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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).


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.


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.


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

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-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.

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