DEDICATION

This work is dedicated to my family and friends who have been supportive socially, emotionally

and financially throughout this journey. I thank you all, May God bless you.

ii

ABSTRACT

Pseudomonas syringae pv. Glycinea is the causal agent of bacterial blight plant disease in

soybean (Glycine max). A study was undertaken to test for transmission of Pseudomonas

syringae pathovar isolates from seed to plant, in order to ascertain the importance of seed as

a primary source of inoculum for bacterial blight disease in soybean (Glycine max). The study

was carried out using five commercial soybean varieties (SC Santa, SC Sequel, SC Squire,

SC Signal, and SC Saga) and three soybean landraces (Chivhu, Harare and Guruve). Seed

samples (40 per variety) were artificially inoculated with pathogen isolates from different

geographic origins (Chivhu, Harare and Guruve) by soaking the seeds in buffered phosphate

saline infested with 1.5 x 107 (CFU)/ml of the respective pathogen. Seeds were then planted

in germination pots. Seedling emergence, disease incidence and seed transmission frequency

(disease incidence + symptomless infection) were compared among three isolates. Seed-to-

plant transmission occurred in all soybean varieties. Transmission was confirmed by bacterial

counts of isolated pathogen from diseased and symptomless plant tissue bacterial counts on

selective medium: Modified Sucrose Peptone which ranged from 6.9 x 106 to 1.2 x 107 on day

12 after germination. The level of disease incidence and seedling emergence, however varied

with different pathogen/seed variety combination. Seedling emergence was generally low in

local landraces (Chivhu, Harare and Guruve) with a low average of 2% germination

percentage. Commercial varieties had a combined average emergence of 52.8 %. The average

disease incidence was 47.6 % in all varieties. The highest disease incidence in commercial

seed varieties was 80%, and was noticed in SC Squire variety/Guruve isolate combination.

The lowest disease incidence was noticed in SC Sequel Variety/Harare isolate (5 %). These

results support seed transmission of Pseudomonas syringae pv. Glycinea in soybean (Glycine

max) and suggest that the distant spread of bacterial blight on soybean may also be due to

seed transmission. The study also confirms latent in planta growth of pathogen in

symptomless plants. This study has shown the importance of seed-borne inoculum of

Pseudomonas syringae pv. Glycinea in disease initiation.

iii

DECLARATION

I, Ndondo Joseph, do hereby declare that the experimental work in this report was carried out

at the Department of Applied Biology and Biochemistry of the National University of Science

and Technology. This reflects my work and effort and has not been reproduced from pre-

existing work and prior to this date, has not been submitted to any local or international

University, Examination board or publication.

Ndondo Joseph Kunashe

Signed……………….....

Date…………………

iv

ACKNOWLEDGEMENTS

I wish to express my indebted gratitude and special thanks to the whole Department of Applied

Biology and Biochemistry staff for their unwavering assistance and guidance throughout the

course of this dissertation. Special mention goes to Mr. K. Mushonga, my incomparable

academic supervisor who in spite of being extraordinarily busy with his duties, took time to

assist and keep me on track. I also would like to acknowledge SEEDCO (PVT) LTD, for

kindly providing a major part of the samples used in this projects. I would also like to thank

all the Technologists and lab assistants who guided me and motivated me in my daily

procedures. Special thanks goes to the Applied Chemistry laboratory staff, for allowing me

to use their spectrophotometer. Lastly, I would be remiss if I did not acknowledge a special

extraordinary woman; my mother Margaret for being with me throughout this period; her

motivational support, financial support and nurturing role was and will always be greatly

valuable.

v

TABLE OF CONTENTS

DEDICATION ................................................................................................................................ i

ABSTRACT ................................................................................................................................... ii

DECLARATION .......................................................................................................................... iii

ACKNOWLEDGEMENTS.......................................................................................................... iv

LIST OF FIGURES ..................................................................................................................... viii

LIST OF TABLES .......................................................................................................................... x

ABBREVIATIONS AND ACRONYMS ...................................................................................... xi

CHAPTER 1 ................................................................................................................................... 1

INTRODUCTION ......................................................................................................................... 1

1.1 BACKGROUND ................................................................................................................... 1

1.2 PROJECT RATIONALE ...................................................................................................... 1

1.3 OBJECTIVES ....................................................................................................................... 5

CHAPTER 2 ................................................................................................................................... 6

LITERATURE REVIEW .............................................................................................................. 6

2.1 SOYBEAN (Glycine max (L.) merril) ............................................................................. 6

2.2 HISTORY AND DISTRIBUTION .................................................................................. 6

2.3 SOYBEAN PLANT DESCRIPTION .............................................................................. 6

2.4 SOYBEAN PRODUCTION ............................................................................................ 9

2.6 SOYBEAN VARIETIES IN ZIMBABWE ........................................................................ 10

vi

2.7 SOYBEAN BACTERIAL BLIGHT DISEASE ................................................................. 11

2.8.1 BIOCHEMICAL PROPERTIES OF Pseudomonas syringae ......................................... 14

2.9 SEED-TO-PLANT TRANSMISSION OF PATHOGENS ................................................ 15

2.9.1 TYPES OF PATHOGENS IN SEED TRANSMISSION ................................................ 16

2.9.2 FORMS OF TRANSMISSION ........................................................................................ 17

2.9 DISEASE PHYSIOLOGY AFTER TRANSMISSION OF PATHOGEN ......................... 18

CHAPTER 3 ................................................................................................................................. 20

METHODOLOGY ...................................................................................................................... 20

3.1 OUTLINE OF METHOD ................................................................................................... 20

3.2 COLLECTION OF SEED SAMPLES AND BREAKING SEED DORMANCY ............. 21

3.3 SEED SURFACE STERILIZATION ................................................................................. 21

3.4 BACTERIAL STRAINS ..................................................................................................... 21

3.5 ARTIFICIAL INOCULATION OF SEED WITH PATHOGEN ....................................... 22

3.6 PLANTING, GERMINATION AND SYMPTOM DEVELOPMENT .............................. 22

3.7 DETECTION OF SYMPTOMLESS INFECTION AND QUANTIFICATION OF

BACTERIA ............................................................................................................................... 23

3.8 EMERGENCE, INCIDENCE AND TRANSMISSION EFICIENCY ............................... 23

3.9 STATISTICAL ANALYSIS ............................................................................................... 24

CHAPTER 4 ................................................................................................................................. 25

RESULTS ..................................................................................................................................... 25

vii

4.1 SEEDLING EMERGENCE ................................................................................................ 25

4.2 DISEASE INCIDENCE ...................................................................................................... 31

4.3 DISEASE INCIDENCE PATHOLOGICAL RESULTS .................................................... 33

4.2 QUANTIFICATION OF BACTERIA FROM PLANT TISSUE ....................................... 36

CHAPTER 5 ................................................................................................................................. 40

DISCUSSION .............................................................................................................................. 40

CONCLUSION ............................................................................................................................ 45

RECOMMENDATIONS ............................................................................................................. 46

REFERENCES ............................................................................................................................. 47

APPENDIX .................................................................................................................................. 53

viii

LIST OF FIGURES

Figure 1: Symptoms of Soybean (Glycine max) bacterial blight caused by Pseudomonas syringae

pv. Glycinea .................................................................................................................................... 3

Figure 2: Soybean (Glycine max) production and processing in Sub-Saharan Africa ................. 10

Figure 3 Symptoms of Soybean (Glycine max) bacterial blight caused by Pseudomonas syringae

pv. Glycinea. ................................................................................................................................. 12

Figure 4: Bacterial blight of soybean. ........................................................................................... 12

Figure 5: Electron micrograph of Pseudomonas syringae ............................................................ 14

Figure 8: Methodology Flowchart ................................................................................................ 20

Figure 9: Seedling emergence trends of various varieties artificially inoculated with Pseudomonas

syringae pv. isolates. ..................................................................................................................... 25

Figure 10: Seedling emergence ANOVA analyses (p<0.05). ....................................................... 26

Figure 11: Seedling emergence t test analyses between control and Harare isolate (p<0.05). ..... 27

Figure 12: Seedling emergence unpaired t test analyses of Control vs Guruve isolate (p<0.05). 28

Figure 13: Seedling emergence unpaired t test analyses of Control vs Chivhu isolate (p<0.05). 29

Figure 14: Disease incidence as a function of isolate/variety combination ................................. 31

Figure 15: Disease Incidence ANOVA analyses (p<0.05) ........................................................... 32

Figure 16: Early Bacterial light disease symptoms on “SC Status” Soybean variety. ................. 33

Figure 17: Early Soybean Bacterial blight disease symptoms ...................................................... 34

Figure 18 : Lesions incited by Pseudomonas syringae pv. Glycinea on emerged cotyledon ....... 35

Figure 19: Plant tissue bacterial quantification ............................................................................. 37

Figure 20: Isolation and quantification of pathogen from diseased and symptomless plant tissue.

....................................................................................................................................................... 38

ix

Figure 21: Isolation and quantification of pathogen from diseased and symptomless plant tissue.

....................................................................................................................................................... 39

x

LIST OF TABLES

Table 1: Nutritional value of soybean ................................................................................... 8

Table 4.1: Seedling emergence reduction ..................................................................................... 30

Table 4.2:Bacterial population in plant tissue............................................................................... 36

xi

ABBREVIATIONS AND ACRONYMS

ANOVA Analysis of Variance

ARDA Agriculture and Rural Development Agency

CFU Colony Forming Units

FAO Food and Agriculture Organization of the United Nations

GDP Gross Domestic Product

GoZ Government of Zimbabwe

IITA International Institute of Tropical Agriculture

ISTA International Seed Testing Association

ml millilitres

MSP Modified Sucrose Peptone Medium

MT Metric Tones

ZIMSTATS Zimbabwe Statistical Office (formerly Central Statistical Office)

CHAPTER 1

INTRODUCTION

1.1 BACKGROUND

Agricultural production contributes about 20 % to the gross domestic product (GDP) of Zimbabwe

and about 70 % of the country’s population derive its livelihood from the agricultural sector

(Ministry of Agriculture, 2011 and Jagwe et al., 2010). In the Agricultural sector about 90 % of

the world food crops including Soybean (Glycine max (L.) Merrill) are propagated by seed

(Maude, 1996). Seed is the product of fertilized ovule that consists of embryo, seed coat and

cotyledon. The seed is probably the most important single input for arable cultivation. It

determines the potential production and thus productivity of all other inputs (Friis-Hansen., 1995).

However, seeds can be passive carriers of pathogens that may be effectively transmitted to the

plant and cause disease when the seeds are sown and emerge under suitable environmental

conditions.

1.2 PROJECT RATIONALE

The occurrence of plant disease has always been a major concern for all agricultural stakeholders.

Plant disease outbreaks like soybean (Glycine max) bacterial blight disease caused by

Pseudomonas syringae pv. Glycinea are of prime importance. Bacterial blight has been reported

to cause significant yield losses ranging from 4 % to as high as 40% under extreme conditions

(Mishra and Krishna, 2001). Infection of approximately 1 in 10,000 seeds was capable of causing

an outbreak of blight (Sutton and Wallen et al., 1970). In 1983 in Uganda, there was a bacterial

2

blight outbreak at the main seed multiplication site. This caused the operation to be abandoned and

delayed the release of seed to farmers (Allen and Lenne, 1998). In Colombia, yield losses of 22

and 45% were estimated from natural and artificial infection, respectively (Yoshi et al., 1976).

There is great need to reduce the impact of severe consequence plant disease outbreaks so as to

maintain a high soybean crop production. This can be achieved by understanding seed-to-seedling

transmission as it is the most critical point of plant infection as 90% of the world food crops

including soybean (Glycine max) are propagated by seed (Maude., 1996). Mechanism by which

seeds transmit pathogens is very important in order to formulate a disease management strategy.

Understanding the contamination routes and the localization of bacteria in reproductive organs, is

essential for selecting new varieties and for screening seeds to avoid contaminated seed.

Pseudomonas syringae pv. Glycinea which causes bacterial blight in Soybean (Glycine max), is a

model pathogen in the study of plant-pathogen interaction. The pathogen was voted the top

bacterial plant pathogen in 2012 by the Journal of Molecular Plant Pathology, based on microbial

pathogenicity, and on economically important plant diseases (Mansfield et al., 2012 and Scholthof

et al., 2011). The pathogen affects over 180 plant species (Mansfield et al., 2012).

The disease primarily affects young plant leaves and characterizes as disease lesions that are small

yellow to brown spots on leaves. The lesions dry out, turn reddish brown to black and become

surrounded by a yellowish green halo. The small lesions may enlarge and merge to produce large,

irregular, dead areas. The old lesions sometimes drop out or tear away, resulting in ragged

appearance of infected leaves. The bacteria can also infect stem petioles and pods. If pod infection

occurs, bacterial blight can become seed borne.

3

Figure 1: Symptoms of Soybean (Glycine max) bacterial blight caused by Pseudomonas

syringae pv. Glycinea

In Zimbabwe, most small-scale farmers that venture into Soybean (Glycine max) production use

farmer-saved or bin run seed because certified seed is expensive and most farmers can hardly

afford purchasing it. It has been established that both commercial and small-scale farmers recycle

seeds every 2-4 years (Kananji et al., 2013). Farmer-saved seed is likely to be of poor quality and

infested by pathogens. In most cases, it is likely that seed borne pathogens accumulate in farmer-

saved seeds with time serving as the primary inoculum. Higher chances are that the inoculum can

be effectively transmitted into plants, causing diseases and tremendous yield losses. The project

aims to find out the potential effects of farmer saved seed and commercial certified seed in seed-

to-seedling transmission of Pseudomonas syringae pv. Glycinea in specific Soybean (Glycine max)

varieties grown in Zimbabwe.

The project focuses on contributing and improving plant disease diagnosis and other plant

pathology research in the country by providing up-to-date plant pathology data. Findings and

information on pathogen transmission studies in various soybean (Glycine max) varieties can be

used to establish soybean (Glycine max) variety testing programs. The information obtained from

this program would be availed to soybean (Glycine max) growers and a computerized Soybean

4

(Glycine max) variety selection program can be established through this information. This has been

done for example by the University of Arkansas in America who have a computerized Soybean

(Glycine max) variety selection program entitled SOYVA (Mayhew et al., 1914). With more than

100 Soybean (Glycine max) varieties available, to a lay-farmer selecting a few varieties to plant is

challenging but with programs such as SOYVA it will be much easier. This project will equip

farmers with knowledge in choosing and deciding on the type of cultivar to grow and selecting a

plant variety for planting is the most important management decision a grower can make.

Key seed production and certification companies like Seed Co, Pannar, National Tested Seeds and

the Seed Services Institute, only concentrate on purity analysis, germination tests, moisture

determination and trueness to variety and rarely cover other important routine seed health tests

recommended by the International seed testing association (ISTA) (Mcdonald and Copeland.,

1998). This is due to shortage (brain drain) of trained seed pathologists and shortage of testing

equipment in Zimbabwe. This project will seek to partly address to this issue by augmenting the

work done by seed companies by covering otherwise overlooked aspects of seed health such as

seed-pathogen transmissibility.

Since the 1970s, commercial seed trade has increased tremendously in both volume and frequency

and is now global. Global commercial seed trade was estimated to be US$10 000 in 2010 (Luciani

et al., 2013). Zimbabwe imports grain in its poor-harvest years, from neighboring countries like

Zambia and South Africa. Data from ZIMSTATS show that between February 2010 and April

2010 6,355 Metric Tonnes (MT) of commercial grade soybeans were imported from Zambia.

These imported seed lots may be pathogen-infested and may end up being planted posing a high

risk of seed-to-seedling transmission of pathogens. More knowledge on the basic biology of seed

transmission of pathogens is needed in order to safeguard seed and plant health.

5

Global climate change continues to alter the environmental factors thereby increasingly exposing

pathogens to newer environments which may drive the emergence of dormant or newer diseases.

It is of prime importance to have more knowledge on naturally resistant landraces or varieties of

soybean (Glycine max) crops by studying the transmission of pathogens on various plant cultivars.

This will then facilitate in devising suitable plant-disease management strategies. Farmers will

make informed decisions and choices of cultivar or landrace suitable for their agro-regions.

In summary this study was aimed at reducing the impact of severe consequence plant disease

outbreaks so as to maintain a high soybean crop production, to assisting and improving plant

disease diagnosis and other plant pathology research in the country by providing up-to-date

scientific data and understand seed-to-seedling transmission as it is the most critical point of plant.

1.3 OBJECTIVES

The main objective of this study was to investigate and provide data on seed-to-seedling

transmission of Pseudomonas syringae pathovars in Soybean (Glycine max).

The specific objectives were:

To collect various soybean (Glycine max) variety and landrace seeds available in

Zimbabwe.

To determine and compare seedling emergence of various soybean (Glycine max) cultivar

seeds, artificially infected with Pseudomonas syringae pv. Glycinea

To determine and compare the incidence of bacterial blight as a result of seed-to-seedling

transmission of Pseudomonas syringae pv. Glycinea in Soybean (Glycine max) varieties.

6

CHAPTER 2

LITERATURE REVIEW

2.1 SOYBEAN (Glycine max (L.) Merril)

Soybean (Glycine max (L.) Merril.), also known as Greater bean is a native plant of Eastern Asia.

The progenitor of soybean (Glycine max) grows wild throughout eastern China, Korea, Japan and

the far eastern portion of Russia. It is a leguminous vegetable of the pea family that grows in

tropical, subtropical, and temperate climates. It belongs to the family Leguminoseae and sub-

family Papilionoidae. The crop is considered one of the five oldest cultivated crops and it was

utilized by the Chinese as a source of food before 2500 BC (Hymowitz and Shurtleff, 2005).

2.2 HISTORY AND DISTRIBUTION

Soy bean may have been introduced to Africa in the nineteenth century by Chinese traders along

the east coast of Africa. In Zimbabwe, it was introduced in 1940 (N2Africa, 2014). Before that,

the government of Zimbabwe (Southern Rhodesia, then) used to import fish meal for feeding

livestock.

2.3 SOYBEAN PLANT DESCRIPTION

Soya bean is an annual, leguminous, warm-temperature, short-day plant, normally bushy and erect

(upright growth habit). Usually plant height varies from 40 to 100 cm (Hymowitz and Shurtleff,

2005). Plants are much branched with well-developed roots, and each plant produces a number of

7

small pods containing one to four round, usually yellow to black seeds. It has a round hairy stem

with branches, and it varies in color according to the cultivar.

Soybean (Glycine max) plant is categorized into determinate and indeterminate types. The

determinate types are short and terminate growth with the onset of flowering and the growth tips

end in a pod-bearing raceme. The harvesting can be done in one round because all pods usually

ripen at the same time. The indeterminate types can grow to a height of about 70 cm. They continue

to grow vegetatively and they flower and form pods resulting in seeds or pods of different sizes

that require manual harvesting at different times.

The root system of the Soybean (Glycine max) plant is extensive, with a tap root that may exceed

1, 5 m in length cm (Hymowitz and Shurtleff, 2005). The tap root has lateral roots within a soil

depth of 300 mm. The roots of Soybean (Glycine max) form a symbiotic relationship with

Bradyrhizobium japonicum bacteria, which is commonly referred to as rhizobia. This bacteria is

species-specific and causes the plant to fix nitrogen into the soil independently. A major advantage

of Soybean (Glycine max) is that because of nitrogen fixation, it does not require any nitrogen

fertilizer.

Soybean (Glycine max) leaves are alternate, vary in shape, and are hairy in some varieties. The

leaves can appear dark green or tinted with brown, red or blue lesions. Soybean (Glycine max)

seeds vary in shape but are generally oval. A Soybean (Glycine max) seed consists of a large

embryo enclosed by the seed coat. There are large variations in seed coat color (light yellow, green,

brown, black, mottled), but commercial Soybean (Glycine max) is nearly always yellow. The hilum

(seed scar) is easily visible on the surface of the seed coat and is classified by color (black,

imperfect black, brown, buff and clear).

8

2.4 IMPORTANCE OF SOYBEANS

Soybean (Glycine max) is a cash crop grown for processing into edible oil (oil extraction) but is

also grown to produce a high protein meal for stock feed (poultry and pigs). It is the most highly

nutritious legume seed, and rival milk and meat in food value It contains 44.65 % high quality

protein (as compared to 7% rice, 12% wheat, and 10% maize) (Sanginga et al. 2003). The 40% of

world’s supply of vegetable oil comes from soybean (Glycine max) and 80 per cent which is used

in margarine, salad oil, cooking oil and shortening (Sanginga et al. 2003). Nutritionally, soybean’s

high protein content is of particular importance to the malnutrition-hit parts of Africa as animal

protein is too expensive for most populations. This particular soybean (Glycine max) protein is

rich in the valuable amino acid lysine. Most cereals are deficient in lysine.

Table 1: Nutritional value of soybean

FOOD TYPE WATER ENERGY PROTEIN OIL CALCIUM IRON

Common beans 10 334 25.0 1.7 110 8.0

Peas 10 337 25.0 1.0 70 5.0

Soybean 8 382 40.0 20.0 200 7.0

Meat 66 202 20.0 14.0 10 3.0

Milk 74 140 7.0 8.0 260 0.2

Egg 74 158 13.0 11.5 55 2.0

Source: (Marealle, 1974)

9

2.4 SOYBEAN PRODUCTION

Nigeria is the largest producer of soybean (Glycine max) in sub-Saharan Africa (650,000 ha),

followed by South Africa (245,000 ha), Uganda (147,000 ha), Malawi (79,480 ha), and Zimbabwe

(69,900 ha) (Giller and Dashiell, 2006). In 2009 in Zimbabwe, 71 574 ha were under Soybean

(Glycine max) (GoZ, 2009) and it contributed 50% to the GDP (GoZ, 2008). However, Zimbabwe

still remains a net importer of soybeans as domestic demand is growing faster than local

production, which is hampered by poor yields. Soybean Diseases reduce yields by up to 80%

(Kananji et al., 2013). Recently, Zimbabwe’s monthly domestic demand stood at 9,000 Metric

Tonnes (GAIN, 2011). The majority of Zimbabwe’s imports come from India while some imports

originate from Malawi (GAIN, 2011). Soybean (Glycine max) is however, mostly cultivated by

small-scale farmers in other parts of Africa where it is planted as a minor food crop among

sorghum, maize, or cassava. More than 90% of farms that produce Soybean (Glycine max) are

smaller than five hectares (Technoserve, 2011).

10

Figure 2: Soybean (Glycine max) production and processing in Sub-Saharan Africa (FAO,

2011)

2.6 SOYBEAN VARIETIES IN ZIMBABWE

As at 2010, the total number of soybean (Glycine max) varieties released in Zimbabwe had reached

39 (N2Africa, 2014). PANNAR, Pioneer, ARDA and National Tested Seeds are some key players

in the development of Soybean (Glycine max) varieties. SEEDCO, a public listed company

incorporated in Zimbabwe develops and releases soybean varieties for Zimbabwe, among other

certified crop seeds. SC Siesta, SC Saga, SC Serenade, SC Safari are some common SEEDCO

varieties (SEEDCO, 2010). PAN 1867 is a conventional soybean (Glycine max) cultivar bred by

PANNAR. It was developed in a collaborative breeding programme which included selection

efforts in Zimbabwe and South Africa. The variety is very widely adapted, and is aimed at being

11

marketed in the tropics of southern Africa. It is has a relatively long growing season and is more

suited to hot areas and tropical production. Other varieties grown in Zimbabwe include Pan

891and Bimha.

2.7 SOYBEAN BACTERIAL BLIGHT DISEASE

Soybean (Glycine max) yields are hampered detrimentally by plant diseases such as bacterial

blight. The disease is a primary leaf disease although symptoms can show on stems, petioles and

pods. It is caused by Pseudomonas syringae pv. Glycinea Symptoms of plants infected early in the

growing season are characterized by brown spots on the margins of the cotyledons. Young leaves

are most susceptible to the bacterial infection. Symptoms in later growth stages include angular

lesions, which begin as small yellow to brown spots on the leaves. The lesions dry out, turn reddish

brown to black and become surrounded by a yellowish green halo. The centers of the spots will

turn a dark reddish-brown to black and dry out. Eventually the lesions will fall out of the leaf and

the foliage will appear ragged. Lesions can also occur on the pods causing the seeds to become

shriveled and discolored. If pod infection occurs, bacterial blight can become seed borne. On stems

and petioles, lesions are large and black. When plants are infected early in the season they may

become stunted and die. The disease occurs in the early growing stages and affects leaf surface

resulting in the loss of photosynthetic area. Bacterial blight is promoted by cool, wet weather (21

– 26.7 °C). Infection can occur early but is most common at mid-season and continues until hot

and dry weather limits development. Disease outbreaks often follow windy, rainstorms.

12

Figure 3 Symptoms of Soybean (Glycine max) bacterial blight caused by Pseudomonas

syringae pv. Glycinea (Giesler, 2011).

Figure 4: Bacterial blight of soybean with (A) overall foliar symptoms and (B) water

soaking on leaf underside present at early stages of infection (Giesler, 2011).

Dark reddish-brown spots

Yellow green (chlorotic) halo

Ragged leaf

Brown spots on margin

Yellow to brown spots

Initial symptoms

Later symptoms

Water-soaked underside

13

Bacterial blight of Soybean (Glycine max) caused by Pseudomonas syringae pv. Glycinea occurs

worldwide in the major growing areas. It is usually the first foliar disease to occur on soybean

(Glycine max) in most growing seasons. Diseased plants are usually widespread within a field.

Lesions or dead spots are most obvious on leaves in the mid-to upper canopy. A simple test for

bacterial blight is to hold infected leaves to the light; bacterial blight spots will be translucent.

Bacterial blight causes substantial losses if susceptible host cultivars are grown under favorable

conditions. In 1977, bacterial blight severely affected soybean (Glycine max) in two locations in

the highlands of Kenya (Kaiser and Ramos, 1980). Pseudomonas syringae pv. Glycinea also

caused the greatest dollar-loss per year in the most important Soybean (Glycine max) growing state

Iowa in 1976 (Kennedy and Alcorn, 1976). In the member states of the European and

Mediterranean Plant Protection Organization (EPPO region), the disease appeared and caused

minor losses six years after those countries started to import seed from North America in 1978

(Gasperini et al, 1982). It potentially causes yield losses ranging from 4 % to as high as 40% under

extreme conditions (Lim, 1992; Mishra and Krishna, 2001). In 1983 in Uganda, there was a

bacterial blight outbreak at the main seed multiplication site. This caused the operation to be

abandoned and this delayed the release of seed to farmers (Allen and Lenne, 1998).

2.8 THE CAUSAL AGENT OF BLIGHT: Pseudomonas syringae pv. Glycinea

The causal agent of soybean bacterial blight, Pseudomonas syringae is a gram-negative, rod-

shaped, aerobic and motile γ-proteobacterium possessing several polar flagella. It is a

phytopathogenic bacteria whose host-specific pathovars collectively attack a wide variety of crop

plants. The organism was first isolated in 1902 by van Hall from the common liliac plant, Syringa

vulgaris, from which it got its name.

14

Pseudomonas syringae can be divided into groups of strains that cause diseases on specific plants,

called pathovars. There are over 50 known pathovars of Pseudomonas syringae (Kreig and Holt,

1984). The pathovars are host-specific for example, Pseudomonas syringae pv. Glycinea causes

bacterial blight in soybean (Glycine max) plants. Pseudomonas syringae contains both pathogenic

and non-pathogenic isolates. The key difference between pathogenic and non-pathogenic forms is

based on the presence of the Type III secretion system which operates like a syringe and plunger

to inject or secrete virulence proteins into host plant, altering its cell functioning.

The Type III secretion system is required to inject effectors, known as Hop (Hrp outer protein) or

Avr (avirulence) proteins, into plant cells, which is an essential process in Pseudomonas syringae

pathogenesis (Alfano and Collmer, 2004). Type III virulence proteins contribute to pathogenesis

chiefly through their role in suppressing plant defense by blocking the synthesis/exudation of plant

antimicrobial metabolites and (Jovanovic et al, 2011).

Figure 5: Electron micrograph of Pseudomonas syringae

2.8.1 BIOCHEMICAL PROPERTIES OF Pseudomonas syringae

Pseudomonads, in general, are nutritionally versatile organisms and can utilize a variety of carbon

sources. It utilizes sucrose which leach from the interior of plants using extra-cellular enzymes

15

like levan-sucrase or sucrose-6-phosphate hydrolase to digest the sucrose. This is of distinct

advantage for the bacteria in order to survive and replicate in the usually nutrient-deprived interior

of plant cells. Pseudomonas syringae also produces nucleating proteins (INPs) proteins which

cause water to freeze at fairly high temperatures between -4 °C to -2 °C (Maki et al, 1974). This

ice that Pseudomonas syringae produces can damage the walls of plant cells, allowing the

bacterium to access nutrients from the cells’ interiors. Pseudomonas syringae tests negative for

arginine dihydrolase and oxidase activity, and forms the polymer levan on sucrose nutrient agar.

Pseudomonas syringae pv. Glycinea has a yellow fluorescent appearance when cultured in vitro

on King’s B medium. This fluorescence is due to production of a siderophore called pyoverdin,

which is a small, high affinity iron chelating compounds secreted by microorganisms.

Pseudomonas syringae forms colonies that are circular, raised globose, glistening, light yellow

colonies on Modified Sucrose Peptone medium with a less dense centre.

2.9 SEED-TO-PLANT TRANSMISSION OF PATHOGENS

Seeds are attached by pathogens at various stages:

The pathogen-infected mother plant infects the seed via the vascular system or floral parts.

During processing of seed and at the time of transportation.

During harvest, storage, seed retention and threshing operations.

A pathogen can be seed-borne or seed transmitted, or both. A seed-borne pathogen is located on

the seed coat or between the seed coat and the cotyledon, while a seed transmitted pathogen is

incorporated in the embryo during the development of the seed. Seed-to-seedling transmission

combines the ability of a pathogen to survive outside the host seed prior to infection, multiply on

the host, and disperse and transmit to plant tissue. Once the pathogen gains entry into a plant, a

16

successful infection can only occur if a parasitic relationship between a pathogen and its host has

been established. A parasitic relationship (compatible interaction) leads to symptom development

whereas commensal interaction (incompatible interaction) leads to a hypersensitive reaction (HR),

a form of plant defense mechanism.

Population development must normally occur for many bacteria to survive and form infectious

doses that can infect plants. These bacterial cells chemically communicate with one another

(quorum sensing) and organize themselves in dense growths to form biofilms that tightly adhere

to surfaces, serving as protectants and enabling cells to produce a favorable environment for

survival and spread. The ability of pathogens to transmit by seed has significant advantages for

pathogens in that it promotes their long-term survival, maximum opportunity for progeny infection

and long distance dispersal mechanism. In seed-to-seedling transmission, both the load (dosage)

of inoculum and the rate of transmission are directly correlated with the spread of disease.

The practice of retaining seed, poor certification procedures and lack of seed health testing, all of

which are worryingly common in Zimbabwe, provide avenues for infection of seed and enables

seeds to act as the primary inoculum in pathogen transmission and disease infection. Seeds

carrying such pathogens are detrimental to crop production because those seeds may not germinate

or may germinate into diseased seedlings. This results in a decrease in the seedling population,

seedling death, loss in photosynthetic area and/or low yields.

2.9.1 TYPES OF PATHOGENS IN SEED TRANSMISSION

In pathogen transmission, there are two broad categories of pathogens: Biotrophs and Necrotrophs.

Biotrophs are pathogens that infect but do not kill plant cells. They penetrate the cell wall and

they become incorporated into the plant moving from cell to cell. They modify various aspects of

17

plant physiology such as respiration, photosynthesis, translocation, transpiration, growth and

development. Pseudomonas syringae pv. Glycinea is a typical biotrophic pathogen. Necrotrophs

are pathogens that kill cells before feeding on the cell or cell’s contents and live on dead tissue.

They do this by secreting toxins that diffuse ahead of the advancing pathogen.

2.9.2 FORMS OF TRANSMISSION

There are basically three forms of transmission. These are:

Systemic transmission of biotrophs

Transmission of necrotrophs causing local infection

Transmission of necrotrophs causing vascular infection

Pseudomonas syringae is a biotrophic organism. These organisms transmit systematically. They

can be located deeper in seed tissues like the seed coat or pericarps. When the seed germinates

these pathogens infects the embryo causing infection at seedling stage and move systemically in

the host plants. Symptoms can show up during different stages of development. In the second form

of transmission on the list, necrotrophs are transmitted from the seed coat and pericarp tissues and

cause progressive invasion to the seedling. This results in localized infection resulting in seedling

blight, leaf spots and root rots. In the third form of transmission, necrotrophs are transmitted from

the seed coat (testa) and pericarp tissues of seed, causing progressive vascular infections to the

seedling. These organisms specifically block xylem vessels physically, leading to wilting and

discoloration (Kathaaperumal et al, 2014).

18

2.9 DISEASE PHYSIOLOGY AFTER TRANSMISSION OF PATHOGEN

Biotrophic pathogens like Pseudomonas syringae become incorporated into and modify various

aspects of plant physiology such as respiration, photosynthesis, translocation, transpiration and

growth and development.

The respiration rate of plants invariably increases following infection by bacteria. This higher

rate of glucose catabolism causes a measurable increase in the temperature of infected leaves. An

early step in the plant’s response to infection is an oxidative burst, which is manifested as a rapid

increase in oxygen consumption, and the release of reactive oxygen species like hydrogen peroxide

and the superoxide anion. In resistant plants, the increase in respiration and glucose catabolism is

used to produce defense-related metabolites via the pentose phosphate pathway. In susceptible

plants, the extra energy produced is used by the growing pathogen (Deacon, 1997).

Pathogens also affect photosynthesis, both directly and indirectly. Pathogens that cause

defoliation such as Pseudomonas syringae pv. Glycinea, rob the plant of its photosynthetic tissue.

In diseases like bacterial blight, necrotrophs decrease the photosynthetic rate by damaging

chloroplasts and killing cells.

Translocation is also affected by pathogen invasion. A biotrophic infection site becomes a strong

metabolic sink where nutrients are translocated into infected leaves to satisfy the demands of the

pathogen. This depletion, diversion and retention of photosynthetic products by the pathogen

results in stunted growth.

Pathogens also affect transpiration by rupturing the hydrophobic waxy cuticle which is one of

the structures of the preformed plant defense system. This results in rapid wilting of the infected

19

plant. Pathogens also affect roots and this directly affect the plant’s ability to absorb water by

damaging the root system. Pathogens of the vascular system similarly affect water movement by

blocking xylem vessels.

20

CHAPTER 3

METHODOLOGY

3.1 OUTLINE OF METHOD

The method was divided into seven sections namely:

1. Collection of seed samples and breaking of seed dormancy

2. Seed surface-sterilization

3. Adjustment of pathogen inoculum to 1 x 106

4. Seed inoculation with pathogen

5. Planting, Germination, and symptom development

6. Detection of symptomless infection and quantification of bacteria

7. Statistical analysis

Figure 6: Methodology Flowchart

Seed collection

seed surface-sterilization

Seed inoculation

Planting, Germination, and symptom development

Quantification & Statistical

Analysis

21

3.2 COLLECTION OF SEED SAMPLES AND BREAKING SEED DORMANCY

Three different farmer-saved soybean (Glycine max) landraces were sourced from local farmers in

Chivhu, Guruve and Harare. In addition, SEEDCO PVT LTD kindly provided five commercial

soybean (Glycine max) varieties namely: Sequel, SC Signal, SC Saga, SC Squire and SC Status.

All seeds were partially germinated by soaking in warm water in separate sterilized beakers for 72

hours in order to break seed dormancy.

3.3 SEED SURFACE STERILIZATION

Seeds were surface-sterilized with sodium hypochlorite (0.3% v/v) and thoroughly rinsed in sterile

distilled water.

3.4 BACTERIAL STRAINS

Three Pseudomonas syringae strains isolated from farmer-saved seeds from Guruve, Chivhu and

Harare using three selective media: Medium M71, Medium D4 and Modified Sucrose Peptone

(MSP) were used in the study. All media had the following antibiotics incorporated: Ampicillin,

Kanamycin and Ketoconazole. The isolates were maintained by streaking onto King’s medium

B (A Non selective agar) at 28oC. The isolation and detection procedure was adapted from the

“general protocol for detection of seed-borne pathovars on Soybean (Glycine max) from

Zimbabwe” and from the “International Rules for Seed Testing: Seed Health Testing Methods

(2014)”.

22

3.5 ARTIFICIAL INOCULATION OF SEED WITH PATHOGEN

Seed samples of 120 seeds per variety were directly inoculated with a Pseudomonas syringae

isolate. A culture of bacterial suspension grown in Nutrient Broth (OXOID) was adjusted to a

concentration of 1 x 107 (CFU)/ml in phosphate buffered saline (PBS) using a spectrophotometer.

This was represented by A0.83. Seeds were then immersed in that inoculum of 1 x 107 CFU/ml.

They were immersed in phosphate buffered saline (2 ml inoculum per seed) for 1 hour. Seeds were

then air-dried for 1 hour in a biosafety cabinet at room temperature. Seed contamination/adhesion

levels were confirmed by immersing a sample of 10 seeds per Soybean (Glycine max) landrace in

Phosphate Buffered Saline at 4 overnight. The resulting seed wash was diluted to 10-3 and 10-4 and

100 µl was cultured on Modified Sucrose Peptone (MSP), Medium D4 and Medium M71 plates.

3.6 PLANTING, GERMINATION AND SYMPTOM DEVELOPMENT

Seeds were randomly sampled from the inoculated lot immediately after drying and planted in

black germination plastic pots. The seeds were planted in pots (3 seeds per pot), containing soil

substrate and exposed to sufficient light. Plants were watered three times per week. Seedling

emergence was assessed and evaluated 4-5 d after planting. Once seedlings emerged, they were

carefully watered from below to prevent splash distribution of the pathogen. Seedlings were grown

until the 2 true leaves stage (approx. 14 days after planting). Cotyledons showing symptoms were

counted. Typical bacterial blight symptoms were observed from germination throughout the

seedling period.

23

3.7 DETECTION OF SYMPTOMLESS INFECTION AND QUANTIFICATION OF

BACTERIA

The viable cell count was used to quantify the pathogen population on diseased and symptomless

plant tissues. Only plates that showed colonies not less than 30 and not more than 300 were

considered for this assay. Bacterial populations were assessed on symptom-free trifoliate leaves.

This was done by first surface sterilizing them with sodium hypochlorite, rinsing them thoroughly

with distilled water and the plant tissue was then immersed in 5 to 20 ml of distilled water in a

clean sterilized mortar. A pestle was used to crush and grind the tissue. The mixture was left to

stand for 10 minutes to allow any bacteria to diffuse out of the tissue and into the water. The

mixture was briefly agitated on an automated shaker to suspend bacteria evenly in the water. The

mixture was then allowed to stand until the larger portions of remaining tissue settled to the bottom

of the tube. From the supernatant, 10 fold serial dilutions were made and 100 µl of 10-3 and 10-4

dilutions were plated onto selective medium Modified Sucrose Peptone (MSP) (Mohan and

Schaad, 1987). The medium had the following antibiotics incorporated under sterile conditions at

1 ml per litre: Ampicillin, Kanamycin and Ketoconazole. Bromothymol Blue was also

incorporated (1 ml per litre). Plates were incubated at 28oC for 4-5 days. Samples from control

plants were also plated onto the same selective medium. On MSP after 4 days, P. syringae pathovar

colonies are circular, raised globose, glistening and light yellow colonies on MSP medium with a

less dense centre. The medium around the colony turns light yellow after 3 days. Quantification

assays were done for 12 consecutive days.

3.8 EMERGENCE, INCIDENCE AND TRANSMISSION EFICIENCY

Emergence was assessed as the ratio of emerged seedlings over the total number of plants that

were planted. Disease incidence was measured as the proportion of diseased seedlings over

24

emerged plants. The efficiency of transmission was expressed as a ratio of the number of pathogen-

inoculated seeds to the number of contaminated seedlings. This was simply done by counting the

number of plants that were infected per cultivar. To calculate the efficiency of bacterial

transmission from seeds to seedlings, sample size in all seed landraces contained the same number

of individuals. The rate of transmission was measured as the number of days taken for the

appearance of disease lesions after germination.

% Seed emergence=Number of seeds emerged

Total number of seeds planted x 100

% Disease Incidence=Number of seedlings with visual symptoms

Total number of plants assayed x 100

Transmission efficiency of bacteria =total number of inoculated seeds

Number of contaminated seedlings x 100

3.9 STATISTICAL ANALYSIS

Results were analyzed using Graphpad Prism 6® software. Analysis of variance (ANOVA) was

used to compare the equivalence of mean emergence and mean disease incidence between

artificially inoculated sample means vs the non-inoculated sample means (Controls) at P ≤ 0.05.

Unpaired two tailed t test was also used to test for equivalence between mean seed mergences of

isolates vs their respective controls.

H0: μ = μ0

H1: μ ≠ μ0

H0: Mean (seed emergence or disease incidence) of sample = Mean (seed emergence or disease

incidence) of control.

H1: Mean (seed emergence or disease incidence) of sample ≠ Mean (seed emergence or disease

incidence) of control.

25

CHAPTER 4

RESULTS

4.1 SEEDLING EMERGENCE

The following graph compares seedling emergence among various soybean varieties and

landraces artificially infected with bacterial isolates. Seedling emergence is the emergence of

the hypocotyl hook (epigeous pattern) as it pushes through the soil. It is obtained by the

formulae:

% Seedling emergence=Number of seedlings emerged

Total number of seeds planted x 100

Figure 7: Seedling Emergence trends of various varieties artificially inoculated with

Pseudomonas syringae pv. isolates.

Note: Landrace 1 was obtained from Chivhu, Landrace 2 was obtained from Harare and Landrace

3 was obtained from Guruve.

0102030405060708090

Lan

dra

ce 1

Lan

dra

ce 2

Lan

dra

ce 3

SC S

ign

al

SC S

qu

ire

SC S

equ

el

SC S

tatu

s

SC S

aga

% S

EED

LIN

G E

MER

GEN

CE

SOYBEAN VARIETY

SEEDLING EMERGENCE AFTER 4-5 DAYS

Chivhu Harare Guruve

26

The following graph is an ANOVA analyses graph to compare the equivalence of mean

emergences, between artificially inoculated sample means vs the non-inoculated sample means

(Controls) at P ≤ 0.05. Null hypothesis for the equality of two means:

H0: μ = μ0

H1: μ ≠ μ0

H0: Mean (seedling emergence) of sample = Mean (seedling emergence) of control.

H1: Mean (seedling emergence) of sample ≠ Mean (seedling emergence) of control.

From the figure, the Null hypothesis was true as there was no statistical difference (P<0.05)

between Control vs Isolates. However control plants (Blue bar) had relatively higher mean

percentages of seedling emergence.

s e e d lin g e m e rg e n c y A N O V A a n a ly s e s

% s

ee

dli

ng

em

erg

en

ce

CO

NT

RO

L

CH

IVH

U I

SO

LA

TE

HA

RA

RE

IS

OL

AT

E

GU

RU

VE

IS

OL

AT

E

0

2 0

4 0

6 0

8 0

1 0 0

Figure 8: Seedling emergence ANOVA analyses (p<0.05).

27

The following graph is a T test analyses graph to test the equivalence of mean emergence of

Harare isolate vs Control. Null hypothesis for the equality of two means was:

H0: μ = μ0

H1: μ ≠ μ0 (Two tailed test)

H0: Mean (seedling emergence of control) = Mean (seedling emergence) of Harare isolate.

H1: Mean (seedling emergence of control) ≠ Mean (seedling emergence) of Harare isolate (P<0.05)

significance level.

From the figure the null hypothesis: H0: μ = μ0 was found to be true, that is, there was no statistical

difference (P< 0.05) between Harare vs Control mean emergence.

s e e d lin g e m e rg e n c y t te s t a n a ly s e s (H a ra re v s C o n tro l)

% s

ee

dli

ng

em

erg

en

ce

CO

NT

RO

L

HA

RA

RE

IS

OL

AT

E

0

2 0

4 0

6 0

8 0

1 0 0

Figure 9: Seedling emergence t test analyses between control and Harare isolate (p<0.05).

28

The following graph is a T test analyses graph to test the equivalence of mean emergence of

Control plants vs Guruve-isolate inoculated plants. Null hypothesis for the equality of two means

was:

H0: μ = μ0

H1: μ ≠ μ0 (Two tailed test)

H0: Mean (seedling emergence of control) = Mean (seedling emergence) of Guruve isolate.

H1: Mean (seedling emergence of control) ≠ Mean (seedling emergence) of Guruve isolate

From the figure the null hypothesis: H0: μ = μ0 was found to be false as there was a statistical

difference (P< 0.05) between Guruve vs Control mean emergence.

s e e d lin g e m e rg e n c y t te s t a n a ly s e s (G u ru v e v s C o n tro l)

% s

ee

dli

ng

em

erg

en

ce

CO

NT

RO

L

GU

RU

VE

IS

OL

AT

E

0

2 0

4 0

6 0

8 0

1 0 0

Figure 10: Seedling emergence unpaired t test analyses of Control vs Guruve isolate

(p<0.05).

29

The following graph is a T test analyses graph to test the equivalence of mean emergence of

Control plants vs Chivhu-isolate inoculated plants. Null hypothesis for the equality of two means

was:

H0: μ = μ0

H1: μ ≠ μ0 (Two tailed test)

H0: Mean (seedling emergence of control) = Mean (seedling emergence) of Chivhu isolate.

H1: Mean (seedling emergence of control) ≠ Mean (seedling emergence) of Chivhu isolate

From the figure the null hypothesis: H0: μ = μ0 was found to be false as there was a statistical

difference (P< 0.05) between Chivhu vs Control mean emergence of the respective plants.

s e e d lin g e m e rg e n c y t te s t a n a ly s e s (C o n tro l v s C h iv h u is o la te )

% s

ee

dli

ng

em

erg

en

ce

CO

NT

RO

L

CH

IVH

U I

SO

LA

TE

0

2 0

4 0

6 0

8 0

1 0 0

Figure 11: Seedling emergence unpaired t test analyses of Control vs Chivhu isolate (p<0.05).

30

Table 4.1: Seedling emergence reduction

Pseudomonas syringae pv. isolates

1

CONTROL

2

CHIVHU

ISOLATE

3

HARARE

ISOLATE

4

GURUVE

ISOLATE

VARIETIES Control

emergence

%

Emergence

%

emergence

reduction

%

Emergenc

e

%

emergence

reduction

%

Emergen

ce

%

Emergen

ce

reduction

%

SC SAGA 75 25 67 75 0 68 5

SC SEQUEL 70 65 7 68 3 50 29

SC SIGNAL 95 65 20 81 15 80 16

SC SQUIRE 75 60 20 45 40 45 40

SC STATUS 68 20 71 20 71 25 63

CHIVHU 44 0 44 5 88 0 100

HARARE 39 3 92 0 100 0 100

GURUVE 45

8 82 0 100 0 100

AVERAGE

EMERGENCE

REDUCTION

63.88 30.75 50.38 36.75 52.13 33.50 56.63

31

4.2 DISEASE INCIDENCE

The following graph compares effect of bacterial isolates on disease incidence across all soybean

varieties. From the figure, SC Squire/Guruve combination had the highest disease incidence

(80%). SC Sequel/Harare combination had the lowest disease incidence (5%). Disease incidence

refers to the occurrence of disease among a group of sampled individuals e.g. plants. It is obtained

by:

% Disease Incidence=Number of seedlings with visual symptoms

Total number of plants assayed x 100

D is e a s e in c id e n c e

S O Y B E A N V A R IE T Y

% D

ise

as

e i

nc

ide

nc

e

Gu

ru

ve

SC

Sig

na

l

SC

Sq

uir

e

SC

Se

qu

el

SC

Sta

tu

s

SC

Sa

ga

0

2 0

4 0

6 0

8 0

1 0 0

C h iv h u Is o la te

H a ra re Is o la te

G u ru v e Iso la te

C o n tro l

Figure 12: Disease incidence as a function of isolate/variety combination

Note: There was zero incidence of blight disease for control plants (Purple bar)

32

The following graph is an ANOVA analyses graph to test the equivalence of mean emergence of

Control plants vs isolate inoculated plants. Null hypothesis for the equality of two means was:

H0: μ = μ0

H1: μ ≠ μ0

H0: Mean (Disease incidence) of control = Mean (Disease incidence) of inoculated plants

H1: Mean (Disease incidence) of control ≠ Mean (Disease incidence) of inoculated plants

From the figure, the Null hypothesis was false as there was a statistically significant difference

(P<0.05) between Control vs Isolates in disease incidence. A range of 4 –80% blight incidence

across the varieties and isolates was recorded. The Guruve isolate-infected plants had the highest

disease incidence pattern than both Chivhu and Harare-infected plants.

Disease incidence: Guruve isolate > Chivhu isolate > Harare isolate

D IS E A S E IN C ID E N C E A N O V A A N A L Y S E S

IS O L A T E

DIS

EA

SE

IN

CID

EN

CE

(%

)

CH

IVH

U

HA

RA

RE

GU

RU

VE

CO

NT

RO

L

0

2 0

4 0

6 0

8 0

1 0 0

Figure 13: Disease Incidence ANOVA analyses (p<0.05)

33

4.3 DISEASE INCIDENCE PATHOLOGICAL RESULTS

Figure 14: Early Bacterial light disease symptoms on “SC Status” Soybean variety.

A- Control plant (status variety) showing no blight symptoms on cotyledons and primary

leaf. B and C Early blight symptoms characterized by yellow to brown spots on the margin

of cotyledon C-Brown spots on margins

Status control Status Isolate 1

Status isolate 2 Status isolate 3

Early symptoms

characterized by yellow

to brown spots on the

margins of the

cotyledons

34

Figure 15: Early Soybean Bacterial blight disease symptoms

A-yellow to brown spots on the margin of cotyledon. B-Dark reddish-brown spots on

developing on margins. C-Yellow-green halo (Chlorotic necrosis) on upper leaf surface. D-

Brown spots on leaf margin. E yellow to brown spots on the margin of cotyledon. F-Lesion

on cotyledon incited by P. syringae pv. Glycinea.

Yellow-green halo

Early signs of ragged leaf

Brown spots on margin

Dark reddish-brown spots

35

Figure 16 : Lesions incited by Pseudomonas syringae pv. Glycinea on emerged cotyledon pose

as source of early inoculum that incite secondary lesions on seedling leaves. A- 0.5 cm x 0.2

cm Lesion on soybean cotyledon on plant. B-Insert of soybean cotyledon sampled for

bacterial count.

0.5 cm x 0.2 cm lesions incited by P. syringae

36

4.2 QUANTIFICATION OF BACTERIA FROM PLANT TISSUE

Table 4.2: Bacterial population in plant tissue

VARIETY/

LANDRACE

BACTERIAL

ISOLATE

(CFU)/ml at day

Day 3 Day 6 Day 9 Day 12

SC Saga

1

--

3.4 x 104

3.7 x 106

6.9 x 106

3 5.8 x 104 1.10 x 105 7.8 x 106 9.4 x 106

SC Sequel 1 4.9 x 104 7.7 x 104 8.0 x 106 8.6 x 106

SC Status 1 3.5 x 104 373.7 x 104 8.8 x 106 9.1 x 106

2 6.7 x 104 1.01 x 105 7.5 x 106 8.5 x 106

SC Signal 1 8.9 x 104 9.9 x104 9.4 106 1.1 x 107

SC Squire 1 3.4 x 104 4.8 x 104 3.6 x 106 4.9 x 106

3 8.5 x 104 9.6 x 104 1.1 x 107 1.2 x 107

*Inoculum used to spike seeds = 1.5 x 107 (represented by A 0.83)

37

This graph shows the log bacterial viable counts (CFU)/ml of some soybean plant tissue as at

day 12 after emergence. The plant tissue sampling included both symptom-expressing plant

and also symptomless plant. Results show that transmission from seed to plant occurred in all

soybean varieties and at day 12 the range of bacterial population was 6.9 x 106 to 1.2 x 107.

As can be seen from the graph bacterial population was almost equaling the population used

initially to infect seed before planting.

B a c te r ia l c o u n ts fro m p la n t t is s u e in re la t io n to in it ia l in o c u lu m (a s a t d a y 1 2 )

S O Y B E A N V A R IE T Y

Lo

g (

CF

U)/

ml

at

da

y 1

2

Init

ial

Ino

cu

lum

SC

Sa

ga

1

SC

Sa

ga

3

SC

Sta

tus

1

SC

Sta

tus

2

SC

Se

qu

el

SC

Sig

na

l

SC

Sq

uir

e 1

SC

Sq

uir

e 3

0

2

4

6

8

Figure 17: PLANT TISSUE BACTERIAL QUANTIFICATION

38

Figure 18: Isolation and quantification of pathogen from diseased and symptomless plant

tissue.

6.7 x 104 CFU/ml 8.9 x 104 CFU/ml

5.8 x 104 CFU/ml

39

Figure 19: Isolation and quantification of pathogen from diseased and symptomless plant

tissue.

TTC 3.4 x 104 CFU/ml 4.9 x 104 CFU/ml

40

CHAPTER 5

DISCUSSION

All the three bacterial isolates were transmitted from seed to plant in all tested soybean (Glycine

max) varieties, however the varieties varied in their response to artificial infection with bacterial

isolates with respect to seedling emergence and disease incidence. Seed-to-plant transmission was

a function of disease incidence in symptomatic plants, and was confirmed by bacterial counts of

pathogen isolated from symptomless plant tissue plated on selective media Modified Sucrose

Peptone. Control Plants derived from seeds not artificially inoculated with pathogen did not

develop characteristic blight symptoms and had relatively higher germination and emergence

means than their inoculated counterparts. This shows that Pseudomonas syringae pv. Glycinea

slowed emergence of seeds and in some cases crippled the whole germination process.

The ability of pathogens in affecting germination has been demonstrated in a study where

sterilized soil infested with P. tomato (at concentrations 102-109 propagules/plant prevented

germination. This demonstrated that at high levels of pathogen (106 propagules/plant)

prevented germination and at lower levels of inoculum (<104 propagules/plant), there was a

significant decrease in percentage seed germination. Another similar study in Nigeria

confirmed seedling emergence reduction and it ranged from 66.7 to 80.0% (Okechukwu et

al., 2010). In this study, the Harare, Chivhu and Guruve reduced germination by 52.13%,

50.38 and 56.63%, respectively. The pathogen was inoculated at a concentration of 10 7 and

higher germination reduction percentages could have been obtained had temperatures have

41

been favorable to the pathogen. The decrease in germination and emergence, demonstrated in

this study, may have greater economic significance if more expensive seeds of soybean

cultivars are to be used.

It is probable that seedling emergence was reduced by the growth inhibiting toxin produced

by Pseudomnas syringae pv. Glycinea in lesions on cotyledons (Hoitink and Sinden, 1970;

Sheesman et al., 1969). This Germination-Arrest Factor (GAF) produced by Pseudomonas.

syringae pv. Glycinea is a small peptide produced by the Pseudomonas family which has

herbicidal activity that specifically inhibits germination of seeds of graminaceous and other

species. Furthermore, Pseudomonas syringae produces ice-nucleating proteins (INPs),

which promotes water-freezing causing seed damage (Maki et al, 1974).

Disease incidence results showed that the adverse effect of bacterial infection was pronounced

as early as the cotyledon stage further confirming literature that classifies soybean blight as

an early season disease (Giesler, 2011). A bacterial blight incidence range of 4 to 80%

bacterial blight incidence across the varieties and isolates was recorded in the study. The

highest blight incidence was recorded in SC Squire/Guruve isolate combination (80%) while

the lowest incidence was observed in SC Saga/Guruve isolate combination (8%). The

maximum disease incidence recorded (80%), concurred with maximum incidence in a similar

study which had 83.3 and 70.3% of plants infected (Anaele et al., 1990). In this study the

pathogen produced characteristic symptoms on plant tissue, but most conspicuous were the

early stage lesions on cotyledons that have been shown to be incited by Pseudomonas.

syringae pv. Glycinea in one study (Daft and Leben, 1972). These lesions on seedlings that

continued to grow have been demonstrated to be a major source of early inoculum that incites

secondary lesions on seedling leaves (Wolf, 1920)

42

The degree of seed to plant transmission varied significantly (P<0.05) with variety/isolate

combinations. The varieties varied in their response to artificial infection with bacterial isolates as

shown by the different seedling emergence, disease incidence, and symptomless plant bacteria

counts in different isolate/variety combinations. These variations indicate virulence variation

among Pseudomonas syringae isolates. This is in line with numerous studies that have reported

that variations occur among isolate strains of Pseudomonas syringae with respect to their

pathogenicity (Baca et al, 1987; Gross et al, 1984; Roos and Hattingh 1987; Perlasca, 1960

and Oprea, 1971). The observed variation due to variety/isolate combinations (Host-pathogen

interaction) was similar to pathogenic variation reported in similar work done with isolates of

Xanthomonas campestris pv. Phaseoli in beans (Ekpo and Saettler 1976).

This variation observed in isolate pathogenicity may also be due to their origin Chivhu and

Harare isolates being introduced from the same climatic region (Natural Farming Region)

while the Guruve isolate being derived a different region. Guruve had the highest effects on

seedling emergence and disease incidence. Also, variations in the molecular components of

the isolates of Pseudomonas syringae pv. Glycinea isolated from various geographic areas

may affect isolate/seed compatibility and the degree of each seed to transmiss ion of each

bacterial isolate (Verdier et al, 1998).

Disease incidence variations may also be due to the different levels of susceptibility of

soybean varieties. This is supported by the fact that soybean varieties are known have

different susceptibility levels ranging from 1 to 2.6 (SEEDCO, 2010). Other researchers have

shown that most cultivars have moderate levels of resistance (Lightfoot, 2008) moreover,

differences in susceptibility between cultivars were also observed in a similar study caused

by the pathogen: P. tomato where seeds of the susceptible variety were more affected than the

43

seeds of the resistant one. This may indicate a different mechanism for foliage resistance and

for seed resistance based on host-pathogen interactions. In a similar study in Nigeria on

Cowpea and Xanthomonas campestristris, the variation in plant transmission with

variety/isolate combinations was confirmed (Okechukwu et al., 2010).

The Modified Sucrose Peptone bacterial counts of symptom and symptomless plant tissue

managed to detect greater bacterial population counts. Highest bacterial counts were obtained

from the cotyledons and leaves than stem. This showed a systemic invasion by the pathogen.

Bacterial population increased with time in plant tissue and at day 12 after germination a

bacterial range of 6.9 x 106 to 1.2 x 107 (CFU)/ml was recorded. Previous studies reported

that in planta population size of Pseudomonas syringae pv. Glycinea reach 1.7 x 108 from 1.4

x 106 (CFU)/ml. (Volksh et al, 1991) and they further report that population increased little

from June to July.

Isolation of pathogen from symptomless plant tissue showed that Pseudomonas syringae

could enter plant tissues but symptoms may fail to develop until the next growing season, thus

indicating that latent infection can occur. This has been demonstrated also in work out of

South Africa using a marked strain of Pseudomonas syringae (Roos, and Hattingh, 1987b;

Roos and Hattingh, 1987c). This latent phase also showed that this bacterium survived as an

epiphyte without causing disease on and in the surfaces of many plants, and as such, is in a

position to cause infection should the right environmental conditions develop as previously

shown by (Lindow et al, 1978; Leben, 1965; Shane and Baumer, 1987).

Other researchers have demonstrated that an antibiotic-resistant strain of Pseudomonas

syringae could be recovered from symptomless maple trees for up to 10 months (Malvick and

Moore, 1987). Establishment of Pseudomonas syringae inside symptomless tissues could

44

represent a very important source of primary inoculum and obviously poses a significant

challenge to the idea of controlling the disease. The epiphytic latent Pseudomonas syringae

may be important for survival and secondary spread during the growing season.

The study has shown that over relying on typical symptoms expression may be misleading, as

symptoms elicited by pathogens are functions of environmental, genotypes and the prevailing

weather conditions. The larger and growing pathogen population detected in symptomless

plant tissue (6.9 x 106_1.2 x 107) posed great risk as (Yunis et al (1980b) found that when the

foliage of field tomatoes was highly infected but symptomless, there was a decrease in foliage

weight, plant height and yield. In a similar work it was found that 200 seedlings which

emerged in infested soil and remained symptomless for a further 8 weeks had 20-30% less

foliage. Thus, the yield obtained from these plants, would also be decreased. This shows that

the only efficient way of avoiding plant disease would be using pathogen-free seed.

This study has shown the importance of seed-borne inoculum of Pseudomonas syringae in

disease initiation. Even though seed-borne inoculum is considered insignificant in causing

bacterial blight in areas where the disease has already been established (Veena et al, 1996),

infected seeds are important means of dispersal of the pathogen to disease-free areas.

45

CONCLUSION

The study has demonstrated that seed-to-plant transmission occurs in soybean (Glycine max)

varieties and landraces grown in Zimbabwe. It has also demonstrated that levels of seed-to-plant

transmission vary in different soybean varieties. These variations are largely due to different

variety/isolate combinations. The variety itself has different susceptibility levels to bacterial blight

whilst the bacterial isolate may vary in virulence and pathogenicity due to variations of molecular

components or due to the geographic source of the isolate. The detection of pathogen in

symptomless plant tissue has shown that Pseudomonas syringae pv. Glycinea can invade plant

tissues but symptoms may fail to develop until the environmental conditions are favorable,

thus indicating that latent infection can occur. This latent growth inside symptomless tissues

could represent a very important source of primary inoculum and may be important for

survival and secondary spread of Pseudomonas syringae pv. Glycinea. This has showed that

over relying on typical symptom-expression may be misleading, as symptoms elicited by

pathogens are functions of environmental, genotypes and the prevailing weather conditions.

Thus the use of pathogen-free seeds for planting will help avoid all the deleterious effects of

seedling mortality, foliar blight and stem canker in surviving plants, the cumulative effect of which

may lead to yield losses.

46

RECOMMENDATIONS

My first recommendation is aimed towards improving detection and quantification methods of

seed pathogens. I highly recommend the use of Quantitative PCR and ELISA to detect and quantify

plant related pathogens such as Pseudomonas syringae pv. Glycinea This has benefits of being

rapid, simple and cheap as compared to traditional plating techniques. I also recommend the

Department of Applied Biology & Biochemistry to install a greenhouse for plant pathology related

studies.

47

REFERENCES

REFERENCES

Alfano, J. R., and Collmer, A. 2004. Type III secretion system effector proteins: double agents in

bacterial disease and plant defense. Annual Review Phytopathology 42:385–414

Allen, D.J and Lenné, J.M. 1998. Disease as a constraint to production of legumes in Agriculture.

In The pathology of food and pasture legumes. Edited by Allen, D.J and Lenné, A.M. Pages 1–61.

CAB International, Wallingford, UK.

Allen, D.J. 1991. Outbreaks and new records. Africa. New disease records from legumes in

tropical Africa. FAO Plant Protection Bulletin 39(2-3): 112-113

Baca, S., Canfield, M.L and Moore, L.W. 1987. Variability in ice nucleation strains of

Pseudomonas syringae isolated from diseased woody plants in Pacific Northwest nurseries. Plant

Diseases 71:412.415.

Baker, K. F and Smith, S.H. 1966. Dynamics of seed transmission of plant pathogens. Annual

Review Phytopathology 14:311–332.

Bashany, Y., Okon, Y & Henis, Y. 1978. Infection studies of Pseudomonas tomato, causal agent

of bacterial speck of tomato. Phytoparasitica 6: 135-143

Cappelli, C. 2007. Seeds: pathogen transmission through: In Encyclopedia of Plant and Crop

Science, Boca Raton, Florida: Taylor & Francis pp 1142–1147.

48

Daft, G.C and Leben, C. 1972. Bacterial blight of soybean: Seedling infection during and after

emergence. Phytopathology 62:1167-1170.

Endres, J. 2001. Soy protein products characteristics, nutritional aspects and utilization.

Champaign. IL: AOCS Press.

FAO (Food and Agriculture Organization of the United Nations). 2011. FAOSTAT, Rome

Available from http://faostat.fao.org [accessed on 27/11/14, 17.00]

Friis-Hansen, E. 1995. Seeds for African peasants: Peasants’ needs and agricultural research –the

case of Zimbabwe. The Nordic African Institute pp 227.

Gain. 2011. USDA Foreign Agricultural Service: Global Agricultural Information Network.

Available at www.gain.fas.usda.gov. [Accessed 21/01/15, 12.00]

Giesler, L.J. 2011. Bacterial Diseases of Soybean. Plant Diseases. University of Nebraska–

Lincoln.

Giller, K.E and Dashiell, K.E. 2006. Glycine max (L.) Merr: In Plant resources of tropical Africa

1. Cereals and pulses. PROTA Foundation, M. Brink and G. Belay (eds). Netherlands/Backhuys

Publishers. Leiden, Netherlands pp.76-82.

Hoitink, H.A and Sinden, S.C. 1970. Partial purification and properties of chlorosis inducing to

toxins of Pseudomonas phaseolicola and Pseudomonas glycinea. Phytopathology 60:1236-1237.

Huynh, T.V., Dahlbeck, D and Staskawicz, B.J. 1989. Bacterial blight of soybean: regulation of a

pathogen gene determining host cultivar specificity. Science 245:1374–1377.

Hymowitz, T and Shurtleff, W. R. 2005. Debunking soybean myths and legends in the historical

and popular literature. Crop Science 45:473-476.

49

Jovanovic, M., James, E.H., Burrows, P.C., Rego, F.G., Buck, M and Schumacher, J. 2011.

Regulation of the co-evolved HrpR and HrpS AAA+ proteins required for Pseudomonas syringae

pathogenicity, National Communication 2:177.

Kaiser, W.J and Ramos A.H, 1980. Occurrence of Pseudomonas syringae on bean and soybean in

Kenya. Plant Disease 64:593-595.

Kananji, G.A.D., Yohane, E., Siyeni, D., Mutambo, L., Kachulu, L., Chisama, B.F., Malaidza H.,

Tchuwa, F and Mulekano, O. 2013. A guide to soybean production in Malawi. Department of

Agricultural Research Services. Lilongwe. Malawi

Kathaperumal, A.K., Peeran, M.F and Krishnan, N. 2014. Mechanism of Seed Transmission.

Popular Kheti 2(2):115-118

Kimbrel, J., Givan, S., Halgren, A., Creason, A., Mills, D. 2010. An improved, high-quality draft

genome sequence of the germination-arrest factor-producing Pseudomonas fluorescens WH6.

BMC Genomics 11:522.

Kreig, N. R and Holt, J. G. 1984. Bergey's Manual of Systematic Biology. Baltimore: Williams

and Wilkins pp141–99.

Leben, O. 1965. Epiphytic microorganisms in relation to plant disease. Annual Review of

Phytopathology 3:209-30.

Lightfoot, D.A. 2008. Soybean genomics development through the use of cultivar “Forest”.

International Journal of plant genomics 79: 315-325.

Lindow, W.E., Amy, D.C and Upper, C.D. 1978. Distribution of ice nucleation-active bacteria on

pants in nature. Applied Environmental. Microbiology 36:831-838.

50

Malvick, D.K and Moore, L.W. 1987. Survival and dissemination of an antibiotic resistant

Pseudomonas syringae strain in a maple nursery and application of DNA restriction fragment

analysis for strain identification. Proceedings of the 3rd International working group on

Pseudomonas syringae pathovars, Lisbon, Portugal, 1-4 September.

Marealle, A.L.D. 1974. Tanzania Food Tables, East African Literature Bureau (Kiswahili). Dar es

Salaam. Tanzania

Mathur, S.B., Haware, M.P and Hampton, R.O. 1988. Identification, significance and transmission

of seed-borne pathogens. A Contribution from Danish Government Institute of Seed Pathology for

Developing Countries. Copenhagen, Denmark No. 109. A reprint from World Crops: Cool Season

Food Legumes. Summerfield R.J pp. 351-365

Maude, R.B. 1996. Seed borne diseases and their control. CAB International. Cambridge pp280.

Mayhew, C., Sneller, C., Cocker, D., Dombeck, D and Widick, D. 1914. Variety development,

testing and selection. In Arkansas Soybean Handbook. Arkansas pp 13-16 available at

www.uaex.edu [Accessed 29/12/14, 12.25]

McDonald, M. B. and Copeland L. O. 1998. Seed Production: Principles and practices. CBS

Publishers and Distributors. India pp 749.

Ministry of Agriculture, 2011. Mechanisation and Irrigation Development, Farm Management

Handbook. Jongwe publishers. Harare.

N2Africa, 2014. Better soybean for farmers in Zimbabwe. Available at http://www.N2Africa.org.

[Accessed on 27/11/14, 17.00]

51

Okechukwu, R.U., Ekpo, E.J.A and Okechukwu, O.C. 2010. Seed to plant transmission of

Xanthomonas campestris pv. Vignicola isolates in Cowpea. African Journal of Agricultural

Research 5(6):431-435

Oprea, F. 1971. Pathogenicity of some Pseudomonas morsprunorum and Pseudomonas syringae

strains in stone fruits. Microbiologia 2:67-75.

Perlasca, G, 1960. Relationships among isolates of Pseudomonas syringae pathogenic on stone

fruit trees. Phytopathology 50:889-899.

Roos, I.M.M and Hattingh, M.J, 1987b. Systemic invasion of cherry leaves and petioles by

Pseudomonas syringae pv. morsprunorum. Phytopathology 77:1246-1252.

Roos, I.M.M and Hattingh, M.J. 1987c. Systemic invasion of plum leaves and shoots by

Pseudomonas syringae pv. Syringae introduced into petioles. Phytopathology 77: 1253-1257

Roos, M.M. and Hattingh, M.J. 1987. Pathogenicity and numerical analysis of phenotypic features

of Pseudomonas syringae strains isolated from deciduous fruit trees. Phytopathology 77:900-908.

Sanginga, N., Dashiell, K., Diels, J., Vanlauwe, B., Lyasse, O., Carsky, R.J., Tarawali, S., Asafo-

Adjei, B., Menkir, A., Schulz, S., Singh, B.B., Chikoye, D., Keatinge, D and Rodomiro, O. 2003,

Sustainable resource management coupled to resilient germplasm to provide new intensive cereal-

grain legume-livestock systems in the dry savanna. Agriculture Ecosystems and Environment 100:

305–314.

Scholthof, K.B.G., Adkins S., Czosnek H., Palukaitis P., Jacquot E., Hohn T., Hohn B., Saunders

K., Candresse T., Ahlquist, P., Hemenway, C and Foster, G.D. 2011. Top 10 plant viruses in

molecular plant pathology. Molecular Plant Pathology 12: 938–954.

52

SEEDCO, 2010. “Product Manual 2010/11.” available at http://seedco.co.zw [accessed on

27/11/14]

Shane, W.W and Baumer, J.S. 1987. Population dynamics of Pseudomonas syringae pv. syringae

on spring wheat. Phytopathology 77:13991405.

Sheesman, J.P., Leben, C., Schmitthemer, A.F AND Coyle, E. 1969. Relation of Pseudomonas

glycinea to systematic toxemia in soybean seedlings. Phytopathology 59:1970-1971

Technoserve, 2011. “Southern Africa projections to a regional soybean roadmap final report.

Available at http://f.cl.ly/items/3138103k2w2l2z0w2e3E/tns-bmgf-regional-report.pdf. [Accessed

on 27/11/14, 17.00]

Valarini, P.J., Menten, J.O.M and Oliveira, D.D.A. 1996, Xanthomonas campestris pv. Phaseoli:

Importance of seed inoculum in the epidemiology of the common bacterial blight of beans,

Fitopathologia Brasileira 21:261-267.

Verdier, V., Assigbetse, K., Gopal, K.C., Wydra, K., Rudolph, K., Geiger, J.P. 1998. Molecular

characterization of the incitant of cowpea bacterial blight and pustule Xanthomonas campestris pv.

vignicola. European Journal Plant Pathology 104(6): 595-602.

Wolf, F.A. 1920. Bacterial blight of soybean. Phytopathology 10:119-132.

ZIMSTATS, 2014. Statement of External trade statistics. Available at http://www.zimstat.co.zw/

[Accessed 23/01/15]

53

APPENDIX