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SPRDYG SEEDING, SEED SIZE AND SEED PATHOLOGY
OF NORTH AMERICAN GINSENG (Panax quinquefolius L.)
A Thesis
Presented to
The Facdty of Graduate Studies
of
The uni ver si^ of GueIph
In partial fulfillment of the requirernents
for the degree of
Master of Science
April, 1999
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ABSTRACT
Sprïng seeding, seed sue and seed pathology
of North American ginseng (Panax quinquefolius L.)
Jennifer M. Hobson University of Guelph
Advisor: Professor J.T.A. Proctor
Spring seeding, seed size and seed pathology were investigated to detemine their
effects on ginseng crop establishment. Gibberellins, drying and pre-plant hydration were
used to reduce the seed dormancy period, fiom 18-22 months to 8-9 months. Field trials
gave poor emergence rates (~33%)~ partially due to infenor seed quality. G&+7 treatments
produced the highest levels of field emergence and high seedling yields. Stratified seeds
produced significantly larger ernbryo lengths and seedlùig yields from extra-large seeds
compared with small seeds, however, both these seed sizes produced significantly lower
yields than medium and large sized seeds. Examination of fungi recovered from flowers,
f i t and seed revealed Alternaria spp., Bohytis cinerea, Fusurium rosetrm, Fzrsarizim spp.,
Mucor and Rhizopis spp. Endo- P-mannanase, an enzyme involved in the mobilization of
endosperm reserves, was detected in green and stratified seed. Further GA3 assays revealed
that GA may stimulate the synthesis d o r secretion of endo-P-mannanase in ginseng seed.
DEDICATION
I would like to dedicate th is study to
Derek Bewley
for his wonderfiil enthusiasm,
nutty humour
and incredible courage.
ACKNOWLEDGEMENTS
I would like to thank Dr. John Proctor for his guidance and support throughout this project and for giving me the oppominity to obtain my M.Sc. 1 would also k e to thank my various advisoiy committee members Dr. Derek Bewley, Dr. V i c e Machado and Dr. Rick Reeleder. Their p l h g and advice made this study possible.
My sincere thanks to the organizations that gave hancial support. This project was made possible by the collaborative research agreement between the Ginseng Growers Association of Canada and Agriculture and Agri-Food Canada, under the Matching Initiatives Prograrn of AAFC. Scholarships were received by the University of Guelph, Horticultural Science Department (Hoskins S cholarship).
Many thanks to Bob Kozak, Jeff Rice and the Delhi Research Farm (Southern Crops Protection and Food Research Centre) for providing samples of ginseng for this study.
1 would like to express my appreciation to Jan Schooley for her availability, her enthusiasm and her endless ideas and advice on ginseng production. She is a credit to the industry.
I gratefùlly acknowledge the technical assistance of Dean Louttit for helping me with rnany aspects of this project, to Rick Reeleder for his expertise and supplies, to Richard Bourgault for his time and proficiency, and to William Matthes-Sem and Bill Kokiopoulos f?om the Ashton lab for their statistical advice. Speciai thanks to Rob Grohs at the Delhi Research F m for managing the gardens and for his cute jokes.
1 would like to express my appreciation to Greg Boland for his friendship, advice and encouragement.
Many thanks to my family for their support and encouragement, and for listening and smiling even though they were not really sure what 1 was talking about. 1 am especially grateful to rny sister Chris, she is always there when 1 need her most.
1 would like to acknowledge my peers who made grad school a rnutually supportive and stimulating environment: Andrea Fiebig, Pam Livingston, David Llewellyn, and Mike Peever.
1 would like to express my deep gratitude to Pam Livingston. Her time-saving advice, editorial seMces and constant fiiendship were incredible. I cannot thank her enough.
And to David Llewellyn, 1 would like to express my appreciation for his constant love, support, advice and fkiendship.
5.0 CHAPTER 5: AN ASSAY FOR ENDO-P-MANNANASE IN ..................................................... GINSENG SEED
......................................................................... 5.1 Introduction 5 -2 Materials and Methods ............................................................ 5 -3 Results and Discussion ............................................................ 5.4 Summary ............................................................................
GENERAL CONCLUS IONS .................................................................................. REFERENCE S ........................................................................................................ APPENDIX 1 ....................................................................................
Chapter 3 :
Treatments for seed size experiments during 1 996 and 1 997. ........... Assignment of field plot numbers to treatments and reps of seed size experiment in 1996 and 1997 ...................................... Mean embryo Lengths and S.E. for 1996 Sized Spring seed at
......................................................................... t h e #3.. Probability estimates of successful seed crack with confidence intervals for 1996 Shed Spring seed at time #3 ............................. Mean final emergence and S.E. for 1996 Sized Spring seedlings in
................................................................ the greenhouse.. Mean £inal emergence and S.E. for 1996 Sized Spring seedlings in
........................................................................ the field.. Mean final yield component parameters and S .E- for 1996 Sized Spring seedlings fkom the Delhi field.. ..................................... Mean h a l emergence and S.E. for 1996 Sized S p ~ g two-year olds
........................................................................ in 1997.. Mean final emergence and S.E. for 1996 Sized Spring second year
............................................................ seedlings in l997... Mean ernbryo lengths and S.E. for 1997 Sized Spring seed at
........................................................................ time #3.. Probability estimates of successful seed crack with confidence intervaIs for 1997 Sized Spring seed at tirne #3. . .......................... Mean final emergence and S.E. for 1997 Sized Spring seedlings in
............................................................... the greenhouse.. Mean final yield component parameters and S.E. for 1997 Sized
..................................... Spring seedlings from the greenhouse.. Mean final emergence and S.E. for 1997 Sized Spring seedlings
................................................................. fiom the fiefd.. Mean final yield component parameters and S.E. for 1997 Sized
..................................... Spring seedlings fiom the Delhi field.. Mean embryo lengths and S.E. for 1997 Sized Stratified seed at
......................................................................... planting Probability estimates of successful seed crack with confidence intervals for 1997 Sized Stratifled seed at planting.. ..................... Mean final emergence and S.E. for 1997 Shed Stratified seedlings
............................................................ in the greenhouse.. Mean Tial fiesh root weight and S.E. for 1997 Shed Stratified
................................................ seedlings in the greenhouse.. Mean final dry root weights and S.E. for 1997 Shed Stratified
................................................ seedhgs in the greenhouse.. Mean final emergence and S.E. for 1997 Sized Strati£ied seedlings
.......................................................... fiom the Delhi field.. Mean final yield component parameters and S.E. for 1997 Sized
................................... Stratined seedlings from the Delhi field..
4.3 .g. Percentages of fungi recovered per t h e , 3 year old plants, at times O through 40.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - - . . . . . . . . . . . . . . . . . . . . . . . 130
4.3. h. Percentages of fun@ recovered per explant type, 3 year old piants, attimes 85 through 168 ...................................................... 130
4.3 .i. Percentages of fun@ recovered per media type, 3 year old plants, attimes85through168 ................................................. 131
4.3 .j. Percentages of fiingi recovered per farm, 3 year old plants, attimes85 through 168 ................. . .-...... .. ......... - -..........-.-... 132
4.3 .k. Percentages of fungi recovered per time, 3 year old plants, at times 85 through 168.. . . . . . . . . . . . . . . . . . . . . . . . . . . -. . - - . . . -. . . . . . - - . . . . . . . 132
5.1 Activity @kat) of endo-P-mannanase in stratified ginseng seed.. . . . . . . . 142
Figure
Cha~ter 2:
2.1. 2.2. 2.3. 2.4. 2.5. 2.6.
Cha~te r 3 :
3.1. 3 .2 . 3.3.
Cha~ter 4:
4.1. 4.2. 4.3. 4.4.
4.5 : 4.6 .
Chapter 5 :
5.1 5.2
LIST OF FIGURE3
Description
S p ring ' 97 exp erimental timeihe ............................................ Field plot layout of Spring Seed 1 996 experimental design .............. Field plot layout of Spring Seed 1997 experimental design .............. Green seed and mature stratified seed of N . Amencan ginseng .......... Swelling & degeneration of ginseng endosperm after GA application ..
. ............................... S p ~ g seeding of N American ginseng seed
Field plot Layout of Seed Size 1996 and 1997 experimental design ..... Representatives of the 1996-97 stratified seed size experiments ........ Field plot expenments (Delhi) of 1996 sized spring seed .................
Mean seed rot % of Spring Seed 98 fkom 6 different f m s .............. Temperature fluctuations within a traditional seed stratification box ... Fungi isolated fiom explant cultures of ginseng flowers, h i t & seed .. Fungi recovered fom explant material of ginseng flowers, fruit & seed ..................................................................... Cornmon visual symptoms of rot in stratified ginseng seed .............. Culture and subsequent identification of fùngi recovered nom ginseng flowers, f i t and seed ...............................................
Agarose gel plate for eado-p-manaanase .................................. Assay for endo-P-mamanase of green seed exposed to GA3
Page
........................................................................ over t h e 143
- C W T E R 1-
GENERAL INTRODUCTION
1.1. Historu, Use and Botany:
Ginseng (Panm qziinquefoliis L.) is a herbaceous p e r e ~ i a i that is known widely for
the medicinal properties of its root. North Arnerican ginseng was discovered in the early
l7OO's in Quebec and became a strong trading commodity between the native people and
Europeans (Heliyer, 1984), while Oriental ginseng ( P m ginseng C.A. Meyer) has
played an integral part of Chinese medicine for centuries (Chang, 1995). Recent trends in
western medicine have witnessed the adoption of a holistic philosophy and a surge of
natural remedies in the market place (Hobbs, 1997). Every part of the ginseng plant is
used in Chinese medicine, however, roots are the main commercial product (But et al.,
1995). Also, there has been rising interest in ginseng value-added products (e-g. teas,
capsules, root powder) that are processed, packaged and sold in North America. Ginseng
is taken for the relief of a large range of ailments, some of which include stress, fatigue
and aging. The active ingredients of ginseng, ginsenosides, are a complex mumire of
cornpounds cailed tnterpene saponins and polysaccharides (But et al., 1995; Schooley and
Reynolds, 1998). Ginsenosides have been identified in al1 parts of the ginseng plant.
There are many dBerent types of ginseng, with the two most common being North
Amencan ginseng ( P m quinquefolius L.) and Oriental ginseng ( P m a ginseng C.A.
Meyer). Other plants termed "'ginseng" include: Siberian ginseng [Eleutherococcus
senticosrs (Rupr. Et Maxim.)], notoginseng or sanqi [ P m notoginseng (Burk.) F.H.
Chen], B razilian ginseng ( P f a f a paniculata Kuntze) and women' s ginseng [Angelica
sinenris (Oliver) Diels] (But et al., 1995). In this study, the word "ginseng" refers to
North Arnerican ginseng, unless othenvise stated.
Ginseng belongs to the family Araliaceae, which shares many characteristics with the
Apiaceae family (formerly the Umbelliferae) (Esau, 1 99 1). It is a perennial herb who se
stem dies back at the end of each growing season. Each spring a peremating bud
produces a long stem with a whorl of compound leaves at the top. Most plant parts are
pentanumerous, such as 5 Iedets per Ieafand 5 petals per ffower (Esau, 199 1). Seedlings
emerge with 3 simple leaves and resemble strawberry plants. By the second year, the
plants produce compound leaves, while in subsequent years they c m produce up to 4
compound leaves (although exceptions have been noted) (Proctor and Bailey, 1987). The
flowers are produced at the juncture of the leaf stems, atop a single peduncle. The
inflorescence is an umbel and florets are bisexual, small, white and closely packed. In
Ontario, flowers open in July, and about 30-35% of these flowers can be found bloorning
at one t h e (Fiebig, 1999). The plant pollen is self-compatible but some benefit can still be
attained through the introduction of commercial honeybees to promote cross-pollination
(McCarthy and Scott-Dupree, 1997). Fruit, known as berries, set soon after flowering
begins. Each berry contains at least 2 seeds, although variations do occur (Proctor and
Bailey, 1987). The root of ginseng consists of a rhizome associated with a fleshy tap root,
having smaller lateral branches (But et al., 1995).
1 -2. Crop Production:
In the wild, ginseng can be found growing in the understorey of deciduous forests in
temperate, eastern North Amenca (But et al., 1995). Extensive collection of the herb has
decimated wild ginseng populations. By the late 1 8 0 0 ' ~ ~ ginseng was being cultivated
under wooden lath shade in southern Ontario (Proctor and Bailey, 1987). However, it has
only been in the last two decades or so that ginseng has been cultivated to any great
extent. The recent, rapid increase in ginseng cultivation has resulted in a need for better
cropping systems (Fisher, 1 993).
In Ontario, stratified seed is planted during August/Septernber into fùmigated (e-g.
metam sodium) raised beds, at the rate of 90 to 1 10 pounds per acre (Fisher, 1995). A
straw mulch is applied to the top of the soi1 to help insulate against freezing temperatures.
Ontario ginseng plants emerge about mid-May and grow until approximately mid-October.
The application of pesticides (mainly fùngicides), fertilizer and irrigation are made
throughout the growing season (Schooley, 1997). Roots are harvested after 3 or 4 years of
production, usually with a modified potato harvester (Proctor and Bailey, 1987). Although
4-year-old roots are larger, the crop is commonly harvested &er 3 years of production to
avoid the chance of losing significant portions of the crop to disease, which have been
building up over the cropping years (Fisher, 1995). Root pnces in 1998 ranged from $20
to $3 0 per pound (Schooley, personal communication).
Seed is also an economically important portion of the plant (Proctor and Bailey, 1987).
Seed prices in 1998 ranged from $15 to $20 per pound for green seed and $25 to $30 per
pound for stratified seed (Schooley, persona1 communication). In Ontario, berries are
harvested by hand in late August. They are mechanically depulped and washed free of the
b i t tissue. Subsequently, they are soaked in a 3% solution of Formalin (a commercial
preparation of formaldehyde containhg 37% active ingredient) for 25 minutes. This seed is
mked with mortar sand, at a ratio of 1 :3, respectively, md buned in stratification boxes
that are located in the ground (Proctor and Louttit, 1995). The seed is stratïfïed in this
manner for about one year, until the following August or September. The stratified seed is
removed f?om the box, cleaned, soaked again in a weak Fornalin solution and planted in
raised beds for emergence the foilowing spring.
The seed is the most widely used means of propagation and should, therefore, be the
main focus for crop irnprovement. Fluctuations in the ground temperatures of outdoor
buried seed boxes may have adverse effects on seed maturation. Outdoor stratiiication
boxes are traditiondy shaded and mulched with the same shade structures and straw
mulch that can be found in the gardens (Proctor and Bailey, 1987). Proctor and Louttit
(1995) report an acceptable soi1 temperature range (at a depth of 10-50 cm) of roughly
-2°C to 15"C, based on a three year study. However, in unusually hot summers (as in
1995) elevated soi1 temperatures in underground seed boxes may have detrimental effects
on seed viability and physiology. In the hot, dry months of July and August, temperatures
in one monitored stratification box rose weli above an acceptable 15-1 8°C (Figure 4.1 .).
In this case, the grower reported large clumps of blackened, rotted seed when the
stratified seed was removed for planting that September. Further problems result in years
where annual rainfall accumulations are atypically high, since outdoor seed boxes may
become water logged, despite efforts to increase drainage potential at the tirne of
construction.
Through research focused on embryo growth and temperature (Lee et al., 1985; Stoltz
and Snyder, 1985; Proctor and Louttit, 1995), a controlled temperature protocol has been
developed for the stratification of ginseng seed. Proctor and Louttit (1995) have practiced
strat-g green seed above ground in a controiled temperature storage unit. The seed
was held at alternating optimal temperatures of 15°C and 3-5°C to mimic seasonai
stratification conditions in southern Ontario. It was found that emergence of the above
ground stratified seed was supenor to that of the outdoor, buned seed (Proctor, personai
communication). The ginseng research group at the Delhi Research F m (Agriculture
and Agri-Food Canada) report an average of 85% emergence after stratification at - 1°C
for 6 months, then 15°C for 6 months, then repeating this regime once (Reeleder, personal
communication). A few local growers have adopted tbis idea of controlied temperature
stratification, by using above-ground stratification in the summer term (April to
Septernber) of stratification. Partially-stratified seed is removed and placed in above-
ground storage at 1 5-20°C for the hot summer months. These growers have reported
heaithier stratified seed as a result of the above-ground stratification (Proctor, personal
communication).
1.3. Crop Diseases:
Diseases are a limiting factor in the production of healthy plants and seed (Putnam,
1989; Reeleder and B r m a l l , 1994). The intense cropping system (Le. close plant
spacing), environmental conditions, long cropping perio d and mechanicd cultural
practices make ginseng an easy target for pathogens. A number of fungal diseases are
prevalent in ginseng gardens (Table 4.1 .a.). These fùngi are found commonly on seed,
shoots and roots of cultivated ginseng. Many of the diseases that iafect ginseng are
soilbome, meaning that they have a direct route to the economically important part of the
plant, the root. One of the most discussed diseases in ginseng is disappearing root rot
caused by Cylindrocqon deshuctans (Zinssmeister) Scholten. One of the oldest
published reports of this disease dates back to 1935, in which the rapid colonization and
disappearance of the infected root is described (Hildebrand, 1935). The cultural control
practices mentioned in the study, crop rotation and rigid sanitation, are the same control
practices used in modem ginseng production.
Ginseng do es not grow well in soils previously cultivated with this same crop (Li,
1995). The source of the problem is cornplex. The main explmation for the problem is
that the populations of fungi build up si@cantly over years of production, thus
increasing the amount of primary inoculum available to the newly emerging seedlings.
Fumigation has been unsuccessful in reducing the fungal populations. Fungi belonging to
the genera Cylindrocaipon and Fzisarizim c m produce very thick-walled resting spores,
known as chlamydospores, that are believed to be resistant to chemicals used in soil
fumigation (Schooley, personal communication). Poor growth of asparagus roots in soil
previously cultivated with the same crop was thought to be due to a significant population
of F&um spp. in that soil (Schofield, 1991). Lake et al., (1993) have recently pursued
the theory that the replant problem may be due to the development of autotoxicity of the
asparagus roots. In ginseng, explanations such as allelo pat hy, pesticide residues, low
organic matter content, and low C:N ratios have been proposed (Oliver et al., 1990; Li,
1995)
1.4. Disease Control:
A smdl number of chernicals are available to help slow down the infection of the
common fungal pathogens of Ontario ginseng (Table 4.1 .b.). There are no chemicals
registered for two very devastating fùngi: Cylindrocarpon and Fzisarium. Pre-plant
fumigation is required to help reduce populations of pathogenic microbes and nematodes
in the soil. The problem with most pesticides is that they are often devastating to non-
target organisms, such as beneficial bacteria present in many soils, although these
populations do recover during the growing season. For these reasons, alternative methods
of disease control have been investigated.
A breeding program for naturd resistance to disease has been initiated by Jim Brande
at the Southem Crop Protection and Food Research Centre, AAFC in Delhi. He noticed
the potential for resistance selection with the discovery of variable "Land races" found in
cultivated ginseng (Bai et al., 1997). To date, germplasm collection has been ind der ta ken
dthough no breeding has taken place.
The recent trend towards Integrated Pest Management has initiated projects which
examine possible biological controls for ginseng pathogens. Joy and Parke (1994)
demonstrated the inhibition of Alternaria p a n a Whetzel spore germination by the
bacterium Bzrrkholderia cepacia, therefore presenting a possible biological control for
Alternaria leafblight. The research team has postulated that the bacterium produces
fungicidal secondary metabolites. Field trials are being investigated and the organism has
been most effective when used in conjunction with a mancozeb fungicide, although the
additional effect of B. cepacia was not shown to be significant. During a recent meeting
with Dr. Parke, she stated that further laboratory testing has revealed that the bacterium
has also shown activity against Botrytis cinerea Pers.:Fr. This fungus is responsible for
another significant disease in ginseng, Botrytis blight. As a drawback, it has been
remarked that tbis bacterium is toxic to humans.
Reeleder and Khan (1996) have initiated a biological approach to the control of
ginseng soil-borne diseases (i-e. root diseases). They noticed that composted plant waste
reduced disease populations in cïops grown in other parts of the world. Due to its hi&
organic and nutrient content, composted material is a nch and diverse source of microbial
populations (Quesnel et al., 1997). Some of these compost fùngi and bacteria are
cornpetitive with disease-causing microbes (Hoitink and Grebus, 1994). Reeleder's
research team has had some success with mushroom and pine bark compost. The
composts have been most effective against populations of CyIindrocmpon desh-uctans and
Phytophthora cactowm (Lebert & Cohn) Schrot., both of which cause root rot in ginseng
(Figure 4.1). One drawback of this type of control practice is the difficulty in obtaining
uniform lots of composted matenal, since compost lots diner markedly in their
composition due to differences in processing techniques.
1.5 Chernical Control of Seedbome Diseases:
There are no registered hngicides for the control of specific seed diseases of ginseng
in Canada, however, a formaldehyde soak is a cornmon grower practice. Schooley (1997)
recommends a 25 minute soak in a 3% solution of Formalin (a commercial preparation of
fomaldehyde containhg 37% active ingredient) as a pre-stratification and pre-seeding
treatment to help reduce seed surface populations of infectious microbes. There is
concern over the use of Formalin, both in the interest of the seeds' viabiiity and its impact
on the environment. The formaidehyde cm penetrate the seed coat and may be
destructive to the seeds' intemal tissues if the recommended exposure time and application
rate are exceeded. In addition, formaldehyde is carcinogenic to animais.
For these reasons, other chemicals are being investigated for their effectiveness in the
control of seed rots. These preparations can help prevent the spread of microbes tiom
infected seeds to neighboring healthy seeds, as weli as protecting germinating seedlings
fiom seed pathogens whiie it pushes through the soil. Brammaii (1997) and Schooley
(1997) had moderate success with a 10% solution of household bleach (hypochlorite,
5.25-6% active ingredient). Brammall(1997) suggested the use of general protectants
(e-g. Captan) combined with specific fùngicides (e.g. metalaxyl) for protection against a
select target group of known ginseng pathogens. Caution is advised when using general
fungicides on young, indehisced, green seed. Lee et al., (198 1) found that treating seed
with Captan before stratification delayed maturation and suppressed seed dehiscence,
possibly by reducing the activity of the h g i that nomally soften the endocarp. Ziezold et
al., (1998b) report some success with a pre-plant soak of stratified seed with benornyl. It
decreased the number of plants becoming diseased in the first 6 weeks after emergence
while having no deleterious effects on seedling emergence. Proctor and bis research team
have found some preliminary success with Topas 250E (propiconazole) for protecting the
susceptible gerrninating seedlings fiom fungal diseases (Proctor; personal communication).
1.6. Seed Dormancy:
A long after-ripening penod (18-22 months) is required for the germination of North
Amencan ginseng seed. The seeds are traditionally stratified in the imbibed state, at
temperatures which give the seed altemating penods of warm and cool. Two cool periods
(winters) are required to break the domancy. Though the embryos have reached their
maximum length by the beginning of the second winter, they still require a second chilhg
to promote germination (Stoltz and Snyder, 1985). The long after-ripening period may be
attributed to the low levels of endogenous growth homones, in the early stages of
stratification, or perhaps to the presence of growth inhibitors (Ren et al., 1997; Huang et
al., 1995).
The reduction of stratification, fYom 18-22 rnonths down to 8-9 months could have
significant benefits in ginseng production. The long, moist stratification period is very
conducive to the growth and infection of seedbome h g i (Agrios, 1988). A reduction in
stratification might therefore limit the amount of seed infection by disease. Traditional
stratification methods force growers to plant seed at the time of seed harvest and mot
harvest (Proctor and Bailey, 1987). Reducing the stratification to a fa11 and winter season
codd produce seed that is ready to plant in the spring time (Hovius, 1996), thus reducing
the fail workload.
1.7 Seed Grading;:
The inflorescence of ginseng plants is called a simple umbel. This type of
innorescence, which is characteristic of its f d y , produces flowers starting at the Lower
rows of fi orets, progressing upwards to the top. This results in the growth and
development of the lowermost h i t (seeds) before the uppennost ones. Most ginseng
growers harvest the bemes at one date, rneaning berries (and seed) will be collected at
various stages of development. Previous studies have shown that berries of the lowemost
positions on the innorescence produce the largest seed (Lee et al., 1987). Research on
other umbelliferous crops has shown that seed size has an effect on emergence a n d
seedling weight (Gray and Steckel, l983a; Gray and Steckel 1983b; Thomas, 1996). It is
possible that ginseng seed of different sizes may afEect plant performance in the garden.
1.8. Seed Patholo.gy:
The average emergence rate of ginseng in Ontario is roughly 65% in the fkst
seedling year (Proctor, persona1 communication). Some of the seed may be immature due
to insufficient development on the inflorescence. Seedling diseases such as pre-emergent
and post-emergent damping-off can account for some of this loss in emergence. However,
a significant proportion of emergence failure rnay be attributed to seedborne diseases
(Ziezold, 1997). It is estimated that seed rots and seedborne diseases are responsible for
the majority of losses in germination and subsequent visible emergence failure @rammd,
1 997; Schooley, personai communication; Ziezold et al., l998a). Many of the fungi
isolated from diseased ginseng seed are among the same fungi that can be isolated fiom
the cultivated plant. An understanding of these seedborne diseases and their origin (Le.
through developing seed on parent plant) could help to develop control strategies for such
pathogens.
1 -9. Objectives:
The objectives of this research were to investigate i) methods which decrease the
stratification period required to break seed domancy, ii) the effect of seed size on
emergence and plant performance and iii) the fungi which are present in flowers and
developing seed of ginseng.
-CHAPTER 2-
THE EFFECTS OF GIBBERELLINS, MATURATION DRYING AND A
PRE-PLANT HYDRATION ON THE G E m A T T O N OF GINSENG SEED.
2.1. Introduction
There are many challenges in ginseng production. The production cycle is long, about
3 or 4 years kom emergence to harvest. In addition, the seed exhibits a long dormancy
period of about 18-22 months. Dormancy is described as the condition of a seeci when it
fails to germinate because of intemal conditions, even though environmental conditions
are suitable (Salisbury and Ross, 1992; Rock and Quatrano, 1995). This dormancy penod
is necessary for embryo development and overall seed maturation (Proctor and Louttit,
1995). During dormancy, metabolic activities such as respiration and protein synthesis
occur, but the embryonic axis does not emerge (Bewley and Black, 1994).
The domancy of ginseng seed is complex. Along with many other herbaceous
perennial seed, it requires a chilling period in order to release the seed f?om its dormancy.
Lee et al., (1 983) and Stoltz and Snyder (1985) found embryos of newly harvested seed
measured 0.4-OSmm, and embryos of mature seed measured 5-6 mm. Traditionally, two
winter seasons are included in its seed stratification period. The most rapid period of
embryo growth occurs during the second fall period (about 1 3 to 1 5 months) (Stoltz and
Snyder, 1985). When ginseng h i t abscises fiom the parent plant, the embryo of the seed
is considered immature because it will not germinate, even when excised fiom the
enclosing tissues (Lee et al., 1983; Hovius, 1996; Ren et al., 1997). This type of
dormancy is known as primary dormancy. The seed of ginseng also exhibits what is
known as secondary domancy, since it d l not germinate before a second period of
chilling, even though the seeds are hydrated and the embryos are mature (Bewley and
Black, 1996). The second cold period is needed to break this endogenous type of
dormancy (Stoltz and Snyder, 1985).
However, this tengthy stratification period can lead to problems with seed viability.
The cool-warm-CO 01 temperatures of the air can lead to extreme temperature fluctuations
in the seed box (Chapter 4, Figure 4.2), (Proctor and Louttit, unpublished). Temperatures
approaching and exceeding 20" may have detrimental effects on embryo viability and the
warm-cool moist conditions under which the seed is stratifÏed favour the growth of many
seed rotting pathogens (Agios, 1988; Brammall, 1997; Reeleder, 1994). The seed is
traditionally planted in late summer/early fall, just before the busy season of root harvest.
Since ginseng seed exhibits characteristics of a recalcitrant seed, the hot and dry
conditions at planting c m be detrimental to the seed's germination viability.
Plant hormones are often used to cause changes in germination, growth and
morphology of many crop species. The classical definition of a plant hormone is as an
organic substance that is synthesized in one tissue in small quantities and translocated to
another, where it exhibits its effect (Salisbury and Ross, 1992). A discrepancy exists in
this def i t ion since some hormones (e.g. ethylene) promote their effects in the same
tissue or cell where they were synthesized (Rock and Quatrano, 1995). There are 5 major
groups of plant hormones: Abscisic acid, auxins, cytokinins, ethylene and gibberellins
(Kende and Zeevaart, 1997). All of these hormones play a role in or may innuence seed
germination (Bewley, 1997a; Hilhorst, 1995 ; Khan and S amimy, 1 9 82).
Growth regulators such as gibberellic acid and cytokinins are associated with the
enhancement of germination (Hilhorst and Karsseq 1992). They are known, dong with
ethylene, to belong to a group called germination "promoters" (Khan and Samimy, 1982).
By 1997, 112 GAs had been isolated and identined fiom biological organisms (plants,
kngi etc.) @samatsu et al., 1997). Over 40 gibberellins have been isolated from seeds
and these GAs are more abundant and structurally diverse than those found in shoot tissue
(Sponsel 1995). In general, levels of GA in ripe seeds are generally low when compared
to immature seeds (Hilhorst and Karssen, 1992). This could suggest either that the
sensitivity to GA increases as the seed matures, meaning lower levels of the hormone are
needed to illicit a response or that GAs play more of a role in promoting germination than
overcoming dormancy (Hihorst and Karssen, 1992; Bewley, 1997a).
Ren et al., (1997) found that endogenous levels of GAs increase over time in
stratikation, and that exogenous application of this hormone stimulates increased leveIs
of endogenous IA4 and zeatin. Gibberellins stimulate the secretion of a range of
hydrolytic enzymes, a major one being a-amylase (Hïlhorst and Karssen, 1992; Jacobsen
et al., 1995). Bewley (199%) has shown that GAs also ehcit the production of another
important enzyme, endo-P-mannanase in the seed of some vegetable crops. Endo-P-
mannanase is responsible for the degradation and weakening of endosperm tissue. The
presence of endo-P-mannanase in ginseng seed was investigated and is discussed in
Chapter 5. Tian et al., (1992) analyzed Oriental ginseng seed for its response to GA and
found that the hormone increased respiration rate within the first few weeks of
stratification. Collectively, hydrolytic enzymes are responsible for the mobilization of
stored endosperm reserves which provide the growing embryo and seedling with a nutrient
~ P P ~ Y .
Supported theories and research on the biosynthesis and effects of cytokinins are much
more equivocal than those for GAs (Horgaq 1984). Although most current studies on
cytokinins include the elucidation of their synthesis and metabolism, little progress has
been made on their action (Davies, 1995). Cytokinin is present in actively dividing cells,
and, therefore, it may be involved in this function (Davies, 1995). Cytokinins (especidy
zeatin) have been found in the endosperm of many developing seeds, and even in the
suspensor of the embryo (Davies, 1995). Pinfield and Stobart (1972) were able to detect
two distinct phases of seedling development of Acer in response to GA and kinetin
application. They found that elongation of the radicle was enhanced by 5x1 O" M kinetin
and that elongation and unfolding of cotyledons was promoted by 5x10~ M GA3. Ren et
al., (1997) found endogenous levels of zeatin concentrations in embryos of American
ginseng seed to follow the pattern of embryo growth (low-high-low). Therefore, Ren et
al., (1997) hypothesized that zeatin may be responsible for ce11 division of the embryo.
This study was based on previous research testing the accelerated germination of North
American ginseng using hormones and controlied temperature, a method dweloped by
Hovius (1 996). Through the use of controlled temperatures and growth regulators (G&),
she was able to reduce seed dormancy £kom 18-22 months to 8-9 months (Figure 2.1).
The resulting seed was termed "spring seed", since it is planted in May instead of the
traditionai fdl planting. An added benefit to the grower is the shifting of work load to the
spring, away £kom the busy fa11 season. Germination and emergence were expected to
increase because of a shorter exposure t h e to the disease-causing stratification conditions
and through the planthg of seed under cooler, more moist spring conditions. Although
some of the germination promothg treatments of this three year study were successful, the
best final emergence (47%) was not commercialiy acceptable. The best emergence was
accomplished by the application of 100 pprn of GA3 at t h e 1 in the spring seed
production system (Figure 2.1 .).
Other research groups have investigated the accelerated stratification of both North
American ginseng and Oriental ginseng (P. ginseng). Ren et al., (1996) found that 80
ppm GA3 and 80 ppm GA3 with 25 ppm ABA accelerated the germination of American
ginseng seed, after 222 days of strafication. Li et al., (1992) found some success using
controlled temperatures to produce Amencan ginseng seeds capable of germination after
only 8 months of stratification. Li et al., (1991) found that controlled temperatures and
GA hastened germination of Oriental ginseng. Chen et al., (1984) reported some success
with the use of GA (200 ppm), kinetin (20 ppm) and ethylene (500 ppm) plus a water soak
to stimulate the germination of Oriental ginseng seeds, d e r only 4 months of warm-cool
stratification. Lee et al., (1983) found GA increased the rate of Oriental ginseng seed
dehiscence.
In this study, additional procedures have been undertaken in an attempt to improve the
emergence rate of spring seed (Table 2.2). The best treatment fiom the Hovius (1 996)
trials, 100 ppm GA3 at M e 1, has formed the basis of treatments in this study. In addition
to this GA3 treatment, more accelerated seed maturation treatments were investigated.
Second and third applications (100 ppm) of GA3 were tested to determine whether
supplementing the level of endogenous GA in the seed
seeds fiorn their dormant state. G&+7 was introduced
16
tissue could release more of the
as an alternative to GA3 and was
applied as a 100 ppm soak at time 1. Since the cost of a 200 ml solution of G&+, is
significantly higher than that of GA3 the use of GA4+7 was lunited to one treatment.
Probert and Brierly (1989) found that a Iunited period of slow drying is effective in
inducing the germination of f?esh seeds of Zirrmia, especiaiiy in conjunction with the use
of dormancy breakhg factors such as GA. Famant et al. (1985) showed that mangrove
seed germination improved in response to desiccation treatments. Preliminary studies on
desiccation tolerance of ginseng seed seem to indicate trends of hproved germination
after 5 hours of dryhg at 23+/- 3 U ~ at 80% relative humidity (laboratory conditions),
(Hovius, 1996). Very gradual maturation d M g was introduced as a germination
stimulant at time 2 for some treatments.
The use of seed priming soaks as weil as priming agents have long been used as pre-
plant treatments to increasa the germination rates in many horticultural crops. The use of
priming agents can improve the earliness and synchronicity of germination of many
vegetable seeds by accelerating imbibition of dry sreds, especially when planted in the
cold, wet soils of spring (Ali et al., 1990). However, since ginseng seed is stratified under
moist conditions at hi& moisture content, prirning agent solutions, which usually increase
seed imbibition to 85%, may actually reduce the water content in seeds. This would result
in the induction of an additional dormancy period. Therefore, water was chosen as the
pre-plant soakhydration treatment (time 3).
The objective of this study was to determine which treatments can shorten ginseng
seed dormancy whiie still demonstrating an acceptable emergence percentage (i-e. one
greater than the 47% as previously obtained by Hovius (1996)). Treatment effects on
germination were measured by recording peak emergence, final embryo length (to
characterize seed matunty) and yield component data such as shoot weights, shoot
lengths, and most ùnportantly, root dry weights.
2.2. Materials and Methods
1996:
Freshly-harvested seed was obtained h m a local grower in September of 1995. It
should be noted that the seed was Later reported to have sat in storage with the h i t tissue
intact an unknown number of days before seed was effectively depulped. The seed was
mixed with water and ailowed to settle, to remove "floaters". The seed was graded using
commercial sized sieves, removing signifïcantly smaller and larger seed fiom the majonty.
To reduce variability within the seed lot, seed of the middle grades (between 5.95 mm and
5.56 mm or between 15/64 inches and 14/64 inches) was used for experimentation.
The seed required for the field and greenhouse trials and those for developmental
measurements cm be calculated as follows:
13 treatments x 4 replications x 300 seedslplot (for field plots) = 15,600
+ 13 treatrnents x 4 replications x 10 seedslpot (for greenhouse plots) = 520
t 13 treatments x 4 re~lications x 10 seedslre~ x 3 measurement dates = 1,560
= 17,680 plus 25% (for floaters, diseased) = 22,100 seeds in total.
AH seeds were stored in labeled, 2 L white, plastic containers for controlied
temperature stratification. The seeds were mked with local mortar sand at the
commercial ratio of 1 :3. The percent moisture of the sand was 12- 1 5%. Water was
periodically added to ensure that the seeds did not dry out and the containers werc
covered loosely with plastic bags to slow down moisture loss from the media. Total
weights of the sand, seed and contaiuer were recorded to maintain moisture content
throughout stratification.
Treatments of alternating warm-cool temperatures with the applications of gibberellins
andor cytokinins were given according to Table 2.1. The growth hormones were applied
at up to 3 times during the stratification period (Figure 2.1 .). Fresh GA3 and BA solutions
were prepared at each treatment tirne. The seeds were soaked for 8 hours at room
temperature, removed nom the solution, not rinsed and placed directly back into the
container of sand or germinated (as at T h e 3). At tirne 1, the fieshly-harvested seeds
were stratified at 1 5 ' ~ for 3 to 4 months. At Time 2 the seeds were moved to 3 ' ~ for
another 3 to 4 months. At t h e 3, seeds were taken out of storage and planted out in the
field and the greenhouse. Embryo length and seed coat crack measurements were taken at
al1 three times during the stratification to monitor development (Figure 2.4.). The
methods for these measurements were undertaken according to the description provided
by Proctor and Louttit (1 995).
Three hundred seeds from each replication were used to seed each 1 metre bed plot.
The beds were located at the ginseng research gardens at the Agriculutre Canada
Research Station in Delhi, ON (Figure 2.6.b.). The experiment was laid out in a
Randomized Complete Block Design, as in Figure 2.2. Each plot included a 1 metre
length of bed row. The seeds were hand sown to a depth of 3 to 4 cm, into raised beds
with 10 rows per bed (Figure 2.6.a.). Inter-row spacing was roughly 2.5 cm. The seeding
took place during the first week of May. M e r seeding, the remaining soi1 was raked over
the seeds and the beds were mulched with oat straw to a depth of 5 to 7 cm. These field
plots were maintained in the same manner as is suggested for other commercial ginseng
gardens, in terms of fertiliiation, fumigation, disease control and irrigation (Schooley,
1997).
Seeds were also sown in the Greenhouse Research faciiities in the Plant Agriculture
Department of the Universisr of Guelph, Guelph, ON (Figure 2.6.c.). The seeds were
sown by hand into 2 L white, plastic containers of Pro- to a depth of 2-3 cm. The
containers were placed on raised benches that were covered with the same polyethylene
shade matenal that is used in the field. The containers were arranged in a Completely
Randornized Design. The media was kept moist by hand watering as needed. The
temperatures in the greenhouse were kept at 21 3 ' ~ during the day and 16 ' 3 ' ~ at
night. It should be noted that seed for field and greenhouse trials were not floated pnor to
planting .
Field and greenhouse plots were used to monitor seedling emergence and growth.
Emergence (when the shoots emerged f?om the soil) was measured oves time in the
greenhouse. Peak emergence was chosen as the final emergence measurement for ail
analysis in this experiment, since new seedlings were lost to disease and environmental
stress (in the greenhouse). M e r about 40 days in the greenhouse and at the end of the
growing season (before senescence) in the field, a random selection of 6 to 10 seedlings
per treatment per plot were sampled randomly. These samples were used to give yield
component data to the experimental results. Al1 root material was gently rinsed in warm
tap water to dislodge soiVmedia particles. They were laid out on paper towels to d w for
2 minutes on each side, before measurements were taken. Yield component data collected
include: leaf area (LA); stem length (STL); root shoulder diameter (RSHLD); fresh shoot
weight (FSW); dry shoot weight (DSW); fiesh root weight 0; dry root weight
@RW); dry root to shoot ratio (DRWODSW). Only the best treatments fkom the 1996
trials were selected for yield component data andysis (Table 2.5.e.).
1997:
Freshly-harvested seed was obtained from a local grower in September of 1996 and
handled similarly as the seed for 1996 trials (above). The previous trials of spring seed
have shown that an application of GA plus BA did not give greater embryo lengths,
emergence or yields than those with GA alone- Therefore, it was reasoned that BA does
not play an important role in the promotion of ginseng seed germination and 1997 trials
did not include BA applications in the treatments. Treatments of aitemating warm-cool
temperatures with the applications of gibberellin (GA3,GA4+7) were administered,
according to Table 2.2. The growth hormone was applied at 3 specific times dunng the
stratification period. Fresh GA3 andlor GA417 solutions were prepared at each treatment
time. The GA3 was added directly to water since it dissolves easily. The GA4+7 had to be
combined with a small amount of ethanoi (95%) first, before it would combine with the
solution, but the ethanol was unlikely to influence the seed @ e r k et al. , 1994). The
solutions were aerated (air was bubbled through the solution via plastic tube) to help
eliminate the detrimental effects of anaerobic conditions. The seeds were soaked for 8
hours at room temperature, removed fiom the solution, without ~ s i n g , and placed
directly back into the container of sand or germinated (as at Tirne 3). Due to its sigdicant
expense, Ghc7 was used only once in this experiment (treatment 2 ).
A treatment c d e d "maturation drying" was carried out at time 2, on treatments 7, 9
and 10. The slight drop in moisture content (about 9 to 10% by weight) was achieved by
placing the seed in a glass desiccator held at OC to reduce microbial growth and
contamination. Seeds were washed fiee of their stratification sand, blotted dry and
weighed (initial fiesh weight). The seeds were then placed in single layers on wire screen
holders which were aacked, ailowing for maximum airtlow, in the desiccator. The
positions of these four holders were shuffled around ùiside the desiccator. A saturated
solution of KCL was poured into the bottom 4 cm of the desiccator and kept uniform
through the use of a magnetic stir bar and magnetic stir plate, placed underneath the
desiccator. The KCL solution regulated the intemal relative humidity of the container to
about 86%, ailowing for slow, non-destructive drying of the sensitive seeds. It took
approximately 48 hours to dry the seeds d o m to the desired 90 to 91% of their initiai
fresh weight. Seed weights were sampled to estimate overall seed moisture loss.
Moisture loss was calculated by the loss of mass after incubation:
desiccated seed (d xl O0 = % of fresh weight
initial fiesh weight (g)
M e r weight measurements were taken, seed were soaked in the appropriate solution:
GA3 or distilled, deionized water. The desiccated seeds were soaked for 8 hours to dlow
re-hydration.
At time 1, the fieshly-harvested seeds were stratined at 1 5 ' ~ for 3 to 4 months. At
tirne 2 the seeds were moved to 3 ' ~ for another 3 to 4 months. At t h e 3, seeds were
taken out of storage and planted in the field and in the greenhouse. Embryo length and
seed coat crack measurements were taken at all three times during stratification to monitor
development (Figure 2.4.). The methods for these measurements were undertaken
according to the description provided by Proctor and Louttit (1995).
Seedling, emergence and samphg for yield component data analysis were performed
similarly to 1996 trials (above).
For the purpose of this study, ccgermination" refers to radicle protrusion frorn the
endospedtesta measuring at least 2 mm. Seedling "emergence" refers to the appearance
of the stem and leaves fiom the surface of the soiVmedia in which it is grown. In this
study, emergence was the response variable that was measured to indicate successfùl
germination.
Experimental Design and Aridvsis:
To optimize the precision of the field experiment at the Delhi Pest Research Station, a
Randomized Complete Block Design was chosen with 4 blocks and 10 (1997) or 13
(1 996) treatments (Figures 2.1. and 2.2.). The assigmnent of the treatments to the
experimental units (1 rn plots) was randomized according to the Random Permutations of
Table 15.7 of Cochran and Cox (I992), (Tables 2.3. and 2.4.). The blocking of land has
grouped the experimental units into more homogenous sections, thus reducing the
experimental error variance (Keuhl, 1994). Each block represents 10 or 13 metres of
neighboring bed plots. Two metre buffer zones, containing no experimentation, were
added to the ends of each bed. Since ginseng is cultivated under shade, these buffer stxips
help to alleviate the problems of the foliar buming that occur at the edges of a garden,
codounding results.
A second set of triais were performed in the University of Guelph research
greenhouses. The 10 to 13 spnng seed treatments were replicated 10 times in 2 L plastic
pots. Each of the 10 pots were planted with 10 seeds. The 100 or 130 pots were set out
in a completely randomized design and were watered and monitored for 30 to 40 days
(until emergence and growth leveled off).
The General Linear Models procedure of the SAS statisticai package (SAS Institute,
Cary, N.C.) was used to andyze the experimental data. A one-way ANOVA was used to
analyze and summarize results. Normality was assessed using the Residual Anaiysis
portion of the SAS output,(e.g. stem and leafplots). In addition to these, a Shapiro-Wilk
W-test (Applied Statistics, 44547-541, 1995) was conducted to give fiirther co&dence in
the assessment. AU "non normal" data was successfuily normalized by eliminating extreme
outliers, as described by the SAS output. Transformations were not needed to give
nomality in most instances and were unsuccessful when attempted in a few cases. Due to
the sigdicant number of treatments, pairwise comparisons of the treatments means were
made using Tukey's Honestly Significaot Difference. Unlike other types of multiple
comparisons, Tukey's (HSD) tests are more stringent in detecting true differences
between means since this procedure helps prevent Type I errors (Kuehl, 1994). Due to
the small number of treatment comparisons, yield cornponent data fiom 2-year-old plants
of 1996 spring seed was analyzed according to the Least Significant DiEerence method.
The level of sigdcance used for all tests was ~ 6 . 0 ~ ~ udess othenvise stated. Standard
errors were cdculated using the individual variance for each treatment group. In the
tables, means followed by the sarne letter were not siDonificantly different.
Seed crack was measured as an indication of embryo maturity. To Save time, any
crack width measuring greater than 0.6 mm was considered a successfùlly cracked seed.
In this study, crack data follows a binomial distribution pattern (Le. number of
observations on the number of successes in independent trials). Logit transformations and
residual analysis are commonly used to improve the normality of binomial data sets
( K e a , 1994). Therefore, a Iogit transformation was applied to crack results. The
Generai Models procedure (PROC GENM0D)of the SAS statistical package (SAS
Institute, Cary, N.C.) was used to analyze the experimentai data. The parameter estimates
and standard errors in the output were used to compute confidence intervals.
2.3, Results and Discussion
Embrvo L e n d and Seed Crack:
Embryo length and seed coat crack increased linearly with t h e (data not shown),
s idar to other results of this type are reported on ginseng (Lee et al., 1983; Stoltz and
Snyder, 1985; JO et al., 1988; Proctor and Louttit, 1995).
a. 1996
The two treatments with a 100 pprn GA3 plus 50 pprn BA application at time 1 (HO)
and a 100 pprn GA3 application at tirne 1 (#2) gave the greatest mean embryo lengths at
time 3 (Table 2.5.a.). According to studies by Proctor and Louttit (1995), the embryo
Iengths of the top two treatments 5.38 and 5.00 mm, indicate physiological maturity of
this seed. These two treatments were significantly longer than the eight Iowest ranking
treatments but not tiom the control (#1). Application of 100 pprn GA3 plus 100 pprn BA
at time 1 (#12) gave the third greatest mean embryo length but was only signi6cantly
greater than the two lowest ranking treatments. Other treatments were not statistically
difEerent fiom each other.
The top three treatrnents giving the highest percentage of cracked seeds at time 3 were
those treatments giving 100 ppm GA3 plus 50 ppm BA at time 1 (#IO), 100 pprn GA; at
time 1 (#2) and 100 ppm GA3 plus 100 ppm BA at time 1 (#12) (Table 2.5.b). These
treatrnents were not signincant £tom each other or the control treatment (#1). Treatment
10 gave signincantly more cracked seeds than the 7 Iowest rankùig treatments.
Treatments 2 and 12 had signincantly more cracked seeds than the 6 lowest raking
treatments. Other treatments were not statistically different fkom each other.
b- 1997
The top three treatments resulting in the longest embvos at time 3 included a G&+7
application at time 1 (#2), a GA3 application at time 1 with a maturation drying treatment
at t h e 2 followed by a water soak at time 3 (#9), and the treatment giving GA3 at time 1
followed by maturation drying at time 2 ($7) (Table 2.6.a.). The embryo lengths (EL)
attained by the top three treatments, 5.36 to 4.19 indicate possible physiologicai maturity
of the seed (Proctor and Louttit, 1995). These three treatments were statistically similar
to each other, and greater than the control treatment (#1). GA4+, was the best treatment
(#2), gïving statistically longer ELs than the five lowest ranking treatments. Al1 of the
treatments with maturation drying made it into the top five and were ail statistically longer
than the control.
The three treatments giving the highest percentage of cracked seed at time 3 were
those which applied GA+, at time 1 (#2), GA3 at time 1 plus maturation dIying at time 2
(#7), and GA3 at time 1 with maturation drying at time 2 followed by a water soak at time
3 (#9) (Table 2.6.b.). There were no significant dserences between the top seven
treatrnents, although they were al1 higher than the control treatment.
The original best treatment (treatment #3) fiom 1996 and previous studies (Hovius,
1 996) was outperfoxmed by the GA 4+7 and the GA3 with maturation drying treatments, in
embryo length and seed crack data from 1997.
Embryo Iength can be used to predict embryo maturïty and seed gerrninabiiity and, in
addition, is a relatively simple measurement to obtain (Proctor and Louttit, 1995).
Ren et al., (1997) found that the GA3 treatments stimulated embryo growth and embryo
weight. They aiso show thzt GA3 treatment at the beginning of stratification produced an
average embryo length of 6.5 mm, about 1 .O mm longer than the control mean (5.4 mm).
Final emerpence:
a. 1996
Greenhouse emergence of sprhg seed in 1996 was poor and gave uncharacteristic
treatment effects (Table 2.5.c.). To illustrate, the control treatment (#1) gave the highest
percent germination, at 3.17 plants per pot. In addition, the control gave significantly
better emergence when compared with treatments (#2, #IO) that were previously amongst
the best in studies by Hovius (1996). Since the control treatment does not include the use
of any germination promoting treatments, the results of this greenhouse trial are considered
inconclusive.
Field emergence of spring seed 1996 was aiso poor, but ranking of treatment
performance gave results similar to those of Hovius (1996). The three best treatments
consisted of a 100 ppm GA3 plus 100 ppm BA application at time 1 (#12), a 100 ppm GA;
plus 50 pprn BA application at tirne 1 (#IO), and a 100 ppm GA3 application at time 1 (#2).
These three treatrnents gave si@cantly higher mean emergence than most other
treatments. The substandard seed quality and subsequently poor seedling emergence was
due, in part, by poor handling practices of the grower. For example, failing to de-pulp seed
soon after harvest.
b. 1997
Greenhouse emergence triais of spring seed in 1997 produced poor stands of emerged
seedlings with few statistical differences (Table 2.6.c.). Treatment ranking did not follow
the order displayed by embryo length and seed coat crack data, which is unusual since these
parameters are commonly used to rnonitor embryo development and mahiity of ginseng
seed (Proctor and Louttit, 1995). The three highest treatments included those treatments
which applied GA3 at aiI three times (#6), applied GA3 at all three times plus maturation
drying at time 2 followed by a water soak at time 3 (HO), and applied G&+7 at t h e 1 (#2).
The top six treatments were statisticaily similar. Treatment #6 was significantly greater
than the k u r lowest ranking treatments. Only treatments #6, #10 and #2 were statistically
greater than the control(#1).
In addition to greenhouse trials, field trials of spring seed in 1997 gave unacceptable
rates of final emergence, with limited statistical significance between most treatments
(Table 2.6.h.). The treatments which gave the best mean emergence were those that
applied at time 1 (#2), GA3 at time 1 and time 3 (#5), and GA3 at alI three times
during stratification (#6). Treatment #2 gave significantly higher seedling emergence than
ail other treatments. The next seven ranking treatments (#5, 6,4, 10, 9, 7 and 3) were
similar and sigrüficantly greater than the control (#l) and treatment #8. Al1 treatments
displayed better final emergence thm the control.
These results show some correlation with other ginseng research. Chen et al. (1984)
reported some success with the use of GA kinetin and ethylene plus a water soak to
stimulate emergence in 42.5 to 46% of Oriental ginseng seeds, after only 4 months of
wann-cool stratification. Ren et a[.,(1997) found that GA3 treatments produced
germination percentages of 85% and GA3 plus ABA treatments produced germination
percentages of 77.3%, but only GA3 treatment was significantly higher than the control
(74%). These results were atypical of American ginseng seed under these conditions.
Yield component data:
a. 1996
Greenhouse trials of 1996 spring seed produced limited arnounts of seedling plant
material due to poor emergence. Leafarea material was crisp, brown and stunted due to
the hot air temperature and subsequent media temperaturss. There was not enough plant
material remaining at the end of the greenhouse trial to permit analysis of yield component
data.
Yield component data were taken fiom 2-year-old plants in the second year of
production (1997) (Table 2.5.e.). Of the three best treatments (# 2, 10 and 12) compared,
there were no significant differences for mean values of leaf area, stem length, root
shoulder diameter, fresh and dry shoot weight, fresh and dry root weight and root to shoot
ratio. This seems to agree with the findings of Hovius (1996), who stated that most
treatment afEects were Lost by the second year of plant production.
b. 1997
Greenhouse trials of spring seed in 1997 produced poor stands of seedlings and
subsequently gave inferior yields for analysis (Tables 2.6.d through 2.6.g.). The reduced
quality made it diEcult for analysis to detect dBerences in the response variables. For all
variables measured, there were no statistical dBerences between the germination
promoting treatments (#2 through # 10). However, al1 treatments were significantly
greater than the control.
Field trials of the 1997 spring seed treated seed gave lower emergence rates than those
found in the ginseng industry (approximately 60-75%) (F'roctor, personal communication).
However, the plants that did grow were acceptable in t e m s of appearance and yield
(Tables 2.6.i. through 2.6.1.). This translates into some significant differences in plant
performance between treatments.
Those treatments involving the application of GA3 at times 1 and 3 (#5), GA3 at time 1
plus maturation drying (#7) and GA3 at ail three times (#6) gave the largest leafareas.
Aithough these treatments were similar, they were signiûcantly greater than the 5 lowest
ranking treatments (including the control).
The G&+7 treatment (#2), the treatment which applied GA3 at al1 three times (#6) and
an combination of al1 treatments, except G&+7 (#IO) produced seedlings with the longest
stems. These treatments were similar but they were sigrilficantly longer than the three
lowest ranking treatrnents, including the control.
Root shoulder diameter (KSKLD) displayed uncharacteristic treatment eEects. The
control treatment (#1) renilted in the largest measurernents but it was only significantly
larger than the treatment giving GA3 at times 1 and 3 (#5). There were no other
sigrilficant differences between rshld for any other treatments.
The original "best" treatment fkom Hovius (1 W6), application of GA3 at time 1 (#3),
produced seedlings with the largest dry root to shoot ratio (drwodsw). Treatment #3,
however, was only significantly greater than the lowest three ranking treatments (#6, 5
and 2). The GA4+7 treatment displayed the lowest DRWODSW, but was statistically
similar to most other higher ranking treatments. Treatments giving all possible
applications (#IO) and treatments which gave GA3 at time 1 plus a pre-plant water soak
exhibited the next largest DRWODSW, but these were oniy sigrilficantly larger than the
lowest ranking treatment (#2).
There were oniy a few difTerences detected in eesh and dry shoot weight response.
The Ci&+, treatrnent (#2) produced seedlings with the largest FSW and DSW. The next
two largest values of FSW came fkom the treatment dispensing ai i possible applications
( H O ) and the treatment applying GA3 at time 1 with maturation drying plus a pre-plant
water soak (#9). Only treatment #2 was found to have sipnificance over another
treatment, specifically, GA3 at time 1 plus a pre-plant water soak (#8). The second and
third ranking treatments for dsw were those which applied GA3 at t h e 1 plus maturation
drying (#7) and which applied GA3 at a l l 3 t h e s (#6). Most treatments were found to
have sigdcantly larger DSWs than treatment #a.
DEerences in fkesh and dry root weight (FRW, DRW) response gave lirnited statistical
distinctions. Treatments that applied GA3 at times 1 and 2 (#4) and those which applied
GA3 at time 1 plus maturation drying (#7) produced plants with the largest FRW, but
these were only significantly greater than treatment #8, GA3 at time 1 plus a pre-plant
water soak. The GA3 at tirne 1 treatment (#3) and the treatrnent applying GA3 at time 1
plus maturation drying (#7) produced seedlings with the Iargest DRWs. However, these
two treatments were significantly larger than treatment #8 only.
General discussion:
GA3 has been used widely to encourage growth and development of plant tissues for
research and horticultural purposes. GA3 was the £irst commercially available form (Khan
and SamUny, 1982) and can be produced fairly economicaüy, naking it the GA of choice.
Other GAs exist with even greater potential of biological activity (e-g. GAI, GA4) (Davies,
1995; Kende and Zeevaart, 1997). An increase in emergence response was evident in this
study through the use of G&+7 in treatment #2. However, the high cost and limited
availability of this GA formulation has confined its use to highly specific research activities
(biochemical and biosynthesis studies), thereby making it an unsuitable aid in the large-
scale germination of crop seeds.
The application of GA3 at time 1 proved more effective than those applications at time
2 and 3 in the 1996 trials. Ren et al., (1997) found exogenous GA3 application more
effective if applied at the beginning of stratification. However, they did find some
promotion of GA when applied in the middle of an 8 month accelerated stratification.
Treatments with maturation drying (#7, 9 and 10) were among those which gave the
largest embryo lengths, cracked seed percentages, greenhouse germination and yield
component data. Although they were in the top three, they were often only significantly
greater than the control. Ln studies of orthodox seed, drying triggers developmentai
changes (Bewley and Black, 1994). Evans et al., (1 975) theorized that this drying
increases tissue sensitivity to endogenous levels of hormones. Nautiyal and Purohit
(1985) suggested that the increase in germination response may be due to reduced
sensitivity of the embryo to endogenous levels of abscisic acid. Farrant et a1.(1985)
showed that mangrove seeds (a recalcitrant species) responded to gradua1 desiccation
treatments with the initiation of cell division and tissue growth. Probert and Brierly
(1989) found that a small reduction in moisture content of Zizania, a recalcitrant species,
increased the proportion of fresh seeds able to gerrninate when exposed to G&+7. They
exposed fkeshly harvested seeds to 1 SOC and 15% relative humidity for 3 days followed by
a soak in 50 pM solution of G&+7. In this study, a GA3 application at thne 1 (#7) before
maturation drying ( t h e 2) was more effective than a GA3 application following maturation
dmg, both at time 2 (# 10).
The effects of GA are quite similar to the effects of moist chilling and light for inducing
germination in dormant seeds (Khan and Samimy, 1982). Carpita et al., (1 979)
demonstrated that both GA and red light decreased the water potential of the embryo of
lemice seed by a sirnilar quantity. This was the result of the accumulation of solutes
(namely, sugars) that are the produced fkom the hydrolysis of storage materials in the
endosperm, resulting in the rapid inflow of water into the endosperm tissue of some of the
seed. Seeds with severely swollen endosperm were encountered during measurements of
embryo length and coat crack taken throughout this study (Figure 2.5.A.). This hyper
swoiien tissue became translucent, obviously losing its structural integrity and leaving it
vulnerable to attack by microbes present among the seed (Figure 2 - 5 8 . ) (Lee et al., 198 1;
Zhang et al,, 1989; Ziezold et al., 1998a). It is estimated that at least some of the seed rot
may have been due to this event.
Emergence and growth of seedliogs in the greenhouse were limited. The media was
exposed to warmer air temperatures and increasing sub strate temperatures compared with
those found in the field. Root growth has be s h o w to be limited by exposure to extrerne
soi1 temperatures (Tindall et al., 1990; Farias-Larios et al., 1994). Limitations in root
growth and development hinder their ability to coilect and store water and nutrients. This
places great restrictions on growth and development of the overd plant, often leading to
early senescence and sometimes death (Salisbury and Ross, 1992). There are no published
reports of commercial ginseng grown successfùlly in the greenhouse.
High seed rot percentages were a sigdicant problem in the production of spring seed.
Hovius (1996) cited seed rot as one of the major obstacles in tissue culture of ginseng
seed. While monitoring embryo length and seed crack at time 3, seed rot rates in excess
of 40% and 50% were noted in this study. Seed soirrce may play a role in the
unsystematic quality of seed that can be purchased fiom ginseng growers in Southem
Ontario (Figure 4.1.). It is likely that poor seed handling practices, such as excessive
amounts of time between h i t harvest and depulping of seed, could increase the
introduction of pathogens in andor on the seed. Poor seed quality for 1996 trials may be
attributed to the extreme heat during its development on the parent plant in 1995
(McElhone, 1995). Ziezold et al., (1998b) had some success with the use of benomyl as a
tùngicidal seed treatment. The use of chernical protectants and the periodic removal of
diseased seed from storage could help reduce the amount of inoculum available for
infection. Hovius (1 996) found no differences between numbers of fungi recovered from
spring seeded versus traditionaily fali planted ginseng seed.
The rapid switch from warm (15 '~ ) to cool ( 3 ' ~ ) temperature stratification may put
stress on the seed, resulting in loss in viability or the onset of imposed domancy. Other
successfÙl germination trials with ginseng seed used a larger range of temperatures and
stages for stratification @en et al., 1996; Ren et al., 1997).
Previous research articles about phytohormone activity established that the
relationship between the application of a hormone and plant response is not directly
related. For exarnple, in aquatic plants, ethylene accumulations in submerged tissues can
be detected prior to the stem elongation response (Salisbury and Ross, 1992). Musgrave
et al., (1972), have shown that the ethylene acts by increasing the tissue's responsiveness
to GA and that it is the endogenous GA that promotes (cell) the stem elongation response.
In addition, Hoffmann-Benning and Kende (1992) found ethylene reduced the amount of
endogenous AB& which inadvertently increased the sensitivity to GA (since B A is its
known antagonist) (Mayer and Poljakoff-Mayber, 1 989; Hilhorst and Karssen, 1 992).
Furthermore, GA likely has more than one site of action (Khan and Samimy, 1982; Kende
and Zeevaart, 1 997).
In that case, it seems possible that ethylene may help to promote sensitivity to GA in
other tissues as well, such as Living endosperm of ginseng seed. Indeed, it rnay take quite
an assortment of germination promoters to successfully hasten the maturation of ginseng
seed. Khan and Samimy (1982) suggested the use of GA (GA4+7), a cytokinin and
ethylene to work synergistically together as advocates of germination. Mahiration drying
showed some promise in the results of the 1997 spring seed trials. Therefore, it may aiso
be advisable to include a physical treatment such as this, with future treatrnents for spring
seed .
2.4. Surnmary
GAs are highly effective at improving the germination of many seed species. Their
presence signals the mobilization of storage reserves and the decrease in mechanical
strength imposed on the embryo (Hilhorst and Karssen, 1992). Their role in the breaking
of both primary and secondary dormancy in ginseng seed expresses the necessity for this
hormone to be included in germination promoting treatments. Cytokinins are also
effective germination promoters, however, the added benefit of BA with GA as a
treatment (1996 trials) did not si&cantly improve germination compared to GA alone.
In 1 996, the treatments which applied GA3 and GA3 plus BA had the longest embryos,
coat crack percentages and germination percentages, however, only field emergence was
significantly greater than the control. The G&+, treatment, which produced the most
field emergence, ranked arnong the top performing treatments for yield parameters such as
dry root weight and dry root to shoot ratio. The best treatment (GA, at time 1) from
previous spring seed trials (Hovius, 1996), produced some of the longest embryos and
coat crack percentages. Those treatments with multiple applications of GA3 gave some of
the highest emergence and yield component variables, and were ofien significmtly greater
than the control ody. Treatments involving maturation drying (at time 2) led to increased
embryo lengths, improved seed cracking, higher germination and better yields in many
cases. However, statistical significance was not high. Pre-plant hydration did not have
any promoting effects on the response variables measured.
The statistical differences between treatments were dficult to discern as plant
performance was poor in both the 1996 and 1997 trials of spring seed. Seed lots may
have varied in quaiity depending upon seed handling practices by the grower and the
populations of seedbome pathogens. Through changes in osmotic potential, GA caused
the rapid intlux of water into treated tissues, facilitating the degeneration and infection of
the swollen endosperrn tissue. Premature sprouting led to s i m c a n t losses and rots
during the latter portion of the stratification penod and the exposed radicle is subject to
breakage during seed processing and planting procedures. Perhaps future spring seed
treatments should avoid the use of largest seed grades, since they are more likely to sprout
before planting (chapter #3).
Field trials and greenhouse trials of spring seed gave unacceptable emergence rates
(33% at best) than those found in the industry. With thorough attention to seed quahty
control, maturation drying and the addition of promoters (such as ethylene), perhaps a
better protocol c m be developed for spring seed.
1 Table 2.1. Treatments for 1996 Spring Seed. Temperature = stratification temperature. '~p~lication Ume corresponds to "Time # Application" in Figure 2.1. 3 GA3 = Gibberellic Acid. 4~~ = Beqladenine.
Treatment 1 ' ~ e m ~ e r a t u r e 1 2 Application 1 GA^ @pm) 1 4 E 3 ~ ( P P ~ )
(reproduced f?om Hovius, 1996)
Table 2.3. Assignment of field plot numbers to treatments and replications of Spring Seed experiments in 1996.
Treatment 1 1 1 1 2 2 2 2 3 3 3 3 4 4 4 4 5 5 5 5 6 6 6 6 7 7
Rep 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2
Plot 1 2 3 4 5 6 7 8 9 10 11 12 23 14 15 16 17 18 19 20 21 22 23 24 25 26
Treatment 7 7 8 8 8
Rep 3 4 1 2 3
Plot 27 28 29 30 3 1 32 33 3 4 35 36 37 38 39 -
40 41 42 43 44 45 46 47 48 49 50 51 52
8 9 9 9 9 10 10 10 10 11 11 11 11 12 12 12 12 13 13 13 23
4 I 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4
Table 2.4. Assigoment of field plot numbers to treatments and replications of Spring Seed experiments in 1997.
Treatment 1 Z 1 1 2 2 2 2 3 3 3 3 4 4 4 4 5 5 5 5
Rep 1 2 3 4 1 2 3
Plo t 1 2 3 4 5 6 7
Treatment 6 6 6 6 7 7 7
4 1 2 3 4 1 2 3
7 8 8 8 8 9 9 9
8 9 10 11 12 13 14 15
Rep 1 2 3 4 1 2 3
Plot 21 22 23 24 25 26 27
4 1 2 3 4 I
2 - 3
4 1 2 3 4
28 33 34 35 36 41 42 43
4 1 2 3 4
44 45 46 47 48
16 17
9 10
18 19 20
10 10 10
TREATMENT REP PLOT 13 4 52 6 4 24 7 4 28 12 4 48 8 4 32 3 4 12 9 4 36 1 4 4
1 1 4 44 4 4 16 10 4 40 5 4 20 2 4 8 1 1 3 43 2 3 7 10 3 39 4 3 15 1 3 3
12 3 47 8 3 3 1 5 3 19 6 3 23 9 3 35 7 3 27 3 3 I l 13 3 5 1
TREATMENT REP PLOT
Figure 2.2. Field plot layout of Spnng Seed 1996 Experimentd Design.
TREATMENT REP PLOT
( North-middle)
TREATMENT REP PLOT
3 2 10 9 2 42 1 2 2 7 2 26 4 2 14 10 2 46 8 2 3 4 5 2 18 2 2 6 6 2 22 5 I 17 10 I 45 1 I 1 6 1 21 9 I 41 2 1 5 3 I 9 8 T 33 7 I 25 4 1 13
(South-front)
Figure 2.3. Field plot layout of Spring Seed 1997 Experimental Design.
Figure 2.4. Green seed (lefi) and mature, stratified seed (right) of North American ginseng. The embryo from the green seed measures less than lmm where as the mature embryo measures 5 to 6rnm on average.
Figure 2.5. A depicts swelling of ginseng endospem due to change in osmotic potential caused by GA application to green seed at Time L. Note that the peripheral endosperm tissue turns transparent, likely due to cell rupture upon swelling. B depicts subsequent degeneration and rot of swollen endosperm.
Figure 2.6. Spring seeding of North Amencan ginseng seed. Seeding was accomplished by hand (A) in boîh field plots at Delhi, ON (B) and in greenhouse trials (C).
Table 2.5 .a. Mean embryo lengths (mm) and standard errors of the means for 1996 S p ~ g seed at Tirne #3.
Treatment 1 2 3 4 5
7 8
1 Significance (letter) denotes significant dBerences (Pc0.05) according to the Tukey Honestly Significant Difference ( 2 ~ ~ ~ ) method of testing.
Mean 3 -70 5.00 3 -27 3 -39 3 -29
1 I 12
Table 2.5.b. Probabiliity estimates of successful seed crack (crack > -6mm) with codidence intervals of those probabilities according to logit transformation, for 1996 Spring seed at Tirne #3.
3.21 3 -23
Standard Error 1 '~i~nificance ('HSD)
2.64 4.52
-43 2 -3 87 -457 -47 1 -425
.466 -435
Treatment 1 2
abcd a
bcd bcd bcd
bcd bcd
-424 .400
5 6 7 8 9 10
cd abc
Mean -63 8 -690
.622
.574 -607 -630 -574 -697
'LCI .598 -653
2~~~
-676 -725
.582 -533 .566 .590 .53 3 -66 1
11 1 .5 66
1 LCI = lower confidence interval 'UCI = upper confidence interval
-66 1 -6 15 -646 -668 .6 15 .73 1
.525 -63 O -533
12 13
-607 -704 -6 15
-668 .574
Table 2.5.c. Mean final emergence (out of 10) and standard errors of the means for 1996 Spring seed in the greenhouse.
/ Treatment 1 Mean 1 Standard Error 1 L~ienificance ('HSD)
2 3 4 5
-
6 7 8
1 SignLfïcance (letter) denotes significant differences (P<O.05) according to the Tukey Honestly Significant Differeoce ( 2 ~ ~ ~ ) method of testing.
10 1 1 22 13
Table 2.5.d. Mean final emergence (out of 300) and standard errors of the means for 1996 Spring seed in the field (Delhi).
-
bc abc
C
a
-- - - - - - - -
0-33 .2 1 1
--
2-33 0.33 1.67
1 .O0 O* O0 2-83
9 1 0.33 -- - -
O. 17 2.17 1.17 2.33
.5 16 0.00 -856
- - - - - - - -
.760
.2 1 1 -667
Treatment 1 2 3 4 5 6 7 8 9
10 T 1 12 13
1
-
ab bc abc
-2 1 1 - -
-167 -543 -3 07 -667
bc -
bc abc abc ab
Signincance (letter) denotes significant daerences (P<0.05) according to the Tukey Honestly Significant Difference (*HSD) method of testing.
Mean 43 .O0 66.75 22.25 54.75 44.3 3 33.00 43.75 26.25 48.25 75.75 24.75 86.67 20.25
Standard Error 4.71 4-66 0.48 1.80 7.3 3 3 -72 2.2 1 2.8 1 2.29 5.57 2.14 9.1 1 5.14
L~ignificance ('HSD) def bc g cd de efg de -
fs de ab
a
f4
Table 2.5.e. Mean finai yield component parameters and standard errors of the means for 1996 Spring two-year-olds in 1997, from the field (Delhi).
1 Parameter 1 Leaf Area (cm2)
Dry Shoot Weight (g) Fresh Root Weight (g)
Dry Root Weight (g) Dry Root:Dw Shoot
1 SE = Standard Error c
Treatment 2 Treatment 10 Treatment Mean 'SE 'LSD Mean SE LSD Mean SE 101.98 6.04 a 97.09 4.49 a 95.37 4.66
12.94 0.44 a 12.23 0.37 a 12.72 0.36 12.29 0.39 a 12.13 0.34 a 11.77 0.31
f the mean. 2 Significance (letter) denotes significant clifferences (P<O.05) according to the Least Significant DEerence (LSD) method of testing.
Table 2.6. a. Mean embryo lengths (mm) and standard errors of the means for 1997 S pring seed at Time #3.
- -- -
Treatment 1 Mean Standard Error 1 1 Significance ( 2 ~ ~ ~ )
- - I
.. - - 1 I
1 Sia&cance (letter) denotes significant dEerences (Pc0.05) according to the Tuke
LSD
~ o n e s t l ~ Significant Difference ( 2 ~ ~ ~ ) method of testing.
Table 2.6.b. Probability estimates of successful seed crack (crack > -6mm) with confidence intervals of those probabilities according to logit transformation, for 1997 Spring seed at Time #3.
1 Treatment 1 Mean 'LCI 1 L ~ C ~
1 LCI = lower confidence interval 'UCI = upper confidence internai
Table 2.6.c. Mean final emergence (out of 10) and standard errors of the means for 1997 Spring seed in the greenhouse.
Treatment I 2 3 4
- . .
6 1 2.00 7 0.90 8 1 1.10
Honestly S i w c a n t Difference ( 2 ~ ~ ~ ) rnethod of testing.
Mean 0.00 1.50 0.70 0.90
5 1 0.80 0.365 0.233 0.233
9 20
Standard Error 0.000 0.373 0.300 0.3 24
a abc abc
1 Significance (letter) denotes sigrilficant differences (Pc0.05) according to the Tukey
0.300 0.267
-- -
0.70 1.60
1 Significance CBSD) C
ab bc abc
0.200
bc ab
bc
Table 2.6.d. Mean final leaf area and stem length with standard errors of the means for 1997 Spring seed in the greenhouse.
Stem Length (cm)
'S.E. I 2 Significance Treatment
1 2
3 4 5 6 7 8 9 1 O
1
y Significant Difference ()HSD) method of testing.
S.E. = Standard Error of the mean 2~ignificance (letter) denotes significant differences ( W O . 05) according to the Tukey Honest
Mean
0.00 7.80 6-85 7.37 8.02 7.40 6.70 6.42 6.50 6.93
Lenf Arer (cm2) Mean
0.00 15.06 10.1 1 13.23 12.20 12.17 12.74 10.31 11.19 13.07
1 S.E.
0.000 3 .O7 1.81 2.29 1.73 2.25 2.48 1.67 1.61 2.20
Significance 2
( 3 ~ ~ ~ )
b a a a a a a a a a
Table 2.6.e. Mean final root shoulder diameter and stem length and root to shoot ratio with standard errors of the means for 1997 Spnng seed in the greenhouse.
Root Shoulder Diameter (mm) Mean 1 S.E. 2 Significance Mean
PHSD)
Root to Shoot Ratio 1 S.E. 2~ignificrnce
P H S D ~
;d Error of the mean letter) denotes significant differences (P<0.05) according to the Tukey Honestly Significant Difference ( 3 ~ ~ ~ ) method
I S.E. = Stand 2 Significance
of testing.
Table 2.6.f. Mean final fresh shoot weight and dry shoot weight with standard errors of the rneans for 1997 Spring seed in the greenhouse.
Dry Shoot Weight (g) 'S.E. I 2~ignifîcance Treatmen t
'S.E. = Standard Error of the mean '~i~nificance (letter) denotes significant differences (P<O.OS) according to the Tukey Honestly Significant Difference (%sD) method of testing.
Fresh Shoot Weight (g) Mean 1 'S.E. 1 2~ignificance Mean
Table 2.6.h. Mean h a 1 emergence (out of 300) and standard enors of the means for 1997 Spring seed in the field (Delhi).
3 1 39.25 1 3 -77 1 bcd
Treatment 1 Mean Standard Error 1 1 Significance ('HSD)
1 Signincance (letter) denotes significant daerences (P<0.05) according to the Tukey Honestly Signincant DBerence (*HSD) method of testing.
1
4 5 6 7 8 9
10
-
46.00 50.75 50.75 40.50 3 1-50 41 .O0 44.00
- - - - 7
473 6 4.03 0.95 4.1 1 3.95 3 .O3 5.2 1
bc b b
bcd cd bcd bcd
Table 2.6.i. Mean final leaf area and stem length with standard errors of the means for 1997 Spring seed in the field (Delhi).
--
1 S.E. = Standard Error of the mean '~ignificance (letter) denotes significant differences (P<O.OS) according to the Tukey Honestly Significant Difference ( 3 ~ ~ ~ ) method of testing.
Treatment
1 2 3 4
Lesf Area (cmz)
5 6 7 8 9 10
Stem Length (cm) Sigiiifïcance 2
( 3 ~ ~ ~ )
cd bc
bcd cd
Mean
13.53 14.22 14.08 13.30
Mean
12.28 13.88 13 .52 12.52
16.21 1510 16,16 12.09 13.73 14.3 1
1 S.E.
0.42 0.47 0.46 0.55
1 S.E.
0.32 0.33 0.27 0.30
0.45 0.44 O. 55 0.70 0.42 0.49
2 Significance ( 3 ~ ~ ~ )
c a
abc bc
a bc b d ------- cd bc
13.31 14.1 1 13.27 12.34 13.30 13.67
0.3 1 0.32 O. 29 0.37 0.26 0.33
abc a
abc c
abc ab
Table 2.6.k. Mean final fiesh shoot weight and dry shoot weight with standard errors of the means for 1997 Spring seed in the field (Delhi).
o f testing.
Treatmcnt
1 2 3 4 5 6 7 8 9 1 O
'S.E. = Standard Error of the rnean 2~ignificance (letter) denotes significant differences (P<0.05) according to the Tukey Honestly Significant Difference ( 3 ~ ~ ~ ) method
Fresh Shoot Weight (g) Dry Shoot Weight (a) 2~ignifica nce
SHSD) ab a ab ab ab ab ab b ab ab
Mean
O. 1 06 0.1 16 O. 1 03 O. 1 09 O, 1 04 0.1 10 O. 113 0.087 0.098 0,101
Mean
0.490 0.510 0.491 0.479 0.464 0.485 0.473 0,436 0.496 0.501
S.E. 1
0.014 0.01 8 0.015 0.016 0.015 0.014 0.0 14 0,02 1 0.014 0.01 5
1 S.E.
0,003 0,004 O. 003 0.005 0.003 O, 003 0.004 0.005 0.003 O, 003
2 Signifieance (=HSD)
ab a ab ab ab ab ab c
bc abc
Table 2.6.1. Mean final Fresh root weight and dry root weight with standard errors of the means for 1997 Spring seed in the field (Del hi).
Fresh Root Weight (g) .. Treatment Mean Signifîcancc Mean 1 S.E. 2
I~HSD) IP<0.10)
1 S.E. = Standard Error of the rnean '~i~nificance (letter) denotes significant differences (P<0.05) according to the Tukey Hones of testing.
Dry Root Weight (g) 1 S.E. 2 Signifïcance
SHSD) (P<0.05) 0.010 ab
/ Significant Difference ('HSD) method
- C W T E R 3-
THIE EFFECT OF SEED SIZE ON EMBRYO LENGTH,
EMERGENCE AND PLANT YIELD OF GINSENG.
3.1. Introduction
The idorescence type that occurs on ginseng plants is called a simple umbel. This
type of inflorescence is characteristic of its family, Araliaceae (Woodland, 199 1). The
blooming pattern progresses inward and upward dong the umbel towards the youngest
central floret. This type of blooming pattern is termed indeterminate (Esau, 1977). The
result is the growth and development of the lowermost h i t (seeds) before the uppermost
ones. Once ripe, these lower bemes will begin to abscise before the topmost bemes.
Most ginseng f m e r s perform a "once-ove? harvest of the bemes, usually late August in
Ontario. This results in a varied mixture of seed in tenns of size and development. Lee et
al., (1987) found that seed size increased with lower (primary) positions on the umbel. A
number of studies with other umbelliferous crops have found that the heavier seeds were
produced by primary umbels in compound umbel type plants (Gray and Steckel, 1983;
Thomas, 1996). Most ginseng seed fiom any given lot fa11 into 4 main sizes. These four
sizes are outlined in Table 3.1.
Seed size has been used successfully as a screening method in determining seed
quality in a wide variety of species. Even a modest increase in seed size c m have
significant consequences on seedling establishment and performance (Fenner, 1992). It
is questioned whether an increase in plant vigor and a better overall plant stand would
result £tom the use of the larger ginseng seed. Hovius (1996) noticed that with ginseng
seed, the larger seed sizes contained the largest embryos and that the embryo length can
be used (with some confidence) to predict the matunty of that seed. One would expect
the larger seed to exhibit faster and better emergence as well as an increase in seedling
size and vigor.
Thomas (1996) studied the relationships between the umbel position and seed yield,
germination and seedling development for some other umbeliferous species. He noted
that the rate of emergence decreased with the use of smaller seed found on the higher
umbel order positions. Early maturing, large seeds fiom f i t order urnbels gemiinated
more rapidly and produced larger more vigorous seedlings. However, the earliest-
produced, larger seed generaily exhibited a lower germination percentage under optimal
conditions.
The objective of this study was to determine whether a diEerence in emergence,
seedling vigor and overall plant stand would result fiom the use of the various sizes of
seed. The success of each size treatment was measured in terms of emergence rate, final
emergence (i.e. established plant stand), final embryo length (to characterize seed
maturity) and yield component data (shoot weights, shoot lengths, root shoulder
diameters, and most importantly, root dry weights).
3.2. Materials and Methods
1996: spring seed
Freshly-harvested seed was obtained nom a Local grower in September of 1995. It
should be noted that the seed was later reported to have sat in storage with the fruit
tissue intact an unknown number of days before the seed was effectively depulped. The
seed was rnixed with water and allowed to settle, to remove "floaters". It was then
graded using commercial sized sieves and divided ioto 4 sizes (Figure 3.2.A) These four
sizes were used as the treatments and are outlined in Table 3.1.
The seed required for the field and greenhouse trials and those for developmental
measurements can be caicufated as follows:
4 treatments x 4 replications x 300 seeddplot (for field plots) = 4,800
+ 4 treatments x 4 replications x 10 seeddpot (for greenhouse plots) = 160
+ 4 treatrnents x 4 replications x 10 seeds/rep x 3 rneasurement dates = 480
= 5,440 plus 25% (for floaters, diseased) = 6,800 seeds in total.
AU seeds were stored in 2 L white, plastic containers for controlled temperature
stratification. The seeds were stored in containers, labeled according to their treatrnent
number. They were mixed with local mortar sand at the commercial ratio of 1 :3. The
percent moisture of the sand used was 1245%. The total weight of the sand and seed
and pail was recorded and water was added periodically to ensure that the seeds did not
dry out. The containers were covered loosely with plastic bags to slow down moisture
loss of the media.
The seed was treated with alternating warm-cool temperatures and the application of
gibberellin at time 1, according to Table 2.1. These practices were camied out according
to the materials and methods of chapter 2. Embryo length and seed coat crack
measurements were taken at al1 three times d u ~ g the stratification to monitor
development of spring seed (see Table 2. l), but only at pre-plant for stratined seed. The
methods for these measurements were undertaken according to the description provided
by Proctor and Louttit (1995). At time 3, seeds were taken out of storage and planted
out in the field aad the greenhouse.
Three hundred seeds &om each replication were used to seed each 1 metre bed plot.
The beds were located at the ginseng research gardens at the Agriculture Canada
Research Station in Delhi, ON (Figure 3.3 .). The experiment was laid out in a
Randomized Cornplete Block Design, as in Figure 3.1. Each plot included a 1 metre
length of bed row. The seeds were hand sown to a depth of 3 to 4 cm, into raised beds
with 10 rows per bed. Inter-row spacing was roughly 2.5 cm. The seeding took place
during the fist week of May. Mer seeding, the remaining soil was raked over the seeds
and the beds were mulched with oat straw to a depth of 5 to 7 cm. These field plots were
maintained in the same manner as is suggested for other commercial ginseng gardens, in
terms of fertilization, fumigation, disease control and imgation (Schooley, 1997).
Seeds were also sown in the Greenhouse Research facilities in the Plant Agriculture
Department of the University of Guelph, Guelph, ON. The seeds were sown by hand into
2 L white, plastic containers of ProMx. The containers were placed on raised benches
that were covered with the same polyethylene shade material that is used in the field. The
containers were arranged in a Completely Randomized Design. The media was kept
rnoist by hand watering as needed. The temperatures in the greenhouse were kept at 21 '
3 ' ~ during the day and 16 ' 3 ' ~ at night time.' It should be noted that seed for field and
greenhouse trials were not floated prier to planting.
To indicate successful germination, emergence (when the shoots emerged fiom the
soil) was rneasured penodically until it leveled ofE Peak emergence was chosen as the
final emergence measurement for analysis in this experiment. After about 40 days in the
greenhouse and at the end of the growing season (before senescence) in the field, a
selection of 6 to 10 seedlings per treatment per plot were randomly chosen for sub-
sampling. Along with shoots, these samples were used to obtain yield component data.
Al1 root material was gently rinsed in warm tap water to dislodge soiUmedia particles.
They were laid out on paper towels to dry, for 2 minutes on each side, before
rneasurements were taken. Yield component data collected include: leaf area (la); stem
length (stl); root shoulder diameter (rshld); fiesh shoot weight (fsw); dry shoot weight
(dsw); fiesh root weight (fnv); dry root weight ( d m ) ; dry root to shoot ratio (dnvodsw).
1997:
Seed size trials for 1997 used i) spring seed and ii) stratified seed. The same
treatment for 1996 spring seed (#3) was used in 1997, the seed was planted in May. The
spring seed was treated and stratified according to the above mentioned methods. The
only difference between sized spring seed 96 and sized spring seed 97 is the seed size and
the fact that the 97 seed was aerated (mixed) by hand biweekly throughout stratification.
StratZed seed was obtained from a local grower in 1996 and seed was graded according
to the above mentioned methods. The stratified seed was hand seeded in October of
1996 for germination in 1997.
Expenmental Design:
To optimize the precision of the field experiment at the Delhi Pest Research Station, a
Randomized Complete Block Design was chosen with 4 blocks and 4 treatments (Figures
3.1 .). The assignment of the treatrnents to the experimental units (1 m plots) was
randomized according to the Random Permutations of Table 15.7 of Cochran and Cox
(1992), (Table 3.2.). The blocking of land has grouped the experimental units into more
homogenous sections, thus reducing the experimental error variance (Keuhl, 1994). Each
block represents 4 of neighboring bed plots. A two metre buffer zone was added to the
ends of each beci where there will be no experimentd plots. Since ginseng is cultivated
under shade, these buffer strips help to alleviate the problems of the foliar burning (the
confounding of results) that one can find at the edges of a garden.
A second set of trials were perfomed in the University of Guelph research
greenhouses. The 4 seed size treatments were replicated 10 h e s in 2 L plastic pots.
Each of the 10 pots will be planted with 10 seeds. The 40 pots were set out in a
completely randomized design and were watered and monitored for 30 to 40 days (until
emergence and growth leveled O@.
The General Linear Models procedure of the SAS statistical package (SAS hstitute,
Cary, N.C.) was used to analyze the experimental data. A one-way ANOVA was used to
analyze and summarize results. Normality was assessed using the Residual Analysis
portion of the SAS output, (e-g. stem and leaf plots). In addition to these, a Shapiro-
Wik W-test (Applied Statistics, 44547-541, 1995) was conducted to give fùrther
confidence in the assessment. Al1 "non normal" data was successfùlly normalized by
eliminating one or two extreme outliers, as described by the SAS output.
Transformations were not successful and were not needed to give normality in most
instances. Treatrnents means were compared ushg the Least Significant Difference
(LSD) method of testing. The level of signincance used for al1 tests was ~ ' 0 . 0 5 , unless
otherwise stated. Standard errors were calculated ushg the individual variance for each
treatment group. Mean values in the Table columns followed by the same letter are not
significantly different .
Seed crack was measured as an indication of embryo maturity. To Save time, any
crack width measuring greater than 0.6 mm was considered a successfully cracked seed.
In this study, crack data foliows a binomial distribution pattern (Le. number of
observations on the number of successes in independent trials). Logit transformations
and residual analysis are commoniy used to improve the normality of binomial data sets
(Keuhi, 1994). Therefore, a logit transformation wiil be used for crack results. The
General Models procedure (PROC GENMOD) of the SAS statisticai package (SAS
Institute, Cary, N.C.) was used to anaiyze the experimental data. The parameter
estimates and standard errors in the output were used to compute co&dence intervals, by
back-transformation.
Though the treatments for sized s p ~ g seed in 1996 and 1997 were the same, a one-
way anova analysis showed that the year was very signincant (Pc 0.05) for the response
variables. Therefore the analysis of these two data sets was performed separately.
3.3. Results and Discussion
Embrvo Lengh and Coat Crack:
Embryo Length and seed coat crack increased linearly with tirne throughout the nine
month stratification penod. Other research groups have reported results on ginseng
similar to time 1 and 2 fiom this study (Lee e t al., 1983; Stoltz and Snyder, 1985; JO et
al-, 1988; Proctor and Louttit, 1995; Hovius, 1996). Therefore, data fiom time 1 and 2
have not been shown.
1996:
Final mean embryo lengths ranged fiom smallest to largest according to the size of the
sprhg seed fiorn which it was measured (Table 3.3 .a.). Extra large seed produced
embryos that were significantiy longer than those h m medium and small. The
proportion of seeds that were cracked at this time (about 70%) were not different
between treatment sizes (Table 3 -3 .b.).
1997:
The final mean embryo lengths of spring shed seed increased as seed size increased
(Table 3 -4.a.). Embryos fiom extra large seed were signiticantly larger than those for
s m d and medium, whereas, large seed embryos were larger than those f?om small seed
only. The final percentages of successfùl seed coat crack for small seed were significantiy
less than ali other treatments (Table 3.4.b.).
The final mean embryo lengths for stratified seed also increased as seed size increased
(Table 3 -5.a.). Distinct dif5erences were noticed among the treatments. Extra large seed
gave sigdcantly longer embryos than al1 other treatments. Embryos of large and
medium sized seed were greater than small seed embryos. The percentages of cracked
seed increased as the treatment size increased (Table 3 -5.b). However, there was no
dif5erence in cracking response between treatments.
Final Emergence:
1996:
Mean final emergence of spring seed was poor in the greenhouse (Table 3 . 3 ~ ) ~ with a
maximum of 1.5 plants emerging out of a possible 10. The extra large and large
treatments resulted in sigdicantly more plant emergence than either medium or small
sized seed. Emergence in the field gave better numbers, with a maxirnum of 141.5
seedlings out of 300 resulting from large seed (Table 3 -3 .de) (Figure 3.3 .). However,
these differences were not significant between the treatments.
Mean final emergence for spnng seed planted in 1996, was also measured in 1997,
when the seedlings reached two-years-old (Table 3 -3 .E). There were no signincant
differences between the number of emerged plants for those treatments. Ln addition, the
mean final ernergence for seedlings that emerged in the second year of production were
recorded (Table 3.3 g.). Again, there were no signifïcant differences between the
treatments.
1997:
Mean final emergence of sized spring seed was poor in the greenhouse trials (Table
3 -4.c.). To illustrate, a maximum of 2 out of 10 possible plants were produced fiom
extra large seed. Extra large seed produced significantly more emerged seedlings than
those fiom small seed. No other significant differences existed between treatments.
Emergence in the field trials was also lirnited (Table 3.4.e.), resulting in no significant
daerences.
Stratified seed produced acceptable mean final emergence in the greenhouse (Table
3.5 .c.). Extra large seed produced significantly lower numbers of emerged seedlings,
compared to dl other treatments. Field trials produced poor mean final emergence of
seedlings (Table 3.5.f ). Extra large seed gave the second lowest emergence, sig&cantly
lower than al1 but the small treatment. Large seed produced a signiticantly higher nurnber
of seedlings than small seed. The emergence of medium seed was signincantly greater
than the emergence of small and extra large seed.
Yield Com~onent Data:
1996:
Sized sprhg seed did not grow weil in the greenhouse, leaving inadequate amounts of
plant material for yield component data. In the field, sized spring seed produced
seedlings with varying yields (Table 3.3 .e.). Extra large seed produced plants with
greater leaf areas, root shoulder diameters, fresh and dry shoot weights, and dry root
weights than those seedlings fiom the srnall seed. Large seed gave larger leaf areas, stem
lengths and fiesh shoot weights than srnall sized seed. Medium sized seed gave plants
with larger leaf areas than those of small sized seed.
1997:
Sized spring seed gave seedlings yields that varied with seed size in the greenhouse
(Table 3.4.d.). Seedlings fkom extra large seed produced signincantly longer stems than
those for srnall seed, however, yields of extra large seed did not differ much f?om those
of other seed sizes. Large seed produced seedlings with significantly longer stems, dry
shoot weights, dry root weights and dry root to shoot ratios than medium and small sized
seed.
Field trials generated adequate plant yields of sized spring seed for analysis (Table
3.4.E) (Figure 3.2.B.). Extra large seed oflen produced the highest yields cornpared to
medium and small sized seed. Seedlings 5om large seed had significaotly larger shoot,
root and root to shoot ratios than those seedlings from medium a d o r srnall seed.
Medium seed produced significantly larger leaf areas and shoot weights than small seed.
The seedlings produced by stratified sized seed had limited root and shoot growth
(Tables 3 -5 .e. and 3.5.f ). By the end of their cultivatioq the leaves became crispy and
brown. For these reasons, yield data was Limited to root weights, which were often
uncharacteristic. Both fiesh and dry root yields nom extra large seed were significantiy
srnalier than aIl other treatrnents,
Field trials of stratified seed produced seedhgs of varying yields (Table 3.5 .g.) .
Extra large seed produced significantly larger seedlings than those of medium and small
seed, for most yield parameters. Large seed produced seedlings with signifïcantiy higher
yields than small, and sometbes medium sized seed. Yields of seedlings nom medium
sized seed were fiequently sigdicantly higher than those of smali sized seed.
General discussion:
Embryo length is a fkequently measured parameter in studies on ginseng seed (Lee et
al., 1983; Stoltz and Snyder, 1995; JO et al., 1988; Hovius, 1996). Hovius (1996) found
some correlation between embryo length, seed coat crack and germinability of North
knerican ginseng seed. According to this snidy, embryo length and seed crack increased
as seed size increased over tirne. Some seeds have sipificantly larger embryo lengths
than other seeds (Le. extra large seeds had sigoificantly longer ernbryo lengths than
medium and small seeds in 1996 spring seed). Quite ofteg the same trends were found
with final emergence rates (Le. extra large seeds produced significantly higher final
emergence than medium and smail seed, in 1996 spring seed). However, the reverse was
found for 1997 greenhouse and field emergence, with extra large stratified seed having
emergence percentages significantly Iower than those from other seed sizes. The
treatment effect of emergence was lost in the second year (1996 spring seed in 1997).
Similar results can be found in related crops having similar seed size variability (due to
umbel-type inflorescence and harvesting practices). Gray and S teckel (1 983 b) found the
measurement of varïabiiity in embryo length to be a usefùl indication of plant
performance. They noticed embryo iength increased (hearly) as seed weight increased.
They also noticed an inverse relationship between embryo length and seedling emergence
percentage. Thomas (1996) found that although emergence rate and seedling size were
sigdcantly larger, heavier parsley seeds were less viable and seedling emergence was
signincantly lower for these larger seeds. He hypothesized that pollinator populations
might be low at the beginning of flowering, leading to embryo-less seeds or poor embryo
quality.
Find emergence was poor in the greenhouse and in the field for both 1996 and 1997
spring and stratified seed. This resulted in limited statistical dserences between
treatment means. High seed rot percentages were a signincant problem in the production
of seed and seedlings in these trials. In addition, greenhouse trials produced seedlings
with poor yields, most noticeable as early shoot die back. A discussion of seed rot and
greenhouse production of ginseng is given in the general discussion of chapter 2.
Precocious germination has been seen in many ginseng seeds removed from
stratification containers in the fall, withùi the traditional planting system Proctor and
Louttit, 1995). Sprouting of seed before the end of the spring seed stratification was
noted during measurement of embryo length and coat crack for extra large and large
spring seed in 1997. It is possible that this size of seed is responsible for the precocious
germination. It is likely that some emergence losses are due to breakage of the radicle
from these precocious seeds during planting.
Extra large and large seed quite often gave the largest sked seedlings (yields).
However, these large seedlings sometimes had the lowest emergence (i-e. stratified sized
seed trials in 1997). Studies on other umbeuiferous species have shown that the
variability in plant and root weight was positively correlated to embryo size, but that
emergence of the Iarger seed was often reduced when compared to that of medium sized
seed (Gray and Steckel, 1983a; Thomas, 1996). Counts and Lee (1991) found a
signifiicant relationship between mean seed size and seedling size of wild rice. They also
noticed that the largest seed exhibited a greater period of domancy.
Curtis and McKersie (1984) found that larger le-e seeds gave faster seedling
growth and longer axial lengths than the smaiier seeds. The faster rate of seedling
elongation in the large seed was thought to be encouraged by a rapid increase in axis dry
weight and by the larger quantity of stored reserves in the larger seeds. In vitro
experiments indicated that lirnited axial growth in smaller seed is not due to limitations in
volume of nutrient reserves (endosperm), rather that the axes of the larger seed simply
have a greater ability to make use of the storage reserves and accumulate dry matter.
Therefore, the dserences in seedling growth may be a result of genetic dflerences or
physiological properties of the axis between the two seed sizes, and not the quantity of
endosperm itself.
Ginseng gardens lose about 66% of the original plant stand by the fourth year of
production, due to diseases, cornpetition and mechanical damage (Schooley, persona1
communication). The use of extra large seed may produce gardens with slightly larger
plants but plant populations may be significantly iower than gardens planted with large
and medium sized seed. This may result in the inefficient use of costly bed space and
sigdicant reductions in root yields (Proctor and Bailey, 1987). Seed size effects on the
piant yields for subsequent years of production may be lost over time. This should be
tested in future trials of older "seed size" gardens. In addition, the use of s m d sized seed
often produced seedlings with significantly srnalier ernbryo lengths, emergence and yields
across a l l trials. Therefore, it may be reasonable to grade seed when harvested and
elirninate any seeds of the extreme sizes from use in planting, in the hopes of improving
seedling emergence.
3.4. Sumrnary
Due to inflorescence type, ginseng seed develops in a non-symmetrical pattern. A
once-over harvest results in seed lots with great variability in size. The effect of seed size
on the emergence and plant yields of ginseng in the seedling year c m also be seen on other
umbelliferous crops.
The final mean embryo lengths of seed were larger as the seed size increased. The
embryo length of extra large and small seed were often significantly larger and smailer
(respectively) than other treatments. Stratified seed field trials of 1997 showed that extra
large and small seed fkequently produced significantly lower seedling emergence.
However, in greenhouse trials, emergence rates of extra large spring seed was
significantly higher than for medium and small seeds. These conflicting results make it
difficult to fonn conclusions about the effect of seed size on ginseng emergence.
Yields of seedluigs fiequently increased as seed size increased. Extra large seed often
produced seedlings with sigruficantly greater yields (e.g. dry root weights, leaf area) but
did not always produce the highest emergence percentages. The effect of plant size may
be lost in subsequent years of production, simila. to the loss in the eEect of spring seed
treatments (chapter 2). Even if the effect of seed size remained constant throughout the
3 or 4 production years, and if these extra large roots were considered more valuable
than srnailer roots, the reduced plant stands (populations) could result in signtficantly
Lower root yield per acre. Future trials of seed size should be conducted to evaluate these
proposals.
Table 3.1. Treatments for Seed Size experiments during 1996 and 1997.
1 Treatment 1 Size Designation 1 Seed Diameter (mm) 1 Commercial Grade
1 Medium I 4.8 - 5.2 i (12-13)
Large 1 5.2 - 5.6 1 (13-14)
Table 3 -2. Assignment of field plot numbers to treatments and replications of Seed Size
4
experiments in 1996 and 1997.
1 Commercial grade = size of hole (e.g. 1 1/64 inches) in commercial sorting screen.
X-Large
Treatment 1 1
5.6 - 6.0 (1 4- 1 5 )
- -
Replication 1 2
Plot 1 2
TREATMENT REPLICATION PLOT 4 4 16 2 4 8 1 4 4 3 4 12 4 3 15 3 3 11 1 3 3 2 3 7 3 2 10 2 2 6 1 2 2 4 2 14 2 1 5 4 1 13 3 1 9 1 1 1
Figure 3.1. Field plot layout of Seed Size 1996 and 1997 expenmental design, a Randomized Complete Block Design.
Figure 3.2. Representatives of the 1996-97 stratified seed size experirnents. A depicts the four seed sizes (letter) classified by diameter of seed coat. B depicts seedlings grown from sized seed (e.g. 11/64 to 12/64 inches) at the end of the first growing season.
Table 3 -3 .a. Mean embryo lengths (mm) and standard errors of the means for 1996 Shed Spring seed at Time #3.
Treatment Mean Standard Error '~ignificance ('LSD) 1 4-51 -3 96 b
- - - - - - 1 - 1 Significance (Ietter) denotes sibonificant dflerences (P<0.05) according to the Least S i e c a n t Dinerence ( 2 ~ ~ ~ ) method of testing.
Table 3 -3 .b. Probability esthates of successful seed crack (crack > -6m.m) with confidence intervals of those probabilities according to Iogit transformation, for 1996 Sized Spring seed at Time #3.
1 LCI = lower confidence interval 2~~~ = upper confidence interval
Treatment 1
Table 3 -3 .c. Mean final emergence (out of 10 plants) and standard errors of the means for 1996 Sized Spring seedlings in the greenhouse.
1 Treatment 1 Mean 1 Standard Error 1 '~ienificance ('LSD) 1
Probability -68
'~ignificance (Ietter) denotes significant ciifferences (Pc0.05) according to the Least Signincant DBerence ( 2 ~ ~ ~ ) method of testing.
Table 3.3.d. Mean final emergence (out of 300 plants) and standard errors of the means
'LCI -652
for 1996 Sized Spring seedlings from the field (Delhi).
2~~~
-7 12
1 Treatment 1 Mean Standard Error 1 '~i~nif icance ('LSD)
1 Si@cance (Ietter) denotes signifïcant differences (P<0.05) according to the Least Significant DEerence ('LSD) method of testing.
Table 3.3 .f. Mean final emergence (out of 300 plants) and standard errors of the means for 1996 Sized Sprhg two-year olds in 1997, fiom the field (Delhi).
Treatment 1 Mean 1 Standard Error 1 '~ ip i f i cance ( 2 ~ ~ ~ )
1 Significance (letter) denotes significant ciifferences (F'<O.OS) according to the Least Sigoificant DifGerence ( 2 ~ ~ ~ ) method of testing.
Table 3 -3 .g. Mean final emergence and standard errors of the means for 1996 Sized Spring second year seediings in 1997, from the field (Delhi).
Table 3.4.a. Mean embryo lengths (mm) and standard errors of the means for 1997 Sized
Treatment 1 2 3 4
Spring seed at Time #3.
1 Sibonificance (letter) denotes signincant differences (P<0 .OS) according to the Least Significant Difference ('LSD) method of testing.
Mean 76.00
1 4 I 5.2 1 1 .117 1 a 1 1 Signincance (letter) denotes significant differences (W0.05) according to the Least
Treatment 1
Significant D ifference ( 2 ~ ~ ~ ) method of testing.
Standard Error 17.607
'~ i~n i f i cance ('LSD) a
Mean 3 -43
a a
68.50 63.50
8.261 2-63 O
Standard Error -265
50.50
'~ i~n i f i cance ('LSD) c
5.3 15 1 a
Table 3 -4.b. Probability estimates of niccessful seed crack (crack > -6mm) with confidence intervals of those probabilities according to logit transformation, for 1997 Sized Spring seed at Time #3.
- - - -
1 LCI = lower confiidence in teha 'UCI = upper confidence interval
Treatment 1 2
Table 3.4.c. Mean £inal emergence (out of 10) and standard errors of the means for 1997 Sized Spring seedlings in the greenhouse.
Probability -668 -72 1
3 1 -71 1
1 Signincance (letter) denotes significant differences (P<0.05) according to the Least Signifcant Difference (*LSD) method of testing.
~LCI -648 .702
Treatment 1 2 3 4
J
W C 1 -688 -739
-692 -729
Mean 1 .O0 1.20 1.80 2.00
Standard Error -476 -327 -327 -3 65
'~ignificance ('LSD) . b ab ab a
Table 3.4.d. Mean final yield component parameters and standard errors of the means for 1997 Sized Spring seedlings, from the greenhouse.
' testing.
Parameter ~ e a f ~ r e a ( c m ~ )
Stem Length (cm) Root Shoulder (mm)
Fresh Shoot Weight (g)
Dry Shoot Weight (g) Fresh Root Weiglit (g) Dry Root Weight (g) DryRoot:DrySlioot
Table 3.4.e. Mean final emergence (out of 300) and standard errors of the means for 1997 Sized Spring seedlings from the field (Delhi).
1 SE = Standard Error of the mean. '~i~nificance (letter) deiiotes significant. differences (P<0.05) according to the Least Significant Difference (LSD) method of
'~i~nificance (letter) denotes significant differences (P<O.O5) according to the Least Significant Difference (*LSD) method of testing.
Treatment 1
Treatment 1 2 3
Mean 10.46 7.56 3 .O5 0.2 1 5 0.055 0.209 0.049 0.88
Treatment 2
Mean 60.00 75.75 67.5
Mern 10.65 6.56 2.59 0,190 0.059 0.141 0.039 0.61
'SE 1.008 0.213 0.3 14 0.02 1 0.003 0.035 0.007 0.106
2~~~
a a a a b a b ab
Treatment 3
Standard Error 4.42 6.52 4.35
SE 1.411 0.262 0.302 0,032 0,007 0.029 0.008 0.073
Mean 11.36 7.46 3.50
0,256 0,067 0.307 0,072 1.00
Trea tmeri t 4
1 Signifieance (*LSD)
a a a
LSD a b a a
ab a b b
Mean 7.79 7.41 3,12 0.230 0.059 0.257 0.061 0.90
SE 1.831 0.280 0.396 0.026 0.010 0.073 0.018 0.162
LSD a a a a a a a a
SE 1.644 0.450 0.328 0.035 0.008 0.090 0,021 0.151
LSD a a a a ab a ab ab
Tabie 3.4.f Mean final yield component parameters and standard errors for 1997 Sized Spring seedlings, fiom the field (Delhi).
Parameter Leaf Area (cm2)
Stem Length (cm) Root Shoulder (mm)
Fresh Shoot Weight (g) Dry Shoot Weight (g) Fresh Root Weiglit (g) Dry Root Weight (g)
Dry Root:Dry Shoot 1 SE = Standard Error *significance (letter) denotes significant differences (PK0.05) according to the Least Significant Difference (LSD) method of testing.
Treatment I
)f the mean.
'LSD c b b c
, c b b a
Treatment 2 Mean 8.81
11.38 5.28 0.344 0.072 0,426 0.137 1.90
Mean 10.74 12.71 5.11 0,400 0,085 0.437 0.142 1.64
'SE ,433 .359 .153 ,014 ,003 .O26 ,008 ,068
Treatment 3 SE ,390 ,337 ,209 O ,003 ,034 .O10 .O98
Mean 11.70 12,23 5.39
0,448 0.095 0.593 0.184 1.89
Treatment 4 LSD
b a b b b b b b
Meaii 12.25 12.95 5.78
0,468 0.095 0.617 0.195 2,03
SE ,488 .368 .127 ,018 ,003 ,038 ,010 ,089
LSD ab ab ab a a a a a
SE ,450 ,253 .161 ,018 ,003 ,035 ,010 ,068
LSD a a a a a a a a
Table 3.5.a. Mean embryo Iengths (mm) and standard errors of the means for 1997 Sized Stratined seed at planting (afler one year of traditional stratification).
1 Treatment 1 Mean 1 Standard Error 1 '~i~nificance ('LSDI 1
' ~igrilncance (letter) denotes significant differences (P<0.05) according to the Least Significant Difference (*LSD) method of testing.
Table 3 S.b. Probability estimates of successfùl seed crack (crack > -6mm) with confidence intervais ofthose probabilities according to logit transformation, for 1997 Sized Stratined seed at planting (after one year of traditional stratification).
1 LCI = Iower confidence interval 'UCI = upper confidence interval
Treatment 1
Table 3.5.c. Mean £inal emergence (out of 10) and standard errors of the means for 1997 Sized Stratified seedlings in the greenhouse.
Probability .657
'Significance (letter) denotes significant dserences (Pc0.05) according to the Least Significant Difference (*LSD) method of testing.
Treatment 1
Table 3.5.d. Mean final fiesh root weight and standard errors of the means for 1997 Sized Stratified seedlings in the greenhouse.
ILCI-
-628
Treatment Mean Standard Error 1 Significance ('LSD)
1 .O66 .O 1 1 b
"CI .685
Mean 6.00
4 1 -140 1 .O13 a 1 Significance (letter) denotes signifTcant differences (Pc0.05) according to the Least Signincant Dserence ( 2 ~ ~ ~ ) method of testing.
Standard Error 5 77
I Significance ( 2 ~ ~ ~ )
a
Table 3 S. e. Mean final dry root weights and standard errors of the means for 1997 Sized Stratified seedlings in the greenhouse.
1 4 .O20 ,003 a I 1 Significance (letter) denotes signiscant differences @'<O.OS) according to the Least
, ~reatment 1 2 3
S imcan t Difference ( 2 ~ ~ ~ ) method of testing.
Table 3 -5.f Mean finai emergence (out of 300) and standard errors of the means for 1 997 Sized Stratified seedlings fkom the field @elhi).
Mean .O12 .O 19 .O2 1
Standard Error .O02 ,002 .O03
Treatment I 2 3
Sigoificant Difference ('LSD) method of testing.
'~ignificance ( 2 ~ ~ ~ )
b a a
I
Mean 45.75 6 1-50 70.75
-- --
4 39.25 5.154 c
Standard Error 4.553 3 -969
14.739
I 1 Significance (letter) denotes significant dserences (P€O.05) according to the Least
'Signifieance ('LSD) bc ab a
ISOLATION OF FUNGAL POPULATIONS FROM
DEVELOPING FLOWERS, FRUIT AND SEED OF GINSENG.
4.1. Introduction
Kigh crop losses in ginseng production are due to the prevalence of at least 8 major
pathogenic genera of fun@ (Table 4.1 .a.). These fûngi can attack ginseng throughout
many stages of plant development and have the potential to infect more than one organ of
that plant. For example, the soilborne fungus Phytophthora can infect the roots and the
shoots of ginseng plants, usually prefening plants in the later years of production
(Dannono and Parke, 1990).
The average emergence rate of ginseng in Ontario c m be as low as 60% in the first
seedling year (Proctor, personal communication). The results are uneven plant stands and
the inefficient use of costly, maintained bed space. Seedling diseases such as pre-emergent
and post-emergent damping-off c m account for some of this loss and may be attributed to
seedbome diseases (Ziezold, 1997). It is estimated that seed rots and seedbome diseases
are responsible for much of the losses in germination and subsequent visible emergence
failure. Ginseng seed rots during the long (1 8-22 months) stratification process, before
germination (Figure 4.5.), @rammall, 1997; Schooley, personal communication; Ziezold
et al., 1998). A portion of seed rot c m be attributed to infenor seed processing practices
at the fm. Practices such as timely de-pulping and short term seed storage conditions
may Vary greatly fiom fami to f m , resulting in variable seed quality (Figure 4.1.). It is
likely that the exposed endosperm (due to dehiscence after 10- 12 months stratification)
makes stratiûed ginseng seed an especially easy target for pathogens. Fluctuations in the
ground temperatures of outdoor buried seed boxes may also help to produce obstacles in
the production of healthy stratified seed, especially during the hot, dry summer months
(Figure 4.2.).
The warm-cool-warm seasonai temperatures and moist conditions of stratification are
very conducive to the growth of many pathogenic fungi. Ziezold (1 997) hypothesized that
seedbome pathogens may be an important source of inoculurn in the fumigated seedling
garden. She stated that these pathogens (e.g. Fz~sarium, Cylindrocmpon) are strategicaily
located to be early colonists of gerrninating and emerging seedlings. Of the fungicides
registered for ginseng production in Ontario (Table 4.1 .b.), none are registered for the
controI of Fusarium and Cylindrocaupon. It is likely that seedborne pathogens are a
major cause of early declines in seedling plant stands, emphasinng the importance of seed
qudity in pro ducing good stands of ginseng. Ziezold et al., (1 998) hypothesized that
quality control methods (i.e. floating stratified ginseng seed before planting) are not
effective enough. They postulated that firm, infected seed sinks and is planted dong with
healthy seed. The result is germination of those diseased seeds but rapid decline w i h the
first 6 weeks of growth.
Brammdl(1997) reported exceptionally high seed rot rates (40 to 50%). He noted that
at the beginning of stratification, there iç a quick initial drop in seed viability of about 5-
10%. This can be explained by natural levels of embryo abortion, quite often due to
environmental stresses dunng ferùlization and developrnent of that seed (Bewtey and
Black, 1994). Viability remains fairly constant for the first winter season, due to sub-
optimal temperatures for the growth of many fin@ (Kendrick, 1992). The following
spring and summer fxnds sigdkant losses in viability due to the warm, wet seed
environment, making it a perfect place for fùngi to grow and infect adjacent seed.
Although some of the primary inocdum is likely carried into stratification odin the green
seed (Schooley, persond communication), it seems likely that a portion o f the pathogens
are introduced via the surroundhg soil, the stratification media (local mortar sand), the
water used to moisten the media and perhaps even the w d s of the stratification box.
Stratification boxes can be thoroughly disinfected with a 10% solution of hypochlorite or
formaldehyde (Schooley, 1997), but the huge arnounts of requked sand and water are not
disinfected so easily. A common method for decontaminating media is steam sterilization,
but this method would be too costiy on such a high volume basis.
Many of the species of fungi which infect ginseng plant organs have ais0 been isolated
fiom stratified seeds (Reeleder and Fisher, 1995a; Ziezold et al., 1998). Brammall(1997)
isolated both fungi and bacteria from rotting stratified seed. He found up to 50-60%
losses after stratification due to seed rot. He recovered species of Botrytis,
Cylindrocarpon, Erwinia and Fzisarium. There is speculation that recovered Erwinia may
indicate a high saprophytic population of that bacterium, and not necessady verify that it
is a causal agent of the seed rot (Reeleder, persona1 communication). Ta date, there are
no published reports of Envinia causing primary disease symptoms in American ginseng.
However, Envinia carotovora pathovar crnotovora causes a root rot in Korean ginseag
(Putnam, 1989).
Only three research groups have been undertaken studies to ident* the seed rot
pathogens of North American ginseng (Lee et al., 198 1; Zhang et al., 1989; Zhang and
Chen, 1992; Zeizold et al., 1998;). Zhang et al., (1989) have isolated Alternaria,
AspergrgrlZis, Cylindrocarpon, Drechslera, Fzcsmium and Trichodema from stratified seed
coats and endospenn surfaces. In a later study, Zhang and Chen (1992) isolated common
ginseng pathogens &om young Panm: quinquefulius seeds in immature fruits, whiie still
develo ping on the parent plant. The initial contamination percent age (Alternaria p m ,
Cylindrocarpon spp.) of the newly harvested seed was quite s m d , about 3 4 % .
However, after stratification in cool, moist conditions, the percentage of contaminated
seeds increased significandy (about 50%) as the fiingï spread to neighboring seeds in that
lot. Ziezold et al., (1998) were also able to isolate common ginseng pathogens through
studies of ginseng seed pathology. Fusarium, Chaetomium, Mucor, Altemaria and
Zopfiela were the most abundant genera isolated fiom the endosperm halves of stratified
seed. Lee et al., (198 1) studied the microflora of Oriental ginseng ( P m ginseng) and
isolated species of Altemaria, Aspergillis, Bo~ytis, Fzmn-ium, Rhizocfonza, Rhizopzcs and
Trichodema fiom the endocarp.
Recently it has been proposed that these pathogens may be entering the seed while it is
still developing on the parent plant (Proctor, persona1 communication), (Schooley,
persona! communication), (Zhang and Chen, 1992). Perhaps the infection is not being
expressed until Later on, as a seed rot during stratification or as a damping off disease in
the garden. Another theory is that there may be a whole host of pathogens involved, that
the seed contamination may start out as one pathogen which gives way to another
oppominist such as Fusarium. If the origin of this seed rot phenomenon can be
elucidated, there is a much better chance of being able to control seedborne pathogens.
In order to address the seed rot problem, a study of ginseng flower and seed pathology
was undertaken. Sampling of reproductive tissues began at the flower bud stage, followed
through h i t development on the plant and penodically during stratification (1 8-22
months). Since fungi are the predominant pathogens cornmonly found in North American
ginseng seed, this study has been restricted to isolation and identification of fiingi.
4.2. Materials and Methods
1996:
Sampies from ginseng flowers and developing fimit were taken weekly &om the week
of July 14 (when about 30% of the florets were open) to the week of August 27 (when
most of the h i t was ripened and subsequently harvested). Flowers and h i t were
sampled f?om 4-year old ginseng gardens only and at one f m location ( f m A) in
Ontario. Inflorescences were sampled in a random manner fiom 3 dEerent gardens at that
f m . Twelve inflorescences were harvested tiom each garden and ail 36 of the samples
were pooled.
1997:
Sarnples from ginseng flowers and developing fniit were taken biweekly fkom the week
of July 14 (when about 30% of the florets were open) to the week of August 27 (when
most of the fniit was ripened and subsequently harvested). Flowers and fniit were sampled
fiom 4-year old and 3-year old ginseng gardens. The 1997 four-year old garden seed was
cultured as a second replication to compare with the 1996 data. It was taken fiom the
same fimn and site (farm A), and again, f?om 3 gardens. To allow cornparison between
difTerent ginseng production sites in southem Ontario, 3 f m sites (with 3-year-old
gardens) were sampled in 1997: f m b and c. Due to recent trends of seed production in
year 3 and flower rernoval in year 4, only 3-year-old gardens were chosen for sampling in
1997. Inflorescences were sampied Eom one garden at each finu, in a random manner.
Twelve infiorescences were harvested fkom each garden and were kept labeled and
separated by f m site ftom which they were sarnpled.
2996 and 1997:
The following represents a timeline for which the explant material were taken in both
1996 and 1997. These "times" will be referred to in the results and discussion of this
study.
Time Date ("week or')
July 14 /96 and 97
July 22 /96 and 97
August 7 /96 and 97
August 20 196 and 97
August 27 /96 and 97
October 1 1 /96 and 97
January 2 197 and 98
May 16 197
October 18 /97
Stage of Development
~ewl~-openedflowers
Senescing flowers
Developing green miit
Developing green bit
Ripe, red fruit
Green seed
Green seed
Green seed
Stratified seed
Two florets or berries were sarnpled from each idorescence, and pooled for
sterilization. Any flowers, h i t or seed that had any signs of rotting or abnormality were
discarded. The explant tissues were taken from each development stage of ginseng seed,
beginning at the flower bud stage through to stratification as follows:
--
Plant ~ e v e l o ~ g e n t Stage 1 Explant Culture Tissue
newly-opened (intact) flowers
developing fniit 1 o v q tissue, seed or seed half (with
anther, ovary, ovule, petal, stigma
senescing flowers fertilized ovule, ovary tissue, stigma
1 seed coat half (coat)
stratified seed (72 months)
Al1 tissue was handled in culture using sterile techniques. Any tender or very tiny plant
material (i.e. bud halves, ovules, petals, anthers, stigmas) was handled within sterile Nytex
(35 CM) baggies and rinsed with autoclaved, deionized water. AU other plant materid was
sterilized with a 1 minute dip in 95% ethanol followed by 3 minutes in a 10% solution of
household bleach (0.6% sodium hypochlorite) and autoclaved, deionized water. They
were then rinsed three times with autoclaved, deionized water and blotted dry with sterile
~hatman@' filter paper. Most of the explant matenal from the 1996 season (up until
August 20) was dissected in open laboratory conditions and sterilized aftenvards. A
plethora of common, non-pathogenic yeasts and air molds were recovered from these
cultures. Hence, aii dissecting was canied out under sterile conditions (Le. under the
laminar flowhood) £kom that culture date forward.
Stratification of green seed was canied out under semi-sterile conditions. Al1
stratification sand was autoclaved twice, at a depth of 4-5 cm to prevent contamination of
the explant material. Seeds were suspended in the media at a 3 : 1 ratio of sand to seed.
The sand was wetted before mixing and rnoisture content was maintained by monitoring
weight. In addition, mWng was canied out on a biweekly basis in order to improve
embryo), stigma
endosperm with embryo half(e+e)
aeration of the media. Seed lots (according to farm, age of plant etc.) were held in 2L
plastic pots under alternating warm ( 1 5 ' ~ for 4 months) and cool ( 3 ' ~ for 4 months)
temperatures to rnimic seasonal conditions (Hovius, 1996).
Sterilized explants were placed onto agar media inside sterile, plastic petn dishes
(standard, 100x1 S m m , Fisher Scientific Co.). Each petri dish contained approximately 17
m! of agar. Three explants were used for each plate, with a total of 4 plates for each
expiant type (Figure 4.6.A-). This gave a total of 12 replications per explant tissue type at
each culture date for each media type. The PDA and MRBA plates were cultured at 23°C
(+/- 3°C) for 7 days in Light. The remaining plates, whose agar contained antibiotics, were
cultured at 23°C (+/- 3°C) for 7 days in darkness.
A total of 5 dEerent media types were used to isolate fungi from the cultured explants
(see Appendix 1). P D 4 MRBA and WA with antibiotics were used to culture flower
parts and developing fiuit. PDA MRBA and PARP were used to culture stratified seed.
CA was used to replate cultures from the PAEW media. The five dserent media were
used to isolate specific groups of fungi. PDA is a general nutritional media which
supports the growth of many different genera of fun@. MRBA is a more specEc
combination of nutrients and a fungicide that selects for the growth of Fzcsarium spp. and
Cylindrocarpon spp. WA with antibiotics suppresses the growth of bacteria but is non
selective for the growth of fungi. PARP suppresses the growth of bacteria while selecting
for the growth of oomycetes such as Pythilrm spp. aad Phytophthora spp.
Identification and Re~orting:
Fungi were identined according to the descriptions provided in the literature (Malloch,
198 1; Simmons, 1982; von hx, 1987) and under the supervision of Dr. Reeleder. They
were identified according to culture characteristics and spore morphology (Figures 4.3 .,
4.4. and 4.6B). Stock cultures (obtained by the Southem Crop Protection and Food
Research Centre, AAFC, Delhi, ON) were used for cornparison purposes.
Many genera of k g i (and some bactena) were isolated fiom ginseng flowers, fruit and
seed (Figure 4.3.). The most obvious were the common yeasts (especiaiiy red yeast), as
they ofien grew rapidly and colonized the plates before other fungi had a chance to
appear. As yeasts are not considered to be plant pathogenic (Mailoch, 198 l), their
fiequency and distribution were not important to this research and are not presented in the
results.
Penicillium spp. andAspergï lh spp. were also quick to colonize the aga. and were
found throughout this experiment. Penicillizrm spp. are commonly found as the causal
agents of post-harvest diseases of h i t s aad vegetables, but not on growing plants or
seeds. Aspergiillus spp. can cause some decay of grains and legumes after harvest.
However, it grows best at low moisture contents (1 1 - 13%) and is often out-competed by
other fungi at moisture contents such as those of ginseng seed (Agrios, 1988). For these
reasons, these cultures were considered contaminants and are not reported in the results.
Fungal cultures belon,@g to the genera Mucor and Rhiropus were also recovered
throughout ginseng flower, h i t and seed development. They belong to a class of fun@
that are most commonly known as saprophytes, ofien found on processed plant products,
and quite cornmon to almost every terrestrial environment. However, some members of
this group are weak plant pathogens, attacking injured tissues to cause soi3 rots of many
fniits, vegetables, flowers, bulbs and seeds (e.g. sweet corn) in storage (Agios, 1988). As
an exampie, Rhiropzu mrhinrs Fisher causes Pole Rot of tobacco, a post-harvest disease
of the curing leaves (Reeleder, 1993). It remains to be investigated whether Rhizopzrs
produces disease in ginseng seed hence these colonies ("Rhizoptis spp. / Mzrcor spp.")
have been reported in the results of this study.
AU Alternaria cultures were classified as Alternaria spp. Both the Altemaria panax
Whetzel stock cultures (obtained by the Southem Crop Protection and Food Research
Centre, M C , Delhi, ON) and the explant cultures had dïfliculty sporulating under
laboratory conditions. This resulted in an inability to correctiy iden* AItemaria cultures
to species. Brammd (1994) reported that laboratory culture and subsequent sporufation
of A. panax was erratic. It is likely that Altemaria panax Whetzel was recovered within
this category of Aïternaria spp., since many of the Alternaria cultures recovered had very
similar characteristics when compared to the stock culture of A. p a n a Whetzel. Ali of
the Botrytis colonies recovered from the explant cultures belonged to the species Bohylis
cinerea Pers.:Fr. Fzrsmizrm rosezrm Link: Fr. was recovered with many other Fusnrirrrn-
like cultures that exhibited hews of pinks and yellows. It is assumed that at least some of
these cultures belong to other plant pathogenic species of Fusarim, namely F. oxyspomm
and F. solani. Much of the literature is vague on reporting particular species of Fzisariium
(Reeleder, 1994; Parke and Shotwell, 1989). The species identification for this genus has
been limited to F. roseurn since it was recovered with the greatest frequency in a recent
study of Ontario ginseng (Ziezold et al., 1998). The remaining Fz~sarium explant cultures
have been classified as Fusarium spp.
Statistical Analysis:
The Completely Randomized Design was analyzed using the General Linear Models
procedure of the SAS statistical package (SAS Institute, Cary, N.C.). A one-way ANOVA
was used to analyze variable means. Transformations were used (fun@ = log
[(fungi+O.2S)/(I 00-fungi+O.25)] ) , however, the results for untransformed data were
identicai, therefore untransformed data was used in the analysis. Treatments means were
compared using the Least Significant Dittèrence (LSD) method of testing. The level of
significance used for all tests was P=0.05, unless otherwise stated.
"AgeY' (4 year-old gardens and 3-year-old gardens) was found to be highiy significant
in the anaiysis. Also, the replicate variable for 4 year-old data was "year" (96 and 97)
while the repiicate variable for 3-year-old data was " f a r d ( fms 48 and C). For these
reasons, 3-year-old and 4-year-old data were analyzed separately.
4.3 Results and Discussion
Floret explant cultures (tirne O and 8') from 4-year-old plants in 1996 and 1997:
Altemaria spp., B. cinerea and M~icor/Rhizops spp. were recovered f?om floret buds
and newly-opened florets of 4-year-old plants in 1996 and 1997 (Table 4.2.a.). These
explant cultures were taken on the week of July 14,1996 and 1997 (time O). Allernuria
spp. were recovered on petals, anthers and pistils, but no significant dflerences were
found among explant types. B. cinerea was recovered on petals, anthers, pistils and ovary
tissue, however, the recovery percentages were not signincantly different among explant
types. Mucor/Rhizopus spp. were found on pistil cultures only, their recovery was not
significantly different over other explant types.
Akemaria spp. were recovered on ail 3 media types and their percent recovery was not
sipficantly greater among media (Table 4.2.b). B. cinerea was recovered on MRBA and
WA only, however these percentages were not signiticantly greater than those on PDA.
M u c o r ~ i z o p ~ s spp. were recovered on WA only, however, the percent recovery among
media were not signifïcantly daerent. Alfernaria spp. were cultured in both years, and no
differences were found between years (Table 4.2.c). B. cinerea was recovered only in
1996, and its percent recovery was sigmficantly greater in 1 996. Mucor&izoplrs spp.
were recovered in 1996 only, however, the amount were so s m d that there was no
signincant Merence between years.
Floret expiant cultures (tirne O and 8) of 3-vear-old plants fiom 3 farms in 1997:
AIternaria spp. and B. cinerea were recovered fiom floret buds, and petals and
anthers of newly-opened florets fiom 3-year-old plants in 1997 (Table 4.3.a). These
cultures were taken the week of July 22, 1996 and 1997 (tirne a), frorn 3 separate Ontario
farms. Alternaria spp. were recovered on petals and anthers. Their percent recovery was
not significantly dinerent fiom each other. Petals cultures produced significantly greater
percentages of Alternaria spp. than for buds, pistils, ovaries and ovules. B. cinerea was
recovered on buds, petds and anthers. These percentages were not significantly different
fiom each other. The percentages of B. cinerea cultures on petals were si@cantIy
greater than for pistils, ovaries and ovules.
Alternaria spp. was recovered on al1 3 media types and their percent recovery was not
significantiy greater among media (Table 4.3.b). B. cinerea was recovered on PDA and
WA only, however these percentages were not significantly greater than those on MRBA.
Alternaria spp. were isolate fiom all3 farms, however, there was no signincant dEerence
among f m s (Table 4.3 .c). B. cinerea was recovered only on farms B and C, but there
was no difference between those and fanri A.
Floret expiant cultures (tirne O and 8): Discussion and Summary
Altemaria spp. and B. cinerea were recovered fkom explant cultures of buds and
newly-opened florets. This resuit complements the hdings of Brammall(1994), who
States that B. cinerea c m be found infecthg developing flowers of Ontario ginseng. The
fun@ were found most often on petds and anthers, and less fkequently on pistils. This
finding is reasonable since the tender, short-lived petal and anther tissue do not have a
thick waxy cuticle to help prevent the germination of airbome conidia (Esau, 1977). In
fact, Boland and Hall (1987) stated that senescing beau flowers often provide nutrients for
genrilnating spores of Sclerotinia sclerotionrm, sustainhg them until they c m infect the
developing seed.
Fungi were not consistently isolated more often fiom 4-year-old gardens, than 3-year-
olds. However, one would expect lower populations of pathogens in the 3 -year-old
gardens since there is one less year for those populations to build-up in the soi1 and
canopy.
B. cinerea was isolated more oRen in 2 996 than in 1997. The 1996 growing season
was characterized by a long, cool and wet spring followed by lower than normal
temperatures. Since these conditions are most conducive to growth, sporulation and
infection of these fin& higher recovery rates were likely. Culture techniques could also
explain the annual variation. In 1996, explant material was dissected on an open lab bench
top, and sterilized hours later for plathg. Extraneous Bobyfis fbngal spores could have
infected the succulent tissue during those few hours, preventhg them fkom being
discarded during surface sterilization.
Floret through developine seed explant cultures (tirne O to t h e 40) fiom Cvear-old plants
in 1996 and 1997:
Alternuria spp., B. cinerea, F. roseum , Fusurizm spp. and MzrcorlRhizoprs spp. were
recovered fiom senescing florets of 4-year-old plants in 1996 and 1997 (Table 4.2.d.
through 4.2.g.). These cultures were taken on the week of July 14 through to the week of
August 27, 1996 and 1997. Ody one farm site was sampled.
AZtemwia spp. and B. cinerea were isolated significantly more often on pistil cultures
than ovary and ovule cultures (Table 4.2.d.). F. roseum, Fusarizlm spp. and
MzrcorlRhizopus spp. isolated in similar percentages across al1 explant types (Figure
4.2.e). Altemaria spp., F. roseum and Fzisarium spp. were isolated quite evenly across aLl
media types. B. cinerea found sigdcantly more often on PDA and WA than MRBA.
Mzrcor/Rhizopus spp. were recovered more often on WA than PDA. B. cinerea, F.
rosezim and Fzrsarium spp. were isolated in sirnilar amounts between the two years (Table
4.2.f ). Altemuria spp. were found significantly more often in 1997 and MzrcorlRhizopzrs
spp. were isolated sigdicantly more in 1996.
For al1 groups of fungi, percent recovery did not increase with time (Table 4.2.g.).
Altemaria spp. were isolated in significantly greater amounts at time 33 than time O. B.
cinerea was most signifïcantly found at time 33 than any other time. It was isolated
significantly more often at times 20 and 40 than t h e O. The numbers of Fusarizim
cultures were not different over t h e O to 40. However, Mucor/Rhimpus spp. were
recovered more often on time 33 than time 20 or 40.
Floret throuph - to developing seed explant cultures (tirne O to time 40) of 3-vear-old ~ lan t s
fiom 3 farms in 1997:
Altemaria spp., B. cinerea, F. roseum, Fusarim spp. and M u c o r ~ i z o p u s spp. were
recovered fkom senescing dorets of 3-year-old plants in 1997 (Table 4.3.d. through
4.3.g.). These cultures were taken on the week of July 14 through to the week of August
27, 1997. They were cultured on 3 dserent media, &om 3 difEerent Ontario farms.
Aiternaria spp. and B. cinerea were found sigdicantly more often on pistil cultures
than ovary and ovule cultures (Table 4.3.d.). Fzisarium spp. were isolated sign6cantly
more oflen on pistil cultures than ovule cultures. F. rosezim and Mucor/Rhizopzis spp.
were found in similar quantities for each explant type. B. cinerea was isolated more often
on PDA and WA than MRBA (Table 4.3.e.). AU other h g i were recovered sirnilady for
each media type. B. cinerea was isolated signifïcantly more often tiom f m s A and C
than f m B (Table 4.3 .E). Al1 other b g i were recovered fairly coasistently f?om each
f m site. As seen in the 4-year-old garden explant cultures, the amount of recovered
fungi did not increase with tirne (Table 4.3 .g.). Populations of Alternaria spp. and B.
cinerea peaked at time 20. This corresponds to the last day of dissecting without the use
of the flowhood. For Altemaria spp., B. cinerea and Mzicor,.?Uzizopzrs spp., significantly
less fungi were isolated at time O than the later culture dates. However, populations of
Fusarium did not signincantly increase over time.
Floret throueh to develo~ing seed exdant cultures (time O to time 40): Discussion and
Summary
Alternmia spp., B. cinerea, F. roseum, Fusarhm spp. and MucorYRhizopus spp. were
recovered throughout explant cultures from developing fiuit of ginseng in 1996 and 1997.
Alternuria spp. was isolated on ali media types and was found significantly more
eequently on pistil cultures. A. panm has been found infecthg seed heads, in addition to
other aerial portions of the ginseng plant (Putnarn, 1989). The infection of h i t tissue
causes the fruit to blacken and shnvel, often persisting on the umbel. Otherwise, infection
causes abscission of the developing h i t . A. pmm has also been isolated nom diseased
peduncles and pedicels of fniiting umbels in North American ginseng (Bramrnall, 1994;
Parke and ShotweU, 1989). Zhang and Chen (1992) also isolated A. panax from green
f i t tissue of North American ginseng.
It is possible that colonization by Altemaria spp. occurred at the tirne of fertilization,
when the style canal was open to pollen. lnfecting fun@ would have had a few weeks to
spread to neighboring tissue during h i t development, resulting in rising populations of
Alternaria spp. as the season progresses. Alternaria spp. were found on few seed cultures
from 4-year-old plant material and not at al1 on 3-year-old garden explants. Possibly the
growth ofAltemaria spp. was just beginning to travel into the region of the developing
seed as the miit was ripening, and would be expressed in later in green seed.
B. cinerea was isolated on pistil cultures and less on ovary and seed tissue cultures.
BrammaU (1994) reported that B. cinerea infects developing h i t , both green and red,
with the berries turning brown after infection, often developing the fùzzy gray spores of
this mold on their surface. In 1996, recovery of B. cinerea fiom 4-year-old plant matenal
peaked on August 20, then dropped immediately after dissection took place aseptically.
Otherwise, cultures of this fingi were isolated in growing numbers as the season
progressed. Again, it may be possible that the infections took place at the tirne of
fertilization and that infecting h g i would spread to other tissues during f i t
development, resulting in rising populations over time.
F. roseum and Fusarium spp. were recovered inf?equently during fniit development.
This supports the theory that Fusarium may play a role as a secondary invader, whose
disease symptoms are expressed Iater through stratification. F. rosezrm and Fusmium spp.
cultures were isolated mainly on pistil tissue, with just a few on ovary tissue and seed
tissue. Fusarizon may have traveled down the pistil canal after the colonization by
Alfernaria and Bomris, and was growing towards ovary tissue. F. rosezrm and Fz~sarizrm
spp. were isolated as time increased. Fusmiz~rn spp. are often considered secondary
invaders as they are fiequently found colonizing wounded or diseased tissue (Reeleder,
1994). It is possible that they are functioning in the same manner here.
There are few studies on the colonization of seeds by pathogenic microbes. Menzies
and Jarvis (1994) noted infestations of tornato seeds with F. oxysponrm while they were
developing inside the fruit. When flowers were inoculated with a spore suspension, 17%
became infected and 100% of the seeds f?om those fhit were infected with F. oxpporrrm.
Those seeds coming fiom plants with diseased crowns or peduncles gave a seedborne
infection rate of 0.03%.
MucorLRhizopus spp. were isolated infkequently during this period of flower and fhit
development. No real trends could be detected as to their development in ginseng. Since
Mucor/Rhizopus spp. are not mentioned in the literature for ginseng plant diseases and
innequently for seed diseases, it seems to follow that they would not be detected in large
amounts Eom developing fniit.
The fùngi were found most often on pistil tissue. This is interestkg since pistils are
persistent throughout flower and fniit development of ginseng, on the parent plant. Since
pistil tissue appears firm and strong, it is not likely that their structure facilitates the
infection of h g i . Perhaps the fungi are able to infect this organ through the same canal
that pollen tubes foliow and may occur simultaneously.
There were few Merences between the recovery percentages between 1996 and 1997,
for 4-year-old material. However, these clifferences were not consistent for the years and
therefore, they were likely due to chance. Fungi were bund in sirnilar amounts across all
f m s . Those dserences that did exist were not consistent for any one f m and therefore
ignored. Greater numbers of fungal colonies were found up to time 20 in 4-year-old
garden explant cultures. This may be explained by the use of the aseptic technique
beginning on time 33 in 1996.
The question remains, how are these fungi entering h i t and seed tissue? Could the
fun@ enter through the flower or is the pathogen translocated f?om vascular tissue,
entering via the peduncle and pedicel on the umbel. Since the predomioant fungi here are
Altemarin and Bohytis, and these fun@ are not usually documented as systemic, it seems
likely that the pathogens are entering through the flower. Gupta and Raychaudhuri (1988)
suggest that spores germinate on the inflorescence and penetrate into the seed while it is
developing on the parent plant. Perhaps the fungi are able to infect the stigma through the
same canal that pollen tubes follow, possibly occurring at the same time as fertilization.
Esau (1977) States that the pollen tube passes through the style, through the micropyle,
passed the integument and on towards the ovule. Pathogenic fun@ might also follow this
route. The fact that mainly pistil and o v q tissue cultures harboured hingi in this study
can be explained. Those ovules or developing seeds that were idected early on were
aborted, leaving only healthy fruit and those k i t slightly iafected with fungi. Perhaps the
funiculus can play a role in transmitting the pathogens fiom the ovary tissue into the
developing seed, later in bit development. The recovery of pathogens from endosperm
cultures of newly-harvested green seed would give this theory some strength.
Green seed cultures (time 85 and 168) fkom 4-vear-old plants in 1996 and 1997:
Altemaria spp., B. cinerea, E roseum. Fusartiim spp. and Mzicor/Rhizopz~s spp. were
recovered from green seed of 4-year-old plants in 1996 and 1997 (Tables 4.2.h. through
4.2.k.). Sampling dates were the week of October 1 1 and the week of January 2, for 1996
and 2997. "Green seed" refers to seed h m the time it was harvested until 12- 14 months
of stratification.
The e+e (endosperm with embryo) cultures had fungal growth of al1 groups stated thus
far and al1 but B. cinerea was found growing on seed coat cultures (Table 4.2.h.).
Fzrsarium spp. were recovered signincantly more ofken on e+e cultures than seed coat
cultures. Othenvise, ail other fungi were recovered with similar eequency on the two
explant types. In general, PDA and WA had more fungal growth than MRBA (Table
4.2 .i.). Alternaria spp . and Fz~sarizm spp. were isolated significantly more O fien fiom
WA than MRBA and PDA. Al1 other fungi were recovered in similar nurnbers fiom each
media type.
Significantly more F. roseum and Fzrsmium spp. were found in 1 997 cultures than 1 996
(Table 4.2.j.). Al1 0 t h fun@ were recovered with similar fiequency in both years. In
general, more fungi were isolated at time 168 than time 85 (Table 4.2.k.). F. roseton and
Fusarizim spp. were isolated sigdlcantly more often on tirne 168 than 85. Populations of
other h g i were not sigdicantly dEerent between the two culture times.
Green seed cultures (tune 85 and 168) of 3-year-old plants fiom 3 fanns in 1997:
Alternaria spp., B. cinerea, F. roseurn, Fusarium spp. and MucorIRhiropus spp. were
recovered from green seed of 3-year-old plants from 3 fanns in 1997 (Tables 4.3 .h.
through 4.3.k.). Seeds were sampled on two dates, the week of October 1 1 and the week
of January 2 for both years.
AU species of fungi were recovered corn explant material. In general, e+e tissue
explants had larger populations of fiuigi (Table 4.3. h.). B. cinerea and Mzrcor/?ihizopus
spp. were found in significantly greater amounts in e+e cultures than coat cultures. The
fun@ were recovered on al1 types of media (Table 4.3.i.). Alternaria spp. and B. cinerea
were isolated from PDA at significantly higher percentages than on MRBA and PARP.
Fuwriium spp. and Mucorh?hizops spp. were recovered in sigdlcantly greater amounts
£kom MEü3A.
Al1 fungi was recovered from al1 three f m sites in 1997 (Table 4.3 .j.). Altemarin spp.
were isolated significantly more often fiom farm A, while Mucor/lZhizopzrs spp. were
found more often from farm C. Fungi were isolated in sirnilar quantities at both culture
times in 1997 (Table 4.3.k.). However, Fz~sarium spp. were found in signifïcantly greater
amounts on time 168 than 85.
Green seed cultures (tune 85 and 168): Discussion and Summary
Altemaria spp., B. cinerea, F. rosezim, Fzisarizm spp. and MzrcorlRhizoptrs spp. were
recovered £tom green seed of 3-year-old and 4-year-old plants in 1996 and 1997. They
were culhired the week of October 1 1 and January 2, for both years.
Alternaria spp. were recovered on endosperm and seed coat cultures. This trend was
seen in both years, at both plant ages and across ail famis. Lee et al., (1 98 1) recovered
Alfernaria spp. from the endocarp of green seed. A l t e r n i a spp. have also been isolated
on many important vegetable crops (e-g. carrot, onion and tomato) (Gupta and
Raychaudhuri, 1988). The number of Alternaria spp. cultures that were isolated
increased as the time under stratification progressed, but the daerences were not
significant. The w a d c o o l and wet conditions of stratification provide a great
environment for fùngal growth and sporulation. A l t e r n i a spp. do not seem to have
speci6c requirements for temperature (Agrios, 1988) and would therefore be expected to
grow well under the warrn and cool periods of stratification.
B. cinerea were isolated less ftequently during this period of developrnent, and only on
e+e tissue cultures. It was recovered quite eveniy from the different years and different
f m s . Lee et al., (198 1 ) isolated Botrytis on the endocarp of green ginseng seed. In
general, B. cinerea has not been well documented as a causal agent of seed rots in any
crops. It is, however, a formidable pathogen that quickly invades leaftissue, stem tissue,
flower tissue and fniit tissue in vegetables and fruit crops al1 over the world (Agrios,
1988). Because it is so common to the ginseng canopy, it follows that it can be found in
the reproductive tissues as well. The nurnber of B. cinerea cultures that were isolated
fioin Cyear-old gardens did not s i e c a n t l y increase as the time under stratification
progressed . The growth and sporulation of B. c i n e m peaks at 18-23°C (Agrios, 1988).
Since the maximum stratification temperatures achieved in this study were 15 OC, it rnight
be that the environmental requirements for fungai growth and sporulation were not met.
However, colonies of B. cinerea recovered fiom the green seed of 3-year-old gardens did
increase over t h e . The dserences seen here are unexplainable.
F. roseum was isolated on most dates nom endosperm cultures of 4-year-old explant
material. It was recovered on e+e cultures more so than on coat cultures, although these
dinerences were not significant. Fusarizm spp. were recovered more often on coat tissue
of 4-year-old expiant material. Both groups of Fuswium were recovered more ofien as
time increased. Zhang and Chen (1992) recovered h g i kom only 3-5% of newly-
harvested ginseng seed in their study. However, they found that the percentage of
recovery increased with time of stratification.
Lee el al., (198 1) isolated F. oxysporurn and F. solani fiom-the ginseng endocarp.
Fz~sarizrm is known as a seed rot pathogen in a significant number of h i t s and vegetables,
causing germination failures and damping-off diseases (Agrio s, 1 9 8 8). F. graminemm
and F. poae were isolated fiom fresh seed of winter wheat over a 3 year study (Clear and
Patrick, 1993). F. oxysponim and F. solani were isolated from newly-harvested seed of
Okra (Singh et al., 1992). The populations of Fusa-izrm cultures increased with time of
stratification of seed, for both F. roseum and F. spp. Growth of this fungus in
stratification was to be expected, given the warm/cool temperatures, moisture and close
proximity of the seed. Fz~smiurn development does not have a specific temperature range
requirement (Agios, 1988) and grew well under the 3 O C to 15 O C temperatures of
stratification in this study.
Mucor~izupus spp. were recovered more often from endosperm cultures thm seed
coat cultures. However, it was isolated intiequently and at low percentages &om
endosperm and seed coat cultures. Mucor spp. andRhizopus spp. are considered weak
parasites (Agrios, 1988) &en causing rots of soft fleshy plant material and processed
plant products. They are not common pathogens w i t b ginseng gardens and therefore
one would expect low levels to be recovered f?om green seed. However, they are a very
common mold in terrestrial environments and their spores are common airbome fun@
(Agrios, 1988). These fun@ were found in flowers and developing f i t tissues, resulting
in their contamination of subsequent green seed seen here. The documented recovery of
Zygomycetes on ginseng seed is lunited, however, Lee et al., (1 98 1) found R
palcilomyces on the ginseng endocarp. The populations ofMucorRhizopus spp. on
green seed did not rise noticeably with stratification.
Trends between fungi isolated fiom individual farm sites were not consistent in 1997.
The same can be said about fungi recovered on a yearly basis for 4-year-old tissue.
Green and stratified seed cultures (tirne 305 and 459) nom 4-year-old plants in 1996:
Altemaria spp., F. roseum, Ftciarium spp. and Mucor/Rhizopzis spp. were recovered
fiom stratified seed of 4-year-old plants in 1996 seed (Tables 4.2.1 through 4-2-11.). These
cultures were taken the week of October 18/97 and January 2/98. A study of stratified
seed fiom 1997 was not possible due to research deadlines.
Most groups of fun@ could be isolated &om e+e and coat explant cultures (Table
4.2.1.). Alternaria spp. and Fusarium spp. were found more numerously on e+e cultures,
but these differences were not significant. B. cinerea could be found more often on coat
cultures, but again, the increase was not significant. Most media provided adequate
nutrients to support the growth of ail b g i , however, B. cinerea was found only on PDA
(Table 4.2.m). PDA supported significantly greater populations of Altemaria spp.
Contrasts between media for other fingi were not s iwcan t ly diff'erent. No cultures of
B. cinerea were isolated at t h e 459 (Table 4.2.11.). Altemaria spp. were found in the
greatest amounts than di other fùngi at these two times.
Green and stratified seed cultures (time 305 to 459): Discussion and Summary
Altemaria sp p., F. roseum, Fzisarium sp p. and Mz~cor/Rhizopis sp p. were recovered
from green seed of Cyear-old plants in 1996, &er 8 (tirne 3 05) and 13 months ( t h e 459)
of stratiiication. "Stratified" seed refers to seed that has undergone straacation or after-
npening for at least 12 months.
Alternmu spp. was recovered in the largest amounts, and more ofien fiom endosperm
tissue than h m seed coat tissue. Zhang et al., (1 989) recovered large amounts of
Alfernaria fiom endosperm cultures of stratined ginseng seed. They also isolated
Altemaria Erom cultures of seed coat surfaces cultures of that seed. Similar results were
obtained in a recent shidy by Ziezold et al., (1998). They isolated Alternaria spp. £kom
10.3% endosperm cultures of healthy stratified ginseng seeds. The recovery of Alternaria
spp. in categories of diseased seed of their study produced smaller percentages.
B. cinerea was isolated fiom green seed but not isolated from any of the stratified seed
tissue at this time. Neither Zhang et al., (1989) or Ziezold et al., (1 998) recovered
cultures of Bo-yfis spp. fkom stratined seed. It has been mentioned previously that B.
cinerea has not been weil documented as a causal agent of seed rots in the bterature.
Perhaps B. cinerea does not perform well in the stratification environment. Challenges
such as poor aeration and the large, heavy structure of the sand particles may hinder its
development.
F. roseum was isolated slightly more often on seed coat tissue than endosperm tissue.
Fusarium spp. were recovered similarly on seed coat tissue and endosperm tissue. Zhang
et al., (1989) isolated Fusarium spp. f?om seed coat çurfaces, endospem d a c e s and
endosperm tissues of stratifïed ginseng seed. Ziezold et al., (1998) recovered F. roseum in
42%, and Fusmium spp. in 46%, of healthy stratified seed. Again, often the fkequencies
of F. roseurn and Fusarizirn spp. in categories of diseased seed were found at lesser
amounts than on the healthy seed. Reeleder (1 994) documented the recovery of Fz~sarizrm
spp. from diseased stratified ginseng seed. M e r witnessing the bright hews of pinks and
yellows of Fusarium mycelium in culture, it seems logical to link this fungus to the yellow
and pink rots of stratified seed.
MucorlRhizopus spp. were found on endosperm and coat cultures fairly evenly.
Ziezold et al., (1998) study also found cultures ofMucor spp., on 20% of healthy
endosperm cultures. Otherwise, the isolation of Mucor and Rhilopics from
diseasedhealthy ginseng seed has not been too signincant in the literature. The
pathogenicity of fungi fkom these genera to intact plants is not clear.
Some of the fùngi recovered from stratified seed were isolated in smaller amounts aod
with less ~equency than the previous sampiing dates. It is possible that fbngal infections
of the green seed resulted in rotting of that seed. Only healthy seeds were used in this
study, hence those diseased seeds would have been discarded. However, fungi would still
be present in neighboring seeds and media, making their way to healthy seed tissue as
populations wodd likely rise again in that stratified seed during the following months.
The absence of Cylindrocaq~on spp. among the isolated fungi in this study was
surprising. Previous studies have isolated Cylzndrocmpon destnictans (Zinssmeister)
Scholten and Cylindrocmpon spp. from ginseng seed, more commonly from stratified seed
(Zhang et al., 1989; Zhang and Chen, 1992; Ziezold et d, 1998). Of most significance is
the study by Zhang and Chen (1992), they isolated C. destntctans from the "host stem7'
dunng the period of early fruit developrnent. They hypothesized that the b g u s can move
&om the root, through the stem, peduncle, pedicels and haily into the bit and seed. In
the late stages of Cylinciocmpon root rot, leafreddening and wilting occur in the shoots
but there are no other reports of the fungus being active in the stem tissue. Since
Cylindoca7pon is known commonly as a soil pathogen, one would expect the main
source of inoculum to be the stratification media or stratification box, demonstrating its
prevalence on stratifïed seed (Zhang et al., 1989; Ziezold et al., 1998a). The seed in this
study was stratified in autoclaved sand, partially explainhg the absence of Cylindrcmpon
in the green and stratified seed. Alternative methods of transit to the seed may be through
rain splashes or f m rnachinery.
General Recommendations:
About 90% of the world's crops are established using seeds. The rnajority of
pathogens attacking major world food crops (e-g. barley, beans, corn etc.) are seedbome
pathogens (Richardson, 1979). Ginseng production in Ontario relies solely on seed as a
means of propagation (Proctor and Bailey, 1987). The need for a better understanding of
seed pathology is pivotal for the control of seed diseases.
Fungi recovered fkom ginseng flowers and seeds include some of the same species
which infect parent plants (especially the aend portions) in the garden. Flowering and
subsequent seed production take place in mid to late summer, when many of those fun@
are growing and spodating readily in the garden. Methods to reduce populations of
Alternaria and Bohytis in the canopy should be investigated and carried out.
Chemicai protectants (such as chlorothalod or mancozeb or new candidate materials)
should be applied while seed is developing on the parent plant. Applications should be
made with equipment designed to penetrate the thick canopy, with high liquid volumes to
aliow adequate coverage ofleaf surfaces. Alternaria, Botrytis and Fusarium are
facultative saprophytes, meaning they can live off dead plant material for extended periods
of t h e . In ginseng gardens they overwinter as mycelium, conidia and andior sclerotia on
straw or on soil. Rernoval of old straw and replacement with Fresh materiai at the end of
each season could signincantly reduce populations of inoculum the following spring. Since
humid, wet conditions are required for sporulation, growth and infection of these fùngi,
any improvement in air circulation within the canopy could result in fewer infections.
Some reduction in leaf wetness can be achieved through less intensive seeding rates and
removal of the side shade after rainfall. Wounding of plant tissues has a drarnatic effect on
the elevation of infection rates. Plant darnage cm be avoided by monitoring machinery
operation, manual labour crews and wind exposure (Le. sandblasting) in production areas.
Restriction in the movement of machinery and workers fbrom infested to non-infested areas
can also have sigdicant effects on reducing the incidence of disease.
Joy and Parke (1994) have found some success using a bacterium as a biocontrol for
both Altemaria and Bofrytis. Perhaps novel approaches to cultural controls, such as
introducing a UV light banier in polypropylene shade cloth to help reduce the sporulation
ofAlfernaria, may also play an important role to the control of ginseng diseases.
However, investigations into the effects of W light on ginseng plant growth and
development would need to be assessed first.
There is room for improvement in seed quality, both in its production and its testing.
Can-ying out seed stratification above ground, in a t emperature-controiled storage facility
c m avoid losses due to extreme temperature or moisture extremes. Seed treatments with
effective fungicides, such as benomyl, should be adopted to mlucimize the production of
disease-fiee, s t r a ~ e d seed.
4.4. Summary
Of the known pathogenic fungi recovered fiom diseased ginseng plants in Ontario,
species of Alternuria, Botrytis and Fzisarizcm were recovered from flowers, h i t and seed
in this study. Other fiingi recovered include Mücor and Rhizopus, along with air molds
such as AspergiIlus and Penicillium. Since culturing and stratification were carried out
aseptically and only healthy explants were chosen for culture, the fungi recovered in this
study were borne fkom flower, h i t and seed tissue only.
Alfernuria andBotryiis were among the fungi most ofien isolated 6om ginseng
flowers, fniit and seed. Alternaria spp. were isolated fiequently throughout flower, h i t
and seed development. B. cinerea was found more commonly on ginseng flowers and
developing &t than on green seed and not at ail on stratified seed. F. rosezim and other
unidentified species of Fusarizrm were recovered infrequently on flower tissue and
gradudly more ofien on M t and seed tissue cultures. It is possible that B. cinerea plays
the role of an early colonizer, making way for opportunistic fungi, such as in Fusarium.
The levels of Mzicor/Rhiropus spp. were negligible and idiequent throughout this study.
Cylindrocmpon spp. were not recovered from any tissue in this study. The populations of
fiuigi dropped again after 12 months of stratification. It is possible that earlier infections
had been expressed as seed rots and were thus eliminated from cdture. Since trends
between farm sites were not consistent, it has been concluded there was littie difference
between farm sites of the 3-year-old gardens sampled in 1997. Variation between seed
processed and stratified at different farm sites in Ontario rnay therefore be attributed to
seed handling practices by the individual grower.
The recovery of fungi fiom flower, developing fnùt and seed tissue gives strength to
the theory that fungi are entering the seed early in development. However, the
pathogenicity of these particular fùngi has not been determined and therefore they cannot
be named as the primary cause of seed rot. The question of infection pathway has yet to
be elucidated. It is possible that the kngi are entering through the style canal, thus
rnoving to the seed tissues Iater in development on the parent plant. The feasibiiity of
other pathways, such as through vasculature in the peduncle and pedicel should be
explored through assessrnent of that tissue for genera of fun@ that are cornmonly found in
the seed.
Given the warm-cool-warm, moist conditions of the strati£ication environment and the
close proxirnity of the seeds, it seems reasonable to hypothesize that small quantities of
pathogenic fûngi could produce significant proportions of infected seed, during and after
stratification.
Table 4.1 .a. Fungal diseases of Ontario ginseng2,
Powdery mildew is caused by more than one genus of îungi. The genus specific to ginseng has not yet been isolated.
Fungus Alternaria pamx Botrytis cirwea
Cylildrocarpon destructans Ftcsariim spp
l Pyfhiem spp. Phytophthora cacîorirm
Rhizoctonia solani '~owdery Mildew
Table 4. l .b, Registered chemicals available for control of hngal diseases in Ontario ginsengz.
' Brammall and Fisher, 1993; Curran, 1985; Hildebrand, 1934; Howard et al, 1994; Hudelson and Parke, 1996; Putnam, 1989; Reeleder and Brainmall, 1994; Reeleder and Fisher, 1995a; Reeleder and Fisher, 1995b; Schooley, 1997
Root
rit
* * * I
I Rhizoctoltia 1 pentachloronitrobenzene 1 Quintozene
A Itemaria Botrytis Pythi~rnl
Phythophthora
' Schooley, 1996; Schooley, 1997.
Stem *
*
I
*
mancozeb, chlorot halonil mancozeb, chlorothalonil
metalaxyl, O-ethyl-phosphonate metalaxyl, O-ethyl-phosphonate
Foliage * *
*
*
Dithane, Bravo Dithane, Bravo Ridomil, Mette Ridomil, Aliette
Berry Head * *
*
Damping off
* * rl<
t
*
Figure 4.2. Temperature fluctuations within a traditional seed stratification box taken fiom above ground air, a 10 cm depth, a 50 cm depth and a 100 cm depth. The timeline (x-axis) depicts the typical seed stratification cycle in Ontario ginseng production. (Courtesy of Proctor and Louttit, unpubiished).
Figure 43. Fungi isolated from explant cultures of ginseng flowers, fmit and seed. Of the many cultures recovered in this study, these represent those fiingi which are known pathogens of Ontario ginseng. A, C and E depict A. panax, B. cinerea and F: roseurn (respectively) afier t week of culture. B, D, and F depict A. panax, B-cinerea and E roseum afier 6 weeks of culture.
Figure 4.4. Fungi recovered fiom explant material of ginseng flowm, fruit and seed. Photos taken under compound microscope at 400 x magnification. A. Depicts myceïium of A. panmc with spore (conidia) primordia. B. Depicts fully-fomed conidia of A. panax (courtesy of R. Reeleder). C. Depicts clusters of conidia of B. cinerea. D. Depicts macroconidia of R roseum.
Figure 4.5. Common visual symptoms of rot in stratified ginseng seed Eom Ontario. A. Depicts "milky" seed. B. Depicts "brown" rot of seed. The arrow indicates the hicular end of the seed and the location of the embryo.
Figure 4.6. Culture and subsequent identification of fungi recovered from ginseng flowers, h i t and seed. ADepicts culture of endosperm (with embryo) of green seed ont0 WA with antibiotics, using the sterile technique. B Depicts identification of fùngal cultures using characteristics of cultures, mycetium and spores.
Table 4.2.a. Percentages of fungi recovered per explant type, from al1 media types. Explant material was recovered from flower buds and newly-opened florets of ginseng at time O for 1996 and 1997 flowers. Samples were gatliered from 4-year-old plants at Farm A. N=6.
Explant Type
bud petal
ant her pistil
*Within a column, means followed by the sarne letter are not significantly différent according to LSD test at P=0,05,
Bokytis cinerea
0.0 a 1.3 a
Altcr~~aria spp. 0.0 a* 5.5 a
Table 4.3.a. Percentages of fungi recovered per explant cultures type, from al1 media types. Explant material was recovered from flower buds and newly-opened florets of ginseng at time O for 1996 and 1997 flowers. Samples were gathered from 3-year-old plants at
4.2 a 1.3 a
-- .
ovary ovule
farm A, B, and C. N=9
Fusarilm roselrnt
0.0 a 0,O a
1.3 a 0.0 a
.. .
0.0 a 0.0 a
1.3 a 1.3 a
Fzr sarizun spp.
0.0 a 0,O a
0.0 a 0.0 a
*Witliin a column, riieaiis followed by tlic sanie lettcr are not sigiiificaiitly diffcrent accordirig to LSD test at P-0.05.
Mucor spp. /Rhizopus spp.
0.0 a 0,O a
0.0 a 0.0 a
Explrnt Type
bud
anthei pistil ovary ovule
0.0 a 0.0 a
F~rsarizrni I ' O S ~ Z ~
0.0 a
0.0 a 0.0 a
. . . . . . .
0.0 a 0.0 a
A Z&waria spp. 0.0 b*
1.8 ab 0.9 b 0.9 b 0.0 b
- ---
0.0 a 1.3 a
Bo ti y/is ciiwea 0.9 ab
Fztsarizrnl spp.
0.0 a
Mzicor spp. /Rhizopirs spp.
0.0 a
0.9 ab 0,O b 0.0 b 0.0 b
0.0 a 0.0 a 0.0 a 0.0 a
0.0 a 0.0 a 0.0 a 0.0 a
0.0 a 0.0 a 0,O a 0.0 a
Table 4.2.b. Percentages of fungi recovered from each culture media type. Explant material was recovered from flower buds and newly-opened florets of ginseng at time O for week 1996 and 1997 flowers. Samples were gathered frorn 4-year-old plants at Farm A.
*Wiiliin a column, means followed by the same letter are not significantly different according to LSD test al P=O.O5,
Table 4.3 .b. Percentages of hngi recovered from each culture media type. Explant material was recovered fi om flow er buds and
Fzlsari~rn~ rose l m
0.0 a 0.0 a 0.0 a
Bolrytis cir~erea
1.3 a 0.0 a 1.3 a
Media Type
MRBA PDA WA
newly-opened florets of ginseng at time O for 1996 and 1997 flowers. Samples were gathered from 3-year-old plants at Farms A, B and C . N=18.
AZtemaria spp. 3.5 a* 1.3 a 0.7 a
F~dsarizïn~ spp,
0.0 a 0.0 a 0.0 a
1 PDA 1.3 a 1 0.4 a 1 0.0 a 1 0.0 a 1 0.0 a I
Mucor spp. ni>hizopzis spp.
0.0 a 0,O a 0.7 a
Media Type
MRBA
Table 4.2.c. Percentages of fungi recovered per year. Explant material was recovered from flower buds and newly-opened florets of ginseng at time O for 1996 and 1997 flowers. Samples were gathered from 4-year-old plants at Farm A. N 4 8 .
Alteruaria spp. 1.8 a*
1 WA 1 1.4 a 1 2.7 a 1 0,O a
*Witliin a column, nieans followcd by the sanie letter are iiot significaiitly different itccording to LSD test iit P=O.OS.
Botrytis cinerea
0.0 a
0.0 a 1 0.0 a
Year
l
1996 1997
I 'Witliin a column, means followed by the same letter are not significantly differcnt according to LSD test at P=0.05,
Fusarium rosezm
0.0 a
A /teruaria spp . 1.8 a* 1.8 a
Fusaritrnt
spp. 0.0 a
Bo try iis ciwlea
1.8 a 0.0 b
Mucor spp. /Rhizoptcs spp.
0.0 a
Ftrsarim rose un1
0,O a 0.0 a
Fzisurizrnr
sp p. 0.0 a 0.0 a
Mucor spp. /Rhizopirs spp.
0.4 a 0,O a
Table 4 .3 .~ . Percentages of hngi recovered per farm site. Explant material was recovered fiom flower buds and newly-opened florets of ginseng at time O for 1996 and 1997 flowers. Sarnples were gathered from 3-year-old plants at Farms A, B and C . N=12.
--
Witliin a coluiiin, means followed by the sanie letter are noi significaniiy diiïerent according to LSD test at P=0.05.
Table 4.2.d. Percentages of hngi recovered per explant type. Explant rnaterial was recovered fiom newly-opened florets through developing seed of ginseng, at tirnes O through 40 for 1996 and 1997 seed. Samples were gathered from 4-year-old plants at farm A. N=30,
Farm
A
Botryfis cimrea
0.0 a
A Itemaria spp. 1.3 a*
*Witliin a column, nieans followed by the same letter are not significanlly different according to LSD test at P=O.OS.
Table 4.3 .d. Percentages of fingi recovered per explant type. Explant material was recovered from newly-opened florets through developing seed of ginseng, at times O through 40. Samples were gatliered from 3-year-old plants at farrns A, B and C. W 4 5 .
Ir,rsariztnt rose tmz
0.0 a
Mucor spp. /Rhizoyzrs spp.
0.5 a 0.5 a 0.6 a
firsarizmt spp.
0.0 a
F~rsariutiz rose~it?~
1.4 a 0.8 a 0.8 a
Botrytis cinerea 18.1 a 8.6 b 4,5 b
Explant Qpe
pistil ovary ovule
Mircor spp. /IRhizopr/s spp.
0.0 a
Fzisarilrnz spp .
0,6 a 0.3 a 0.0 a
A Iternaria spp. 8.0 a* 4.1 b 1.4 b
*Niillin a colurnii, nieans follo\ved by the sariie lctlcr are not significantly differerit üccording to LSD test at P=0.05.
Fusari rrnt rose rrrn
0.7 a 0.0 a 0,O a
Botrytis cinwea
7 , 6 a 1.1 b 0,7 b
Ex p h n t Type
pistil ovary ovule
Alternaria spp.
18,3 a* 2.0 b 0,O b
Ft~sarit~nî spp.
0,7 a 0,2 ab 0.0 b
Mircou spp. /Rhizuplcs spp.
0.9 a 0.4 a 0.0 a
Table 4.2.e. Percentages of fungi recovered per media type. Explant matenal was recovered froin newly-opened florets through developing seed of ginseng at times O through 40 for 1996 and 1997 seed. Samples were gathered from 4-year-old plants at farm A. N=30.
*Within a column, ineans followed by the sanie letter arc riot significantly different according to LSD test at P=0.05.
Media Qpe
MRBA PDA WA
Table 4.3 .e. Percentages of fungi recovered per media type. Explant materiai was recovered fiom newly-opened florets through developing seed of ginseng, at times O through 40. Sarnples were gathered from 3-year-old plants at farms A, B and C. N=45.
A Ilen iaria spp. 3.6 a* 4.2 a 5.8 a
WA 1 7.2 a 4.8 a 1 0.7 a 1 0.0 a 0.9 a 1 *Witliin a column, nieans followed by the same letter are not significantly different according to LSD test at P=0.05.
Table 4.2.f. Percentages of fungi recovered per year. Expiant material was recovered from newly-opened florets through developiny seed of ginseng, at times O through 40 for 1996 and 1997 seed. Samples were gathered from 4-year-old plants at farm A. N=45.
Botiylis cir~erea
1.9 b 16.4 a 12.8 a
Mucor spp. /Rhizopzrs s p p.
0.0 a 0.4 a
Fzisarim spp.
O,5 a 0.4 a
Media Type
*Witliin a colutiin, tiicans followed by the saine lcttcr iirc not sigxiiîicantly difîcrent according to LSD test at P=0,05,
Fzuarium rosezim
1.6 a 1.1 a 0.3 a
Alterraaria
F~isarium spp .
0.6 a 0.3 a 0.0 a
B»syfis
Botrytis cirierea 10.6 a
Firsarirrnt rose rrnt
1,3 a
Year
1996
M~tcor spp. LUhizopirs spp.
0.3 ab 0,O b 1.4 a
Alteriraria spp, 2.4 b*
rusezm 0.0 a 0.0 a
Fusariirrn spp,
0.6 a
cirwea 0.2 b 4,4 a
MRBA PDA
Mtrcor spp. /Rhizopits sp p.
1 , l a
spp, 4.6 a* 8.5 a
Table 4.3.f. Percentages of fungi recovered per farm. Explant material was recovered fiom newly-opened florets through developing seed of ginseng, at tirnes O through 40. Samples were gathered from 3-year-old plants at farms A, B and C. N=45.
Table 4.2.g. Percentages of fungi recovered per time. Explant material was recovered f'rorn newly-opened florets through developing seed of ginseng, at times O through 40 for 1996 and 1997 seed. Samples were gathered fiorn 4-year-old plants at farm A. N=l8.
Farm
1
A B C
*Witliin a column, means followed by the sanie letter are not significantly different according to LSD test at P=O.OS.
A /ternaria spp . 7.2 a* 8.5 a 4.6 a
Botryfis cinerea
4.4 a 0.6 b 4.4 a
Time
1
O 8
20 33 40
Fztsuri nnt roseztrn
0.0 a 0.7 a 0.0 a
*Wii.liin a coluinn, imans followed by the sanie letter are iiot significantly different according to LSD test at P=0,05,
A It ernaria spp.
0.4 c* 3.7 bc 6.9 ab 8.8 a
2.8 bc
Fzrsariîinz spp.
0.0 a 0.5 a 0.4 a
Bo/rytis cirîerea
0,9 c 5.1 bc 11.6 b 21.8 a 12.5 b
Mtccoi spp. /.hizopris spp.
0.0 a 1.1 a 0.2 a
Filsariim roseztn~ 0.0 a 2.3 a 0.4 a 1.8 a 0.4 a
Fusariuni spp.
0,O a 0.0 a 0,9 a 0.4 a 0.0 a
Mircor spp. /Rhizopits spp.
L
0.4 ab 0.4 ab 0.0 b 1.8 a 0.0 b
Table 4.3.g. Percentages of fùngi recovered per time. Explant material was recovered from newly-opened florets through developing seed of ginseng, at times O through 40. Samples were gathered from 3-year-old plants at farms A, B and C. N=27.
Time 1 Altemaria ( Botrytis 1 Ftrsari~rm ( Ftrsarizrnl 1 Mzrcor spp.
Table 4.2.h. Percentages of hngi recovered per explant type. Explant material was recovered from green seed of ginseng, at times 85 to 168 in1996 and 1997. Samples were gathered from 4-year-old plants at farm A. N=8.
O 8
20 33 40
I coat 1 3.3 a 1 0.0 a 10.0 a 1 1 1,7 a 1 3,2 a 1 *Witliin a colunin, mcans followd by the m i e lettcr are not significantly difierelit according to LSD test at P=O,OS,
"Witliin a colunin, means followed by tlic saine lettcr arc not significantly diffèrent according to LSD test at P=0,05.
spp . 0.6 b* 7.0 a 11.1 a 9.0 a 6.1 a
Explrnt Type
e t e
Table 4.3.h. Percentages of fungi recovered per explant type. Explant material was recovered from green seed of ginseng, at times 85 to 168 in 1997. Samples were gathered from 3-year-old plants at farms A, B and C. N= 18.
cinerea 0,O b 2.4 ab 4.0 ab 3.1 ab 6.2 a
Alîerr~aria spp. 2.5 a*
*Withiii a columti, nicans followed by the sanie lcttcr are not significantly different according to LSD test at P=0.05,
Expla nt Type
e t e coat
rosetm 0.0 a 0.0 a 0.0 a 1.2 a 0.0 a
Botrytis cirwea
2.5 a
Allermwin spp. 8.3 a* 5 , l a
spp. 0.0 a 0.3 a 0.0 a 0.6 a 0.6 a
Fzisari~int rosem 11.5 a
/7(hizopîis sp p. 0,O b 1.6 a
0.3 ab 0.0 b 0.3 ab
Robytis cirlerecr
8.8 a 0.0 b
Ftrsarinrn spp,
6.5 b
Mucor spp. /Rh zzoplrs ' spp.
4.2 a
Fzlsarizi~z rosezm
8.2 a 5.6 a
Fzrsarizin~ spp.
15.2 a 9.2 a
Mzrcor spp. /Rhizopr rs spp.
3.2 a 0.0 b
Tabie 4.2.i. Percentages of fungi recovered per media type. Explant material was recovered from green seed of ginseng, at times 85 to 168, in 1996 and 1997. Samples were gathered from 4-year-old plants at farms A. N=12.
Witliin a column, rneans followed by the same lettcr are not sigriificantly different according to LSD test at P=O.Q5,
Media Type
MRBA PDA
PARP
Table 4.3.i. Percentages of fùngi recovered per media type. Explant material was recovered fiom green seed of ginseng, at times 85 to 168, in 1997. Samples were gathered from 3-year-old plants at farms A, B and C. N=12.
Media Type 1 Altmaria 1 Botrytis 1 F~rsarizrnz 1 Fusarium 1 Mitcor spp. 1
Alternaria spp, 0.0 b* 2.0 b 10.5 a
1 spp. 1 cirïerea 1 rosez~rn 1 spp. /Rhizoprrs spp. [ MRBA 1 4.9 b* 0.0 b 2.8 a 1 22.9 a 3.5 a 1
Botytis cirlerea
0.0 a 3.1 a 0.0 a
PDA 1 15.2 a 13.3 a 1 8.9 a 5.5 b 1.3 ab 1 PARP 1 0.0 b 1 0.0 b 1 9.0 a 1 8.2 b 1 0.0 b 1
*Witliin a coluniri, means followed by the sanie letter are not significantly different according to LSD test at P=0.05.
Fzm-wiunt rose zm 13.4 a 8,3 a 20,5 a
Table 4.2.j. Percelitages of fungi recovered per year. Explant material was recovered from green seed of ginseng, at times 85 to 168, in 1996 and 1997. Samples were gathered fiom 4-year-old plants at farm A. N= 12.
Year ( Alienlaria 1 Bolrytis 1 fitsarizml 1 Fztsarittm 1 Mltcor spp. 1
Fzrsarizrni spp.
5 .3 b
Mucor spp. /Rhizoprrs spp.
4,2 a
*Witliin a column, nieans followed by ilie sanie lcttcr are not sigriificantly different according to LSD test at P=0,05.
spp .
5.0 b 5,1 a
ciriereu
250 a 1 0,O a
r o s w n 1996 4.1 a I .O a*
spp . i
/Ahizoprcs spp.
2.1 a 4,1 b 4.1 b
Table 4.3.j. Percentages of fùngi recovered per farm. Explant material was recovered fiom green seed of ginseng, at times 85 to 168, in 1997. Samples were gathered from 3-year-old plants at farms A, B and C. N=12.
1 C 1 2.8 b 1 6.3 a 7.6 a 1 11.1 a 4.8 a 1 *Witliin a coluniii, nicans followed by the slinie letter arc not significantly différent according to LSD test at P=0.05.
Farm
A
Table 4.2.k. Percentages of fungi recovered per time. Explant material was recovered from green seed of ginseng, at times 85 to 168, in 1996 and 1997. Samples were gathered fiom 4-year-old plants at farm A. N=10.
A Iternaria spp. 9.7 a*
- - -
*WiOiin a coluriin, ineans followed by the same lctter are not significantly different according to LSD test at P=O,O5,
Time
85
Table 4.3.k. Percentages of fungi recovered per time. Explant material was recovered from green seed of ginseng, at times 85 to 168, in 1997. Samples were gathered fiom 3-year-old plants at farms A, B and C. N=18.
Bolryf is chereu
1.4 a
Aiternaria spp. 0.8 a*
Fusaritrn~ rosem
6.9 a
Bolryt is cinerea
2.5 a
Time
85 168
Ft rsar izcrn spp.
14.4 a
*Within a coluriiri, iiicans fol1owed by the sanie letter are rio[ significaritly differcrit accordirig to LSD test at P=O.OS.
A Iterr îaria spp. 6.1 a* 7.3 a
Botrytis cirierea
3.3 a 5.6 a
Mucor* spp. /Rhizopirs sp p.
0.0 b
Fusariuni roseimi 3,2 b
Fzrsnri~ini rosezrrn
5.1 a 8,7 a
Frtsarizr nt
spp, 4.1 b
Mucor spp. /Rhizop~rs spp.
1.6 a
F~rsarizrni spp.
8.7 b 15.7 a
Mitcor spp, /Rhizoplrs sp p.
1.4 a 1.8 a
Table 4.2.1. Percentages of fingi recovered per explant type. Explant material was recovered fiom green seed and stratified seed of ginseng, at times 305 and 459, for 1996 seed. Samples were gathered from 4-year-old plants at fann A. N=4.
*Within a column, rneans followed by tlie same letter are not significantly different according to LSD test at P=0.05,
Table 4.2.m. Percentages of hngi recovered per media type. Explant material was recovered from green seed and stratified seed of ginseng, at times 305 and 459, for 1996 seed. Samples were gathered from 4-year-oid plants at farm A. N=4.
Fzrsariunt spp.
6.25 a 6.0 a
Fzrsarizrnt rosezm
8.3 a 10.5 a
*~ithinacoluinn, means followed by the sanie letter are not sigiiificantly different according to LSD test at P=O.O5.
Mlrcor spp. ~ t i z o p t i s spp.
4.0 a 6.3 a
Botrytis cirierecr
0.0 a 8.3 a
Explant Type
e-te coat
Table 4.2.11. Percentages of fungi recovered per time. Explant material was recovered from green seed and stratified seed of ginseng, at times 305 and 459, for 1996 seed. Samples were gathered from 4-year-old plants at farm A. N=4.
Alternaria spp .
33.5 a* 20.8 a
Fzrsarizm spp,
8,3 a 4,O a
Media Type
MRBA PDA
Mucor spp. /Rhizopits spp.
8.3 a 2.0 a
Botrytis cirîerea
0.0 a 8.25 a
A lf er-naria spp.
16.8 b* 37.5 a
305 459
Fzrsarizrm roseiint 10.5 a 8.3 a
*Within a colunin, iiieaiis followed by the same Iettcr are not significantly différent according to LSD test at P=O,OS.
spp . 25.0 a* 29.3 a
cirlerca 8 .3 a 0.0 a
rose rini 8.3 a 10.5 a
spp. 10.3 a 2.0 a
/Rhizoptrs spp, . 8.3 a 2.0 a L
-CHAPTER 5-
AN ASSAY FOR ENDO-P-MANNANASE IN GINSENG SEED
5.1. Introduction
In many seeds, dormancy is imposed (at least partially) by the mechanical force of the
tissues surrounding the embryo, thus preventing the emergence of the radicle. This type
of dormancy is termed "coat-enhanced domancy", as the testa is thought to be
responsible for the prevention of radicle emergence (Bewley and Black, 1994). This type
of domiancy exists in ginseng seeds because radicles excised fiom surrounding tissues will
often germinate (Hovius, 1996). However, it is frequently the endosperm tissue
underlying the testa which presents the greatest barrier to radicle growth and emergence
(Bewley, 1997b). Mature, dehisced ginseng seed does not always germinate and perhaps
this is due to the force imposed by the endosperm. It c m be reasoned, therefore, that a
weakening of this tissue could help facilitate radicle protrusion (Bewley, 1997a).
This weakening involves partial enzymatic degradation of the ce11 walls. The
structural component of endosperm cell walls, hernicellulose, is ofien found in the f o m of
galactomannans or mannose polymers within many seeds ( e g tomato, lettuce, Datzrra
ferox) (Bewley, 1997b). These galactomannan reserves are mobilized following
germination to support the growth and development of the emerging seedling. Endo-P-
mannanase is responsible for hydrolyzing the P(1+4)-mannose links in the mannan
backbone. Two other cell wall hydrolases are responsible for fùrther degradation of the
gdactomannan. In some legumes, the aleurone layer is the site of synthesis and secretion
of these ceiI w d hydrolases (Reid and Meier, 1972), while in other endospermic seeds,
the endospexm celis are the sites of hydrolase synthesis and secretion (Seiler, 1977).
BewIey (1997b) found some relationships between the breakhg of domancy and the
weakening of ce11 w d s by endo-P-mannanase in the seeds of tornato, lemice and Datura
ferox. He also found gibberellin as a prornoter of endo-P-mannanase synthesis in some
species, and abscisic acid to be a block. This is interesting since GA stimulates the
production of other hydrolytic enzymes ( e g a-amylase).
The purpose of this study was to i) determine ifginseng seed tissues contain detectable
amounts of endo-P-mannanase and ii) determine ifGA3 has any effect on the amount of
endo-P-mannanase liberated £tom the seed tissues. The first objective was carried out
using mature, stratified seed since this is most Iikely the developrnental stage that would
show endo-P-mannanase activity. The second objective was carried out using immature
seed (containing extremely low levelç of endo-P-mannanase) in order to facilitate the study
of increasing ievels of the enzyme over tirne.
5.2. Materials and Methods
Stratified seed and endo-e-mannanase:
Seeds that have undergone 12 months of stratification were collected fiom a local
grower in September of 1997. This seed required a 3 to 4 month chilling period to
complete its dormancy at this time. In November, seeds of extra-large (5.6 to 6.0 mm)
and large (5.2 to 5.6 mm) grade were sampled for this assay. Ali dissection took place in
the lamioar flowhood using sterile techniques. Intact seeds were sterilized in a 10%
solution of household bleach (0.6% sodium hypochlorite) and rinsed in autoclaved,
deionized water for 3 minutes. They were rinsed 3 times and blotted dry with ~ h a t m a a @
filter paper. The endocarp was sliced along the seam with a sterile steel blade in order to
cut the seed into equal haives and the seed coats were discarded. Excised embryos were
meanired and the endosperm halves were saved and categorized according to the size of
their ernbryo. "Smali" embryos measured less than 1.5 mm, "medium" embryos ranged
fkom 1.5 to 3.0 mm and "large" embryos measured Çom 3 .O to 4.5 mm. Six hdves of
endosperm and 12 embryos were ailocated per sample.
These tissues were ground with a mortar and pestle after being fiozen at -10 '~.
Exactiy 0.2 ml of citrate buffer (pH 5.0) were added, along with 15-20 grains of washed
sea sand to aid grinding. Gradually, 0.7 ml of citrate buffer were added with additional
ghding. The resulting mixture was poured out into an Epindorf tube and 0.5 ml citrate
buffer was used to rime the remaining materid from the instruments into the Epindorf
tube. The homogenate was then centrifuged at high speed for 3 minutes. The supernatent
was pipetted off, topped up with 1.5 r d citrate buffer and held for plating.
Two pl of each sample solution were blotted onto the wells of pre-made agarose
matrix plates containing a locust bean galactomannan substrate, that was stained with
Congo red dye (Downie et al., 1994) (Figure 5.1). Inoculated gel plates were incubated at
2 3 ' ~ + 3 ' ~ for 12 hours. The plates were then washed in citrate buffer (pH 7.0) to
remove extra liquid and enhance the colour of the Congo Red dye. The diameter (mm) of
the clearing zones surroundhg the wells are quantitatively related to endo-P-mannanase
activity in that tissue sample.
Green seed and GA3:
Freshly harvested ginseng seeds were collected f?om a local grower in September of
1997. In November, these seeds of x-Iarge (5.6 to 6.0 mm) and large (5.2 to 5.6 mm)
grade, containing tiny immature embryos (< 1 -0 mm), were sampled for this assay. Al1
dissection took place in the laminar flowhood using steriie techniques. Intact seeds were
sterilized in a 10% solution of household bleach (0.6% sodium hypochlorite) and rinsed
with autoclaved, deionized water for 3 minutes. They were rinsed 3 times and blotted dry
with ~ h a t m a r ? filter paper. The endocarp was sliced dong the seam with a sterile steel
blade in order to cut the seed into equal halves. Seed coats and embryos were discarded
and the endosperm halves were kept for tissue culture. Six halves of endosperm were
used per sample.
The endosperm halves were cultured (6 halves per petri dish) in 5x10'~ M GA3 (2
pprn), 5 x 1 0 ~ M GA3 (200 ppm), 105 GA3 (400 ppm) and O M GA3 (control) to test for
stimulation of endo-P-mannanase synthesidsecretion. The endosperm halves were flooded
with 0.6 ml of GA solution inside each petri dish (mini, 1Ox601nm, Fisher Scientific Co.)
Endosperm and surrounding solutions were sampled at 0, 48, 72 and 96 hours. The
samples of solution were immediately used for enzyme analysis on agarose gel plates. The
endosperm tissues were grounded up and centrifuged according to the methods outlined
for "stratified seed and endo-P-mannanase" experiment above.
Two yl of each sample solution were blotted onto the wells of pre-made gel plates
containing a mamanase substrate (Figure 5.1). Inoculated gel plates were incubated at
2 3 ' ~ + 3 ' ~ for 12 hours. The plates were then washed in citrate buffer (pH 7.0) to
remove extra liquid and enhance the colour of the Congo Red dye. The diameter (mm) of
the well, which was digested by sample's enzymes, were measured to estimate endo-e-
mannanase activity in that tissue sarnple. Two individual trials were averaged together for
analy sis.
5.3. Results and Discussion
Stratified seed and endo-P-mannanase:
The fist set of experiments resulted in significant levels of enzyme activity fiom tissue
samples of ginseng seed (Table 5.1). These prelirninary trials indicated that endo-e-
mannanase can be found in stratified, dehisced ginseng seed. Levels of endo-p-mannanase
were notably greater in endosperm tissue than in embryo tissue. This observation agrees
with findings of Bewley (1997b), who noticed endo-fhnannanase activity to be greatest in
the endosperm cells of tomato and lettuce. Living endosperm tissue cm produce its own
hydrolytic enzymes (Bewley, 1994), therefore one would expect to fïnd more endo-P-
mannanase activity in these tissues. Recent research in the characterization of ginseng
endosperm seems to indicate that it is living tissue (Yu, and KUn, 1992; Yu et al., 1992),
thus supporting the idea that it has a synthesis or secretory role for this enzyme. The
amount of enzyme activity in the larger embryos warrants some simcance since there is
more activity than one might expect to find d f i s e d £kom the endosperm tissue.
Endo-P-mannanase activity was found to be greatest in the largest embryos, and in the
endosperm of those large embryo seeds. Since this enzyme is linked to post-germination
events such as storage reserve rnobilization, it is not surprising to fïnd that the enzyme was
found in greater quantities in seed with more mature and larger embryos. It was
surprising, however, to £hd such large quantities of endo-P-mannanase activity in various
ginseng seed tissues. This suggests that this enzyme may play an important role in seed
development of this species.
Green seed and GA1:
Endo-P-mamanase activity was found in green seed, although the amounts were
extremely srnall compared to activity found in stratified seed (Figure 5.2). Smailer
amounts can be expected since embryo growth and development activities are low during
the first few months of development (Proctor and Louttit, 1995). The solutions described
here were obtained by sampling the liquid mounding the endosperm cultures. These
solutions gave more endo-fi-mannanase activiq than the endosperm tissue samples.
Bewley (199%) stated that most mannanases are soluble and are therefore able to diffuse
easify among cells. This would explain the apparent "drain" of the enzyme from the
endosperm tissue into the surrounding culture solutions. It is possible that the GA not
only stimulates the synthesis of these enzymes but also the secretion of them into
surrounding tissues.
GA3 appeared to have influenced the activity of endo-P-mannanase over time. The
effect was most notable at 105 M GA3 for the solution samples. The highest level of endo-
p-manaanase activity could be found after culturing endosperm in a 10" solution of GA3
for 96 hours. The values of activity found at 5x103 GA aRer 48 hours and O GA d e r 48
hours were atypical. In tomato seeds, gibberek also induced an increase in endo-P-
mannanase activity and led to the initial suggestion that GAs induce germination through
the weakening of endosperm cell walls (Ekwley, 199%). GAs also play a role in the
stimulation of a-amylases for the breakdown of storage reserves in the endosperm cells
(Bewley and Black, 1994).
5.4. Surnmary
Endo- B-mannanase has importance in the post-germination events of some
endospermic seeds. Bewley (199%) found this enzyme to play a role in the break down
and mobilization of ceil w d storage reserves, namely galactomannans in legumes, tomato,
lettuce and Damra ferox. Endo-P-maananase has been found increasingly in many other
seeds (including embryo tissue), as in the case of the green and stratified North American
ginseng seeds in this study.
Gibberellins have been associated with the promotion of the synthesis and secretion of
endo-P-mannanase in some seeds. This study's hd i igs give strength to this concept.
The amount of enzyme activity in embryo tissue may indicate that the ginseng ernbryo is
the natural source for G 4 a signal which passes into the surrounding endosperm ceils
causing their secretion of endo-P-maonanase and the sofiening of neighbonng endosperm
celi walls. Previous research (Yu, and Kim, 1992; Yu et al., 1992) indicated that ginseng
endosperm is living tissue, suggesting that these cells may be the site of endo-P-mannanase
synthesis as weii as its target site of action. Further research into the relationship between
endo-P-mannanase, GA and endosperm may permit a better understanding of ginseng seed
development. In addition, this knowledge may help increase the precision in timing of GA
application to hasten ginseng seed development.
It is possible that this enzyme also plays a role in the sofiening of the seed coat, for
dehiscence during stratification, and the release of coat-imposed domancy. Evaluation of
seed coat tissue for endo-P-maonanase activity should be further investigated.
Figure 5.1. Typical results of an assay for endo-a-mannanase activity. Two microlitre aliquots were placed ont0 the wells of this agarose plate. The plate contains locust bean galactomannan as the substrate for the endo-a-mannanase and Congo red dye for ease of activity detection. The quantity of enzyme activity is related to the diarneter of the clearing zones.
Table 5.1. Activiw @kat ) per seed tissue of endo-P-mannanase in stratified ginseng seed.
Tissue samples include: srnail, medium and large embryos; endosperm f?om srnall, medium
and large embryo seed.
Embryo Tissue PKAT Endosperm Tissue PKAT
Medium 1 94.84 Medium 4158.15
~ Q U Y h l O
aldues anssg lad aseueuueu 40 leyd a 6 w a ~ v
GENERAL CONCLUSIONS
Previous studies have demonstrated that spring seed may be a viable method of
reducing the long dormancy of ginseng seed fiom 18-22 months to 8-9 months.
Gibbereilins, cytokinins, drying and pre-plant hydration were used to accelerate
germination. The application of GA alone gave germination percentages similar to the
application of GA with B A Therefore, the cytokinins did not provide any added benefit
to ginseng germination.
Previous studies have stated that GA4 has greater potential for biological activity than
other gibbereliins. A mixture of two gibberellins (G& and GA7) has the potential to
access two types of receptors in the seed tissue, whereas GA3 may only access one
receptor type. Therefore, GA4t7 would be a better germination promoter for ginseng than
GA3. Unfortunately, the cost of G&+7 is a limiting factor in its applicability to commercial
ginseng production.
Drying had some promothg affects on germination, however, these and other
treatment effects were difficult to detect due to generdy poor emergence (up to 33%) in
1996 and 1997 trials. Low emergence levels were attributed to high seed rot, an effect
that was increased by the osmotic effect of GA. Seed quality plays an important role in
spring seeding. Timely and efficient seed handling and proper stratification procedures
could eliminate environmental stresses and possibly prevent infection of stressed seed.
Irnproved rnethods of quality control should be enforced to prevent the contamination of
seed through the spread of inoculum via diseased seed.
Ginseng fh i t develops non-symmetncdy on the umbel leading to a range of seed
sizes at harvest. Seed size had a significant effect on seed development, emergence and
seedling yield. Since extra-large and small seed sizes produced significantly low seedling
emergence and since Ontario ginseng seed prices have dropped in the last decade, then
perhaps extreme seed sizes should be eliminated before planting. This could maximize
emergence and improve plant stands.
Diseases are a Limiting factor in ginseng production. Many of the same fungai groups
which cause ginseng plant diseases can be found in and on the seed. Examined flower,
f i t and seed samples contained many fimgal species. Altemaria spp. were found more
frequently and in the largest quantities on all sampled tissues. Botrytis cinerea was found
more often on flowering and h i t ing tissue while Fusarium rosezïm md Fzrsarizcm spp.
were isolated more fiom seed. It is possible that Bohytis is an early colonizer of
developing seed, whose population drops off, allowing an oppominist funpi Iike Fzmzrizcm
to colonize. The recovery of these fùngi fiom early flower and seed development as weil
as in seeds supports the theory that pathogens may be entering the seed while it is still
developing on the parent plant. However, without specific pathogenicity testing, the
aforementioned fùngi cannot be labeled as sources of disease in ginseng seed.
Endo-P-mannanase is a hydrolytic enzyme that is thought to be responsible for ce11 wall
degradatioa and liberation of storage compounds after germination of other horticultural
crops. The presence of endo-e-mannanase was detected in green and stratified seed fiom
this study. Since GA is known to stimulate other hydrolytic enzymes (ie. a-amylase), the
application of GA3 at 400 ppm (10-~ M) likely stimulated the production of endo-P-
maonanase in the endosperm tissue and surrounding solution. Further investigation should
be performed to q u a n t e the presence of this enzyme in other seed tissues and to elucidate
the maximum production~secretion for this enzyme in seed tissue at various develo pmental
stages.
Agrios, G.N. 1988. Plant Pathology. 3d ed. Academic Press Lnc. San Diego, CA..
Ali, A., Souza Machado, V. and Hamill, A.S. 1990. Osmoconditioning of tomato and onion seeds. Scientia Horticultwae 43: 2 13-224.
Bai, D., Brandie, J. and Reeleder, R 1997. Genetic diversity in North Amencan ginseng (Panmc quinquefohs L-) grown in Ontario detected by RAPD analysis. Genome 41: 11 1-1 15.
Baranov, A. 1966. Recent advances in o u knowledge of the morphology, cultivation and uses of ginseng (P. ginseng C.A. Meyer). Journal of Economic Botany 20:403-406.
Bewley, J.D. 1997a. Seed germination and domancy. The Plant Cell 9: 1055-1066.
Bewley, J.D. 1997b. Breaking down the walls - a role for endo-beta-mannanase in release from seed dormancy? Trends in Plant Science 2: 464-469.
Bewley, J.D. and Black, M. 1994. Seeds: Physiology of Deveiopment and Germination. 2nd ed. Plenum Press, New York.
Boland, G.J. and Hall, R 1987. Epidemiology of white mold of white bean in Ontario. Canadian Journal of Plant Pathology 9: 2 1 8-224.
Brammall, R 1994. Alternaria Blight. Botrytis Blight. In: Howard, RJ., Garland, J.A. and Seaman, W.L. (ed.). Diseases and Pests of Vegetable Crops in Canada. The Canadian Phytopathological Society and Entomoiogical Society of Canada. Ottawa, ON. pp. 296-297.
Brammall, R 1997. "Seed Quality", a presentation at the h u a 1 Agrospray Ginseng Conference and Trade Show. January 28-29. Delhi, ON.
Brammall, R and Fisher, P. 1993. Bohytis blight of ginseng. Ontario Ministry of Agriculture, Food and Rural Mairs. Order No. 93-07 1.
Brayford, D. 1992. Cylindrocarpon. In: Singleton, L.D. (ed) Methods for Research on Soilbome Phytopathogenic Fun@. pp. 103-106.
Brenner, M.L. and Cheikh, N. 1995. The role of hormones in photosynthate partitionhg and seed fUing. In: Plant Hormones: physiology, biochemistry and molecular biology. (ed. Davies, P.J.) Kluwer Acadernic Publishers. The Netherlands.
But, P.P.H., Hu, S.Y. and Cao, H. The ginseng plant: products and quality. Ln: Bailey, W.G., Whitehead, C., Proctor, J.T.A., Kyle, J.T. (ed) Proceedings of the International Ginseng Conference, Vancouver 1994. pp.24. Simon Fraser University. Burnaby, B.C.
Carpenter, S.G. and Cottam, G. 1982. Growth and reproduction of American ginseng (Panax quinquefolius) in Wisconsin, U.S.A. Canadian Journal of Botany 60: 2692-2696.
Carpenter, W.J. and Ostmark, E.R 1992. Growth regulators and storage temperature govem germination of Coreopsis seed. HortScience 27: 1 190-1 193.
Chang, Y.S. 1995. Ginseng and health In: Bailey, W.G., Whitehead, C., Proctor, J.T .A., Kyle, J. T. (ed) Proceedings of the International Ginseng Conference, Vancouver 1994. pp.23. Simon Fraser University. Bumby, B .C.
Chen, Y., Sun, C. and Zheng, G. 1984. Methods to promote the seed germination of P m ginseng. Kexue Tongbao Vol. 29 No. 3.
Cho, D.H., Park, K.J., Yu, Y.H., Ohh, S.H. and Lee, H.S. 1995. Root-rot development of 2-year old ginseng (Panax ginseng C.A. Meyer) caused by Cylindrcarpon destnrctans (Zinssm.) S cholten in the continuous cultivation field. Korean Journal of Ginseng Science 19: 175-180.
Choi, S.Y. 1990. Electrophoretic variations in protein and some enzymes during stratification of P m ginseng seeds. Korean Journal of Crop Science 35: 3 3 4-3 4 1.
Clear, RM. and Patrick, S.K. 1993. Prevalence of some seedborne fûngi on soft white winter wheat seed fiom Ontario, Canada. Canadian Plant Disease Survey. 73: 143-149.
Corne, D. and Thevenot, C. 1982. Environmental control o f ernbryo dormancy and germination. In: K h q A. (ed) The Physiology and Biochemistry of Seed Development, Dormancy and Germination. pp 272-297. Elsevier Biomedical Press.
Counts, RL. 199 1. Germination and early seedling growth in some northem wild rice (Zizania palustris) populations differing in seed size. Canadian Jomal of Botany 69: 689-696.
Curran, D.F. 1985. The ginseng disease and Pest reference guide. Copyright D.F. Curran.
Curtis, K. and McKersie. 1984. Growth potential of the axis as a determinant of seedling vigor in Birdsfoot Trefoil. Crop Science 24: 47-50.
Darrnono, T.W. and Parke, J.L. 1990. Chlamydospores of Phytophthora cactonm~: their production, structure, and infectivity. Canadian Journal of Botany 68: 640-645.
Davies, P.J. 1 995. Plant Hormones: P hysiology, Biochernistry and Molecular B iology. Kluwer Academic Publishers, The Netherlands.
Derh., M.P.M., Vermeer, E. and Karssen, C.M. 1994. Gibberellins in seeds of Arabidopsis thalimia: biological activities, identification and eEects of light and chilling on endogenous levels. Plant Growth Regdation 15: 223-234.
DiNola, L., Mischke, C.F. and Taylorson, RB. 1990. Changes in the composition and synthesis of proteins in cellular membranes of Echinochloa crus-galli &.) Beauv. seds during the transition from dormancy to germination. Plant Physiology 92: 427-433.
Downie, B., Hilhorst, H.W.M. and Bewley, J.D. 1994. A new assay for quan-g endo-e-mannanase activity using Congo red dye. Phytochemistry 36: 829-835.
Duke, J. A. 1992. Handbook of phytochemical constituents of GRAS herbs and other economic plants. pp 426428. CRC Press, Inc. Boca Raton, FL.
Dwivedi, S.K. and Dubey, N.K. 1992. Mycoflora associated with seeds of Cyamopszs tetrgonoloba L. (Taub.) Journal of Mycopathological Research 30: 1 53 -1 56.
Esau, K. 1991. Anatomy of seed plants. 2" ed. John Wiley and Sons, Lnc. Toronto, Canada. pp. 550.
Evans, M., Black, M. and Chaprnan, J. 1975. Induction of hormone sensitivity by dehydration is one positive role for drying in cereal seed. Nature (London) 258: 144-1 45.
Faleni, R 1994. Issues of the pharmacologïcal and medicd uses of ginseng. In: Bailey, W.G., Whitehead, C., Proctor, J.T. A., Kyle, J.T. (ed) Proceedings of the International Ginsneg Conference, Vancouver 1994. Pp.76-78. Simon Fraser University. Bumaby, B.C.
Farios-Larios, J O , Orozco-Santos, M. 1994. Colour polyethylene mulches increase fniit quality and yield in watermelon and reduce insect pest populations in dry tropics. Gartenbauwissenschaft 62: 255-260.
Farrant, J.M., Berjak, P. and Pammenter, N.W. 1985. The effect of drying rate on viability retention of recalcitrant propagules of Avicennia marina. South Afncan Journal of Bot;iny 51: 432-43 8.
Farrant, J.M., Berjak, P., Cutting, J.G.M. and Pammenter, N.W. 1993. The role of plant growth regulators in the development and germination of the desiccation-sensitive (recalcitrant) seeds of Avicennia marina. Seed Science Research 3: 55-63.
Fenner, M. 1992. Environmental influences on seed size and composition. Horticultural Reviews 13:183-213.
Finch-Savage, W.E. and Phelps, K. 1993. Onion (AlZizim cepa L.) seedling emergence patterns can be explained by the infhence of soil temperature and water potentiai on seed gemllnation Journal of Experimental Botany 44: 407-4 14.
Fiebig, A. 1999. Inflorescence development of American ginseng: abscission zones and ethephon effects. M. Sc. thesis. University of Guelph, ON.
Fisher, P. 1993. Growing ginseng in Ontario. In: Ontario Ministry of Agriculture, Food and Rural Mairs (ed.) Proceedings of the 1993 Ontario Horticuitural Crops Conference. pp. 116-121.
Fisher, P. 1995. Ginseng production in Ontario. In: Bdey, W.G., Whitehead, C., Proctor, J.T.A., Kyle, J.T. (ed) Proceedings of the International Ginsneg Conference, Vancouver 1994. pp. 56-59. Simon Fraser University. Bumaby, B.C.
Gaur, A. and Dev, U. 1988. Detection techniques for seed-borne hngi, bacteria and viruses. In: Agnihotri, V.P., Sarbhoy, A-K, and Kumar, D. (ed) Perspectives in Mycology and Plant Pathology. pp.59 1-609. Malhotra Publishing House. New D e l k India.
Gray, D. and Steckel, J-RA. 1983a. Some effects of umbel order and harvest date on carrot seed variability and seedling performance. Journal of Horticultural Science 58: 73-82.
Gray, D. and Steckel, J.RA. 1983b. Seed quality in carrots: the effects of seed crop plant density, harvest date and seed grading on seed and seedling variability. Journal of Horticultural Science 58 : 3 93-40 1.
Gray, D. and Thomas, T.H. 1982. Seed germination and seedling emergence as influenced by the position of development of the seed on, and chernical applications to, the parent plant. In: Khaq A.A. (ed.) The physiology and biochemisty of seed development, dormancy and germination. pp. 369-387. Elsevier Biomedical Press.
Gupta, I.J., Schmitthenner, A.F. and McDonald, M.B. 1993. Effect of storage fungi on seed vigour of soybean. Seed Science and Technology 21: 58 1-59 1.
Gupta, RP. and Raychaudhuri, S.P. 1988. Seed-borne diseases of vegetable crops. In: Agnihotri, V.P., Sarbhoy, A.K. and Kurnar, D. (ed) Perspectives in Mycology and Plant Pathology. pp.212-223. Malhotra Publishing House. New Delhi, India.
Hellyer, W.C. 1984. Canadian ginseng production: past and present. In: Proctor, J.T.A. (ed.) Proceedings of the 6<h North American Ginseng Conference. Guelph, ON. pp.3.
Hildebrand, A.A. 1934. Root rot of ginseng in Ontario caused by members of the genus Rumularia. Canadian Journal of Research 12: 82- 1 14.
Hilhont, H.W.M. 1995. A critical update on seed dormancy. 1. Primary dormancy. Seed Science Research 5: 6 1-73.
Hilhorst, H.W.M. and Kansen, C.M. 1992. Seed dormancy and germination: the role of abscisic acid and gibberellins and the importance of hormone mutants. Plant Growth Regulation 1 1 : 225-23 8.
Hisamatsu, T., Koshioka, M., Kubota, S., Nishijirna, T., King, RW., Mander, L.N. and Owen, D.J. 1997. Endogenous gibberehs in Mrmhiola i n c m R Br. : Isolation and identification of GAL 12 (1 2P-hydroq-GA1Z). Phytochernistry
Ho, T.D. 1982. The mechanism of abscisic acid during seed germination. In: Khan, A. (ed) The Physiology and Biochemistry of Seed Development, Dormancy and Germination. pp 3 00-3 19. Elsevier Biomedicd Press.
Hobbs, C. 1997. Ginseng: Facts and Folklore. Herbs for Health 2: 19-20.
Hoffmann-Benning, S. and Kende, H. 1992. The role of abscisic acid and gibberellin in the regulation of growth in rice. Plant Physiology 99: 1 156- 1 16 1.
Hoitink, H.A.J. and Grebus, M.E. 1994. Status of biological control of plant diseases with composts. Compost Science and Utilization 6: 6-12.
Horgan, R 1984. Cytokïnins. In: Advanced Plant Physiology. (ed. Wilkins, M.B.) Pitman, London.
Hovius, M.H.Y. 1996. Spring seeding of Amencan ginseng using temperature and growth regulators to overcome dormaocy. M.Sc. thesis. University of Guelph, Ontario.
Huang, Y., Li, X. and Cui, S. 1995. Growth inhibitors in Amencan ginseng seeds. In: Bailey, W.G., Whitehead, C., Proctor, J.T.A., Kyle, J.T. (ed) Proceedings of the International Ginseng Conference, Vancouver 1 994. pp. 78-83. Simon Fraser University. Burnaby, B.C.
Hudelson, B. and Parke, J. 1996. Control of diseases, pests and weeds in cultivated ginseng in Wisconsin. University of Wisconsin-Madison. Madison, WI.
Jacobsen, J.V. and Pressman, E. 1979. A structural study of germination in Celery (Apium graveolens L.) seed with emphasis on endospem breakdown. Planta 144: 241-248.
Jacobsen, J.V., Gubler, F. and Chandler, P.M. 1995. Gibberellin action in germinated cereai grains. In: Plant Hormones: physiology, biochemistry and molecular biology. (ed. Davies, P.J.) Kluwer Academic Publishers. The Netherlands.
JO, J., Blazich, F.A., and Konsler, T.R 1988. Postharvest seed maturation of Amencan ginseng: stratification temperatures and delay of stratification HortScience 23: 995-997.
Jordon, E.G., Sinclair, J.B., Manandhar, J.B. and Thapliyal, P.N. 1992. Soi1 srpe and other field conditions affecthg seed-borne fùngi in Illinois soyabeans. Seed Science and Technology 20: 6 19-628.
Joy, A.E. and Parke, J.L. 1995. Biocontrol of Altemaria leaf blight on Amencan ginseng by Burkholderia cepc ia AMMD. In: Bailey, W.G., Whitehead, C., Proctor, J.T.A., Kyle, J.T. (ed) Proceedings of the International Ginseng Conference, Vancouver 1 994. pp. 93 - 100. Simon Fraser University. Bumaby, B .C.
Kar, C. and Gupta, K. 199 1 . Effect of triazole-type plant growth regulators on sunflower and safflower seed viability. Canadian Journal of Botany 69: 1344- 1348.
Karssen, C.M. 1982. Seasonal patterns of dormancy in weed seeds. In: Khan, A. (ed) The Physiology and Biochemistry of Seed Development, Dormancy and Germination. pp 244-268. Elsevier Biomedical Press.
Kende, H. and Zeevaart, J.A.D. 1997. The five ccclassical" plant hormones. The Plant Ceil 9: 1 l97:12lO.
Kendrick, B. 1992. The Fifth Kingdom. 2"* ed. FOCUS Information Group Inc. Newburyport, MA..
Khan, A. A. 1982. Gibberellins and seed development. In: Khan, A. (ed) The Physiology and Biochemistry of Seed Development, Dormancy and Germination. pp 1 12- 13 1. Elsevier Biomedical Press.
Khan, A. A. 1997. Quantification of seed dormancy: physiological and molecular consideratioos. HortScience 32: 609-6 14.
Khan, A. A. and Samimy, C. 1982. Hormones in relation to primary and secondary seed dormancy. In: Khan, A.(ed) The Physiology and Biochemistry of Seed Development, Dormancy and Germination. pp 203 -23 7. Elsevier Biomedical Press.
Kim, J.H., Lee, M.W. and Kim, G.P. 1974. Studies on the root rot of ginseng (IV) distribution of fungi and Fusarium sp. Population in ginseng cultivation soil. Korean Journal of Mycology 2: 15-1 9.
Konsler, T.R 1986. Effect of stratification temperature and time on rest fu&ent and growth in American ginseng. Journal for the American Society of Horticultural Science 111: 651-654.
Konsler, T.R and Shelton, J.E. 1990. Lime and phosphorus effects on American ginseng: 1. Growth, soi1 fertility, and root tissue nutrient status response. Joumal of Amencan Society for Horticultural Science 1 15: 5 70-5 74.
Laidman, D.L. 1982. Control rnechanisms in the mobiiization of stored nutrients in germinating cereals. In: Khan, A. (ed) The Physiology and Biochemistry of Seed Develo pment, Dormancy and Germination. pp 3 7 1-405. Elsevier B iomedicd Press.
Lake, RJ., Falioon, P.G. and Cook, D.W.M. 1993. Replant problem and chemicd components of asparagus roots. New Zealand Joumal of Crop and Horticultural Science 21: 53-58.
Lau, 0. and Yang, S.F. 1974. Synergistic effect of calcium and kinetin on ethylene production by the mungbean hypocotyl. Planta 118: 1-6.
Lee, H.S., Kim, S.W., Lee, K.W., Eriksson, T. and Liu, J.R 1995. Agrobacterizm- mediated transformation of ginseng (Panm ginseng) and mitotic stability of the inserted P-glucuronidase gene in regenerants fkom isolated protoplasts. Plant CeU Reports 14: 545-549.
Lee, J.C., Ahn, D.J., Byen, J.S., Jang, J.G. and Hwang, K.J. 1987. Effect of seed postion on seed size, contents of ginsenosides, free sugars and fatty acids in P m ginseng (P. pseudoginseng). Korean Joumal of Crop Science 32: 330-335.
Lee, J.C., Byen, J.S. and Proctor, J.T.A. 1983. Effect of temp on embryo growth and germination of ginseng seed. Proceedings of the Fifth North Ameiïcan Ginseng Conference. Lexington, Kentuclq. pp 1 1-2 1.
Lee, J.C., Chung, Y.R, and Ohh, H.P.S.H. 198 1. Influence of seed dressing with Captan WP on the dehiscence of Pana ginseng seeds. Korean Journal of Ginseng Science 28: 262-266.
Lee, J.C., Strik, B.C. and Proctor, J.T.A. 1985. Dormancy and Growth of Amencan ginseng as iduenced by temperature. Joumal of the American Society for Horticultural Science 110: 319-321-
Lee, M.W. 1975. Studies on the Pseudomonasf7uorescens causing root rot of ginseng. Korean Journal of Microbiology 13: 143-1 56.
Lee, M.W. 1977. Studies on the root rot of ginseng (VII) on pathogenic microbial population in continuous culture and first-time culture of ginseng. Korean Journal of Microbiology 15: 20-30.
Lenton, LR, Ap pleford, N.E. J. and Cro ker, S. J. 1 994. Gibberellins and a-amylase gene expression in germinating wheat grains. Plant Growth Regdation 15: 26 1-270.
Lewis, W.H. 1988. Regrowth of a declimated population of Panmc quinquefohun in a Missouri climax forest. Rhodora 90: 1-5.
Lewis, W.H. and Zinger, V.E. 1982. Population dynamics of the Arnerican ginseng Panax quinquefolium (Ardiaceae). Amencan Journal of Botany 69: 1483 - 1490.
Li, T.S.C. 1995. The effects of chemical and organic treatrnent on ginseng seedlings planted in old ginseng SOL In: Bailey, W.G., Whitehead, C. , Proctor, J.T.A., Kyle, J.T. (ed) Proceedings of the International Ginseng Conference, Vancouver 1 994. pp.2 17-2 10. Simon Fraser University. Bumab y, B .C.
Li, X.E., Chen, Y. and Zhang, J. 199 1. Effect of hormones on fier-ripening in P m m ginseng seeds. Acta Botanica Sinica 33: 385-3 89.
Li, Y., Xu, A., Liu, P., Guo, X., Zhao, C. and Qian, S. 1992. The technique for accelerating after-ripening of American ginseng seeds. Special Wid Economic Animal and Plant Research 4: 20-2 1.
Lian, Y.Q. and Shen, J.H. 1989. Embryological studies on ginseng (Puna ginseng C.A. Meyer). Acta Botanica Sinica 31: 653-660.
Liu, M., Li, R.J. and Liu, M.Y. 1993. Adaptive responses of roots and root systerns to seasonal changes. Environmental and Experimental Botany 33(l) : 1 75- 1 8 8.
Malloch, D. 198 1. Moulds: Their Isolation, Culfivation and Identification. University of Toronto Press, Toronto.
Mayer, A.M. and Poljakoff-Mayber, A. 1989. The Germination of Seeds. Pergamon Press. Odord, England.
McCarthy, J. and Scott-Dupree, C. 1997. Pollination of American ginseng ( P m pinquefolium L.). Bee Biz. 8: 24-27.
MeElhone, H. 1995. Green seed prices skyrocket. Canadian Tobacco Grower 44: 14.
Menzies, J.G. and Jarvis, W.R. 1994. The infestation of tomato seed by Fusarium oxysponrm fsp. radicis-lycopersici. Plant Pathology 43 : 3 78-3 8 6 .
Musgrave A., Jackson, M.B. and Ling, E. 1972. Caiiitriche stem elongation is controlled by ethylene and gibberelh. Nature New Biology 238: 93 -96.
Nautiyal, A.R and Purohit, A.N. 1985. Physiological and biochernical aspects of seed development in Shorea robusta. Seed Science and Technology 13: 59-68.
Oliver, A., Van Lierop, B. and Buonassisi, A. 1990. American ginseng culture in the arid climates of British Columbia, Province Of British Columbia, Ministry of Agriculture Fishenes and Food.
Parke, J.L. and Shotwell, KM. 1989. Diseases of Cultivated Ginseng. University of Wisconsin-Madison. Coilege of Agriculture and Life Sciences. Agricultural Bulletin no. A3465
Persons, W.S. 1995. Amencan ginseng farming in its native woodland habitat. Ln: Bailey, W.G., Whitehead, C., Proctor, J.T.A., Kyie, J.T. (ed) Proceedings of the International Ginseng Conference, Vancouver 1994. pp.78-83. Simon Fraser University. Burnaby, B.C.
Pill, W.G.9 Frett, J.J. and Morneau, D.C. 1991. Germination and seedling emergence of pnmed tomato and asparagus seeds under adverse conditions. HortScience 26: 1 160-1 162.
Pinfield, N.J. and Stobart, A.K. 1972. Hormonal regulation of germination and early seedling development in Acer pseudoplafms (L.). Planta 104: 13 5- 145.
Pinfield, N.J., Stutchbury, B.A., Bazaid, S.A. and Gwarazimba, V.E.E. 1990. Abscisic acid and the regulation of embryo domancy in the genus Acer. Tree Physiology 6: 79-85.
Pressman, E. and Shaked, R 199 1. Interactive effects of GAs, CKs and growth retardants on the germination of celery seeds. Plant Growth Regdation 10: 65-72.
Probert, RJ. and Brierley, E.R 1989. Desiccation intolerance in seeds of Zizmia paZusfris is not related to developmental age or the duration of post-harvest storage. bals of Botany 64: 669-674.
Proctor, J.T.A. 1995. Ginseng: old crop, new directions. Horticultural Conference, October 1995. Indianapolis, Indiana.
Proctor, J.T.A. and Bailey, W.G. 1987. Ginseng: industq, botany and culture. Horticuitural Reviews 9: 1 8 8-23 6.
Proctor, J.T.A. and Louttit, D. 1995. Stratification of Amencan ginseng seed: embryo growth and temperature. Korean Journal of Ginseng Science 19: 17 1-1 74.
Putnam, M. 1989. Ginseng pathogens. In: Duke, J.A. (ed) Ginseng: A concise handbook. pp. 150- 163. Algonac, Michigan.
Quesnel, Fey Khan, N. and Reeleder, RD. 1997. Evaluation of composts for control of soilbome diseases of ginseng. Annuai Meeting of the Amerkari Phytopathological Society, August 9- 13. Rochester, NY.
Reeleder, RD. 19%. Laboratory assays for evaluation of products for control of Pole Rot of tobacco caused by Rhizops mrhims. Tobacco Science 37:34-3 8.
Reeleder, RD. 1994. Damping-off, root rot, seed decay. In: Howard, R.J., Garland, J.A. and Seaman, W.L. (ed.). Diseases and Pests of Vegetable Crops in Canada. The Canadian Phytopathological Society and Entomological Society of Canada. Ottawa, ON. pp. 296-297.
Reeleder, RD. 1996. An o v e ~ e w of ginseng diseases. Horticulhiral Crops Conference. November, 1996. Quebec.
Reeleder, RD. and Brammal, RA. 1994. Pathogenicity of Pythum species, Cylindrocarpon destructans, and Rhizoctonia solani to ginseng seedfings in Ontario. Canadian Journal of Plant Pathology 16: 3 1 1-3 16.
Reeleder, RD. and Fisher, P. 1995. Diseases of ginseng. Ontario Ministry of Agriculture, Food and Rural Mairs. Order No. 95-003.
Reeleder, RD. and Fisher, P. 1995. Rhizoctonia disease of ginseng. Ontario Ministry of Agriculture, Food and Rural Mairs. Order No. 95-00 1.
Reeleder, RD. and Khan, N.I. 1996. Use of biowaste amendments to control ginseng root rots caused by Cylindrocarpon destructans and Phytophthora caaonirn. Annual Meeting of the Canadian Phytopathological Society. (conference abstract).
Rees, M. 1993. Trade-offs among dispersal strategies in British plants. Nature 366: 150- 152.
Reid, J.S.G. and Meier, Et. 1972. The function of the aleurone tayer during galactomannan mobilixation in germinating seeds of fenugreek (Trigonella foemm- graecum L.), crimson clover (Trigonella Nicantatzrm L.) : a correlative biochernical and ultrastructural study. Planta 134: 209-22 1.
Ren, G., Chen, F., Lian, Hay Zhao, J. and Gao, X. 1997. Changes in hormone content of Panar pinquefolim seeds during stratification. Journal of Horticulhiral Science 72: 901-906.
Ren, G., Chen, F., Lian, H., Zhao, J., Gao, X. and Guo, C. 1996. Effecrs of GA3 and ABA application on after-ripening of P m pinquefoliunt seeds during stratification. Korean Journal of Ginseng Science 20: 83-87.
Richardson, M.J. 1979. An Annotated List of Seedbome Diseases. 3* ed. Commonwealth Mycological Institute, Kew, England. pp. 3 20.
Rock, C.D. and Quatrano, RS. 1995. The role of homones during seed developrnent. In: Plant Hormones: physiology, biochemistry and molecular biology. (ed. Davies, P.J.) K h v e r Academic Publishers. The Netherlands.
Savage, J. 199 1. Amencan ginseng culture in the arid climates of British Columbia. Korean Joumal of Ginseng Science 15: 41-73.
Salisbury, F.B and Ross, C.W. 1992. Plant Physiology- 4rth ed. Wadsworth Inc. California, US.
Schooley, J. 1996. Ginseng production in Ontario. Ontario Ministry of Agriculture, Food and Rural Mairs.
Schooley, J. 1997. Ginseng Pest Control Recomrnendations For 1997- 1998. Ontario Ministry o f Agriculture, Food and Rural Affairs. Publication no. 6 1 0.
Schooley, J. and Brammal, R 1998. Ginseng Diagnostic Workshop. Promoculture Canada Ltd. Hensall, ON.
Schooley, J. and Reynolds, L.B. 1998. The Effect of Production Practices On The Quality of Ginseng Roots. Ontario Ministry of Agriculture, Food and Rural Mairs. Publication no. 98-067. AGDEX 260/20.
Scott, RK., Harper, F., Wood, D.W. and Jaggard, K.W. 1974. Effects of seed size on growth, development and yield of monogerm sugar beet. Journal of Agricultural Science 82: 5 17-530.
Seiler, A. 1977. Galactomannabbau in keimenden Johannisbrodsamen (Ceratorlia siliqzra L.). Planta 134: 209-221
Simmons, E.G. 1982. Altemaria themes and variations (W-X). Mycotaxon 14: 17-43.
Singh, K., Tandon, M.P. and Shukla, D.N. 1992. Some pathogenic fungi from vegetable and b i t crops. National Academy Science Letters 15: 3 17-3 18.
Smalle, J. and Van Der Staeten, D. 1997. Ethylene and vegetative development. Physiologia Plantarum 100: 593-605.
Smith, A. 1995. North Amencan ginseng production: the grower' s perspective. In: Bailey, W.G., Whitehead, C., Proctor, J.T.A-, Kyle, J.T. (ed) Proceedings of the International Ginseng Conference, Vancouver 1994. pp.292-294. Simon Fraser University. Bumaby, B .C.
Sponsel, V.M. 1995. The biosynthesis and metabolism of gibberellins in higher plants. In: Plant Hormones: physiology, biochemistry and rnolecular biology. (ed. Davies, P.J.) Kiuwer Academic Publishers. The Netherlands.
Stoddart, J.L. 1983. Sites of gibbereuin biosynthesis and action. In: The Biochemistry and Physiology of Gibberellins. Vol. 2. (ed. Crozier, A-) Praeger, New York.
Stoltz, L.P. and Snyder, J.C. 1985. Embryo growth and germination of American ginseng seed in response to stratification temperatures. HortScience 20: 26 1-262.
Thomas, T.H. 1 996. Relationships between position on the parent plant and germination characteristics of seeds of parsley (PetroseZiniztrn crispzcm Nym.). Plant Growth Regdation 18: 175-181.
Tian, Y., Li, X., Yang, JO, GUO, R and Liu, J. 1992. Effects of gibbereilin on the physiological donnancy of ginseng seed. Korean Journal of Ginseng Science 16: 252. Ab stract.
Tindall, LA., Beverly, RB. and Radcliffe, D.E. 1990. Mulch effect on soi1 properties and tomato growth using micro-injection. Agronomy Journal 83: 1028- 1034.
Tirajoh, A. and Punja, Z.K. 1995. Tissue culture and Agmbacterium-mediated transformation of American ginseng (Panax qzcinqt<efoliztnz L.). In: Bailey, W.G., Whitehead, C., Proctor, J.T .A., Kyle, J.T. (ed) Proceedings of the International Ginseng Conference, Vancouver 1994. pp. 146- 1 5 8. Simon Fraser University. Bumaby, B.C.
Usha, CM., Patkar, ILL., shetty, H.S., Kennedy, R and Lacey, J. 1993. Fungai colonization and mycotoxin contamination of developing rice grain. Mycological Research 97: 795-798.
von Am, J.A. 1987. Plant Pathogenic Fungi. Cramer, Berlin.
Wareing, P.F. 1982. Hormonal regulation of seed dormancy-past, present and future. In: Khan, A. (ed) The Physiology and Biocbernistry of Seed Development, Domancy and Germination. pp 1 85-200. Elsevier Biomedical Press.
Wiegrefe, S. J. 1994. Overcoming dormancy in maple seeds without stratification. Landscape Plant News 5: 8-13.
Wulff, RD. 1986. Seed size variation in Demodizim panindahm II. Effects on s e e d h g growth and physiological performance. Journal of Ecology 74: 99-1 14.
Yu, S.C. and Kim, W.K. 1992. Structural changes and histochemical study of endosperm on Panm ginseng CA- Meyer dwing embryo development. Korean Journal of Ginseng Science 16: 3 7-43.
Yu, S.C., No, M. J., Lee, C.S. and Kim, W.K. 1992. Lipid and lipase distribution on endosperm ceil of Pmax ginseng seed for the electron microscope. Korean Journal of Ginseng Science 16: 12% 13 7.
Yu, Y.H. and Ohh, S.H. 1995. Problems and present status of research on ginseng diseases in Korea. In: Bailey, W.G., Whitehead, C., Proctor, J.T.A., Kyle, J.T. (ed) Proceedings of the International Ginseng Conference, Vancouver 1994. pp. 12 1 - 13 2. Simon Fraser University. Bumaby, B.C.
Zhang, T. and Chen, W. 1992. A preliminary study of seed pathology of P m quinqz~efoliz~m. Korean Journal of Ginseng Science 16: 244-245. Abstract.
Zhang, T., Zhang, Y., Chen, W. Zhang, C. 1989. An analysis of seedborne fùngi of imported seeds of Panax quinquefolium. Acta Phytopathologie Sinica 19: 134.
Ziezold, A.M. 1997. Chemical control of disappearing root rot of ginseng caused by CyZindrocmpon destructans. M. Sc. thesis. University of Guelph, ON.
Ziezold, A.M., Reeleder, R, Hall, D.R and Proctor, J.T.A. 1998a. Seedbome fiiogi and fùngicide seed treatment of ginseng. Journal of Ginseng Research 22: 229-23 6.
Ziaold, A.M., H d , D.R, Reeleder, R and Proctor, J.T.A. 199%. Effect of drenching soi1 with benomyl, propiconazole and fluazuiam on incidence of disappearing root rot of ginseng. Joumal of Ginseng Research 22: 237-243.
-Appendïx 1- Culture Media Preparation For The Isolation Of Fungi
From North American Ginseng
Potato Dextrose Agar (PDA)
To prepare 1 L of media: potato dextrose agar 3 9 g lactic acid 1.2 ml deionized water 1 L
Combine agar and water in two 1L Erlenmeyer flasks. Seal openings with cheesecloth and M O L Autoclave for at 100 kPa, 121°C for 20 minutes.
Rose Bengal Agar (RI3PB5&) (MRBA)
To prepare 1 L of media: Difco Bacto Agar 20 g Rose Bengal Dye 0.05 g Anhydrous Dextrose 10 g Bacto Peptone 5 g KH2 PO4 l g MgS04-'7J&O 0.5 g deionized water 1 L Botran 0.5 g
Combine all ingredients (except B otran) in two 1 L Erlenmeyer flasks. Seal openings with cheesecloth and tinfoil. Autoclave for at 100 kPa, 12 1°C for 20 minutes. When cooled to about 5S°C, add Botran. Swirl flask to keep Botran particles well dispersed throughout the mixture.
Water Agar with Antibiotics (WA)
To prepare 1 L of media: Sigma Agar 20 g (could substitute Difco Bacto Agar) Chlorotetracyciine 0 -05 g Streptomycin Sulfate 0.05 g deionized water 1 L
Combine all ingredients (except antibiotics) in two 1L Erlenmeyer flasks. Seal openings with cheesecloth and tinfoil. Autoclave for at 100 kPa, 12 1°C for 20 minutes. When cooled to about 55"C, add antibiotics.
Cornmeal Agar with Aotibiotics (PARP)
To prepare 1 L of media: Commeal Agar 17 g Ampicillin 0.25 g Pirnafùcin 0.2 mL Rifamp icin 0.01 g Terraclor O. 134 g deionized water 1 L
Combine all ingredients (except antibiotics and fungicide) in two 1L Erienrneyer flasks. SeaI openings with cheesecloth and tinfoii. Autoclave for at 100 kPa, 12 1°C for 20 minutes. When cooled to about 55"C, add antibiotics and fungicide. Swirl flask to keep Rifmpicin particles well dispersed throughout the mixture.
Cornmeal Agar "Replating" Media (CA)
To prepare 1 L of media: Conuneal Agar 17 g Streptomycin 0.1 g deionized water 1 L
Combine al1 ingredients (except antibiotic) in two 1L Erlenmeyer flasks. Seal openings with cheesecloth and tinfoil. Autoclave for at 100 kPa, 121°C for 20 minutes. When cooled to about 55"C, add antibiotic.