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MANAGING FOLIAR BLIGHTS ON SPECIALTY CROPS

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BRYAN JAY WEBSTER

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MANAGING FOLIAR BLIGHTS ON SPECIALTY CROPS
By

Bryan Jay Webster

A THESIS

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

MASTER OF SCIENCE
Department of Plant Pathology

2005

ABSTRACT
MANAGING F OLIAR BLIGHTS ON SPECIALTY CROPS
By
Bryan Jay Webster
Specialty crops contribute 40 billion (about 40%) of all annual agricultural sales in
the US. Botrytis cinerea and Alternaria panax are common pathogens on specialty
crops. Control of these foliar blights includes fungicides such as chlorothalonil, a B9.
carcinogen and an industry standard. However, consumers are interested in products that
are environmentally friendly and safe to humans. An in vitro bioassay was used to assess
the ability of biopesticides/reduced risk fungicides to protect geraniums from B. cinerea
infection. Polyoxin D zinc salt and azoxystrobin were effective in limiting germination
to < 4.0 %, limiting appressorial development to < 3.0, and preventing germ tube
elongation from reaching > 5.0 pm over ﬁve incubation intervals. The efﬁcacy of
biopesticides/reduced risk products for B. cinerea control was evaluated under
greenhouse conditions on geranium and ginseng. Azoxystrobin and polyoxin D zinc salt
signiﬁcantly minimized disease severity on geraniums. Polyoxin D zinc salt, boscalid,
ﬁuazinam, and fenhexamid signiﬁcantly limited number of lesions, disease progression,
and disease severity on ginseng. The efﬁcacy of biopesticides/reduced risk products
was evaluated for A. panax control on a ginseng ﬁeld plot. Polyoxin D zinc salt,
fluazinam, and boscalid provided signiﬁcant control against defoliation and disease
severity. The ﬂuazinam, boscalid, and polyoxin D zinc salt treatments provided

signiﬁcantly better yields of the dried, marketable product than the untreated.

DEDICATION

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iii

ACKNOWLEDGEMENTS

First and foremost, I’d like to thank Dr. Mary Hausbeck for her support, guidance,
and overall inﬂuence as a mentor throughout my graduate project. I feel extremely
fortunate for the experience and preparation I received from Mary and the Hausbeck lab
in order to succeed in this program. Thanks to my committee members: Dr.’s
Hammerschmidt, Schilder, and Byme.

I’d also like to acknowledge the limitless technical support from Blair Harlan and
the assistance given by Jeff Woodworth. Additional thanks go to Sheila Linderman and
Brian Cortright for their vast knowledge and experience; to Ryan, Catarina, and Buck for
their insights and willingness to always offer helpful suggestions; and lastly to Amanda
and Shaunta for their endless, daily contributions and their patience for putting up with

my misanthropic views.

iv

TABLE OF CONTENTS

List of Tables ........................................................................................................... vi
List of Figures ......................................................................................................... viii
LITERATURE REVIEW .......................................................................................... 1
Introduction ........................................................................................................ 1
Botrytis cinerea .................................................................................................. 2
Classiﬁcation ...................................................................................................... 3
Pathogen life cycle ............................................................................................. 4
Infection process ................................................................................................ 5
Management strategies ....................................................................................... 7
Chemical control ................................................................................................ 9
Alternaria panax .............................................................................................. l 1
Disease cycle .................................................................................................... 1 1
Control ............................................................................................................. 12
Fungicide alternatives ...................................................................................... 12
EXPERIMENTAL RESEARCH
INTRODUCTION ................................................................................................... 1 4
MATERIALS AND METHODS ............................................................................. 17
In vitro bioassay on ‘Orbit White’ geranium ................................................... 17
F ungicide greenhouse trial on ‘Orbit White’ geranium ................................... 21
Fungicide greenhouse trial on American ginseng ............................................ 24
F ungicide ﬁeld trial on American ginseng ....................................................... 27
RESULTS ................................................................................................................ 30
In vitro bioassay on ‘Orbit White’ geranium ................................................... 3O
Fungicide greenhouse trial on ‘Orbit White’ geranium ................................... 38
Fungicide greenhouse trial on American ginseng ............................................ 43
Fungicide ﬁeld trial on American ginseng ....................................................... 47
DISCUSSION .......................................................................................................... 52
LITERATURE CITED ............................................................................................ 57
APPENDIX A: REDUCED RISK AND BIOPESTICIDE STUDIES IN 2004 ..... ()3

LIST OF TABLES

Table 1. Products tested in vitro for efﬁcacy on excised ‘Orbit White’ geranium discs
against Botrytis cinerea infection .................................................................................... 18

Table 2. Products tested for efﬁcacy on ‘Orbit White’ geraniums against Bot/jvtis
cinerea infection .............................................................................................................. 22

Table 3. Products tested for efﬁcacy on American ginseng against Botrytis cinerea
infection ........................................................................................................................... 25

Table 4. Environmental conditions for post harvest drying procedure on American
ginseng ............................................................................................................................. 28

Table 5. Analysis of variance for percent germination of Botrytis cinerea conidia on
‘Orbit White’ geranium leaf discs .................................................................................... 31

Table 6. Analysis of variance for appressorial development of Botrjytis cinerea conidial
germ tubes on ‘Orbit White’ geranium leaf discs ............................................................ 31

Table 7. Analysis of variance for mean length of Botiytis cinerea conidial germ tubes on
‘Orbit White’ geranium leaf discs .................................................................................... 31

Table 8. The effect of foliar fungicides applied to ‘Orbit White’ geraniums on number of
lesions, disease severity, and on area under the disease progress curve caused by Botrytis
cinerea. Values are according to last rating date for trial ............................................... 39

Table 9. Analysis of variance for average number of lesions caused by Botrytis cinerea
on ‘Orbit White’ geranium ............................................................................................... 40

Table 10. Analysis of variance for area under the disease progress curve (AUDPC)
caused by Botrytis cinerea on ‘Orbit White’ geranium ................................................... 40

Table 11. Analysis of variance for disease severity caused by Botrytis cinerea on ‘Orbit
White’ geranium .............................................................................................................. 40

Table 12. The effect of foliar fungicides applied to American ginseng on number of
lesions, disease severity, and on area under the disease progress curve (AUDPC) caused

by Botrytis cinerea. Values are according to the last rating date for trial ...................... 44

Table 13. Analysis of variance for average number of lesions caused by Botiytis cinema
on American ginseng ....................................................................................................... 45

Table 14. Analysis of variance for area under the disease progress curve (AUDPC)
caused by Botrytis cinerea on American ginseng ............................................................ 45

vi

Table 15. Analysis of variance for disease severity caused by Botrytis cinerea on
American ginseng ............................................................................................................ 45

Table 16. The effect of foliar fungicides on number of American ginseng plants infected,
number defoliated, and disease severity caused by Alternaria panax ............................. 48

Table 17. Analysis of variance for number of American ginseng plants infected by
Alternaria panax .............................................................................................................. 49

Table 18. Analysis of variance for number of American ginseng plants defoliated from
infection by Alternaria panax .......................................................................................... 49

Table 19. Analysis of variance for disease severity on American ginseng plants caused
by A ltemaria panax ......................................................................................................... 49

Table 20. The effect of foliar fungicides against Alternaria panax on yield of American
ginseng roots .............................................................................................................................. 50

Table 21. Analysis of variance for American ginseng root yield infected by Alternaria
panax ................................................................................................................................ 50

Table 22. The effect of foliar fungicides applied to ‘Emperor’ geraniums on number of

lesions, number of lesions with sporulation, and on disease severity caused by Botrytis
cinerea. Values are according to last rating date for trial ............................................... ()5

vii

LIST OF FIGURES

Figure 1. Conidial germination stages of Botrytis cinerea. (A,B) Emergence of a germ
tube from B. cinerea conidium at 6 and 12 h, respectively. (C,D) Enlarged, rounded
ends of germ tubes visible on developing appressoria from B. cinerea. Black scale bars =
20 um. Images in this thesis are presented in color ........................................................ 32

Figure 2. Mean percent of Botrytis cinerea conidia germinated at ﬁve incubation
intervals and treated with one of four fungicides. Bars indicate standard error of the
means ............................................................................................................................... 33

Figure 3. Mean number of Botrytis cinerea germ tubes that developed appressoria at
ﬁve incubation intervals and treated with one of four fungicides. Bars indicate standard
error of the means ............................................................................................................ 35

Figure 4. Mean germ tube length (um) of Botrytis cinerea at ﬁve incubation intervals
and treated with one of four fungicides. Bars indicate standard error of the means ....... 36

Figure 5. Evaluation of biopesticides/reduced risk ﬁingicides on ‘Orbit White’ geranium
against Botrytis cinerea. (A) Untreated control; (B) Chlorothalonil; (C) Polyoxin D zinc
salt; (D) Azoxystrobin. Phytotoxic response observed on: (E) Pyrimethanil and (F) neem
oil extract. Images in this thesis are presented in color .................................................. 42

Figure 6. Evaluation of biopesticides/reduced risk fungicides on American ginseng

against Botrytis cinerea. (A) Untreated control; (B) Chlorothalonil; (C) Polyoxin D zinc
salt; (D) F enhexamid; (E) Boscalid; (F) F luazinam. Images in this thesis are presented in
color ................................................................................................................................. 46

viii

Literature Review

Introduction

Specialty crops (or minor crops), as deﬁned by the Food Quality Protection Act,
are those which are grown on S 300,000 acres nationally, and include crops produced for
niche markets such as some fruits, vegetables, nuts, herbs, and nursery plants
(Pennsylvania State Univ., 2004). Together, these specialty crops generate more than
$40 billion of all annual agricultural sales and account for over 50% of agricultural sales
in 23 states, including Michigan (NASS, 2004).

Specialty crops include greenhouse-grown geraniums (Pelargom'um x horror-um)
and ﬁeld-grown ginseng (Panax quinqefolium). Michigan is the third largest producer of
floriculture crops in the nation with an estimated wholesale value of $342 million in
2004. Approximately 20% of the US. geranium market is supplied/produced by
Michigan growers (NASS, 2004). Ginseng plants, grown and harvested for their roots,
are a high value crop in Michigan and Wisconsin and have an estimated value of over
$100 million. These two states provide approximately 90% of the cultivated ginseng in
the US. and contribute 10% of the global supply. In the last decade, Michigan has
developed a new ginseng industry with signiﬁcant acreage located in the Upper Peninsula
and represents a Michigan inventory of over $50 million. This ginseng is produced under
a natural forest canopy and has a higher market value (up to 10 times) than that of
cultivated ginseng in Wisconsin, which implements artiﬁcial shade cloths over
agricultural soils (Hausbeck, 2004).

Both geranium and ginseng growers have reported leaf and ﬂower blighting

caused by Botrytis cinerea, commonly known as gray mold (Hausbeck, 2004). Dense

canopies, close plant spacing, and senescing ﬂower petals provide an ideal environment
for Botrytis blight in geranium production. For ginseng, cool, wet weather and
cultivation under shade cloths promote infection of ﬂowers, foliage, and berries. Botrytis
blight hinders healthy plant growth, resulting in poor quality ginseng roots with reduced
market value. Developing seed within the berries may also become infected, thereby
hindering future seed production and seedling gardens (Hausbeck, 2004).
Botatis cinerea

Disease Cycle. Affecting a wide variety of omamentals, vegetables, fruits, and other
crops, Botrytis species cause numerous diseases in the form of blights, cankers, leaf
spots, damping-off, and rots of fruits, stems, corms, tubers, bulbs, and roots (Agrios,
1997). Some specialty crops, including greenhouse-grown geraniums and ﬁeld-grown
ginseng, are particularly susceptible to Botrytis blight because of propagation methods
and production environment. Poor air circulation, close plant spacing, promotion of
dense canopies, and high levels of relative humidity increase the crop’s vulnerability to
leaf blight. Blight on these leaves may provide secondary inoculum for subsequent
ﬂower and stem blight (Hausbeck & Moorman, 1996).

The blight or gray mold disease caused by B. cinerea becomes established under
cool, humid conditions. Improper aeration, relative humidity _>_ 85% (DeLozier, 1980),
and temperatures ranging from 15-23 °C provide the ideal conditions for infection,
sporulation, and germination (Agrios, 1997; Jarvis, 1977; Salinas, 1995). Once
established, B. cinerea produces a profusion of gray mycelium and long, branched
conidiophores bearing clusters of one-celled, ovoid, asexual conidia, that resemble a

cluster of grapes. Under humid conditions, conidia are readily released and transported

by air currents. Spore germination can occur on all parts of the host plant above the soil
line. Irregularly shaped, ﬂat, black sclerotia (masses of mycelia which serve as restingor
overwintering structures) oﬁen develop and germinate by way of mycelia] threads which
can infect plant tissues (Coley-Smith et al., 1980). On rare occasion, conidia and
sclerotia yield a Botryotinia perfect stage in which sexual ascospores are produced in an
apothecium. Gerrninating sexual and asexual spores invade tissues through wounds or by
penetrating old ﬂower petals or dying leaves. In ﬁeld crops, B. cinerea overwinters in the
soil as mycelium in decaying plant material or as sclerotia (Agrios, 1997).

Classification. The classiﬁcation of B. cinerea has been difﬁcult due to the inability of
obtaining the perfect (sexual) state from the imperfect (asexual) state under controlled
conditions (Coley-Smith et al., 1980). Ascomycetes belonging to the subphylum
Ascomycotina (class Hyphomycetes) are further categorized according to the
morphology of their sexual (teleomorph) and asexual (anamorph) structures. Members of
Sclerotiniaceae (order Leotiales) have distinctive anamorphs, while the teleomorphs are
relatively uniform. Despite classiﬁcation based on the teleomorph form, genera have
been distinguished by characteristics of their anamorphs, as well as named after them.
Thus, the sexual form, Botryotiniafuckeliana (de Bary) Whetz., produces the anamorph
Botrytis cinerea Pers.:Fr. (Coley-Smith et al., 1980; Farr, 1989; Kohn, 1979). To further
describe the holomorph, Morgan (1971) (as cited by Jarvis, 1977), was able to recognize
two forms of B. cinerea in a numeric analysis of twelve Botrytis taxa; Type A was
characterized by gray or dark brown colonies, luxuriant spore production, and few to no
sclerotia, and Type B colonies were cream or white, with sclerotia] formation preferred at

the expense of conidial proliferation. Munoz et a1. (2002) conﬁrmed that B. cinerea is

made up of two groups, where both have been genetically isolated and could be
considered two distinct species.

Pathogen Life Cycle. Botrytis cinerea has four important infective propagules: conidia,
mycelia, ascospores, and sclerotia (Coley-Smith er al., 1980). The asexual stage
(producing conidia and sclerotia) occurs through the spring and summer and is the
primary inoculum source. On extremely rare occasions, the sexual stage (ascospore
development) may take place with the onset of cold weather in the fall.

The conidiophores arise from mycelia as straight, cylindrical columns, which
develop terminal branches of conidiogenous cells. At maturity, some branches inﬂate
into spherical ampullae, with 8x15 um, smooth, elliptical conidia attached by a ﬁne
denticle. Gray-brown conidia appear as a grape-like cluster under microscopic
examination. Dry and hydrophobic upon release, one to ﬁve germ tubes may gemiinate
from each conidium in the presence of free water (Coley-Smith et al., 1980).

Botryotim’a ascospores are produced within apothecia. The Botryotinia apothecia
are open, disk- or cup-shaped structures, 1-5 mm in diameter, which may or may not be
stalked, and have the asci home in a palisade layer within the cup. Eight ovoid
ascospores develop within the apothecia and are discharged under conditions of high
relative humidity (Coley-Smith et al., 1980). It should be noted that these sexual
structures are not typically found in nature, but have been seen and documented on onion
and bean crops (Ellerbrock & Lorbeer, 1977; Polach & Abawi, 1975).

BOtIyIiS cinerea frequently produces a dense, compact mass of mycelia to create
ﬂat, blackened, 1-2 mm, irregularly-shaped sclerotia which are ﬁrmly attached onto or

just below the host cuticle. Sclerotia may germinate for direct infection of tissue during

the growing season or overwinter in the soil. Although it appears that seeds are not
typically susceptible to infection by B. cinerea, seed lots may become contaminated by
sclerotial bodies that are of the same size and dimension of seeds of ornamental crops
(Agrios, 1997; Coley-Smith et al., 1980).

Infection Process. Sclerotia germinate and produce ﬁlamentous mycelial growth, which
yields conidia-producing sporocarps. Conidia, mycelia, and ascospores all have the
capacity to initiate the infection process by invading senescent leaves and ﬂowers
through stomata or wounded tissues in the presence of free water. Although conidia,
mycelial fragments, sclerotia and ascospores are all considered inocula, the conidia of B.
cinerea act as the primary infection propagules during the repeated asexual life cycle
throughout the host plant’s growing season. The conidial infection process involves
several distinct phases: (i) attachment to the host surface, (ii) germination of the spore
and penetration of the plant surface, and (iii) colonization of the plant tissue (Agrios,
1997)

Proper and secure attachment to the host surface by conidia is critical for
subsequent infection. Doss et al. (1995) categorized two phases, in which immediate
adhesion takes place once the conidia comes in contact with the surface tissue.
Immediate attachment occurs in the presence of free water and hydration of the spore(s).
Delayed adhesion takes place several hours after the initial inoculation and increases the
strength of adhesive forces. These adhesive forces are carried out via a secreted
mucilaginous material consisting of glycoproteins and/or enzymes (Nicholson, 1996).

Attachment is followed by conidial germination, during which one or more germ

tubes develop along the tissue surface. The length of the developing germ tube and the

formation of an appressorium, or penetration structure, are contingent on the availability
of nutrients in the surrounding microenvironment. It has been observed that in the
presence of nutrients, the length of germ tubes are much longer than those that germinate
in the absence of exogenous nutrients (Clark and Lorbeer, 1976; Cole et al., 1996;
Salinas and Verhoeff, 1995; Van den Heuvel and Waterreus, 1983).

Immediate penetration and delayed penetration are two mechanisms by which
Botrytis spp. invade the host tissue. Botrytis cinerea commonly displays delayed
penetration with extensive hyphal growth along the host surface prior to appressorial
development. The hyphal tips may differentiate into appressoria over the top of
epidermal cells or anticlinal wall junctures and afﬁx to the infection surface in a similar
method as the germ tube (Clark and Lorbeer, 1976). Doss et al. (1995) reported that
germ tubes are encased in a sheath that is involved in adhesion and cannot be removed
with proteases or carbohydrate-degrading enzymes. Moreover, Cole et a]. (1996)
revealed that a mucilaginous substance secreted from appressoria formed a pad of matrix
material that assists in attachment to the penetration site. They also noted that spores
germinating under wet conditions became adhered with a ﬁbrillar-like matrix, while dry
conditions cause the spores to produce a condensed, amorphous matrix adhesion pad.

Once an appressorium is attached, turgor pressure increases within the rounded
structure from imbibing external water by osmosis. This forces a hyphal peg out from the
bottom of the appressorium to penetrate the host tissue (Agrios, 1997). Most penetration
may be aided by the release of fungal cutinases and cell wall-degrading enzymes (Struck
et al., 1998). Following penetration of the cuticle, the penetration peg differentiates into

swollen infection hyphae within the epidermis. Infection hyphae grow into adjacent cells

and cause further degradation. As infection progresses, the epidermis swells and lesions
become evident on plant surfaces. Advanced lesions eventually sink and form cavities
from the loss of structural integrity of the epidermal and mesophyll cells (Pie and De

Leeuw, 1991). Clark and Lorbeer (1976) pointed out that this swelling of the epidermal
wall (up to 2-3 times its normal thickness) and ensuing cavity formation may occur in
advance of internal hyphal development. The expression of swelling and cavity
formation on host tissue is dependent upon the pathogen’s ability to release pectic and
cell wall-degrading enzymes or toxins (Coley-Smith et al., 1980). Once the structural
integrity and physiological processes of the host tissue are altered at the invasion site,
necrotic lesions develop and may coalesce on leaf and ﬂower tissue. Fruit, tuber, comi,
bulb, and root tissues may become soft while woody stems may develop cankers (A grios,
1997)

Management Strategies. Manipulating environmental parameters is important for the
successful production of crops under greenhouse or greenhouse-like conditions. Densely
planted ginseng plots and closely spaced geranium potted plants are methods which
maximize production space. However, these characteristics create a favorable
environment for colonization by B. cinerea due to a lack of airﬂow within ginseng
canopies and extensive dieback of the lower leaves in geranium crops. Colonized,
senescent leaves produce sufﬁcient sporulation to successfully inoculate healthy plants
that are in direct contact. Conidia of B. cinerea are released and carried on air currents
and/or from vibrations received by the host plant, as Hausbeck and Pennypacker (1991b)
illustrated in a study on the inﬂuence of grower activity during the production of

geranium propagation. Under conditions suitable for disease incidence, the environment

surrounding newly harvested cuttings was analyzed for peak conidial concentrations
(PCCs) throughout the propagation process. Irrigation, shipment, sanitation, and cutting
placement all provided opportunities for the increased occurrence of PCCs. Thus, it is
recommended that minimal activity take place in the propagation area and that different
stages of the process be segregated into distinct greenhouses. Hausbeck and Pennypacker
(1991a) suggest that plant spacing be increased to encourage better air circulation and
light penetration to reduce senescence of the lower leaves and create an unsuitable
microenvironment for sporulation and germination of the pathogen. Increased plant
spacing also allows for better fungicide coverage.

Sirjusingh and Sutton (1996) looked at wetness duration and temperature in
relation to ﬂower and leaf infection by B. cinerea. Since it is common for older, infected
ﬂower petals to fall onto lower leaves and facilitate new infections under free water
conditions, Sirjusingh and Sutton (1996) quantiﬁed the relationship of wetness duration
and temperature during the wetness period to infection. Sporulation of B. cinerea on
geraniums was higher when wetness on ﬂowers and leaves persisted for more than 4 to 6
h at 21 to 30°C, or greater than 6 to 8 h at 15°C. The authors suggest that periods of
wetness greater than 4 h and overhead irrigation be avoided, and that controlling
humidity with proper heating, ventilation, and air circulation be used to prevent infection
on geranium ﬂowers and leaves.

Hausbeck et al. (1996) investigated the effect of low humidity on disease by
testing the beneﬁts of forced heated air, along with plastic mulch on greenhouse- grown
geranium stock plants. Individual treatments of heated air and plastic mulch were not as

effective in disease management as the two in combination. When used alone, the plastic

mulch was not cost effective and was impractical unless the plants remained in one
greenhouse for the duration of the growing season. Forced heated air seemed beneﬁcial
in systems where plants were moved around within one growing season. Combining both
strategies may reduce initial infection prior to the onset of stem blight and subsequent
disease progression.
Chemical control. Despite signiﬁcant research on managing B. cinerea, controlling this
pathogen has been difﬁcult because it can infect host crops at almost any stage of growth,
and can infect all plant parts. Treatments which are designed and implemented for
protection of a speciﬁc plant part (i.e., ﬂowers) are usually insufﬁcient for providing
protection to all other susceptible parts (i.e., stems, leaves) (Coley-Smith et al., 1980).
Despite the inability of targeting Botrytis management on whole plants, fungicides have
been relied upon for management on ﬁeld and greenhouse crops for several decades.
Due to extensive use of fungicides, B. cinerea has evolved resistance to some of the
traditional chemicals, eliminating the efﬁcacy of two entire fungicidal classes altogether.

Vali and Moorman (1992) showed that frequent and consistent use of a
dicarboximide, such as vinclozolin, produced increased proportions of resistant strains of
the pathogen due to B. cinerea’s adaptive capabilities. As a result, management goals
have focused on reducing spray frequencies per year, combining fungicides or alternating
fungicides that provide different modes of action, and timing applications with ﬂowering
or fruiting periods.

Two classes of fungicides that have become ineffective for Botrytis management
include: the benzimidazoles (inhibits tubulin formation) and the dicarboximides (affects

cell division, DNA and RNA synthesis and metabolism). Resistance to dicarboximides

(vinclozolin, iprodione and procymidone) has been reported in greenhouses and
vineyards in the U. S. (Moorman & Lease, 1992b; Vali & Moorman, 1992), Canada
(Northover & Matteoni, 1986), the U. K. (Locke & Fletcher, 1988), Italy (Gullino er al.,
1982), Israel (Katan & Ovadia, 1985), and Greece (Panayotakou & Malathrakis, 1983).
Following the discovery of such extensive Botrytis resistance to benzimidazoles in
Pennsylvania greenhouses, it was determined that benzimidazoles alone could no longer
be recommended for Botrytis blight management.

Since the early 19903, experiments have been conducted to test the effectiveness
of applying a mixture of different pesticides with separate and distinct modes of action.
In addition, tests were run with biological control agents and with chemicals with multi-
site activity to address concerns of pathogen resistance. Although results varied, they did
suggest that pathogen resistance was likely when the same treatment(s) were used
consistently over time (Elad et al., 1992; Faretra & Pollastro, 1993; Li & Leifert, 1994;
Moorman & Lease, 1992b, 1995; Pollastro et a1, 1996; Vali & Moorman, 1992).

The difﬁculty for many ornamental producers in avoiding the repeated exposure
of plants to chemicals of the same modes of action has been with the production process
itself. Omamentals are commonly grown in more than one greenhouse throughout their
production cycle. Each greenhouse facility exercises its own fungicide regime and does
not pass along application records as the crop is moved from one phase of production to
the next (Moorman & Lease, 1992a). For ginseng production, polyoxin D zinc salt
recently became registered to manage B. cinerea; some additional control can be
achieved with products registered for Alternaria. Crisis exemptions or yearly Speciﬁc

Exemptions to Section 18 of FIFRA have enabled ginseng growers from Michigan and

10

Wisconsin to use fungicides effective in controlling foliar blight outbreaks (Hausbeck,
2004)
Alternaria ganax

A ltemaria panax is a fungal pathogen that causes blights on shoots, stems. and
leaves of both American and Asian ginseng, as well as multiple members of the
Araliaceae family that are indigenous to Hawaii and southern Florida (Garibaldi er al..
2004; Hausbeck, 2004; Uchida, 2003). Of the few foliar pathogens on American
ginseng, A. panax can cause severe losses for ginseng growers in the US. by infecting
the foliage, stems, and berries. If not controlled, the pathogen can reach epidemic
proportions within a month of hot and humid conditions (Hausbeck, 2004). Altemaria
blight ﬁrst appears as water-soaked lesions or spots on plant foliage. As the disease
progresses, these lesions turn tan-colored with dark brown margins and are sometimes
accompanied by yellow, chlorotic haloes surrounding the lesion. The tissue in the center
of the lesion may dry up and fall out, leaving a “shot-hole” appearance. Stems and
petioles may also become infected and collapse. Diseased leaves and stems may develop
dark brown, fuzzy patches of conidia which are released by wind and rain to spread the
epidemic to healthy plants (Hausbeck, 2004; Uchida, 2003).
Disease cycle. Alternaria panax overwinters in plant debris from the previous growing
season. In the spring, the asexual spores become active and can infect newly emerging
plants by wind and rain-splash. These asexual spores are born on conidiophores and
released for dissemination. Each spore is multicellular and resembles a drumstick in
appearance. Once the spores become adhered to host tissue, it will germinate in the

presence of moisture/free water via germ tubes. These germ tubes will penetrate the

11

tissue surface and invade leaf cells. As the fungus develops within the leaf tissue,
conidiophores arise and produce new conidia to infect healthy plants and start the disease
cycle again (Agrios, 1997; Hausbeck, 2004; Uchida, 2003).

Left uncontrolled, Altemaria blight may develop into a signiﬁcant epidemic in
less than 30 days. If most or all of the foliage is destroyed, there is decreased root
development and grth which results in a yield reduction at harvest. Subsequent
outbreaks in the following harvest years can reduce future yields drastically. Reports of
yield losses from growers range from 50-100% if the epidemic is left unmanaged. In
addition, Altemaria blight can ruin seed production on mature plants, thus reducing
healthy seed supplies for future growing seasons (Hausbeck, 2004).

Control. Cultural control is a good way to prevent ideal conditions for the pathogen to
thrive. Increasing the spacing between plants and avoiding weeds and shrubs around the
garden perimeter improves airﬂow throughout crop canopies. Drip irrigation is also
encouraged to maintain dry foliage. Sanitation of diseased leaves can prevent the spread
of a sudden outbreak. Chemical control is a necessary means to prevent infections for
ginseng growers. Fungicides such as mancozeb and chlorothalonil are used frequently
for the duration of the growing season for adequate protection. In order to delay the
development of pathogen resistance to a particular chemical, it is recommended that the
chemical fungicides mentioned above be alternated with differing modes of action
(Hausbeck, 2004; Uchida, 2003).

Fungicide alternatives. Recently, reduced-risk fungicides have/will become available
for use against foliar pathogens. The strobilurins are an example of a group of fungicides

that offer some degree of control for ornamental growers while reducing safety concerns

12

for applicators, non-target organisms, and the environment (Hausbeck et al., 2003). In
addition to the toxicity risks associated with industry standard chemicals (i.e.
chlorothalonil and thiophanate-methyl), the production of these chemicals is costly, time
consuming (up to seven years for testing and approval), and involves extensive research
and development. This risky process can cost an agrichemical company an estimated $40
to $60 million in production costs before it’s reviewed for its ﬁrst registration. It is
estimated that only one in twenty thousand new chemicals actually survive the screening
process for new registration (Bischoff, 1993). Alternatively, reduced-risk fungicide
registration is expedited by the EPA because of the lower toxicity to human health and
natural resources.

Another class of products that poses lower risks is that of biopesticides or
biofungicides. These are naturally-based microbial or biochemical products which are
effective in reduced concentrations and decompose quickly. Alternating standard
chemicals with differing modes of action and reduced-risk fungicides may provide
effective management of foliar pathogens while reducing the risk of pathogen resistance
(Environmental Protection Agency, 2004).

In the following pages, several reduced-risk and biopesticide products are tested
against foliar infection at different stages of development. This has been carried out by:
i) in vitro bioassay to observe conidial germination and the early initiation of the
infection process on treated leaf tissue, ii) greenhouse trials on specialty crops geranium
and ginseng to measure efﬁcacy of products under a Botrytis cinerea epidemic in a
controlled environment, and iii) a ﬁeld plot trial on ginseng to measure efﬁcacy of

products under an Alternaria panax epidemic in a Wisconsin research garden.

13

INTRODUCTION

Specialty crops include greenhouse-grown geraniums (Pelargonium x /I()I‘IOI‘IIIII)
and ﬁeld—grown ginseng (Panax quinqefolium). Michigan is the third largest producer of
ﬂoriculture crops in the nation and supplies approximately 20% of the US. geranium
market (NASS, 2004). Ginseng plants are a high value crop, grown and harvested for
their roots. Total crop value in Michigan and Wisconsin combined is estimated at over
$100 million. These two states provide approximately 90% of the cultivated ginseng in
the US. and contribute 10% of the global supply (Hausbeck, 2004).

Botrytis cinerea causes leaf and ﬂower blight on these two specialty crops during
their production. Dense canopies, close plant spacing, and senescing ﬂower petals
provide B. cinerea with an ideal environment for infection on geraniums (Hausbeck and
Pennypacker, 1991a). Cool, wet weather and cultivation under shade cloths promote
infection of ﬂowers, foliage, and berries of ginseng plants (Hausbeck, 2004). In response
to B. cinerea infection, growers implement fungicidal applications into their production
schedule. Among the available products, chemical fungicides provide adequate control
as long as B. cinerea hasn’t developed resistance to the product’s single mode of action.
In the past, B. cinerea has developed resistance to two ﬂingicide classes: dicarboximides
and benzimidazoles (Gullino et al., 1982; Katan & Ovadia, 1985; Locke & Fletcher,
1988; Northover & Matteoni, 1986; Panayotakou & Malathrakis, 1983). The use of
products from either category is therefore not recommended (Moorman & Lease, 1992b;
Vali & Moorman, 1992). In addition to resistance issues, chemical fungicides pose risks
to the non-targeted environment and health risks to the applicators (Hausbeck et al. ,

2003). Reduced-risk fungicides and biopesticides are becoming increasingly available

14

and are registered more quickly than chemical products because of the lower toxicity to
human health and natural resources (Environmental Protection Agency, 2004)).
Alternating standard chemicals with differing modes of action and reduced-risk
fungicides may provide effective management of B. cinerea and A. panax while reducing
and/or prolonging the risk of pathogen resistance.

To assess the ability of fungicides to protect geraniums from Botrytis cinema. a
leaf bioassay was developed for Pelargonium x hortorum ‘Orbit White’ plants (Byme.
1996; Dwyer, 2004). Treatments included an untreated control, a biopesticide, a
biocontrol agent, a reduced-risk fungicide, and an industry chemical standard. By
quantifying spore germination (%), appressoria formation, and average length of germ
tubes, the bioassay may reveal how and when these products impact the infection process
on the ‘Orbit White’ cultivar. Using similar products, a greenhouse trial was conducted
to observe the efﬁcacy of biopesticides and reduced-risk fungicides in a controlled
environment similar to geranium production conditions. Data collected determined
disease severity and was assessed for AUDPC values and lesion counts on ‘Orbit White’
geraniums.

American ginseng (Panax quinquefolium) is grown in shaded gardens which
create greenhouse-like conditions that are suitable for Botrytis blight (Hausbeck, 2004).
Because of this moderated microenvironment surrounding ginseng canopies, a second
trial was carried out in a greenhouse to mimic these controlled conditions. The trial
consisted of products from similar categories as stated above to observe efﬁcacy against
B. cinerea infection. Data collected was a measure to determine disease severity and was

assessed for AUDPC values and lesion counts on two-year ginseng plants.

15

Alternaria panax is a common pathogen of ginseng, causing foliar and stem
blights to crops. If left untreated, rapid dissemination of asexual spores is possible under
ideal conditions and may incur losses ranging from 50 to 100% of production yield
(Hausbeck, 2004). Very few fungicides are approved for use on ginseng against A.
panax, which increases the risk of the pathogen developing resistance to the few
approved products. A ﬁeld trial was conducted on a research plot in Wausau, WI to test
the efﬁcacy of biopesticides and reduced-risk products against A. panax. An identical
protocol of fungicides used for the ginseng greenhouse trial described above was also
used for this ﬁeld trial. Data collected determined disease severity and was assessed for

lesion counts and defoliated/dead plants on two- to three-year ginseng plants.

16

MATERIALS AND METHODS

In vitro bioassay on ‘Orbit White’ geranium:
Inoculum preparation and procedure. Botrytis cinerea was isolated from zonal

(Pelargonium x hororum) and ivy (Pelargonium peltatum) geraniums obtained from the
Michigan State University Demonstration Gardens. Cultures of B. cinerea were grown
on potato dextrose agar (Difco Laboratories, Detroit, MI) + Streptomycin in 100 x 15 mm
Petri dishes under cool white ﬂuorescent light. Spore suspensions were made by ﬂooding
cultures with 10 mL of sterile distilled water (deO) and gently agitated with a brush to
release spores. The suspension was ﬁltered through one layer of cheesecloth to remove
mycelial fragments. The concentration was adjusted to 1 x 10° conidia/mL using a
hemacytometer (Buck, 2002).

Treatment gpplication and inoculation. Leaves at approximately the second to third node
were tagged on mature, seed-propagated geraniums (cv. Orbit White) that were
maintained in a research greenhouse at Michigan State University. Five treatments
(Table 1) were applied to respective plants: a biopesticide (Endorse), a biocontrol agent
(Bacillus subtilis), a reduced-risk fungicide (azoxystrobin), a standard chemical
protectant (chlorothalonil), and an untreated control. Twenty-four hours after treatment,
tagged leaves were removed and 12 mm discs were excised with a sterilized cork borer
and placed into corresponding Petri dishes. All dishes contained a plastic mesh grid
(sterilized in a 10% bleach solution) that separated leaf discs from ﬁlter paper that was
saturated with sterile distilled water (deO). Leaf discs were placed adaxial surface up
and inoculated with 20 ul of spore suspension with 0.2% of Tween 20 in the center of
each of six discs per plate. Petri dishes were wrapped in Paraﬁlm, to maintain humidity

and avoid evaporation, and incubated in a diurnal growth chamber for 3, 6, 12,

17

Table 1. Products tested in vitro for efﬁcacy on excised ‘Orbit White’ geranium discs
against Botrytis cinerea infection.

 

 

Active Formulation Used .

Product Igredient Manufacturer (per lOMons) Classiﬁcation"
Dacoéttiilkﬁgather chlorothalonil Sygigibirrfﬁgc' (625202313) BZ carcinogen
Endorsezswp PolyofagtDm Arygggffgisgfecimp- (9372;13g) Biopesticide
Heritage SOWG azoxystrobin Syg§::l:bi::?§g)c' (Zgz'ggzg) Reduced risk
[113113,ng Bacillus subtilis Aggigifséinc' (185301211) 13 iupestic ide

 

’ Human risk assessment: 82 carcinogen = likely human carcinogen; Reduced risk = reduced pesticide risk
to human health, non-target organisms, and environmental resources; Biopesticide = naturally based
microbial or biochemical product with low toxicity.

18

24, and 36 h at 16 °C under continuous illumination. A separate preliminary test was
conducted twice to determine the length of time (hours) for conidial germination and to
monitor the effects of using a biocontrol agent under procedural conditions. Similar
methods as mentioned above were used with the exception of longer incubation time
intervals.

Observations: Conidial observations were made with a compound light microscope at

 

400x (Olympus America Inc., Melville, NY). Thirteen conidia were randomly selected
from one of four quadrants on each disc to determine percentage of germination and
subsequent appressorial development. Individual conidia were considered germinated if
the germ tube length measured at least half the width of the conidium (Lacy, 1994). The
average length of germ tube (um) was determined by randomly selecting ﬁve germinated
conidia from each disc. Observations for the parameters listed above were only made on
single conidia having one germ tube; conidia in pairs or clusters were not considered.
Experimental design and stgtistical analysis: This experiment was arranged in a
randomized block design with two separate trials carried out. Percent germination and
appressorial development were analyzed using the same statistical model, though the
residuals for appressorial development were not grouped by treatment because of the low
number of observations. The trial by treatment interaction was a random effect due to
high variability of treatments for each of the two runs. The time by treatment interactions
were ﬁxed effects. Analysis of variance (ANOVA) was conducted using the MIXED
procedure of the Statistical Analysis System (SAS Institute Inc., Cary, NC.) Differences
were determined by pairwise comparisons within interaction terms using Least Square

means (P > 0.05). Square root transformations were made on percent germination and

19

appressorial development data; results were back-transformed. A separate model was
used for germ tube length, with each trial treated as a random effect. Data were averaged
across each disc and log... transformations were used because of the high quantity of
zeros. All data were back-transformed for reporting analysis of variance and pairwise
comparisons using Least Square means (P > 0.05). Lower and upper conﬁdence intervals

were used instead of standard errors on back-transformed data.

20

Fungicide greenhouse trial on ‘Orbit White’ geranium:
Plant material. A 288-cell ﬂat was seeded with ‘Orbit White’ geraniums (Pelargonizun x

 

liortorum) in September, 2004. Six weeks after seeding, seedlings were potted in 6-inch
plastic pots containing Baccto high porosity professional planting mix (Michigan Peat
Company, Houston, TX) and maintained in a research glass greenhouse at Michigan State
University. Plants were fertilized with 250 ppm Peter’s 20N-20P-20K liquid feed
fertilizer (The Scotts Company, Marysville, OH) three times a week, and fertilized with
acid treatment (1100 ml diluted phosphoric acid) once a week. Glasshouses were
maintained at 24 °C for both day and night. No supplemental lighting was used.
Inoculum preparation and procedure. Botrytis cinerea was isolated from zonal
(Pelargonium x hororum) and ivy (Pelargom’um peltatum) geraniums obtained from the
Michigan State University Demonstration Gardens. Cultures were grown on potato
dextrose agar (Difco Laboratories, Detroit, MI) amended with 5.0 g Streptomycin sulfate
(Sigma Chemical Co., St. Louis, MO)/ L of FDA solution in 100 x 15 mm Petri dishes
under cool white ﬂuorescent light. Spore suspensions were made by ﬂooding cultures
with 10 mL of sterile distilled water (deO) and gently agitated with a brush to release
spores. The suspension was ﬁltered through one layer of cheesecloth to remove mycelial
fragments. The concentration was adjusted to l x 10" conidia/mL using a hemacytometer
(Buck, 2002).

The trial consisted of nine treatments (Table 2) including an untreated control.
Eight fungicide treatments were applied using a hand-pressurized spray bottle to runoff.
Plants were placed in individual humidity chambers immediately after fungicidal

treatment and allowed to dry. Four hours after treatment, plants were inoculated with the

21

Table 2. Products tested for efﬁcacy on ‘Orbit White’ geraniums against Botrytis

 

 

cinerea infection.
Active Formulation Used
Product Ingredient Manufacturer (per 100 gallons) Classiﬁcation‘
Daconil Weather . Syngenta Crop Proc. 22 ﬂ oz . ,
S tik 6F chlorothalonil Greensboro, NC (650.6 ml) 82 carcrnogen
. . Arysta LifeScience
Endorse 2.5WP polyoxsgiltD zrnc Corp. (929'; 5b,) Biopesticide
San Francisco, CA ' 5
. . Syngenta Crop Proc. 8.0 oz ‘ ‘ , '
Heritage 50WG azoxystrobrn Greensboro, NC (226.8 g) Reduced risk
. 0 Gliocladium Kemira Agro Oy 100.0 oz . .. . ‘. 3
Prtmastop l oWP catenulatum Helsinki, FIN (2,835 g) BlopLSllleL
Rhapsody ‘. ' . . AgraQuest Inc. 8.0 qt . ) . ‘.
1.34%AS Bacillus subtlhs Davis, CA (7570 ml) BlOpcSllUdC
. . . Citrex, Inc. 12.9 ﬂ oz
Citrex 100L ascorbic acrd Miami, FL (381.5 ml) -
‘ . . Bayer Crop Science 1.6 pt ‘ :1 .
Scala 4008C pyrimethaml Research Triangle, NC (7571 ml) Reduced risk
. ‘ . Olympic Hort. Prod. 1 gal .
Triact 70EC neem 01] Bradenton, FL (3,790 ml) BIOpLSlllee
Messenger ha in rotein EDEN Bioscience 9.0 02 Bio esticide
3WDG ’p P Bothell, WA (255.1 g) p

 

7' Human risk assessment: BZ carcinogen = likely human carcinogen; Reduced risk = reduced pesticide risk
to human health, non-target organisms, and environmental resources; Biopesticide = naturally based

microbial or biochemical product with low toxicity.

22

B. cinerea conidial suspension using a spray bottle to runoff. The plastic bags were
immediately sealed and reopened only for subsequent treatment and inoculum
application. After each treatment, plants were reinoculated four hours later using the
method previously described. Six replicates per treatment were arranged in a completely
randomized design in a research glass greenhouse at Michigan State University. This
experiment was conducted twice; Messenger was substituted for Primastop in the second
trial of the experiment.

Disease assessment and statistical analysis. Disease ratings were taken weekly (28 Dec,
4, ll, 18, 25 Jan, and 1 Feb: trial 1; 17, 24, 31 Mar, 7, 14, 21, and 28 Apr: trial 2). This
included disease severity, percentage of infected leaves (number of leaves with Botrytis
blight divided by total number of leaves for each plant), number of lesions and number of
lesions with sporulating B. cinerea. Plant disease severity was visibly assessed using a
scale: 1 = healthy plant; 2 = 5% infection; 3 = 15%; 4 = 25%; 5 = 40%; 6 = 60%; 7 =
75%; 8 = > 75% + partial leaf abscission; 9 = > 75% + leaf abscission; 10 = plant death.
The area under the disease progress curve (AUDPC) was calculated to express the
progression of lesion incidence on plant foliage using the method of Shaner and Finney

(1977):

AUDPC = :1 [(Y,-+,,l + Y,-)/2][X;+1-X,-]
where Y,- = number of lesions per plant at the ith observation, X,- = time (days) at the ith
observation, and n = total number of observations. Data for all disease assessments were
analyzed with analysis of variance (ANOVA) using the GLM procedure of the Statistical
Analysis System (SAS Institute Inc., Cary, NC). Differences among treatment means

were determined by pairwise comparisons using Fisher’s protected LSD (P > 0.05).

23

Fungicide greenhouse trial on American ginseng:
Plant material. Two-year old, ﬁeld-grown ginseng (Panax quinquefolium) roots were

dug from commercial Wisconsin ﬁelds in the fall of 2003 and 2004 and stored at
approximately 3 °C for 100 days in silica sand for each respective year. Roots were
transplanted into 1 gallon pots containing Baccto high porosity professional planting mix
(Michigan Peat Company, Houston, TX) and placed under 63% shade (to mimic
production systems) in a research glass greenhouse at Michigan State University. Plants
were fertilized with 250 ppm Peter’s 20N-20P-20K liquid feed fertilizer (The Scotts
Company, Marysville, OH) three times a week, and fertilized with an acid treatment
(1 100 ml diluted phosphoric acid) once a week. Glasshouses were maintained at 24 °C
for both day and night. No supplemental lighting was used.
Inoculum preparation and procedure. Botrytis cinerea was isolated from infected
ginseng (Panax quinquefolium) plants from a Michigan State University research
greenhouse. Cultures were grown on potato dextrose agar (Difco Laboratories, Detroit,
MI) amended with 5.0 g Streptomycin sulfate (Sigma Chemical Co., St. Louis, MO) / L of
PDA solution in 100 x 15 mm Petri dishes under cool white ﬂuorescent light. Spore
suspensions were made by ﬂooding cultures with 10 mL of sterile distilled water (ngO)
and gently agitated with a brush to release spores. The suspension was ﬁltered through
one layer of cheesecloth to remove mycelial fragments. The concentration was adjusted
to 1 x 10" conidia/mL using a hemacytometer (Buck, 2002).

The trial consisted of eleven treatments (Table 3) including an untreated control.
Ten fungicide treatments were applied using a hand-pressurized spray bottle to runoff.
Plants were placed in individual humidity chambers immediately after fungicidal

treatment and allowed to dry. Four hours after treatment, plants were inoculated with the

24

Table 3. Products tested for efﬁcacy on American ginseng against Botrytis cinerea

 

 

infection.
Active Formulation Used
Product mredient Manufacturer (per 100 gallons) Classiﬁcation’
Bravo Weather . Syngenta Crop Proc. 2.0 pt 7 .. v )
S tik 68 C chlorothalonil Greensboro, NC (946.4 ml) B- carcinogen
polyoxin D zinc Arysta LifeScience Corp. 2.2 lb . j g . ‘.
Endorse 2.5WP salt San Francisco, CA (9979 g) Biopesticide
* y - . Arysta LifeScience Corp. 1.5 lb 1 .. .. . .
Elex ate 50WDG fenhexamid San Francisco, CA (680.4 g) Reduced risk
‘. thiophanate- Cerexagri, Inc. 21.8 ﬂ oz . .. U ,
Topstn 4.5L methyl King of Prussia, PA (644.7 ml) 132 carcinogen
. . . . AgraQuest Inc. 10.0 lb . . .
. o -. . . . .
Serenade lO/oWP Bacillus subttlzs Davis, CA (4,536 g) Biopesticide
‘ . . _ . Syngenta Crop Proc. 0.75 oz .) ' . ‘ . .. -
Actigard 50WDG acrbenzolar Greensboro, NC (213 g) l lant actix ator
. BASF Corp. Research 6.8 oz .
Endura 70WG boscalid Triangle, NC (192.8 g) Reduced risk
PlantShield Trichoderma BioWorks, Inc. 100.0 02 Bio :sti ‘ide
1%WP harzianum Fairport, NY (2,835 g) 'pt I
Messenger ha in rotein EDEN Bioscience 9.0 oz Bio ‘8“ ‘ide
3WDG rp p Bothell, WA (255.1 g) p“ ‘
. , . Syngenta Crop Proc. 1.0 pt ‘ ‘ ‘ _. ‘ .
Omega 500F ﬂuazrnam Greensboro, NC (4732 ml) Reduced risk

 

’ Human risk assessment: 82 carcinogen = likely human carcinogen; Reduced risk = reduced pesticide risk
to human health, non-target organisms, and environmental resources; Biopesticide = naturally based

microbial or biochemical product with low toxicity; Plant activator = activates plant’s defense
mechanisms against diseases/pests.

25

B. cinerea conidial suspension using a spray bottle to runoff. The plastic bags were
immediately sealed and reopened only for subsequent treatment and inoculum
application. After each treatment, plants were reinoculated four hours later using the
method previously described. Six replicates per treatment were arranged in a completely
randomized design in a research glass greenhouse at Michigan State University. This
experiment was repeated two times.

Disease assessment and statistical analysis. Plant ratings were taken weekly (19, 26 May
and 2, 9, 16, 23, 30 Jun: trial 1 in 2004; 12, 19, 26 May and 2 Jun: trial 2 in 2005) and
assessed for disease severity, percentage of infected leaves (number of leaves with
Botrytis blight divided by total number of leaves for each plant), number of lesions and
number of lesions with sporulating B. cinerea. Plant disease severity was visibly
assessed using a scale where l = healthy plant; 2 = 5% infection; 3 = 15%; 4 = 25%; 5 =
40%; 6 = 60%; 7 = 75%; 8 = > 75% + partial leaf abscission; 9 = > 75% + leaf
abscission; 10 = plant death. The area under the disease progress curve (AUDPC) was
calculated to express the progression of lesion incidence on plant foliage using the

method of Shaner and Finney (1977):

AUDPC = :IKYA,” + Y,)/2][X,~+1-X,-]
where Y, = number of lesions per plant at the ith observation, X,- = time (days) at the ith
observation, and n = total number of observations. Data for all disease assessments were
analyzed with analysis of variance (ANOVA) using the GLM procedure of the Statistical
Analysis System (SAS Institute Inc., Cary, NC). Differences among treatment means

were determined by pairwise comparisons using Fisher’s protected LSD (P > 0.05).

26

Fungicide field trial on American ginseng:
Plant material. Field-grown ginseng (Panax quinquefolium) roots were planted in the fall

 

of 2002 and grown for two seasons in a research garden in Marathon County, WI under
wooden laths. Weed control and fertilization were done according to commercial
production standards. Inoculation of plant material was not necessary due to Alrernaria
panax occurring naturally in the ﬁeld. Ginseng roots were harvested on 14 and 28 Sep,
2005, had foliage removed, and were lightly rinsed with distilled water to remove excess
soil. Fresh weights from each treatment block were recorded immediately following
harvest. Roots were then laid out to dry for 14 days on 4 x 10 ft screens and subjected to
forced heated air to mimic standard harvesting/drying procedures by ginseng growers
(Table 4). Weights were recorded on dried roots, as this is the marketable product.
Experimental design and statistigl anglysis. The trial consisted of eleven treatments
(Table 3) including an untreated control. All plants were grown on 4-tt wide, raised beds
with 1 it in between bed rows. F ungicide treatments were replicated four times and
arranged in a randomized complete block design. Treatment blocks were 10-ft sections
of bed rows with a 2 it buffer on each end. Ten fungicide treatments were applied using
a C02 backpack sprayer with a 4 it boom equipped with four 8006 nozzles spaced 18 in
apart, operating at 40 psi, and delivering 100 gal/A. Treatments were applied at 7-day
intervals from 1 Jun through 4 Aug. Plants were assessed for disease severity, the
number of plants infected and the number of plants with partial to complete defoliation
from Alternaria panax on 28 Jun, 11 Jul, and l, 10 Aug. The ﬁnal two ratings are being
reported from 1 and 10 Aug. Plant disease severity was visibly assessed using a scale
where 1 = no visible lesions; 2 = 25% infected; 3 = 50% infected; 4 = 75% infected; 5 =

100% infected + some defoliation; 6 = 25% defoliation; 7 = 50%

27

Table 4. Environmental conditions for post harvest drying procedure on American

 

 

 

ginseng.
Temperature Relative Humidity

Day High Low High Low
1 69.0 49.6 98 47
2 67.0 48.1 100 57
3 68.4 62.2 99 55
4 67.7 64.2 100 87
5 86.6 60.1 92 53
6 90.3 86.6 59 43
7 96.4 89.6 55 39
8 102.7 94.8 51 34
9 103.5 95.6 39 21
10 102.7 95.6 22 21
11 103.5 95.6 27 21
12 102.7 94.8 28 21
13 100.3 93.3 26 21
14 115.6 87.4 21 21
15 1 17.4 63.5 68 21
16 68.4 67.7 36 27

 

28

defoliation; 8 = 75% defoliation; 9 = 95% defoliation; 10 = complete defoliation and
plant death. Harvested roots were weighed for yield data. Fresh weights were taken on
the harvest date, and dried root yields were recorded two weeks later, as this is the
marketable product. Data for all disease assessments were analyzed with analysis of
variance (ANOVA) using the GLM procedure of the Statistical Analysis System (SAS
Institute Inc., Cary, NC.) Differences among treatment means were determined by

pairwise comparisons using Fisher’s protected LSD (P > 0.05).

29

RESULTS

In vitro bioassay on ‘Orbit White’ geranium:
GerminzLion of conidia: The effects of time and treatment on conidial germination were

signiﬁcant at P < 0.05 (Table 5). Conidial germination occurred between three and six
hours at 16 °C (Fig. 2). Germination on untreated leaf discs increased dramatically and
reached 16.2 % after six hours of incubation, but then decreased after 12 hours.
Germination rose again to 14.3 % at the 24 h interval and slowly increased to a peak of
16.4 % at the 36 h incubation time.

Alternatively, conidia exposed to chlorothalonil had a slight increase in
germination at 6 h, but did not germinate at any of the other time intervals and was
statistically better than the untreated control at 3, 6, 24, and 36 h intervals. The
azoxystrobin treatment limited germination almost as effectively as chlorothalonil and
was signiﬁcantly better than the untreated control at time intervals: 3, 6, 24, and 36.
Conidia treated with B. subtilis rose to 1.8 % germination at the 12 and 24 h intervals, but
decreased by the 36 h interval to differ signiﬁcantly from the untreated control.
Germination was limited to < 1 % by Polyoxin D zinc salt treatment except for at the 24 h
incubation interval where it reached approximately 3 %. However, this treatment
decreased by 2.2 % at 36 h to be signiﬁcantly better than the untreated control. Each of
the four fungicides tested were statistically better than the untreated control at the 36 h
incubation interval (P = 0.05).

Appressorial development: The effects of time and treatment on appressorial

development were signiﬁcant at P < 0.05 (Table 6). Appressorial structures began

30

Table 5. Analysis of variance for percent germination of Botrytis cinerea conidia on
‘Orbit White’ geranium leaf discs.

 

Effect Num DF Den DF F Value Prob>F
TRT 4 5 7.67 0.0232
Time 4 270 8.49 <.0001
TRT*Time 16 270 2.85 0.0003

 

Table 6. Analysis of variance for appressorial development of Botrytis cinerea conidial
germ tubes on ‘Orbit White’ geranium leaf discs.

 

Effect Num DF Den DF F Value Prob>F
TRT 4 5 3.99 0.0808
Time 4 270 5.41 0.0003
TRT*Time 16 270 2.57 0.0010

 

Table 7. Analysis of variance for mean length of Botrytis cinerea conidial germ tubes on
‘Orbit White’ geranium leaf discs.

 

Effect Num DF Den DF F Value Prob>F
TRT 4 24 33.70 < 0.0001
Time 4 24 7.58 0.0004
TRT*Time 16 24 4.1 1 0.0009

 

31

 

Figure 1. Conidial germination stages of Botrytis cinerea. (A,B) Emergence of a germ
tube from B. cinerea conidium at 6 and 12 h, respectively. (C,D) Enlarged, rounded
ends of germ tubes visible on developing appressoria from B. cinerea. Black scale bars =
20 um. Images in this thesis are presented in color.

 

25
+ Untreated control
0 Polyoxin D zinc salt

20 L —'v— Bacillus subtilis
V Azoxystrobin

J
1

   

 

 

 

 

 

 

 

 

g + Chlorothalonil
I;
m 15 ~
.5 i
E i
h -1
a:
m -4
+0 10 n
t: .
a:
u i
l- 4
a:
a .

5 _

i
o- I

 

 

I I I I I

3 6 12 24 36

Time (hours)

Figure 2. Mean percent of Botrytis cinerea conidia germinated at ﬁve incubation
intervals and treated with one of four fungicides. Bars indicate standard error of the
means.

33

 

developing from germ tubes between 3 and 6 h (Fig. 3). Untreated conidial germ tubes
had a rapid increase in appressorial development between 3 and 6 h, and then rose
steadily to the 24 h time interval. Development of appressoria had a slowed increase
from 24 to 36 b. Although leaf discs treated with either chlorothalonil or azoxystrobin
limited appressoria from developing to < 0.2 % at all incubation intervals, the differences
were not statistically signiﬁcant from the untreated control until the 24 and 36 h intervals.
The polyoxin D zinc salt treatment allowed some penetration structures to develop with a
slow increase that peaked at 24 h, but then limited development to < 0.2 % at 36 h.
Bacillus subtilis controlled appressorial development at 3 h, but saw a rise in
development between 6 and 12 h, followed by a slow decline from 12 to 36 h intervals.
Polyoxin D zinc salt and B. subz'tlis did not differ signiﬁcantlyfrom the untreated control
until the 36 h incubation interval. All fungicide treatments were statistically better and
had fewer appressoria developed than the untreated control at 36 h (P = 0.05).

Mean germ tube length: The effects of time and treatment on mean germ tube length
were signiﬁcant at P < 0.05 (Table 7). Due to the lack of germination and subsequent
growth of infection germ tubes at the 3 h time interval, signiﬁcant elongation of germ
tubes was not seen until the 6 h interval (Fig. 4). The mean length of B. cinerea germ
tubes on untreated geranium discs slightly increased between 3 and 6 h, and remained
constant from 6 to 12 b. An increase in average germ tube length took place between the
12 and 24 incubation intervals and then rapidly increased to 55.25 pm in mean length at
the 36 h interval. Minimal elongation took place on discs treated with polyoxin D zinc
salt, azoxystrobin, and Bacillus subtilis. Only discs treated with chlorothalonil were

signiﬁcantly different from the untreated control. At 12 h, B. subtilis and polyoxin D

34

 

20

+ Untreated control
0 Polyoxin D zinc salt -—

+ Bacillus subtilis "

15 r v Azoxystrobin

 

 

 

 

a
'5 + Chlorothalonil
in
in
a:
a
Q 10 —
<
I.—
O
I.
at
.n
E 5 ~
3
Z
0 ..

 

 

 

 

I I I I I

3 6 12 24 36

Time (hours)

Figure 3. Mean number of Botrytis cinerea germ tubes per disc that developed
appressoria at ﬁve incubation intervals and treated with one of four fungicides. Bars
indicate standard errors of the means.

35

100

 

i + Untreated control
: O Polyoxin D zinc salt
80 A —-v— Bacillus subtilis

 

 

 

 

1% v Azoxystrobin "
V —I— Chlorothalonil
5 .
g, 60 1
g .
a:
.a
3 .
"" 4O 4
E .
L-
at
or
5 20 -
a: .
E .
0 _

 

 

 

l I V I r

3 6 12 24 36

Time (hours)

Figure 4. Mean germ tube length (pm) of Botrytis cinerea per disc recorded at ﬁve
incubation intervals and treated with one of four fungicides. Bars indicate standard error
of the means.

36

zinc salt increased in mean germ tube length; no treatments were signiﬁcantly different
from one another. The 24 h incubation interval saw a decline in mean length for discs
treated with B. subtilis. Chlorothalonil and azoxystrobin were statistically better than the
untreated control. At 36 h, mean length of germ tubes slightly rose for those treated with
azoxystrobin. Discs treated with one of the four fungicide treatments were all

signiﬁcantly better with shorter germ tubes than the untreated control.

37

Fungicide greenhouse trial on ‘Orbit White’ geranium: In 2004, untreated plants
showed symptoms of Botrytis blight and infection with 5.8 lesions per plant at the last
rating date (Table 8). Treatment with chlorothalonil limited lesion development to 1.3
per plant, but was not signiﬁcantly better than the untreated control. Plants treated with
polyoxin D zinc salt, a biopesticide, and azoxystrobin, a reduced risk fungicide, had
fewer lesions than the untreated control. However, the reduction was not signiﬁcantly
different. The two biocontrol agents, Bacillus subtilis and Gliocladium catenulatum, did
not limit disease and plants treated with these products had signiﬁcantly more lesions
than the untreated control. According to AUDPC values, chlorothalonil, azoxystrobin,
and polyoxin D zinc salt resulted in less disease than the control plants, but the
differences were not signiﬁcant (Table 8). However, all other treatments resulted in
AUDPC values signiﬁcantly greater than the untreated control. At the ﬁnal rating date,
untreated control plants received a disease severity value of 4.7. Chlorothalonil, pol yoxin
D zinc salt, and azoxystrobin had lower severity ratings which were statistically better
than the untreated control. All other products had severity values > 4.7. Pyrimethanil
and neem oil were statistically worse than the untreated control.

In 2005, heavy disease pressure was evident with 16.0 mean lesions per plant
(Table 8). Chlorothalonil was signiﬁcantly better than all other treatments in limiting
disease at the last assessment date. Plants treated with Polyoxin D zinc salt, ascorbic
acid, and azoxystrobin were not statistically better than the untreated control plants. The
biocontrol agent, Bacillus subtilis, did not provide effective control and was signiﬁcantly

worse than the untreated control. AUDPC calculations show that chlorothalonil and

38

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39

Table 9. Analysis of variance for average number of lesions/plant caused by Botrytis

cinerea on ‘Orbit White’ geranium.

 

Source DF SS MS Prob>F
Total for Experiment 1 53 2299.43

Total for Treatment 8 1556.93 194.62 <.0001
Error 45 742.50 16.50

Total for Experiment 2 53 3614.15

Total for Treatment 8 2133.48 266.69 <.0001
Error 45 1480.67 32.90

 

Table 10. Analysis of variance for area under the diseased progress curve (AUDPC)
caused by Botrytis cinerea on ‘Orbit White’ geranium.

 

Source DF SS MS Prob>F
Total for Experiment 1 53 131985583

Total for Treatment 8 105969033 132461.28 <.0001
Error 45 260165.50 5781.46

Total for Experiment 2 53 159598013

Total for Treatment 8 105620226 132025.30 <.0001
Error 45 539777.88 11995.06

 

Table 11. Analysis of variance for disease severity caused by Botrytis cinerea on ‘Orbit

White’ geranium.

 

Source DF SS MS Prob>F
Total for Experiment 1 53 294.59

Total for Treatment 8 242.26 30.28 <.0001
Error 45 52.33 1.16

Total for Experiment 2 53 133.64

Total for Treatment 8 97.48 12.19 <.0001
Error 45 36.17 0.80

 

40

azoxystrobin provided adequate control of Botrytis blight with values that were
statistically better than the untreated control. Polyoxin D zinc salt and ascorbic acid had
relatively low AUDPC values, but did not signiﬁcantly differ from the untreated control.
AUDPC values of Bacillus subtilis and neem oil were signiﬁcantly higher than that of the
untreated control and several other treatments. Disease severity values for untreated
plants were given a 5.5 at the ﬁnal rating date. Plants treated with chlorothalonil or
polyoxin D zinc salt received low disease values that were statistically better than the
untreated control. Azoxystrobin and ascorbic acid received rating values that were less
than the untreated control. However, their rating values were not signiﬁcantly different.
Pyrimethanil was signiﬁcantly worse than the untreated control.

In both trials, phytotoxicity was observed on all plantstreated with pyrimethanil
and neem oil extract. Brown spots, chlorosis, leaf curl, and subsequent partial abscission
of leaves was observed on treated foliage (Fig. 5). Dessicating leaf margins and an
overall decline in plant vigor provided an opportunistic environment for B. cinerea to

infect and cause a greater degree of disease progression than the untreated control.

41

 

Figure 5: Evaluation of biopesticides/reduced risk fungicides on ‘Orbit White’ geranium
against Botrytis cinerea. (A) Untreated control; (B) Chlorothalonil; (C) Polyoxin D zinc
salt; (D) Azoxystrobin. Phytotoxic response observed on: (E) Pyrimethanil and (F) neem
oil extract. Images in this thesis are presented in color.

42

F ungicide greenhouse trial on American ginseng: In 2004, untreated plants had a
mean of 5.0 foliar lesions per plant at the last rating date (Table 12). No treatment in this
trial was statistically better than the untreated control. However, chlorothalonil and
boscalid limited lesion development to fewer than 0.3 lesions/plant. The biocontrol
agent (T richoderma harzianum) and the reduced risk fungicides (fenhexamid, ﬂuazinam,
and polyoxin D zinc salt) resulted in S 1.5 lesions/plant. Similar results were observed
according to AUDPC values (Table 12), where chlorothalonil and the reduced risk
products (fenhexamid, ﬂuazinam, and polyoxin D zinc salt) had lower values than the
untreated control plants. In terms of disease severity, fenhexamid, chlorothalonil,
fluazinam, and boscalid received values that were all signiﬁcantly better than the
untreated control rating (5.8). All other treatments received disease severity ratings <
5.8, but were not statistically different.

In the 2005 trial, heavy disease pressure was observed on untreated plants (8.6
lesions/plant). F luazinam, a reduced risk fungicide, differed signiﬁcantly from the
untreatedcontrol and prevented disease development. Plants treated with chlorothalonil,
boscalid, fenhexamid, and polyoxin D zinc salt had statistically fewer lesions than the
untreated control. AUDPC values for treatments reﬂect similar results. A disease
severity rating of 6.4 was received by the untreated control group in this trial. All other
treatments had disease severity ratings < 6.4, but only fenhexamid, chlorothalonil,
ﬂuazinam, polyoxin D zinc salt, boscalid, and B. subtilis were signiﬁcantly better than

the untreated control. No phytotoxicity was observed on any plants (Figure 6).

43

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44

Table 13. Analysis of variance for average number of lesions/plant caused by Botrytis
cinerea on American ginseng.

 

Source DF SS MS Prob>F
Total for Experiment 1 59 1716.733

Total for Treatment 10 306.08 30.61 0.4078
Error 49 1410.65 28.79

Total for Experiment 2 54 1125.75

Total for Treatment 10 499.26 49.93 0.0018
Error 44 626.48 14.24

 

Table 14. Analysis of variance for area under the disease progress curve (AUDPC)
caused by Botrytis cinerea on American ginseng.

 

Source DF SS MS Prob>F
Total for Experiment 1 61 392263.97

Total for Treatment 10 102672.33 10267.23 0.0828
Error 51 289591.63 5678.28

Total for Experiment 2 58 302385.23

Total for Treatment 10 121972.74 12197.27 0.0029
Error 48 180412.49 3758.59

 

Table 15. Analysis of variance for disease severity caused by Botrytis cinerea on
American ginseng.

 

Source DF SS MS Prob>F
Total for Experiment 1 59 492.18

Total for Treatment 10 133.32 13.33 0.0816
Error 49 358.87 7.32

Total for Experiment 2 54 302.44

Total for Treatment 10 138.92 13.89 0.001 1
Error 44 163.52 3.72

 

45

 

Figure 6: Evaluation of biopesticides/reduced risk fungicides on American ginseng
against Botrytis cinerea. (A) Untreated control; (B) Chlorothalonil; (C) Polyoxin D zinc
salt; (D) Fenhexamid; (E) Boscalid; (F) Fluazinam. Images in this thesis are presented in
color.

46

Fungicide field trial on American ginseng: Substantial infection by Alternaria panax
was observed on ginseng plants due to above average hot and humid conditions in
Marathon County, WI in 2005.

Untreated plots had an average of 212.5 infected plants (approximate mean
number of plants/plot = 235) on 1 Aug rating (Table 16). Plots treated with ﬂuazinam
and boscalid had the lowest mean of infected plants and had statistically fewer diseased
plants than the untreated control. Polyoxin D zinc salt and chlorothalonil were also
signiﬁcantly different from untreated control plots. F enhexamid and thiophanate methyl
had fewer infected plants than the untreated control plots. However, the differences were
not statistically different. Plants that became defoliated due to A. panax infection
averaged 191.0 defoliated plants/control plot on the 1 Aug rating. Plots treated with
ﬂuazinam, boscalid, polyoxin D zinc salt, and chlorothalonil had the lowest means,
respectively, and were signiﬁcantly better than the untreated control. F enhexamid,
thiophanate methyl, and acibenzolar treated blocks had fewer defoliated plants, but were
not signiﬁcantly different from the untreated. F luazinam boscalid, polyoxin D zinc salt,
chlorothalonil, and fenhexamid received the lowest disease severity ratings, respectively,
which were statistically lower than the untreated control.

Disease pressure had increased at the 10 Aug rating date. Untreated blocks had
an average of 216.0 infected plants/plot (Table 16). Plots treated with boscalid and
fluazinam had the lowest number of infected plants and were signiﬁcantly better than the
untreated control. No other treatments were statistically better. Of those infected with A.
panax, plants that became defoliated averaged 215.8 in the control plots. Those treated

with ﬂuazinam, boscalid, polyoxin D zinc salt, and chlorothalonil had signiﬁcantly less

47

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48

Table 17. Analysis of variance for number of American ginseng plants infected by

 

 

Alternaria panax.

Source DF SS MS F Value Prob>F
Total for 1 Aug 42 389106.42

Model 13 295932.81 22764.06 7.09 < 0.0001
Error 29 93173.62 3212.88

Total for 10 Aug 42 268836.60

Model 13 165815.35 12755.03 3.59 0.0020
Error 29 103021.25 3552.46

 

Table 18. Analysis of variance for number of American ginseng plants defoliated from

infection by Alternariapanax.

 

 

Source DF SS MS F Value Prob>F
Total for 1 Aug 41 468065.90

Model 13 312549.40 24042.26 4.33 0.0006
Error 28 155516.51 5554.16

Total for 10 Aug 42 456615.07

Model 13 321306.93 24715.92 5.30 < 0.0001
Error 29 135308.14 4665.80

 

Table 19. Analysis of variance for disease severity on American ginseng plants caused

 

 

by Alternaria panax.

Source DF SS MS F Value Prob>F
Total for 1 Aug 42 409.07

Model 13 335.34 25.87 10.32 < 0.0001
Error 29 72.73 2.51

Total for 10 Aug 42 361.67

Model 13 278.84 21.45 7.51 < 0.0001
Error 29 82.83 2.86

 

49

Table 20. The effect of foliar fungicides against Alternaria panax on yield of American

ginseng roots.

 

 

 

Root ield lbs
Treatment and rate/Az Fresh 1 ( ) Dried’
Untreated control ....................... 4.1 c 0.9 e
Omega SOOF 1 pt ....................... 10.8 a 2.3 ab
Endura 70WG 6.8 oz ................. 10.4 a 2.6 a
Endorse 2.5WP 2.2 lb ................ 7.0 b 2.2 abc
Bravo Weather Stik 68C 2 pt 6.0 be 1.4 bcde
Elevate 50WDG 1.5 lb .............. 4.8 bc 1.1 de
Serenade 10%WP 10.0 lb .......... 4.9 be 1.1 de
Actigard 50WDG 0.75 oz .......... 5.6 be 1.3 bcde
PlantShield 1%WP 100.0 oz ...... 5.3 be 1.2 cde
Topsin 4.5L 21.8 ﬂ oz ............... 7.2 b 2.0 abcd
Messenger 3WDG 9 oz ............. 6.3 bc 1.4 bcde

 

’ The treatments were applied on seven-day intervals from 1 Jun through 4 Aug.
" Dried root yield taken two weeks following harvest.
‘ Column means followed by the same letter are not significantly different (Fisher’s Protected LSD;

P=0.05).

Table 21. Analysis of variance for American ginseng root yield infected by Alternaria

 

 

ﬁnax.
Source DF SS MS F Value Prob>F
Total for fresh 32 265.76
yield
Model 12 205.47 17.12 5.68 0.0003
Error 20 60.29 3.01
Total for dried 32 19.71
yield
Model 12 11.78 0.98 2.48 0.0353
Error 20 7.93 0.40

 

50

defoliation than the untreated control at the ﬁnal rating date. F enhexamid and
thiophanate methyl had fewer plants defoliated than the untreated control, but did not
differ statistically. A disease severity rating of 10.0 was given to the untreated control
plots at the ﬁnal rating date. Again, ﬂuazinam, boscalid, polyoxin D zinc salt, and
chlorothalonil had the lowest severity ratings which were signiﬁcantly less than the
untreated control. No other treatments were statistically different from the untreated
control at the ﬁnal rating date.

Root yield of American ginseng was assessed after the ﬁnal rating date. Fresh
weight was taken at time of harvest, and dried weight was recorded two weeks post-
harvest (Table 20). Fresh and dried root yield on the untreated control plants were the
lowest of all treatment plots, with 4.05 and 0.9 lb, respectively. Root yield for plants
treated with ﬂuazinam, boscalid, polyoxin D zinc salt, and thiophanate methyl had the
highest recorded fresh and dried weights and were statistically higher than the untreated
control plots. No other treatment plots yielded signiﬁcant differences from the untreated

control.

51

DISCUSSION

In addition to cultural methods, fungicides are a primary method for controlling
foliar blights caused by Botrytis cinerea, and are necessary to minimize infection and
prevent an epidemic during production. Chlorothalonil is the current industry standard
and continues to show strong efﬁcacy in controlling foliar blights by using a multi-site
mode of action. However, this active ingredient is considered a B2 carcinogen and poses
high toxicity risks. Due to the increasing desire to register less toxic fungicides for
specialty crop use, other products offering different modes of action may show similar
efﬁcacy to this chemical standard.

Azoxystrobin is a reduced risk fungicide currently registered for use on turf grass
and vegetable crops to control numerous pathogens including B. cinerea. It’s efﬁcacy on
‘Orbit White’ geraniums in the greenhouse trial in 2004 was better than most products
evaluated aside from chlorothalonil. Though not statistically different from the control in
terms of lesion counts per plant, the active ingredient slowed disease progession and
signiﬁcantly minimized disease severity compared to untreated plants. Similar efﬁcacy
by azoxystrobin has been demonstrated in recent years on other geranium cultivars
(Hausbeck et al., 20003; Hausbeck et al., 2000b), as well as poinsettia (Benson & Parker,
1999), by consistently limiting leaf lesions and subsequent sporulation caused by Botrytis
blight. In controlling B. cinerea outbreaks, geranium growers currently have a reduced
risk product to incorporate into their [PM programs.

Polyoxin D zinc salt is a biopesticide that was originally registered for use on
multiple turf grass pathogens. Its control against B. cinerea on ‘Orbit White’ geraniums

in 2005 limited number of lesions to even less than azoxystrobin and signiﬁcantly

52

minimized disease severity in 2004 and 2005 compared to the untreated plants. Another
study showed similar effectiveness in controlling Botrytis blight on ‘Red 11’ geraniums in
terms of minimizing diseased foliage and leaves with sporulation from B. cinerea
(Hausbeck et al., 2002). In addition to geranium, American ginseng is a host plant for B.
cinerea infection and foliar blighting is a common issue for ginseng growers in
Wisconsin and Michigan (Hausbeck, 2004). Polyoxin D zinc salt has recently become
registered for use on these specialty crops. The greenhouse trial conducted here shows
that this biopesticide offers good efﬁcacy in controlling foliar blighting by signiﬁcantly
limiting number of lesions, disease progression, and disease severity caused by B. cinema
in 2005. Similar results were observed in a ﬁeld experiment using identical formulations
(Hausbeck & Harlan, 2004a). In addition to this product, other reduced risk fungicides
were tested for efﬁcacy against B. cinerea on ginseng due to the lack of numerous
products registered for use on this specialty crop. The greenhouse trial in 2005 shows
signiﬁcant control of disease severity, progression and lesion development by boscalid,
ﬂuazinam, and fenhexamid. Similarly, signiﬁcant results were observed in controlling
Botrytis blight when these three products were tested by Hausbeck & Harlan (2004c).

In 2005, a ﬁeld study on American ginseng was carried out to test the efﬁcacy of
these biopesticide/reduced risk products in the presence of another foliar pathogen:
A lternarz'a panax. As a result of the unseasonably hot temperatures, Altemaria blight
caused heavy disease pressure on the untreated plants. Visible efﬁcacy of the tested
products was dramatic and differences were seen as early as July. By the last rating date
in August, polyoxin D zinc salt, ﬂuazinam, and boscalid provided signiﬁcant control

against defoliation and disease severity caused by A. panax. Fluazinam and boscalid

53

showed better efﬁcacy than chlorothalonil in this experiment, and limited foliar infection
signiﬁcantly compared to the untreated control. Moreover, harvested ginseng roots from
the ﬂuazinam, boscalid, and polyoxin D zinc salt treatments provided signiﬁcantly better
yields of the dried, marketable product than the untreated. Identical differences were
observed on ginseng plants treated with ﬂuazinam and boscalid against Altemaria blight
in the previous growing season (Hausbeck & Harlan, 2004b).

A simultaneous in vitro assay was carried out on geranium foliage to evaluate the
various modes of action on the infection process of B. cinerea, as has been done in the
past (Buck, 2002). Overall, conidial germination, appressorial development, and
elongation of germ tubes were less than what was observed in the preliminary assay
trials. It should be noted that high variability such as this may be contingent on the
presence or absence of exogenous nutrients (Clark & Lorbeer, 1976), attributed to a latent
infection by B. cinerea (Coley-Smith et al., 1980), or affected by the selection of B.
cinerea isolates used (Buck, 2004).

Since chlorothalonil is the current standard, its efﬁcacy was included for
comparisons to the other products evaluated in this leaf disc assay. Germination was
limited by the chlorothalonil treatment, as was expected. When absorbed into the fungal
body, this chemical disrupts energy production by inhibiting enzymes at several sites
(Syngenta Crop Protection, 2005). Another multi-site fungicide is a biocontrol agent that
uses a patented strain (QST 713) of Bacillus subtilis. The bacterium contains
lipopeptides that puncture pathogen cell membranes, causing conidia and mycelia to
collapse and die (Agraquest fact sheet, 2005). In this experiment, B. subtilis did not

completely limit (< 3%) germination or appressorial development between the 6 and 12 h

54

incubation interval. However, after 12 h, there was a gradual decline in germination and
appressorial development until the last incubation interval. This delay may be indicative
of the antimicrobial activity taking effect and becoming a limiting factor to fungal
development. This treatment was inconsistent in limiting disease by B. cinerea and A.
panax in the greenhouse and ﬁeld trials.

The biopesticide polyoxin D zinc salt, a by-product of a bacterium found in soil,
was also evaluated. The fungicide has an active ingredient that inhibits the development
of chitin in the cell walls of the fungus (Environmental Protection Agency, 2001 ). A
fungus lacking chitin is unable to continue growing and infecting plant cells. This
experiment shows that polyoxin D zinc salt limited conidial germination to < 2.0 0/8
except at the 24 h interval, where germination reached 3.0 %. Overall, this mode of
action was effective in limiting germination to < 4.0 %, limiting appressorial
development to < 3.0, and preventing germ tube elongation from reaching > 5.0 pm over
all incubation intervals.

Azoxystrobin was tested as the reduced risk fungicide. This product prevents
energy production in the fungus by inhibiting respiration of the mitochondria that’s found
inside the nucleus of fungal cells (Syngenta Crop Protection, 2005). In this experiment,
azoxystrobin limited infection by < 1.0 % for all three parameters under the ﬁve
incubation intervals observed. This product provided similar efﬁcacy to chlorothalonil
but has a lower toxicity rating and is non-carcinogenic. Moreover, the three non-
carcinogenic products evaluated here provided signiﬁcant control at various time

intervals observed.

55

Future use of these reduced risk and biopesticide products on geranium cultivars and
American ginseng could provide growers with effective control strategies in managing
epidemics caused by B. cinerea and A. panax without causing toxicity to non-target

organisms and the applicators.

56

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62

APPENDIX A

REDUCED RISK AND BIOPESTICIDE STUDIES IN 2004

63

GERANIUM (Pelargom'um xdomesticum ‘Emperor’) B.J. Webster and MK. Hausbeck

Botrytis blight; Botrytis cinerea Michigan State University
Department of Plant Pathology

East Lansing, MI 48824-131 1

Evaluation of reduced risk fungicides and biopesticides for control of Botrytis blight
of geranium, 2004.

Geranium cuttings were taken from stock plants on 27 Feb and placed into Oasis
strips for rooting. Rooted cuttings were transplanted into 4-in. plastic pots containing a
soilless medium (Baccto Professional Planting Mix, Michigan Peat Company, Houston,
TX) on 19 Mar. Plants were fertilized three times weekly with 250 ppm Peter’s 20-20-20
liquid feed (The Scotts Company, Marysville, OH). Six replicates per treatment were
arranged in a completely randomized design. Botrytis cinerea inoculum was grown on
potato dextrose agar for three weeks under ﬂuorescent light. Plates were ﬂooded with
distilled water and gently agitated with a plastic-bristled brush to dislodge conidia.
Liquid from the plates was strained through cheesecloth, and adjusted to 2.9 x 107
conidia/ﬂ oz. F ungicide treatments were applied with a hand-pressurized sprayer to
runoff. Plants were placed into clear plastic bags supported by wire meshing
immediately after treatment. Four hours after fungicide treatment, plants were inoculated
with the B. cinerea conidial suspension using a janitorial spray bottle to runoff. The
plastic bags were immediately sealed and reopened only for subsequent treatment and
inoculum application. Treatments were applied on 24, 31 Aug and 7, 14 Sep. After each
fungicide treatment, plants were re-inoculated four hours later using the method
previously described. Lesions were counted and disease severity ratings were taken on
21 Sep.

Disease pressure was moderate in this trial. All products signiﬁcantly limited disease
development compared to the inoculated control. Signiﬁcant differences among
treatments were not observed. Several products limited the number of lesions and disease
severity to 3.0 or less and included the following: Endorse 2.5WP, Rhapsody 1.34AS,
Daconil Weather Stik 6F, and Decree 50WDG.

64

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