of 5 replicates.

yFusarium disease rating based of the scale of 1-5 where
1 = healthy seedling, 2 = slightly infected, 3 = moderately infected
4 = severely infected, 5 = dead seedling.

87
FOA and 76% lower for FM. The root dry weights were 54% lower for FOA and

68%lower for FM than the control. The number of lesions per meter of root for the
FOA-inoculated plants increased 169% as the ME2-27-19A concentration increased, a
possible synergistic effect.

The counts of the soil microorganisms were so variable that conclusions could not
be drawn, except that both ME2-27-19A and FOA or FM were present in the respective
treatments. ME2-27-19A produced distinctive growth on chitin agar, allowing it to be
distinguished from other bacteria. This medium also may be useful for identifying
ME2-27-19A in natural soil or for isolating other actinomycetes.

In the second greenhouse experiment, the concentration of ME2-27-19A was
slightly lower and the plants were several weeks older, making comparison between the
two experiments difficult. The ratings for Fusarium-inoculated plants averaged 50%
higher than control, while the lesions/meter were two-fold higher. The root dry weights
decreased 36% for FOA and 25% for FM. The root dry weight of FM-inoculated
seedlings without ME2-27-19A was not different from the non-inoculated control.
However, the dry weight of the FM-inoculated plants with ME2—27-19A were 33 to 50%
lower than the control. The counts of the microflora showed no differences between the

sections, suggesting that ME2—27—19A and Fusarium were not limited to one section.

Video Imaging. Video imaging of the asparagus roots revealed that the line-intersect
method underestimated the root lengths by 67% (Table 4). There were no differences

in the fleshy root lengths, however, the fibrous root lengths were lower with FOA and

 

 

 

88
FM treatments. The fleshy root length of FM-treated swdlings decreased as the

concentration of ME2-27-19A increased.

Root Colonizing Ability. The study of root-colonizing ability revealed that ME2-27-19A
cells colonized the full length of the root in a sterile system, but were not sufficient to

keep the pathogen from invading its host plants.

Maltodextrin. Maltodextrin powder provided a method for storing and applying

ME2—27-19A. At the beginning of the experiment, it had a concentration of 1.8 x 107
cfu/g. After 5 months, it had decreased by 47% (9.6 x 10‘ cfu/g). After 1 year, it had
decreased to 1% of the original concentration (1.5 x 10‘ cfu/ g), which would limit its
effectiveness. Maltodextrin has potential as a formulating agent for this actinomycete,

although its shelf-life is limited, which is common with biofungicides.

In conclusion, ME2-27-19A may be one of the organisms which contribute to the
Fusarium-suppressiveness of soils, but it does not provide sufficient control to be used

as a biological control agent for Fusarium spp. in asparagus.

89

Table 4. Video imaging data from asparagus roots grown in sterilized field soil with
ME2-27-19A and FOA or FM.

Fibrous Fleshy

 

 

ACT FUS (< 1mm) (> 1mm) Fibrous/ Estimated
conc spp Root Root Fleshy T R n
Length Length Ratio Video Line Intersect
(m) (m) (m) (m)
0 0 49.1 13.2 3.8 62.3 21.8
10‘ 46.6 14.0 3.3 60.7 21.0
108 50.5 12.1 4.2 62.5 ‘ 21.1
0 FOA 31.1 10.8 2.9 41.9 13.4
10‘5 27.7 9.2 3.2 36.9 10.5
108 24.1 8.3 3.1 32.4 10.5
0 FM 45.6 12.7 3.8 58.3 21.9
106 37.3 10.7 3.5 48.0 15.2
1118 32.1 8.8 3.7 40.8 12.9
LSD”, 14.0 3.7 0.5 17.3 7.0

‘Means represent the average of 5 replicates.

90
Literature Cited

Cassini, R.C., S. El-Medawar and RP. Cassini. 1985. Biological control technique to
prevent Fusarium Decline in the fields. pp. 228-237. In: E.C. Loughwd and
H. Tiessen, (eds.). Proceedings of the Sixth International Symposium on
Asparagus. Univ. of Guelph, Guelph, Ont. Can.

Damicone, J.P., D.R. Cooley, W.J. Manning. 1981. Benomyl in acetone eradicates
Fusarium moniliforme and F. oxysporum from asparagus seed. Plant Disease
65 :892-893.

Elmer, W.H. and CT. Stephens. 1988. Comparison of techniques for eliminating
contaminants from asparagus seeds. Hortscience 23: 1031-1032.

Hoagland, D. R. and D. I. Arnon. 1938. The water-culture method for growing plants
without soil. Univ. Calif. Agric. Expt. Sta. Circular 347 : 1-39.

Komada, H. 1975. Development of a selective medium for quantitative isolation of
Fusarium oxysporum from natural soils. Rev. Plant Prot. Res. 8:114-125.

Lacy, M.L. 1979. Effects of chemicals on stand establishment and yields of asparagus.
Plant Disease Reporter 63:612-616.

Lahdenpera, M.L., E. Simon and J. Uoti. 1990. Mycostop - a novel biofungicide based
on Streptomyces bacteria. In: Biotic Interations and Soil-bome Diseases.
Proceedings from the First Conference of the European Foundation for Plant
Pathology.

Lingappa, Y. and J .L. Lockwood. 1962. Chitin media for selective isolation and culture
of actinomycetes. Phytopathology 52:317-323.

Mace, M.E., A.A. Belland and CH. Beckman. 1981. Fungal Wilt Diseases of Plants.
Academic Press, New York.

Misaghi, LL 1990. Screening bacteria for root colonizing ability by a rapid method. Soil
Biol. Biochem. 22:1085-1088.

Smith, J., A. Putnam and M. Nair. 1990. In vitro control of Fusarium diseases of
Asparagus ofi'icinalis L. with a Streptomyces or its polyene antibiotic,
Faeriefungin. J. Agric. Food Chem. 38: 1729-1733.

Smucker, A.J.M. 1990. Quantification of root dynamics in agroecological systems.
Remote Sensing Reviews 5:237-248.

91

Stephens, C.T., R.M. DeVries and K.C. Sink. 1989. Evaluation of asparagus species for

resistance to Fusarium oxysporum f. sp. asparagi and F. moniliforme. HortScience
24:365-368.

Stephens, QT. and W.H. Elmer. 1988. An in vitro assay to evaluate sources of

resistance in Asparagus spp. to Fusarium crown and root rot. Plant Disease
72:334-337.

Stephens, C.T., S. Garrod and W.H. Elmer. 1985. A comparison of techniques for
evaluateion of Fusarium spp. resistance in asparagus, pp. 156-167. In: E.C.
Lougheed and H. Tiessen, eds. Procwding of the Sixth International Symposium
on Asparagus. University of Guelph, Guelph, Canada.

Tennant, D. 1975. A test of a modified line intersect method of estimating root length.
J. Ecol. 63:995-1002.

SUMMARY AND CONCLUSIONS

Asparagus decline syndrome presents some difficult challenges. Asparagus is a
deep rooted, perennial crop, so temporary controls of Fusarium spp. have limited effect.
Unlike most other commercial crops, asparagus has no natural resistance to FOA and
FM. In addition, asparagus produces autotoxic compounds that predispose it to infection
by F OA and FM.

This dissertation examined two biological agents which have been identified as
possible controls for Fusarium spp. Brassica residues have been used to control
Fusarium oxyspomm f. sp. conglutinans in cabbage and Aphanomyces root rot in pea.
However, the effective compounds were volatile, which would have limited influence on
FOA or FM in asparagus. Brassica residues also are phytotoxic which would prevent
intercropping with asparagus or other sensitive crops. Solarization was not effective for
reducing Fusarium inoculum.

Several soil-home microorganisms have been isolated that compete with
F. oxysporum or reduce infection in susceptible plants. ME2-27-19A may be one of the
many organisms competing with Fusarium spp. for the limited supply nutrients, but its
inhibitory compounds were not effective at eliminating FOA or FM.

Introducing microorganisms into the soil can be expensive because of the

excessive quantities of inoculum or partial sterilization required to overcome the natural

92

 

 

 

93

population. Yet, introduction of microorganisms may be necessary when a suitable
antagonist is not present.

While the biological agents examined here were not effective at controlling FOA
or FM, the basic hypothesis remains valid: to modify the soil environment so that
asparagus and the naturally suppressive microorganisms have an advantage. Practices

.which may maintain asparagus health include liming heavily at planting, preventing

drought stress, and preventing mechanical damage.

 

APPENDICES

 

 

 

95
APPENDIX A

Effects of brassica cover crops on disease rating and phytotoxicity in the field.

 

 

 

1993
Asp.
Brassica Brassica Stand
Asparagus Stand Fresh Count
Disease thtgxigity‘" Countw Weightw (plants/
Treatment Ratingzy Tomatgf Cucumber“ Cabbage" (pl agts/m’) (kg m2) row)w
Sudan 2. 3 2. 0 13 2. 3 23.3
Kale 1.9 1.8 1.8 2.0 104 4.6 24.3
Turnip 2.0 3.0 2.2 2.3 104 5.7 23.3
’Global’ 2.1 2.0 2.4 2.3 87 3.9 26.5
’Glacier’ 2.2 2.2 2.0 1.8 116 5.2 25.1
Mustard 2.1 2.8 2.0 1.8 175 1.9 25.5
’Dwf Essex’ 2.1 3.0 2.6 2.8 101 . 4.0 24.3
’Humus’ 2.0 2.4 1.6 1.8 142 4.2 24.6
LSD”, ns ns ns ns 46 1.1 ns

zMeans represent the average of 3 subsamples from 5 replicates. There were no
significant differences for the three sampling dates, so the data were combined
for presentation.

yFusarium infection rating based on the scale of 1-5, where
1 = healthy seedling (0-10 lesions), 2 = slightly infected (10-30 lesions),
3 = moderate infected (30-40 lesions), 4 = severely infected (40+ lesions),
5 = dead seedling.

"Phytotoxicity ratings based on the scale of 1-4, where
1 = green foliage with a dense canopy, 2 = green foliage, but sparse canopy,
3 = chlorotic foliage, 4 = dead or dying foliage, or poor plant stand.

wMeans represent the average of 5 replicates.

 

96
APPENDIX B

Effects of brassica cover crops on microflora populations in field soil (in cfu/ g).

 

 

Total Total
"Pi—m BMW—Ema
Fall 1989 sudan 230 120 4080 9060
kale 180 140 4230 9000
turnip 80 110 3090 7160
’Global’ 130 100 4980 10238
’Glacier’ 130 130 3850 6800
mustard 60 200 41 10 7380
’Dwf Essex’ 140 230 3070 7030
’Humus’ 180 310 3810 11710
LSD”, ns ns ns ns
Spring 1990 sudan 40 100 3070 8160
kale 70 60 3210 16240
turnip 40 30 3070 10980
’Global’ 20 80 2700 7850
’Glacier’ 40 50 3360 8830
mustard 50 90 2470 8570
’Dwf Essex’ 40 30 1990 9030
’HumuL’ 0 40 3690 9170
LSD“ ns ns ns ns

‘Means represent the average of 5 replicates.

 

 

97
APPENDIX C

Growth as a percentage (%) of excelsior control for asparagus, cress, wheat,
cucumber or cabbage roots following incorporation of brassica residues
(2% of soil dry weight).

 

 

 

 

 

 

Asparagus ~
W 0 weeks 2 wmb 4 weeks 12 weeks
sudan 5.3 40.7 50.2 66.4
kale 0.8 44.8 41.8 70.1
turnip 59.1 32.8 63.8 114.3
’Global’ canola 5.5 44.0 29.6 147.7
’Glacier’ canola 15.4 28.9 44.8 116.8
mustard 67.8 82.5 93.6 148.9
’Dwf Essex’ rape 56.3 30.8 61.2 108.1
’Humus’ rape 0.0 28.8 49.9 117.7
LSD”, (treatment within time) = 44.1
Curly CreL
sudan 4.2 27.1 53.4 99.2
kale 1.4 24.1 69.2 101.6
turnip 7.3 38.5 71.4 66.8
’Global’ canola 7.2 34.4 42.4 145.2
’Glacier’ canola 3.2 23.7 40.9 78.3
mustard 7.7 32.7 82.8 165.2
’Dwf Essex’ rape 3.0 60.6 108.3 81.9
’Humus’ rape 0.0 16.8 60.0 44.3
LSD,“ (treatment within time) = 76.6
Wheat

sudan 26.5 81.3 78.3 123.6
kale 41.6 53.9 79.9 98.5
turnip 33.3 59.5 85.1 135.5
’Global’ canola 24.3 75.8 71.2 118.3
’Glacier’ canola 62.8 57.6 78.9 106.2
mustard 27.7 47.5 86.8 112.0
’Dwf Essex’ rape 36.0 49.0 81.3 103.3
flumus— 24.8 42.0 86.4 102.9

LSD”, (treatment within time) = 47.8

 

 

   

 

98

, APPENDIX C (continued)

 

 

 

 

 

Cucumber
Treatment 0 weeks 2 weeks 4 weelg 12 week;
sudan 9.8 90.8 107.2 90.8
kale 17.8 86.8 96.6 85.4
turnip 15.0 71.9 105.1 95.8
’Global’ canola 37.9 76.4 84.1 92.2
’Glacier’ canola 22.0 67.6 108.8 90.2
mustard 58.8 63.7 98.2 94.6
’Dwf Essex’ rape 23.7 65.7 105.7 96.0
W 6.9 58.4 96.5 103.4
LSD.” (treatment within time) = 30.5
Cabbage

sudan 1.5 75 .2 86.1 106. 1
kale 5.8 53.8 88.1 89.2
turnip 14.7 41.0 81.7 100.3
’Global’canola 0.0 42.2 87.9 106.6
’Glacier’ canola 2.3 15.6 81.9 98.6
mustard 4.9 32.3 82.2 107.0
’Dwf Essex’ rape 11.6 15.7 96.2 95.0
’Humus’ mire 1.2 37.0 93.1 31.9

 

LSD,” (treatment within time) = 33.1

 

99
APPENDIX D

Ratings of Fusarium infection of asparagus seedlings following incorporation of fresh
or dry brassica residues (2% of soil dry weight) in the greenhouse.

 

Dry Dry Dry Fresh Fresh

Residue Residue Residue Residue Residue
mm +FOA +FM +0 +FOA +FM
sudan 2.6 2.0 1.4 1.6 2.6
kale 2.0 2.6 1.8 2.0 2.2
turnip 2.4 2.4 1.8 1.6 2.2
’Global’ canola 2.2 2.2 2.0 2.4 2.6
’Glacier’ canola 2.2 2.2 1.2 2.4 1.8
mustard 2.0 2.4 2.0 1.6 2.0
’Dwf Essex’ rape 2.2 2.8 1.6 2.2 2.0
’Humus’ rape 1.8 2.4 1.2 2.0 2.2
excelsior 1.6 1.4 1.0
no residue 24 2.2

 

Root growth (cm) of asparagus seedlings following incorporation of brassica residues
(2% of soil dry weight) in FOA- or FM-inoculated soil.

 

Dry Dry Dry Fresh Fresh

Residue Residue Residue Residue Residue
T_reatment +FOA + PM + O +FOA +FM
sudan 431 749 626 759 543
kale 550 1031 610 369 361
turnip 541 717 522 357 575
’Global’canola 799 1031 280 305 551
’Glacier’ canola 761 974 525 514 833
mustard 682 1065 893 415 410
’Dwf Essex’ rape 774 1396 482 644 489
’Humus’ rape 523 783 416 306 252
excelsior 451 399 414
no residue 5 13 613

 

LSD.05 = 346

 

100
APPENDD( E

Effects of ’Humus’ cover crop and solarization on the disease rating and growth of
asparagus in the field.

Root Seedling
Disease Length Fresh

 

 

_eat_¢_tsTr m n W
No brassica, No cover 2.4 593.2 5.1

No brassica, Cover 2.7 669.3 3.6
Brassica, Cover 2.7 480.7 3.3
Brassig. No carver 2.7 442.2 4.9
LSD.05 ns ns ns

'Fusarium infection rating based on the scale of 1-5, where
1 = healthy seedling, 2 = slightly infected, 3 = moderately infected,
4 = severely infected, 5 = dead seedling.
YMeans represent the average of 6 subsamples from 3 replicates.

 

Effects of ME2-27-19A crude extract on asparagus seedlings inoculated with FOA or

 

101

APPENDIX F

 

FM in vitro.
ACT Root Shoot
FUS Concn Disease Dry Wt Dry Wt.
pg-ml" Rating” (mg)z (mg)z
O 0 1.0 6.7 19.4
100 1.3 4.4 16.9
200 1.5 5.3 16.6
FOA 0 2.5 4.9 18.9
100 2.7 4.6 13.4
200 3.3 4.8 22.7
FM 0 2.7 5 .0 19.0
100 4.3 3.4 16.5
200 3.7 6.0 20.2
LSD.05 1.3 2.5 8.0

zMeans represent the average of 4 replications.

yFusarium infection rating based on the scale of 1-5, where
1 = healthy seedling, 2 = slightly infected, 3 = moderately infected,

4 = severely infected, 5 = dead seedling.

 

 

 

 

 

102

APPENDIX G

Effects of benomyl on asparagus seedlings inoculated with FOA or FM in sterilized

 

 

 

field soil.
Fusarium Bacteria Number Shoot Shoot
CMPD Concn FUS Pop. Pop. of Shoots Length Rating
Jamil) “We of“) tern)
0 0 1.5‘ 7.2‘ 2.6y 9.9y 1.6 "‘
Ben 100 0.0 7.5 3.6 8.6 1.6
250 0.0 8.0 2.2 9.1 1.6
500 0.0 7.7 3.2 7.5 2.0
1000 0.0 7.5 3.8 6.2 2.6
0 FOA 5.9 7.6 1.6 4.8 5.0
Ben 100 2.4 8.2 3.2 10.1 2.4
250 1.6 7.9 3.0 10.4 2.2
500 1.0 8.0 4.4 7.7 3.0
1000 0.0 5.7 3.2 5.9 2.6
0 FM 5.6 7.9 2.0 7.5 4.6
Ben 100 0.0 8.0 3.6 5.9 2.8
250 0.0 8.0 4.2 6.2 2.6
500 0.0 7.6 4.0 6.1 2.4
1000 0.0 7.9 2.8 7.6 2.4

 

zMeans represent the average of 4 replicates.
yMeans represent the average of 5 replicates.
"Shoot rating based on a scale of 1-5, where

= healthy seedling, 2 = slightly stunted, 3 = moderately stunted,

4 = severely stunted or diseased, 5 = dead seedling.

 

 

103
APPENDIX H

Effects of ME2-27-19A applied as a root-dip (dip) or surface-application (drench) on
asparagus inoculated with FOA or FM in vitro.

Drench Drench

 

 

 

 

Disease Disease Root Shoot
ACT FUS Rating Rating Dry Wt. Dry Wt.
l_cim con_cn inn—WM
1 0 0 1.0 1.0
10‘ 1.0 1.0 no data no data
108 1.0 1.0
0 FOA 2.5 2.8
10‘ 2.3 2.0
108 2.3 2.3
0 FM 2.8 3.0
10‘ 2.8 3.0
108 3.0 3.0
LSD.05(interaction) 0.8 0.9
2 0 0 1.0 1.0 5 .3 24.6
10‘ 1.0 1.0 3.9 10.4
103 1.0 1.0 4.0 14.6
0 FOA 4.0 4.7" 1.5 11.1
106 2.8 4.3 3.7 8.4
108 3.5 2.7 2.8 7.8
0 FM 3.3 3.7" 3.0 10.3
106 3.3 3.7 2.3 9.8
108 2.5 3.0 3.3 9.7
LSD.05(interaction) 1. 1 1.2 1 .7 7.6

zMeans represent the average of 4 replicates.

yDisease rating based on scale of 1-5, where l = healthy seedling, 2 = slightly
infected, 3 = moderately infected, 4 = severely infected,
5 = dead seedling.

"Linear F-test significant at P _<_ .05

104
APPENDD( H (continued)
Drench Drench
Disease Disease Root Shoot
ACT FUS Rating Rating Dry Wt. Dry Wt.
Ti on n i 2’ rench m m
3 0 0 1.0 6.0" 19.2
106 1.0 3.1 19.3
108 1.0 3.6 18.9
0 FOA 4.0 5.3" 12.7
10‘5 4.0 2.7 14.7
103 3.0 2.1 13.1
0 FM 3.0 4.5" 14.5
10" 2.8 3.4 17.1
108 3.0 2.1 13.0

 

LSD_05(interaction) l . 3 2. 0 4. 9

 

 

105
APPENDIX I

UV-spectrum of Fusarium-inhibitory compound from ME2-27-19A (0.23 mg)
in methanol (10 ml).

 

 

 

 

 

 

 

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N N m m e v

Wavelength (nm)

 

106
APPENDIX J

1H-NMR spectrum (500 MHz) of Fusarium- inhibitory compound from
ME2-27-19A in dg-DMSO.

 

 

 

 

 

 

 

 

 

 

 

 

f‘é
‘3'
H

 

PPm

 

 

iii