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thesis entitled

VIRULENCE AND FUNGICIDE SENSIVITY OF
PHYTOPHTHORA CACTORUM ISOLATED FROM
AMERICAN GINSENG

presented by
Shaunta Nichelle Hill

has been accepted towards fulfillment
of the requirements for the

MS. degree in Plant Pathology

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Virulence and fungicide sensitivity of Phytophthora cactorum
isolated from American ginseng

By

Shaunta Nichelle Hill

A THESIS
Submitted to
Michigan State University
In partial fulfillment of requirements
for the degree of
MASTER OF SCIENCE
Department of Plant Pathology

2005

Abstract
Virulence and fungicide sensitivity of Phytophthora cactorum isolated from
American ginseng
By

Shaunta Nichelle Hill

Foliar blight and root rot of American ginseng (Panax quinquefolium) caused by
Phytophthora cactorum has been managed with the fungicide mefenoxam. During 2003
and 2004, commercial ginseng gardens were monitored in Wisconsin (WI) and Michigan
(MI) as well as research gardens at Michigan State University (MSU). Following periods
of cool, wet weather, reddening of leaflets, root rot, plant wilting and death were
observed. Diseased plants were sampled and P. cactorum was isolated. A minimum of
104 P. cactorum isolates were recovered each year. Isolates were screened for sensitivity
to mefenoxam by measuring radial mycelial growth on lOO-ppm mefenoxam-amended
V8 agar. For 2003, 79, 48 and 85% of isolates collected from MSU, MI and WI plants
were resistant to mefenoxam, respectively. In 2004, 83 and 91% of isolates recovered
from MSU and W1 plants and seedlings were resistant to mefenoxam, respectively.
Isolates were tested for virulence using an apple fruit bioassay. All were virulent.
Alternative products were tested against P. cactorum. Mefenoxam was the least effective
while dimethomorph, mancozeb with zoxamide and copper hydroxide were the most
effective against P. cactorum. Phytophthora cactorum is an important pathogen in MI
and W1 gardens. The development of isolates resistant to mefenoxam is a new finding.

Currently un-registered fungicides may be useful in managing P. cactorum diseases.

Dedication

To my circle of support: Jacqueline, Anita, Shirley, Sebren and Deja. Words
can’t express my gratitude for your presence, support and love. To the teachers and
mentors from my past, who helped to guide my path and Ms. Lenora Ashford, thanks for

the science talk!

iii

Acknowledgements -

I would like to thank the people who contributed to the completion of this
research. To my major professor, Dr. Mary Hausbeck, thank you for your guidance and
the opportunity to study plant pathology. To Dr. Annemiek Schilder and Dr. Joe Vargas,
my committee members; thank you for your support towards this degree and knowledge
that you have shared. I wish to also thank Dr. Marcia Ewers for insisting I apply to Grad
School and Blair R. Harlan, Sheila Lindennan and Jeffrey A. Woodworth who offered
technical assistance.

I would like to acknowledge the USDA Cooperative State Research, Education,
and Extension Service; Pest Management Alternatives Program and the IR-4:
Biopesticide Research Grant as funding sources for the duration of this research. Also, I
would like to acknowledge the Ginseng Growers of Michigan, Ginseng Board of
Wisconsin and Wisconsin Ginseng Growers Association for their commitment and
patience with this project.

A special acknowledgement to my family and friends. Thank you for your love

and support thought my graduate program and in everyday aspects of my life.

iv

TABLE OF CONTENTS

List of Tables ................................................................................................... vi
List of Figures .......................................................................... vii
LITERATURE REVIEW ................................................................................. 1
History of Ginseng ................................................................................. 1
Botanical Description ............................................................................. 3
Cultivation ............................................................................................ 5
Environmental and nutrient requirements .............................................. 5
Chemistry and Medical Uses ................................................................. 7
Phytophthora cactorum disease of Ginseng .......................................... 9
Disease Management ........................................................................... 12
INTRODUCTION .......................................................................................... 1 6
MATERIALS AND METHODS ................................................................... 19
Isolation and culture of P. cactorum .................................................... 19
Laboratory screening for mefenoxam sensitivity ................................. 20
Screening for virulence ........................................................................ 22
Efficacy of fungicides using apple fruit and ginseng seedlings ........... 22
Detection of P. cactorum on ginseng seed ........................................... 27
RESULTS ...................................................................................................... 30
Isolation and culture of P. cactorum .................................................... 30
Laboratory screening for mefenoxam sensitivity ................................. 30
Screening for virulence ........................................................................ 31
Efficacy Screening of Alternative Chemical Products ........................ 31
Detection of P. cactorum on ginseng seed ........................................... 32
DISCUSSION .......................... ' ...................................................................... 4 3
LITERATURE CITED .................................................................................. 48

LIST OF TABLES

TABLE

1. Products tested for efficacy on apple fruit against P. cactorum recovered
from American ginseng in experiment 1 and 2 .............................................. 24

2. Source and number of P. cactorum isolates recovered from ginseng
in 2003 and 2004 ............................................................................................ 34

3. In vitro mefenoxam sensitivity screening of P. cactorum from ginseng
in 2003 and 2004 ............................................................................................ 34

4. Virulence screening of P. cactorum isolates from ginseng using
‘McIntosh’ apple fruit .................................................................................... 37

5. Efficacy of registered and unregistered fungicides for control of a
mefenoxam-insensitive P. cactorum isolate using ‘McIntosh’ apple fi'uit in
experiment 1 ................................................................................................... 38

6. Efficacy of registered and unregistered fungicides for control of a
mefenoxam-insensitive P. cactorum isolate using ‘McIntosh’ apple fruit in
experiment 2 .................................................................................................. 39

7. Evaluation of registered and unregistered fungicides for control of a
mefenoxam-insensitive P. cactorum root rot of ginseng seedlings in
experiment 1 and 2 ......................................................................................... 40

8. Incidence of fungal pathogens recovered from stratified

American ginseng seed when directly plated on water agar amended with
2% ampicillin (2 ml/L). .. ............................................................................... 42

vi

LIST OF FIGURES

FIGURE

1. Ginseng plant disease symptoms caused by Phytophthora cactorum.
Wisconsin ginseng gardens exhibiting foliar blight and plant death (A-C).
Advanced stage of root rot (D). Images in this thesis are presented in color.
........................................................................................................................ 35

2. Laboratory in vitro screen for mefenoxam sensitivity in P. cactorum.
Panel 1A indicates growths on the V8 control ( no agar amendment); 1B
indicates limited P. cactorum grth of a mefenoxam-sensitive isolate on
amended V8 agar. Panel 2A depicts P. cactorum growth on a control plate
in comparison to 2B which exhibits grth of a mefenoxam insensitive
isolate on mefenoxam-amended agar ............................................................ 36

vii

Literature Review

History of Ginseng

Ginseng has long been used as a “cure-all” medicine by the Chinese. The first
reference to ginseng appeared in Chinese literature in the year 1 BC. (Putnam, 1984). In
1709, the first Westemer, French missionary Father J artoux, became aware of both the
ginseng plant and the great esteem in which it was held (Putnam, 1984).

Father J artoux recorded his memories of China and descriptions of ginseng in the
Memoir of the Royal Academy of Paris in 1714 which was translated into English and
published in the Philosophical Transactions of the Royal Society of London (Hardacre,
1968; Putnam, 1984). In 1716, Father Joseph Francis Lafitau observed American
ginseng in his hometown of present day Montreal and confirmed his identification based
on descriptions by Father J artoux (Beyfuss, 1998). After ginseng was found in Canada,
the search for it began in the United States. In 1751, ginseng was found in New York,
Massachusetts and Vermont (Nash, 1898; Beyfuss, 1998) and soon after the harvesting of
ginseng for trade began. In 1773, the ship "Hingham" sailed from Boston to China with
55 tons of wild ginseng on board (Keller, 1998). The first shipment of wild ginseng to
China after the American Revolution was reported to have been made by John Jacob
Astor from New York in 1782 (Keller, 1998).

With its rising popularity and the amount exported increasing yearly, the wild-
ginseng population began to dramatically decline and cultivation became the focus. In
1870, Abraham Whisman of Virginia tried unsuccessfully to cultivate ginseng. It wasn’t
until 1885, that George Stanton was successful. George Stanton is credited as being the

“father of ginseng cultivation”, not only because he was able to keep his gardens healthy,

but because he was able to understand the shade requirements and stratification needs of
the plant (Harding, 1936). With the cultivation of ginseng the trade market remained
viable and flourished. In the 19th century, nearly 21,000 tons of American ginseng were
exported between 1821 and 1983 (Baranov, 1966; Keller, 1998). From 1992 to 2001, the
export value was over 1.8 billion dollars and in 2002, exports rose to 33.6 million, up
34% from 2001 (USDA Tropical products, world markets and trade, 2003).

With exports of ginseng increasing in the US. since commercialization, the
number of ginseng farms also increased. The first census report to include ginseng was
taken in 1954. At that time the US. Census of agriculture reported only 5 ginseng
growers with a total of 21 acres (Carlson 1986). The next census report that included
ginseng was published in 1992 and included 824 ginseng farms (US. 1997 Census of
Agriculture — State Data). In 1997, the number of farms harvesting ginseng increased to
1,081. In 2002, the number of farms harvesting ginseng decreased to 588 (U .S. 2002
Census of Agriculture — State Data).

To protect wild populations of American ginseng, the US. government regulates
its harvest. The Convention on International Trade in Endangered Species of Wild Fauna
and Flora (CITES) was initiated by the United States in 1977. It regulates the
commercial trade of endangered species and monitors the trade of species that are at risk
of becoming endangered (Beyfuss, 1998). Under the terms of CITES, the US. Fish and
Wildlife Service monitors wild ginseng populations and issues permits for wild and
cultivated ginseng importing and exporting (Pritts, 1995).

Since the start of CITES, wild ginseng has been deemed endangered in Maine and

Rhode Island, threatened in Michigan and New Hampshire, of “special concern” in

Connecticut, Tennessee, Massachusetts and North Carolina and “vulnerable” in New

York and Pennsylvania (USDA, NRCS, 2002).

Botanical Description

Ginseng is a member of the Araliaceae family and found in the genera Panax and
Eleutherococcus. Siberian ginseng (Eleutherococcus senticosus) is not true ginseng
(Pritts, 1995). Panax is derived from the Greek word panakos (a panacea) meaning “cure
all”. The common name ginseng literally means "root of man" because the root of this
plant often resembles the shape of a human body (Mindell, 1992). Of the several species
of ginseng included in the Panax genus, only Oriental ginseng (Panax ginseng) and
North American ginseng (Panax quinquefolium) are important to the ginseng market.
Oriental ginseng refers to plants native to North Korea and China while American
ginseng is native to Canada and the eastern and midwestem United States.

Ginseng is a unisexual erect perennial plant with a canopy that can reach 30-40
cm in height and requires only 30% of full sunlight (Proctor, 1996). The total number of
leaves on a ginseng plant corresponds to the plant’s age (Proctor, 1996). The canopy of a
mature ginseng plant consists of three or four compound leaves arranged in a whorl.

Each leaf contains five ovate, saw-toothed leaflets. The upper three leaflets are larger
than the lower two with the leaves referred to as prongs. There are five leaflets on each
leaf. An immature plant has one or two prongs and adds an additional leaf every few
years until maturity. A first year seedling has one leaf with three leaflets. In the
Midwest, the cultivation season for ginseng is late April through late October (Polczinski,

1982; Pritts, 1995; Hausbeck, 2004).

Ginseng has a single umbel inflorescence that forms at the terminus of the stem
(Putnam, 1984). The umbel has 5-50 small greenish-white perfect flowers (Putnam,
1984) that are self-fertile and each capable of producing a small green kidney-shaped
berry after fertilization. The berries ripen and turn crimson red in late summer or early
fall (Harding, 1936; Pritts, 1995). Each berry contains two seeds approximately 0.5 cm
in diameter and is slightly longer than it is wide. Stratification is needed for ginseng seed
and is generally eighteen to twenty-two months in duration (Proctor and Bailey, 1987).
During stratification, the endocarp of the berry remains tightly closed, opening slightly
along its suture a month or so before germination (Putnam, 1984). The series of warm
and cold temperatures over the 18-22 month stratification period is necessary for the
embryo to grow and mature (Putnam, 1984; Proctor and Louttit, 1995; Harrison et al.,
2000)

Ginseng has a fibrous taproot, which can be 7-13 cm long (Anonymous, 2000).
The outer skin of the root is generally light to golden brown and the interior is white to
light yellow in color. The root is also forked, fleshy, ringed, necked and wrinkled, thus
its nickname of “old man’s roots”. The neck of ginseng refers to its rhizome at the crown
of the root. It is on this rhizome that the bud for the next year’s shoot develops during the
spring; with the bud expanding so that the stem, rather than the leaves, is seen first
(Putnam, 1984). This bud remains dormant during the winter months (Harrison et al.,
2000). Root age can also be determined by counting the rings (scars from winter

dormancy and annual abscission of the foliage) on the neck or crown of the root.

Cultivation

Ginseng can be grown as wild, wild-simulated, woods-cultivated, and cultivated.
Wild ginseng is found in its native habitat, usually a hardwood forest. Wild-simulated
ginseng is seeded and grown under approximately 70% natural shade. Woods-cultivated
ginseng is similar to wild simulated ginseng; however, the area is first cleared by
removing plants and leaf litter that would naturally compete with or hinder ginseng
growth. Cultivated ginseng is grown under shade cloth in a cleared field. Both cultivated
and woods-grown ginseng require intensive maintenance including mulch such as straw
or hay, weeding, and fungicide use.

Ginseng grading and selection for market is based on its cultivation method and
its appearance. Wild or woods-grown ginseng is preferred over cultivated ginseng
because of its rarity. Therefore, the bulk of the market consists of ginseng cultivated
under shade-cloth. Roots that appear old, large, and thick are preferred. The root should
also be coarse in texture and have many rings, a white interior and golden brown exterior
(Pritts, 1995).

Ginseng roots are generally harvested after four years under shade cloth, six to
eight years when woods-cultivated, and after eight to fifteen years of growing in a wild-
simulated system (Pritts, 1995). It is illegal to harvest wild ginseng without special
permits. Ginseng can be harvested by hand or by use of mechanical diggers and once
roots are retrieved, they are washed, dried and prepared for market.

Environmental and nutrient requirements

Ginseng grows best in loamy, light textured, well-drained soil with a pH of 5.5 to
6.5 (Pritts, 1995). The crop also requires 20 to 60 inches of precipitation annually and a

minimum cold period (<49° F) of sixty days to break dormancy each spring (Pritts, 1995).

Mulch is typically added to the plant beds afler seeding (Putnam, 1984) and is
used for frost protection. Mulch also helps to maintain the soil moisture and prevent soil
temperature from rising too rapidly in the spring (Polczinski, 1982). Mulch suppresses
weed growth, acts as an organic fertilizer by helping retain the soil humus level, and

plays a role in preventing soil erosion (Polczinski, 1982).

Nitrogen requirements for ginseng gardens increase with the age of the garden at
approximately 40, 60, 80 and 100 kg N/ha for 1, 2, 3, and 4-year gardens, respectively
(British Columbia Ministry of Agriculture, Food and Fisheries, 2003). Nitrogen is
responsible for foliar growth of the plant and nitrate is the preferred and most readily
available form. Symptoms of nitrogen deficiency include slow growth and pale green to
yellow leaflets. (British Columbia Ministry of Agriculture, Food and Fisheries, 2003;

Curran, 1985).

The recommended rate of phosphorus for ginseng is based on the recommended
amount used for root vegetables (15 lb phosphate) (P205) /acre (Harrison et al., 2000).
Symptoms of phosphorus deficiency include purpling or reddening of the leaflets, and
reduced growth (British Columbia Ministry of Agriculture, Food and Fisheries, 2003).
The recommended rate of potassium is also based on the amount used for root vegetables
(60 lb potash) (K20) lacre, (Harrison et al., 2000). Potassium deficiency symptoms
include slow growth, mottling, bronzing and drying of leaf tips and margins (British

Columbia Ministry of Agriculture, Food and Fisheries, 2003).

Chemistry and Medicinal Use

The traditional western attitude has been that ginseng varieties offer similar
benefits (Pritts, 1995). However, the Chinese believe that the two types (American and
Oriental) represent yin and yang. American ginseng is believed to be yin, which in
Chinese tradition is negative, cold, and dark (Duke, 1989). Oriental ginseng is
considered to be yang, which is positive, warm, and light (Duke, 1989). Traditionally,
an overly yang person was encouraged to eat more yin food and an overly yin person
should have more yang food (Duke, 1989). This practice was believed to establish a
natural balance, considered to be important for the body, whereas all diseases were
considered to result from imbalances (Chen, 2002).

Ginseng contains at least eight or ten different saponins or ginsenosides, which
are the main active ingredients. The range of saponins and ginsenosides vary and
distinguishes the varieties. Other constituents of ginseng include panaxic acid, essential
oils, vitamins, saccharides and various kinds of amino acids. American ginseng contains
slightly more saponins but less proteinous and oily substances than Oriental ginseng
(Hou, 1978).

Ginseng in the United States was not considered to have medicinal value until
about 1905 (Pritts, 1995). Ginseng has been studied as a treatment option for heart
disease, cancer, obesity and more recently diabetes. Research conducted at St. Michael’s
Hospital and the University of Toronto concludes that American ginseng reduces blood
sugar (V uksan et al., 2000; Ireland, 2000; Devitt, 2000 and 2003). Also Sotaniemi et a1.
(1995) reported that ginseng might be a useful therapeutic adjunct in the management of

non-insulin-dependent diabetes. Sotaniemi et a1. (1995) also stated that ginseng therapy

elevated mood, improved psychophysical performance, reduced fasting blood glucose,

and body weight.

Diseases of ginseng
Ginseng is susceptible to a number of pathogens that can cause foliar blights, stem

blights and root rots. Some of the most devastating pathogens include Alternaria panax,
Botrytis cinerea, F usarium spp., Cylindrocarpon destructans, Rhizoctonia solani and
Phytophthora cactorum.

Alternaria panax can cause leaf blight and stem rot. A typical symptom of
Alternaria blight is spotting and yellowing of the leaves (Davis and Shoemaker, 1999).
The spots, in which clusters of Alternaria conidia form, appear on the leaves in rings of
yellowed tissue. Infection may also occur at the base of the stern resulting in premature
stem dieback or defoliation and browning or reddening of the stem. Elongated, yellow to
light brown spots on the stems may also be present. Disease is favored by humid and wet
conditions. The pathogen is capable of overwintering in plant debris.

Another important foliar blight of ginseng is caused by Botrytis cinerea. Botrytis
produces sclerotia that are capable of over wintering. Symptoms of Botrytis blight
include an obvious gray fuzzy mat of conidia on the leaves, lesions, and water soaking
(Hausbeck, 2004). Disease is favored by continuous leaf wetness and a dense canopy
(Brun, 1999). Under wet conditions, lesions can expand rapidly, completely cover
leaflets and blight healthy leaflets. Conidia are readily produced on diseased tissue and
may be spread by wind, splashing rain, or human and machinery movement within the
garden.

Cylindrocarpon destructans can cause damping off, crown and root rot of ginseng

(Hausbeck, 2004). Infection normally starts near the root tip and progress upward often

destroying the entire root. A classic symptom of C. destructans root rot is “disappearing
root”, whereby the root tissue disintegrates due to rot. Another symptom of C.
destructans rot is a reddish-brown to dark-brown rusty colored canker on the root. These
cankers are often dry and cracked (Hudelson, 2002).

Rhizoctom'a solani infection of ginseng results in symptoms similar to
Cylindrocarpon and includes a rusty discoloration of the root and crown with reddish -
brown cankers on the roots. Infection by F usarium spp. can cause stem and crown
infections, although root rots are most common resulting in red streaking of the tissue.
When F usarimn root rot is advanced, the foliage wilts and becomes reddened.

Phytophthora cactorum
Phytophthora cactorum (Lebert & Cohn) J. Schro't, is considered to be the most

devastating fungal disease on ginseng because it can destroy entire ginseng gardens in a
few weeks (Darmono et al., 1991).

Phytophthora cactorum is a homothallic oomycete with a wide host range. This
species infects more than 200 plant species in 150 genera representing 60 plant families
(Erwin and Ribeiro, 1996). Phytophthora cactorum was first reported from rotting cacti
(Cereus giganteus and Melocactus nigrotomentosus) in Czechoslovakia by Lebert and
Cohn in 1870 (Erwin and Ribeiro, 1996). On ginseng, P. cactorum can cause leaf blight
and/or root rot. Van Hook first documented root rot of ginseng by P. cactorum in the
United States in 1906, (Putnam, 1984; Erwin and Ribeiro, 1996). In 1915, Rosenbaum
published an in-depth treatise on Phytophthora disease of ginseng (Putnam, 1984). In
1915, Phytophthora root rot was one of the most serious diseases confronting ginseng

growers (Duke, 1989). During 1984 in Wisconsin, up to 50% of plants in some gardens

were lost to P. cactorum (Duke, 1989). Phyt0phthora cactorum is also known for
causing leather rot in strawberry and collar rot in apples.

Phytophthora cactorum has aseptate mycelium. The optimum growing
temperature is 25° C. Phytophthora cactorum produces oospores, sporangia, zoospores,
and chlamydospores, all of which require free water for germination. Oospores are
sexual resting spores and are generally 15-36 pm in size (Erwin and Ribeiro, 1996).
When conditions are favorable for germination, oospores produce mycelium, which can
then form sporangia. Germination in the field begins after a dormant period during the
winter at soil temperatures of about 75° C and then increases rapidly within 22 days as
the soil temperature increases to 13° C (Erwin and Ribeiro, 1996).

Sporangia are asexual structures, and can germinate directly. The Sporangia are
ovoid, papillate and borne terminally (Erwin and Ribeiro, 1996). They can also
germinate indirectly by division of the contents within the spore into an indefinite
number of zoospores (Ribeiro, 1978). Zoospores are released from the sporangium
through a pore at the tip (Ribeiro, 1978). Producing zoospores gives P. cactorum an
increased potential for epidemics. They may also be dispersed passively in flowing water
over long distances, contaminating growing sites and irrigation water (Erwin and Ribeiro,
1996). Zoospores are asexual, kidney-shaped spores that move by using one tinsel and
one whiplash flagellum.

Zoospores exhibit strong attraction to certain amino acid exudates produced by
roots (Ribeiro, 1978). Wounds, succulent tissues, and shoot tips are the most susceptible
to infection. When zoospores come into contact with an appropriate surface, they

become circular, lose their flagellum, secrete a cell wall (encyst) and form a germ tube to

10

infect the plant and initiate mycelial growth if conducive conditions exist (Ribeiro, 1978).
Zoospores typically penetrate a host directly through roots or through stomata and they
can form spherical haustoria (Ribeiro, 1978).

Chlamydospores are round to ovoid vegetative asexual resting spores, which
germinate by germ tubes (Tucker, 1967) and can form within or at the tips of hyphae.
Historically, chlamydospores of P. cactorum have been observed infi'equently. Darrnono
and Parke (1990) have suggested that this may be the result of in vitro conditions. The
authors reported that the optimal temperature for chlamydospores production by P.
cactorum was at 4° C and although chlamydospores were produced at 24 and 28° C, they
were less abundant. Darinono and Parke (1990) suggested that chlamydospores may be
able to form during the winter months under natural conditions and contribute to
overwintering survival of P. cactorum thereby serving as an inoculum source for the
following growing season.

An early symptom of Phytophthora infection on ginseng is a drooping leaflet
(Erwin and Ribeiro, 1996). Dark green water-soaked lesions may appear on leaflets
(Duke, 1989; Proctor, 1996). Purpling or reddening of the leaflets may be observed if
infection occurs late in the season. Phytophthora leaf blight can also resemble symptoms
resulting from heat and drought injury, Alternaria leaf blight, or frost damage.

Infection of ginseng foliage is promoted by wet conditions (Pritts, 1995). Spores can
be splashed along with soil particles to adjacent plants, and contact with tools can also move
spores (Pritts, 1995). Infections initiated on leaves can move down the stem, causing
collapse and separation from the root crown.

Excess water and poorly drained soils can predispose the roots to rot. High

moisture and cool temperatures favor infection of stems by P. cactorum, which moves

11

rapidly down to the root (Erwin and Ribeiro, 1996). Initial indications of root rot caused
by P. cactorum include root discoloration and a spongy texture. Secondary decay often
occurs due to invasion of soft rot bacteria. Instead of having a white and firm interior,
the root turns brown with a soft or liquid texture, a sign of the vascular tissue breakdown.
Secondary roots may separate or be discolored. Late season infections in mature gardens
may go unnoticed until the roots are harvested and dried (Pritts, 1995).

Disease Management

Primary control of P. cactorum begins with sanitation. By using clean seed,
destroying diseased plants and using good cultural practices, P. cactorum infections can
be reduced. Any equipment used in diseased beds should be thoroughly washed and
disinfected with a soap or bleach solution and allowed to dry before moving it to healthy
beds (Pritts, 1995). Diseased plants should be removed from a garden and should include
healthy plants within eighteen inches of the infected plants (Pritts, 1995). When disease
pressure is high, fungicide applications may be necessary.

Fungicide options for P. cactorum have been available for use on ginseng for
approximately the past 20 years. Metalaxyl (trade name: Ridomil ®; Syngenta Crop
Protection Inc., Greensboro, NC), a systemic fungicide specific against Pythium and
Phytophthora spp. was introduced in 1977 (Duke, 1989). Metalaxyl is in the acylamide
class and affects RNA synthesis.

Preliminary research by Putnam and Mitchell (1984) showed that metalaxyl could
reduce disease incidence by 55-66% on three-year-old plants. Laboratory studies verified
that metalaxyl inhibited both grth and reproduction of P. cactorum when grown on an

artificial medium (Putnam and Mitchell, 1984; Duke, 1989). Metalaxyl also increased

12

yield by 50% with a single spray in early June at an application rate of 0.25 kg a.i./ha
(Putnam and Mitchell, 1984; Erwin and Ribeiro, 1996).

Mefenoxam, (trade name: Ridomil Gold ®; Syngenta Crop Protection Inc.)
introduced in 1997, is the R-enantiomer of metalaxyl. Like metalaxyl, mefenoxam is a
systemic fungicide specific against Pythium and Phytophthora spp. Mefenoxam also
inhibits RNA synthesis, which results in the prevention of spore production and
inhibition of mycelial grth (Syngenta Crop Protection Inc., 2004).

Two formulations of Ridomil Gold® are available for use on ginseng. Ridomil
Gold® EC (emulsifable concentration) and Ridomil Gold GR (granular). The labeled
rate for Ridomil Gold® BC on ginseng is 12 ounces per acre as a drench in 100-400 gal.
of water (Syngenta Crop Protection Inc., 2004). For the Ridomil Gold® GR on ginseng
the rate is 15 lbs. per acre applied uniformly to the soil surface in the spring before the
plants begin growing (Syngenta Crop Protection Inc., 2004).

F ungicide Resista_11_c_e_

Dekker (1987) defines fungicide resistance as a condition where strains of a
sensitive species, become (usually by mutation) significantly less sensitive to a fungicide.
Application of the fungicide selects for the resistant strains, and thus favors their
multiplication. Problems with fungicide resistance have increased since selective
fungicides were introduced (Dekker, 1987). Resistance to mefenoxam among some
Phytophthora spp. has been documented on crops such as cucumbers, potato, and
soybean (Lamour and Hausbeck, 2000; Taylor et al., 2002; Malvick, 2003).

Development of fungicide resistance by a firngus may increase based on the

chemical and its use over time and depends on the reproductive mode of the fungus.

l3

Reproduction in oomycetes occurs with two gametangia per mating strain: an oogonium,
which can hold one or several eggs, and an antheridium, which fertilizes the oogonium
(Heffer, 2002). Homothallic fungi such as P. cactorum have one mating type for
reproduction. A strain sensitive to a fungicide will produce sensitive progeny and a
resistant strain will produce resistant progeny.

Methods for deterrrrining fungicide resistance can be accomplished via laboratory,
greenhouse and field tests. For all tests, potentially fimgicide-resistant strains must be
compared with the sensitive wild-type fungus (Dekker, 1987). A common laboratory
technique for resistance screening is to use a radial growth of mycelium on fungicide-
amended agar medium. This technique uses small agar blocks with mycelium taken from
the periphery of an actively growing fungal colony that are placed on either agar media
amended with the fungicide (test plate) or on agar media with no fungicide (control)
(Dekker, 1987). The toxicity of the fungicide is evaluated by measuring and comparing
the diameter of the colony grth on the test and control plates (Dekker, 1987).

Utkhede and Gupta (1988) studied resistance to metalaxyl by in vitro selection of
P. cactorum strains exhibiting resistance. Single spore isolates were maintained on corn
meal agar (CMA) with fimgicide and incubated at 18° C for 4 weeks after inoculation
with a 5mm P. cactorum agar plug. Fungicide concentrations of the test plates were 100,
150, 200 and 250 ug- ml'1 of metalaxyl. All plates were measured after 4 weeks at 18° C.
Utkhede and Gupta (1988) determined that exposure to a gradual increase in metalaxyl
concentration resulted in the development of isolates that exhibited in vitro resistance to
metalaxyl. Further, a linear relationship exists between the concentrations of metalaxyl

and the inhibition of P. cactorum growth. Inhibition (%) of P. cactorum on CMA plates

14

amended with 50, 100,150, 200 and 250 ug- ml'1 of metalaxyl was 18:1: 7, 32 i 10, 351: 8,
42$ 8, and 33d: 9, respectively.

Jeffers and Schnabel (2004) reported that P. cactorum isolated from strawberry
roots and crowns was resistant to mefenoxam. Mefenoxam sensitivities were determined
by monitoring the radial mycelial growth of the isolates on clarified V8 juice agar
amended with lOO-ppm mefenoxam. All isolates were resistant to mefenoxam with
growth on fungicide-amended plates obtaining 73- 89% of the unarnended control plates.
Prior to the research presented in this thesis, research has not been conducted to

determine if resistance is a problem with P. cactorum isolated from ginseng.

15

Introduction

American ginseng (Panax quinquefolium) is an unisexual, erect, perennial plant
(Proctor, 1996). Ginseng has been used extensively in oriental countries as a traditional
medicine (Anonymous, 2000). Today it is more common to the find the root ground into
a powder or used as an extract for use in cosmetic and food products including
toothpaste, lipstick, soft drinks, energy drinks, tea, coffee, baked goods and vitamins
(Pritts, 1995).

Ginseng is propagated by seed. A stratification period of 18 — 22 months is
required for growth and maturation of the embryo (Putrnan, 1984; Proctor and Louttit,
1995; Harrison et al., 2000). Ginseng requires shade and is commonly cultivated using a
natural or artificial canopy. More than 95% of the commercial ginseng grown in the US.
is cultivated in Wisconsin (Adam, 2004). Currently, Wisconsin’s 400 ginseng growers
cultivate 2,100 acres of ginseng producing 500 to 1,700 lb/acre, which represents 10% of
the world’s supply of ginseng (Hausbeck, 2004). At approximately $20 to $40/lb,
ginseng is a high value crop. Michigan has a relatively new ginseng industry with
significant acreage established in 1995 (Hausbeck, 2004). Most of the production is
located in the Upper Peninsula and is grown under a natural forest canopy. Based on
current prices, woods-grown cultivated ginseng represents a Michigan inventory of over
$50 million (Hausbeck, 2004).

Phytophthora cactorum (Lebert & Cohn) J. Schrot, causes foliar blight and root
rot, which may destroy an entire ginseng garden in a few weeks (Darmono et al., 1991)

(Figure l). Phytophthora cactorum was first documented in association with ginseng in

16

the US. in 1906 (Van Hook, 1906; Erwin and Ribeiro, 1996) and has become an
increasing concern among Wisconsin and Michigan ginseng growers (Hausbeck, 2004).

Phytophthora cactorum is a homothallic oomycete and produces oospores,
chlamydospores, sporangia, and zoospores, all of which require free water for
germination. Oospores (15-36 pm) and chlamydospores (39.7 um) are potential
overwintering structures (Darmono and Parke, 1990; Erwin and Ribeiro, 1996).
Germination of oospores in the field generally occurs during temperatures of 75° C to
13° C (Erwin and Ribeiro, 1996). Ovoid Sporangia can undergo a division of contents
into swimming zoospores. Zoospore production increases the potential for disease
epidemics. They may be disseminated passively in flowing water over long distances,
contaminating growing sites and irrigation water (Erwin and Ribeiro, 1996).

Cool temperatures and high moisture favor foliar infections of ginseng (Darmono
et al., 1991; Pritts, 1995). During periods of warm, sunny weather when the diseased
tissues dry rapidly, the disease is temporarily arrested (Rosenbaum, 1915; Erwin and
Ribeiro, 1996). Foliar infection results in dark green water-soaked lesions and wilting
and reddening of leaflets. When the stem becomes infected, disease can progress rapidly
down the stalk (Erwin and Ribeiro, 1996). Root rot symptoms include root discoloration
and a spongy texture. Secondary roots may separate and also be discolored.

Phytophthora cactorum infects more than 200 plant species in 150 genera
representing 60 plant families, and isolates are generally not host specific (Erwin and
Ribeiro, 1996). Darmono et al. (1991) determined that isolates of P. cactorum from
ginseng gardens and forest soils in Wisconsin were pathogenic to ginseng. Darmono et

al. (1991) also investigated the potential of using apple cotyledons and ginseng leaflets as

17

baits for P. cactorum. All of the selected garden and forest isolates colonized both the
apple cotyledons and the ginseng leaflets in vitro; however, the recovery of P. cactorum
on either bait was not a good predictor in assessing pathogenicity or the degree of
virulence to ginseng seedlings. Tucker (1967) used wounded apple fruit to conduct
pathogenicity studies with isolates of P. cactorum recovered from beech (F agus sp.),
yellow Turk Cap lily (Lilium candidum), apple (Pyrus malus), rhubarb (Rheum
rhaponticum), peony (Paeonia sp.) and pine (Pinus sp.). Apple fruit may also be used for
P. cactorum detection and isolation (Erwin and Ribeiro, 1996).

Historically, P. cactorum diseases (foliar blight and root rot) have been managed
with frequent use of the registered fungicides, metalaxyl and/or mefenoxam (Putnam and
Mitchell, 1984). Recently, Jeffers and Schnabel (2004) indicated that P. cactorum
isolated from strawberry roots and crowns was resistant to mefenoxam. Resistance to
mefenoxam among other Phytophthora spp. has been documented on cucumbers, potato,
and soybean (Lamour and Hausbeck, 2000; Taylor et a1, 2002; Malvick, 2003). Other
products registered for Phytophthora foliar blight and root rot of ginseng include fosetyl-
a1 (Aliette) and salts of phosphorous acid (Phostrol), which offer a moderate level (60—
74%) of control against P. cactorum (Hausbeck, 2004).

The objectives of this research were to: 1) determine the prevalence of P.
cactorum in Wisconsin and Michigan ginseng gardens, 2) screen P. cactorum isolates for
sensitivity to mefenoxam, 3) determine the pathogencity and virulence of the isolates, and

4) screen alternative chemical products for efficacy against P. cactorum.

18

Material and Methods

Isolation and culture of P. cactorum:
From April to August 2003, American ginseng plants with P. cactorum symptoms

(reddened leaflets and spongy roots) were collected from 34 cultivated and woods-grown
commercial ginseng gardens in Wisconsin and Michigan, and from ginseng research plots
at the Plant Pathology and Horticulture farms located at Michigan State University, East
Lansing, MI. From May to August 2004, six cultivated commercial ginseng gardens
were sampled in Wisconsin and one research ginseng garden was sampled at the MSU
Plant Pathology research farm. In addition, greenhouse seedlings growing in a research
greenhouse on the campus of Michigan State University exhibiting wilting, blackened
stems and damping off were sampled.

Samples (seedling and 2,3-year old plants) were washed with distilled water and
then sprayed with ethanol (70%). Tissue samples (approximately 2x2 mm) of leaves,
stems and roots were embedded in 2% water agar (Sigma-Aldrich Corp., St. Louis, MO)
amended with ampicillin (2 ml/L) (Sigma-Aldrich Corp.) in 100x15 mm petri dishes
(VWR International, West Chester, PA). Cultures were incubated at room temperature
(23-25° C) and evaluated for characteristic P. cactorum hyphal grth and Sporangia
(Erwin and Ribeiro, 1996) after 24 hours. Hyphal tips were transferred to 4% V8
(Campbell Soup Company, Camden, NJ) agar until axenic cultures were obtained.
Cultures were incubated at room temperature under lab lighting for 4-5 days to stimulate
Sporangia production for zoospores. Single-zoospore isolates were obtained by flooding
agar plates with 20 ml of 10° C sterilized distilled water, incubating the plates at room
temperature for 25 minutes, and plating 5 drops of the zoospore suspension onto water

agar plates (100x15 mm). The zoospore suspension was spread onto water agar plate

19

with a sterile hockey stick. Single germinating zoospores were transferred to 20% V8
agar for long-term storage.

All cultures were stored at 13° C in 1.5 ml sterile micro-centrifuge tubes (Dot
Scientific Inc., Burton, MI) containing 1 ml of sterilized distilled water and one hemp
seed (Carolina Biological Supply Co., Burlington, NC). Cultures were also placed into
long-term storage at 13° C on V8 (20%) agar media amended with benomyl (0.050 g)
(Du Pont, Wilmington, DE), rifampicillin (2 ml/L) (Si gma-Aldrich Corp.) and ampicillin
(2 mI/L) in petri plates (35x10 mm) (V WR International).

Laboratory screening for mefenoxam sensitivity:

Isolates were removed from long-term storage, transferred to V8 (20%) agar petri
plates (100x15 mm), and allowed to grow at room temperature (23-25° C) until cultures
were a minimum of 40 mm in diameter. Four agar plugs (7 mm) were removed from
each culture plate. One agar plug was placed in the center of each of two V8 (20%) agar
plates and each of two V8 (20%) agar plates amended with lOO-ppm mefenoxam
(Syngenta Crop Protection Inc, Greensboro, NC). The plates were incubated at room
temperature for 3 days and the radial mycelial growth (cm) recorded. Thirty-five isolates
recovered in 2003 and 13 recovered in 2004 were also screened on lOOO-ppm
mefenoxam at 3 and 5 days with the radial mycelial grth diameter (cm) measured.

During the mefenoxam screening, ten isolates produced mycelial growth between
23 and 50% on the mefenoxam-amended agar relative to the unamended control plate.
Because the sensitivity of the isolates was determined by the presence or absence of
grth on 100-ppm mefenoxam, an additional assay using apple fruit was designed to

determine the mefenoxam sensitivity. Standards including a known mefenoxam resistant

20

(growth 100% of the control) and mefenoxam sensitive (0% of the control) isolates were
used.

Isolates were grown on V8 (20%) agar at room temperature until cultures reached
40 mm in diameter and then agar plugs (7 mm) were removed. Five commercial
‘McIntosh’ apple fruit were used to test each isolate. Prior to inoculation, apples
(approximately 110 g) were washed in warm water with Joy dish soap (Procter &
Gamble, Cincinnati, OH) to remove residue. The apples were wounded to the depth of a
10 mm approximately 45 mm down from the stem by a pushpin dipped in ethanol (70%).
Three of the five apples were treated with mefenoxam (0.75 pt/100gal) until runoff with a
hand-pump compressed air sprayer. The remaining two apples served as controls. After
the treated apples” dried, one plug of the appropriate P. cactorum isolate was placed over
the wound site. Agar plugs (and wound sites for control apples) were then covered with a
1.5 ml micro-centrifuge tube and secured to the apple with petroleum jelly.

Inoculated apples were placed in clean aluminum foil pans with paper towels
saturated with water. The pans were covered with plastic wrap and the apples were
incubated for one week at room temperature under fluorescent lights. The average
temperature inside the trays was 30° C and was monitored using a data logger (Watch
Dog lOO-Temp 2K, Spectrum technologies, Inc, Plainfield, 11.). After one week, the
diameter (in cm) of lesion coverage for each apple was recorded and averaged.
Phytophthora cactorum infection was confirmed through isolation from the apple tissue

following the termination of the experiment.

21

Screening for virulence:

Isolates of P. cactorum were tested for virulence on apple fi'uit (Tucker, 1967)
and compared to other PhytOphthora spp. including P. citricola, P. gonapodyides and an
uninoculated control. Phytophthora citricola and P. gonapodyides were chosen because
they cause root and mu rot to apple, respectively (Erwin and Ribeiro, 1996). Each
Phytophthora isolate was screened for virulence on four commercial ‘McIntosh’ apple
fruit (Erwin and Ribeiro, 1996) arranged in a completely randomized design. Apples
were prepared and wounded as previously described. Agar plugs (7 mm) were made for
each P. cactorum isolate and Phytophthora spp. standard. One agar plug was then placed
over each wound site and covered with a 1.5 ml micro-centrifuge tube. The micro-
centrifuge tubes were secured to the apple with petroleum jelly.

The apples were incubated in the same manner as described for the mefenoxam-
sensitivity apple fruit assay. After one week, the area of the fruit covered with a necrotic
lesion (%) was estimated as 0, 25, 50, 75 or 100%. The virulence ratings of the apples
were averaged for each isolate and the trial was conducted twice. After rating for
disease, the apples were surface disinfested by wiping with ethanol (70%). Tissue
samples (approximately 2x2 mm) were removed from the apple and embedded into water
agar to confirm P. cactorum as the causal agent of the observed disease.

Efficacy of fungicides using apple fruit and ginseng seedlings:

McIntosh apple fruit. Commercial ‘McIntosh’ apples were used to assess the ability of
various chemical treatments (Table 1) including reduced risk products and biopesticides
to protect against disease caused by a mefenoxam-resistant isolate of P. cactorum.

Apples were prepared and wounded as previously described. Immediately following

22

wounding, the apples (six per treatment) were treated with fungicide until runoff with a
hand-pump compressed air sprayer. Treated apples were allowed to dry before they were
inoculated and placed in aluminum foil pans for incubation. Inoculation and incubation
were accomplished as previously described. Apples were arranged in a completely

randomized design.

23

Table 1. Products tested for efficacy on apple fruit against P. cactorum recovered from
American ginseng in experiment 1

 

Active Formulation Used
Product Ingredient Manufacturer (per 100 gallons) Classification‘
Acrobat 50 WP dirnethomorph BASF Corp. Research 6.4 oz E chemical
Triangle, NC (189.2 ml)
Aliette WDG fosetyl-al w Bayer Crop Science, 5 1b -
Research Triangle, NC (2,268 g)
Gavel 75DF mancozeb + Dow Agroscience 2 lb 82 carcinogen
zoxarrridey Indianapolis, IN (907.2 g)
Kocide 2000 copper Griffin LLC 3 lb
DF hydroxidex Valdosta, GA (1,361 g) No rating
Phostrol 4.32EC phosphorus NuFarm Specialty Prod. 4.5 pt Biopesticide
saltsw Lobeco, SC (2,041 g)
Ranman 4008C cyazofamid ISK Bioscience Corp. 1.5 fl 02 Reduced risk
Concord, OH (44.4 ml)
Reason 5008C fenamidone Bayer Crop Science 4 fl oz Reduced risk
Research Triangle, NC (118.3 ml)
Ridomil Gold mefenoxamw Syngenta Crop Proc. 0.75 pt Reduced risk
EC Greensboro, NC (354. 9 ml)

 

wLabeled for ginseng and P. cactorum.

"Labeled for ginseng.
y Section 18. Exemption of federal and state agencies for emergency use.
zHuman risk assessment: B2 carcinogen: Likely human carcinogen; C carcinogen= Possible human
carcinogen for which there is limited animal evidence; D carcinogen: There is inadequate evidence to
determine carcinogenicity in humans; E chemical: Evidence of non-carcinogenicity in humans.

24

Table l con’t. Products tested for efficacy on apple fruit against P. cactorum recovered
from American ginseng in experiment 2

 

 

Active Formulation Used
Product [firedient Manufacturer (per 100 gallons) Classificationz
Acrobat 50 WP dirnethomorph BASF Corp. Research 6.4 oz E chemical
Triangle, NC (189.2 ml)
Bravo Weather chlorothalonil Syngenta Crop Proc. 3 pt 82 carcinogen
Stick 6F Greensboro, NC (1,419.5 ml)
Cabrio EG pyraclostrobin BASF Corp., Research 4 02 Reduced risk
Triangle, NC (118.3 ml)
Curzate 60DF cymoxanil DuPont 8 fl oz E chemical
Wilmington, DE (236.6 ml)
Dithane 75DF mancozeb Dow Agroscience 1.5 lb 82 carcinogen
Indianapolis, IN (680.4 g)
Endorse 2.2WP polyoxin D zinc Cleary Chemical Corp. 2.2 lb Biopesticide
salt Dayton, OH (998.0 g)
Omega 500F fluazinam, Syngenta Crop Proc. 4 oz Reduced risk
Greensboro, NC (118.3 ml)
Quadris azoxystrobinx Syngenta Crop Proc. 12.3 fl oz Reduced risk
Greensboro, NC (363.8 ml)
Pristine 38WG pyraclostrobin + BASF Corp, Research 4 oz Reduced risk
(BAS 516) boscalid Triangle, NC (118.3 ml)
Previcur Flex propamacarb Bayer Crop Science 19.2 fl oz D carcinogen
6F Research Triangle, NC (567.8 ml)
Ridomil Gold mefenoxamw Syngenta Crop Proc. 0.75 pt Reduced risk
EC Greensboro, NC (354. 9 ml)
Tanos 50DF famoxadone + DuPont 10 oz E chemical
cymoxanil Wilmington, DE (295.7 ml)
Zoxium zoxamide Dow Agroscience 5.0 oz Reduced risk
Indianapolis, IN ( 147.9 ml)

 

wLabeled for ginseng and P. cactorum.

"Labeled for ginseng.
’ Section 18. Exemption of federal and state agencies for emergency use.
zHuman risk assessment: BZ carcinogen= Likely human carcinogen; C carcinogen= Possible human
carcinogen for which there is limited animal evidence; D carcinogen= There is inadequate evidence to
determine carcinogenicity in humans; E chemical= Evidence of non-carcinogenicity in humans.

25

After one week, the diameter (in cm) of lesion coverage for each apple was
recorded and then averaged for the six apples per treatment. The number of apples
infected for each treatment was also recorded. Alter rating, apples were wiped with 70%
ethanol solution and samples were taken to confirm infection by P. cactorum. Each
experiment was conducted three times. Data were subjected to analysis of variance
(ANOVA) and when significant treatment effects were indicated, treatment means were
compared by Fisher’s Least Significant Difference (LSD) test at P =0.05 (SAS Institute
Inc. Cary, NC.)

Ginseng seedlings. Stratified ginseng seed (Panax quinquefolium) were mixed in silica
sand and stored at 3° C until germination. Seeds that initiated germination were hand
planted into 72-cell flats containing silica sand in greenhouse seedlings growing in a
research greenhouse on the campus of Michigan State University. Following emergence,
seedlings were carefully removed from the sand and observed for disease symptoms.
Any seedling showing root discoloration was discarded. Healthy seedlings were hand
planted into 89 x 64 mm pots (Hummert International, Earth City, MO) with autoclaved
medium particle vermiculite (Therm-O-Rock Inc., Chandler, AZ) and medium grain sand
(at a 25% to 75% ratio respectively) in the research greenhouses at Michigan State
University. Plants were maintained in the greenhouse under 63% shade cloth (Hummert
International) with temperatures between 17.8 and 256° C and irrigated as needed.

A mefenoxam-insensitive isolate of P. cactorum was grown under lab lighting for
7 days on dilute V8 agar. Six plates with sporulating P. cactorum were flooded with 20
ml of 10° C sterilized distilled water and then incubated at room temperature for 25

minutes to release zoospores. Fungicide treatments were applied as a drench in sufficient

26

volume to displace approximately 10% of the liquid in the pots. Plants were arranged in
a blocked design with 8 plants per treatment. Two experiments were conducted. A 2 ml
zoospore suspension was injected into the soil at the base of each seedling immediately
after the fungicide treatments were applied. For the first experiment, the plants were
treated and inoculated on 26 April and disease ratings were taken on 5, 8, and 10 May.
For experiment 2, the plants were treated and inoculated on 3 May and disease ratings
were taken on 12, 18 and 27 May. Disease ratings were based on a 1 to 5 scale, where
l=hea1thy, 3= collapse of the stem, and 5=dead. Data were subjected to analysis of
variance (ANOVA) and when significant treatment effects were indicated, treatment
means were compared by Student-Newman-Keuls test at P =0.05 (SAS Institute Inc.
Cary, NC.)

Detection of P. cactorum on ginseng seed:
Detection from seedlings. Stratified ginseng seeds were planted in the research

greenhouses at Michigan State University in new 80 mm deep (104 oz) aluminum foil
pans (Handi-foil Corporation, Wheeling, IL) with autoclaved medium particle
vermiculite and medium grain sand (at a 25% to 75% ratio respectively). After
emergence, 200 tissue samples were taken from seedlings exhibiting wilting and
blackened stems. Samples (approximately 2x2 mm) of stems and roots were embedded
in 2% water agar amended with ampicillin (2 ml/L) in 100x15 mm petri dishes. Cultures
were incubated at room temperature (23-25° C) and evaluated for characteristic P.
cactorum hyphal growth and Sporangia (Erwin and Ribeiro, 1996) after 24 hours. All
isolates were prepared and maintained as previously described.

Koch’s postulates were conducted for P. cactorum isolates recovered from

ginseng seedlings. Stratified ginseng seed was germinated in the greenhouse in silica

27

sand. Afier two weeks of grth (height of 25 mm), seedlings with healthy leaves and
roots were selected. Seedlings were aseptically transferred to capped 25 x 70 mm (30 ml)
glass vials containing 5 ml of water agar amended with ampicillin (2 ml/L) (Reid, 2000).
Seedlings were observed for 2 days under fluorescent lights for disease. Several P.
cactorum isolates recovered from ginseng (three collected from mature plants in 2003
and seven collected from seedlings in 2004) were grown on V8 agar until 40 mm in
diameter and then agar plugs (7 mm) were removed. One agar plug of each isolate was
placed at the base of the appropriate seedling stem at the agar line. Each isolate was
tested twice. Seedlings were incubated in the laboratory under florescent light. After one
week, tissue samples were removed from the seedlings and embedded in water agar with
ampicillin (2 ml/L). Phytophthora cactorum exhibiting characteristic growth and
structures on the isolation media (gnarled mycelium, ovoid and papillate sporangia and
oospores with paragynous antheridium) was recovered.

Koch’s postulates were also conducted with seedlings planted into 89 x 133 mm
pots with silica sand. Seedlings were inoculated at the base of the stem as previously
described. Seedlings were placed inside plastic bags with paper towel saturated with
water and incubated in the laboratory. After one week, tissue samples were removed
from the seedlings and embedded in water agar with ampicillin (2 mI/L). Phytophthora
cactorum exhibiting characteristic growth and structures on the isolation media was
recovered.

Detection from seed. Stratified ginseng seeds were obtained from five commercial
ginseng gardens. Twenty-five seeds from each of the sampled seed lots were rinsed in

distilled water to remove sand particles and embedded in 2% water agar amended with

28

ampicillin (2 mI/L) in 100x15 mm petri dishes. After 2 days, plates were examined and

sub-cultured for fungal identification. The study was conducted twice.

29

Results

Isolation and culture of P. cactorum:

Over the course of the study, P. cactorum was a significant problem in Michigan
and Wisconsin and 162 isolates were collected (Table 2). In 2003, 26 ginseng gardens
with symptomatic plants (wilted, reddened leaves and discolored root) were surveyed in
Wisconsin. Pathogens recovered included F usarium spp., Pythium spp, Cylindrocarpon
destructans and P. cactorum. In 2004, six gardens with high P. cactorum disease
pressure were surveyed. Only one of the six had previously been sampled in 2003. In
Wisconsin, 47% (15) of 32 ginseng gardens had incidence of P. cactorum from 2003 and
2004. Phytophthora cactorum was recovered from 83% of the sampled ginseng gardens
in Michigan during 2003. Additional P. cactorum isolates (56) were recovered from
Michigan State University research greenhouse seedlings in 2004 (Table 2).
Laboratory screening for mefenoxam sensitivity:

Insensitivity to mefenoxam was detected in the majority (76 and 88% for 2003
and 2004, respectively) of samples collected. Nearly all isolates (93%) recovered from
the greenhouse seedlings were insensitive to mefenoxam (Table 3). Two isolates
recovered in 2003 from Wisconsin ginseng plants produced mycelial growth at 25 and
49% of their control on the mefenoxam-amended V8 agar. When these two isolates were
screened during the mefenoxam-sensitivity apple assay, they produced lesions at 8 and
26% of their controls respectively. Because growth in vitro and in vivo was less than
50% of the control, both isolates were classified as sensitive to mefenoxam.

Eight isolates recovered in 2004 from Wisconsin seedlings produced mycelial

growth less than 50% of their controls during the in vitro assay. To determine

30

mefenoxam sensitivity of the eight isolates, they were screened on mefenoxam-treated
apple fruit. Four isolates produced mycelial grth of 36, 42, 47 and 44% respectively,
on the mefenoxam-amended V8 agar (relative to the control) produced lesions greater
than 50% of their controls on the apple fruit and were classified as insensitive to
mefenoxam. The remaining 4 isolates with 23, 23, 35 and 38% of mycelial grth on the
mefenoxam-amended V8 agar did not produce lesions of 50% or greater than the controls
on the apple. Lesion coverage was measured at 0% for 3 of the 4 isolates and 24% for
the isolate having 38% growth on amended agar. These isolates were classified as
sensitive to mefenoxam.

Screening for virulence:

Of 111 isolates collected in 2003 that were screened for virulence to ‘McIntosh’
apple fi'uit, 84% produced lesions spanning 25% of the surface fruit area (Table 4). In
2004, 103 isolates were screened and nearly all produced lesions spanning 25% of the
apple fruit (Table 4).

Efficacy of fungicides using apple fruit and ginseng seedlings:
‘McIntosh’ apple fruit. In experiment 1, significant differences (P=0.05) were observed

among the treatment means (Table 5). Dimethomorph (Acrobat), copper hydroxide
(Kocide), mancozeb + zoxamide (Gavel) and fenamidone (Reason) consistently yielded
disease rating less than the untreated control in each replication. In 2 of the 3
replications, phosphorus salt (Phostrol) and cyazofamid (Ranman) provided Significantly
better control than the untreated. No significant differences were detected among the
untreated and the remaining products.

Significant differences (P=0.05) were also observed in the second experiment

among treatments (Table 6). Dimethomorph (Acrobat) provided complete protection

31

against P. cactorum in each replication and was significantly better at managing disease
than the untreated control. In 2 of the 3 replications, mancozeb (Dithane) and zoxamide

(Zoxium) were significantly better than the untreated control.

Ginseng seedlings. Disease pressure was severe in both experiments, with 75 and 87.5%
of the untreated inoculated plants dead at the final observation. Copper hydroxide
(Kocide) completely prevented plant death in each experiment. Dimethomorph (Acrobat)
limited plant death to 25% (Table 7).

Plant death was limited to 25% of total plants for phosphorus salts (Phostrol) and
fosetyl - a1 (Aliette) treated plants in the first experiment. However, in the second
experiment, plant death reached unacceptable levels with plant death at 75% or greater.
Mancozeb (Dithane) and mancozeb + zoxamide (Gavel) limited plant death to 37.5% at
the final rating for both experiments. F enarnidone (Reason) limited plant death to 25% in
the first experiment, but was at 50% for the second. Mefenoxam (Ridomil Gold), the
industry standard, performed poorly in both experiments due to the mefenoxam—
insensitivity of the isolate. All other treatments had 250% plant death at the final
evaluation and offered little disease control (Table 7).

Detection of P. cactorum on ginseng seed:
Detection from seedlings. Greenhouse-grown ginseng seedlings were sampled for

disease. Tissue excisions were taken from approximately 195 seedlings, and placed onto
amended water agar. Fifty-six P. cactorum isolates were identified based on
morphological characteristics. Ten isolates (7 of which were recovered from the
seedlings) were selected and used to inoculate healthy ginseng seedlings. After 1 week

of initiating Koch’s Postulates, symptoms (wilting and blacken stems) matching those

32

exhibited on greenhouse seedlings and seedling recovered from the field were observed.
Disease symptoms on seedlings began at the base of the stem at the inoculation site.
Wilting and twisting of stems were observed 3 days after inoculation. Four days after
inoculation, root discoloration was observed. Stems turned brown, and later blackened at
6 days post inoculation. Disease tissue was excised to confirm P. cactorum based on
morphological characteristics (gnarled mycelium, ovoid and papillate Sporangia and
oospores with paragynous antheridium).

Detection from seed. In addition to P. cactorum, four fungi were recovered from direct
plating of 125 (5 seed lots) stratified ginseng seeds: Pythium spp, F usarium spp.
Septonema sp. and Cylindrocarpon destructans. These four fungi are known ginseng
pathogens and were identified by morphological characteristics. In the first experiment,
all seeds yielded growth of F usarium spp. and Cylindrocarpon destructans. Eighty
percent of seeds were infected with Septonema sp. and 60% with Pythium spp.
Phytophthora cactorum was recovered from only 20% of the seeds. In the second
experiment, only 4 pathogens were identified: Pythium spp, (on 40% of seed), F usarium

spp. (<1%), Septonema sp. (1%) and C. destructans (< 1%) (Table 8).

33

Table 2. Source and number of P. cactorum isolates recovered from ginseng in 2003 and

 

 

 

 

2004
Gardens sampled“ Isolates recovered x

Location 2003 2004 2003 2004
Michigan (Upper Peninsula) 6(5) - 23 -
Michigan State University 2(2) 1(1) 19 6
Greenhouse SeedlingsZ - 6(6)y - 56
Wisconsin 26(9) 6(6) 72 42
Total 34(16) 13(13) 114 104

 

WThe number of gardens sampled is followed by the number of gardens where P. cactorum was
recovered.

xDashes represent not sampled.

yThe number of seedling trays with disease symptoms is followed by the number of tray infected with P.

cactorum
zSeedlings were obtained from stratified ginseng seeds produced in WI and germinated in research

greenhouse at Michigan State University.

Table 3. In vitro mefenoxam sensitivity screening of P. cactorum from ginseng in 2003

 

 

 

 

and 2004

Insensitive isolates y Sensitive isolates y
Location 2003 2004 2003 2004
Michigan (Upper Peninsula) 11 - 12 -
Michigan State University 15 5 4 1
Greenhouse SeedlingsZ - 52 - 4
Wisconsin 61 37 1 1 5
Total 87 94 27 10

 

yDashes represent not sampled.
zSeedlings were obtained from stratified ginseng seeds produced in WI and germinated in research

greenhouse at Michigan State University.

34

- - n- l-Il I.

 

. ,7. I-é\'; ,
Figure l: Ginseng plant disease symptoms caused by Phytophthora cactorum. Wisconsin
ginseng gardens exhibiting foliar blight and plant death (A-C). Advanced stage of root rot
(D). Images in this thesis are presented in color.

35

 

Figure 2. . Laboratory in vitro screen for mefenoxam sensitivity in P. cactorum. Panel 1A
indicates growths on the V8 control ( no agar amendment); 13 indicates limited P. cactorum
growth of a mefenoxam-sensitive isolate on amended V8 agar. Panel 2A depicts P. cactorum
growth on a control plate in comparison to 2B which exhibits growth of a mefenoxam insensitive
isolate on mefenoxam—amended agar.

36

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42

Discussion

Phytophthora cactorum is recognized worldwide as a pathogen of numerous crops
(Erwin and Ribeiro, 1996) and can be especially devastating on ginseng (Darmono et al.,
1991). In 1915, Phytophthora root rot was one of the most serious diseases confronting
ginseng growers (Duke, 1989). In 1984, Wisconsin growers lost up to 50% of the plants
in some gardens due to P. cactorum (Duke, 1989).

Darmono et al., (1991) documented the presence of P. cactorum in Wisconsin and
recovered isolates from both forest and ginseng garden soils. In our 2003 and 2004
study, 47% of all sampled symptomatic and asymptomatic plants from Wisconsin
ginseng gardens yielded P. cactorum. Disease pressure was severe in gardens where
Phytophthora was recovered.

Woods-grown ginseng is a relatively new commercial crop in Michigan, with the
first seedlings (Wisconsin transplants) having been planted in 1995 (Hausbeck, 2004).
This is the first report of P. cactorum on ginseng in Michigan. Phytophthora cactorum
was recovered from plants in 5 of 6 gardens sampled during 2003 in the Upper Peninsula
and was responsible for significant plant loss. Additional P. cactorum isolates were
recovered from research gardens established at Michigan State University. These
gardens had been established from transplants of 2- to 3-year-old roots and planting of
seed from Wisconsin in 2003 and 2004. Symptoms were particularly severe during the
cool, wet conditions of early spring; conditions conductive for P. cactorum (Pritts, 1995;
Darmono et al., 1991).

Much of the ginseng sampled during this study were from 2- and 3-year old

plantings collected from failing gardens. Ginseng growers prefer to harvest roots at 3 or

43

4 years old, as this stage is more desired for market. In addition to P. cactorum, other
pathogens were frequently isolated from ginseng plants and include F usarium spp., C.
destructans, and Pythium spp. Competition among these pathogens may have hindered
the recovery of P. cactorum.

Recovery of P. cactorum isolates from ginseng seedlings grown in a sterilized
medium in the greenhouse and from seed directly plated on agar, suggests that P.
cactorum may be disseminated via the seed. Hobson (1999) estimated that seed rots and
seedborne diseases are responsible for much of the reduction in germination and seedling
emergence of ginseng. Pathogens including F usarium and Cylindrocarpon have been
documented on ginseng seed and are thought to be early colonists of germinating and
emerging seedlings (Ziezold, 1997; Hobson, 1999). It was hypothesized that quality
control methods such as floating stratified ginseng seed before planting, were not
effective due in part to the fact that infected seed could sink to the bottom of the float tray
along with the healthy seed (Ziezold et a1. 1998; Hobson, 1999). Since P. cactorum has
only been reported on safflower seed (Zad, 1992), further research on seed transmission
is needed.

All ginseng P. cactorum isolates collected were pathogenic to apple fruit.
Pathogenicity was determined by testing each isolate on apple fruit hosts. Most
Phytophthora spp. grow rapidly on apple tissue and the use of apples for the isolation of
Phytophthora from infected plant parts has proven successful (Tucker, 1967). It has also
been documented that P. cactorum is not host specific and is virulent on apple fruit

(Tucker, 1967; Erwin and Ribeiro, 1996). Although the use of ginseng seedlings for

44

pathogenicity tests is relatively easy and fast (Darmono et al., 1991), healthy stratified
seeds were not available during this study.

After 7 days of incubation the majority of screened isolates produced lesions that
covered 25% of the apple fruit. After 14 days, many lesions expanded and covered 100%
of the apple. The observed virulence in this research did not appear to be associated with
mefenoxam sensitivity or origin of the isolates. Although a quarter percentage was used
for determining the virulence of each isolate; five isolates (3.6 and 0.97% of isolates for
2003 and 2004 respectively) were scored as virulent to apple hit with a maximum
surface lesion of 10% of the apple fi'uit.

Darmono et al. (1991) reported that while apple cotyledons and ginseng leaflets
could be colonized by pathogenic and non-pathogenic isolates of P. cactorum, the
recovery of P. cactorum from each host was not a good predictor of pathogenicity or
virulence to a ginseng seedlings. Because there was little distinction between virulence
of the isolates on the apple fi'uit, the research findings presented should be verified on
ginseng seedlings and mature plants to better estimate the disease potential of each
isolate.

Phytophthora cactorum is highly destructive and fungicides are commonly
required to limit plant loss. Mefenoxam is the primary registered fungicide used for P.
cactorum control on ginseng (Hausbeck, 2004). Resistance to mefenoxam among some
Phytophthora spp. has been documented on crops such as cucumbers, potato, and
soybean (Lamour and Hausbeck, 2000; Taylor et al., 2002; Malvick, 2003). Recently,
resistance to mefenoxam in P. cactorum was identified with isolates from strawberry

roots and crowns (J effers and Schnabel, 2004). In this study, P. cactorum from ginseng

45

was primarily insensitive to mefenoxam. This is the first report of P. cactorum resistant
to mefenoxam in ginseng and the second for any other crop in the United States or
elsewhere.

In addition to metalaxyl and mefenoxam (Ridomil 2E or Ridomil Gold) the
fungicides Aliette (aluminum tris) and Phostrol (phosphorus salts) are labeled for use on
ginseng for Phytophthora control. Utkhede (1987) tested metalaxyl, aluminum tris, and
mancozeb for control of P. cactorum crown rot of McIntosh apple trees. Utkhede
concluded that metalaxyl and fosetyl - a1 could arrest flirther symptom development in
‘McIntosh’ apple trees naturally infected with P. cactorum if treated at an early stage of
disease. An additional study was conducted by Utkhede and Smith (1991) verifying that
metalaxyl and fosetyl - 31 were effective against P. cactorum on ‘McIntosh’ apple trees.

When using inoculated apple fi'uit to test the efficacy of different control
products, the greatest lesion coverage and incidence of infection was observed with the
untreated inoculated and mefenoxam-treated apples. Mefenoxam did not perform well in
managing disease because of the use of a mefenoxam-insensitive P. cactorum isolate.
Although some control was observed with phosphorus salts and fosetyl - al, the number
of infected fruit and percent of lesion coverage on the apples was variable among the
replicates. The most effective products in managing P. cactorum were dirnethomorph,
copper hydroxide and fenamidone. Results from this study, although on apple fruit, may
provide an indication of fungicides efficacy on ginseng foliar blight.

The performance of these products were also examined when used as a soil
drench to protect ginseng seedlings. As with the apple study, the greatest incidence of

infection and subsequently the greatest amount of plant death was observed with the

46

mefenoxam- treated plants. This is again, attributed to the insensitivity of the isolate to
mefenoxam. The efficacy of Phostrol (phosphorus salts) and Aliette (fosetyl - a1) against
P. cactorum was varied among the trials. The most effective products were Kocide
(copper hydroxide) and Acrobat (dimethomorph).

Phytophthora cactorum (Lebert & Cohn) J. Schrot, is considered to be a
devastating fungal disease on ginseng. It appears that P. cactorum is disseminated via
seed, further hindering the ginseng industry. With only three products registered for
Phytophthora control on ginseng, resistance to the fungicide standard, mefenoxam is of
great concern. Additional fiangicides and new cultural strategies (seed stratification and

storage procedures) are needed to maintain this high value export cr0p.

47

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