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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.
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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.
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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…………………
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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.
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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
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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
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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
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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
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Figure 21: Isolation and quantification of pathogen from diseased and symptomless plant tissue.
....................................................................................................................................................... 39
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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
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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
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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.
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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
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(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.
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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.
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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
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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).
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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)
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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).
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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
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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.
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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
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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.
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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
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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
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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]
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APPENDIX