AETIOLOGY AND IMPORTANCE OF FOLIAGE DISEASES AFFECTING CITRUS 
IN THE NURSERY AT THE AGRICULTURAL RESEARCH STATION (A.R.S.).
KADE.
BY
JOSEPH OKANI MONGER
THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON IN 
PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF 
DEGREE IN MJTNL CROP SCIENCE.
JUNE 2004
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S&  CrOg.CS' f t l L  
W\ -t° x  
C - '
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DECLARATION
I hereby declare that except for reference to other peoples work which have been duly 
cited, this work is the result of my original research and that this thesis has neither in 
whole nor part been presented for another degree elsewhere.
JOSEPH HONGER 
(STUDENT)
(SUPERVISOR)
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DEDICATION.
This work is dedicated to 
Mrs. Irene Komley Dan-Okine of The Holy Spirit Cathedral, Accra.
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a c k n o w l e d g e m e n t s
My first and foremost thanks go the Almighty God for His mercies and love that 
sustained me throughout the period of my study at the University.
I would also like to extend my sincerest thanks to my supervisor Prof. K.A. Oduro for 
suggesting this work and following it up with the necessary guidance and support as I 
carried out the research work.
I' am also grateful to Prof. S. K. Offei o f the Crop Science Department o f the University 
of Ghana and Dr.K.G. Ofosu -Budu at A.R.S. Kade for all the help they afforded to me 
both in Accra and in Kade.
To Mr. Eric Cornelius, of the Crop Science Department, University of Ghana, I say a big
thank you for the encouragement and suggestions he gave to me during the period of my
research work. I would also like to mention the invaluable help given to me by Mr.
Jonathan Quaye o f the Animal Science Department who helped me a lot in the handling
me.
of the bacterial part o f the work. I appreciate very much the help offered to by Mr. 
Stephen Torkpo of the A.R.S Kade and Phyllis Mante o f New Era J.S.S.
Finally to all the Lecturers and Workers of the Crop Science Department. University of 
Ghana. I say a big thank you and may (rod bless you all.
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TABLE OF CONTENT
DECLARATION 1
DEDICATION.
ACKNOWLEDGEMENTS IV
TABLE OF CONTENT V
LIST OF TABLES VIII
LIST OF PLATES IX
ABSTRACT X
CHAPTER ONE 1
INTRODUCTION 1
CHAPTER TWO 6
LITERATURE REVIEW 6
2.1 Origin and spread of citrus. 6
2.2 Importance of nurseries in the propagation of citrus. 7
2.3 Diseases as constraints to citrus production. 8
2.4 Importance and aetiology of some foliage disease of citrus 9
2.4.1 Greasy spot. 9
2.4.2. Melanose. 11
2.4.3 Citrus scab 11
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2.4.3 Lime anthracnose
2.4.4 Altemaria brown leaf spot o f mandarins
2.4.5 Altemaria lea f spot of rough lemon
2.4.6 Sooty mould and sooty blotch
2.4.7 Areolate Leaf Spot
2.4.8. Cercospora Leaf Spot
2.4.9 Bacterial blast
2.4.10 Citrus Canker
2.4.11 Tristeza
2.4.12 Citrus exocortis
2.5. Control of foliage diseases of citrus
2.5.1 Regulatory
2.5.2 Cultural methods 
2.5.3. Chemical control
CHAPTER THREE
MATERIALS AND METHODS
3.1 Characteristics of area of study
3.2 Area, location and some practices in the nursery.
3.3 Field survey
3.3.1 Determination of field symptoms of the diseases
3.3.2 Determination of disease incidence and severity
3.3.3 Effect o f diseases on matured citrus trees in the nursery
3.3.4 Incidence of leaf miners
3.3.5 Incidence of foliage diseases in the other nurseries at the station
3.4 Laboratory work
3.4.1 Isolation and confirmation of causal agents of diseased leaves
3.4.2 Alternative methods for the isolation of causal agents of the diseases
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3.4.3 Pathogenicity test o f isolates 35
3.4.4 Characterisation of isolated bacteria 36
3.4.5. Further studies on citrus canker 38
CHAPTER FOUR 40
RESULTS 40
4.1 Varieties of citrus grown in the nursery 40
4.2 Diseases identified in the nursery 41
4.2.1 Brown leaf spot 41
4.2.2 Citrus scab 47
4.2.3 Greasy spot 50
4.2.4 Citrus canker 54
4.3 The influence of method of inoculation on the development of citrus canker 
disease on seedlings 62
4.4 Incidence of leaf miners at the nursery 66
DISCUSSIONS 68
REFERENCE 74
APPENDIX 82
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Table 1. Varieties o f citrus identified in the nursery. 40
Table 2. Host range, incidence and severity o f brown leaf spot in the nursery. 44
Table 3. Host range, incidence and severity o f citrus scab in the nursery 49
Table 4. Host range, incidence and severity o f greasy spot-like disease in the nursery. 53
Table 5. Host range, incidence and severity o f citrus canker disease in the nursery. 58
Table 6. Influence of method of inoculation on canker disease development. 65
LIST OF TABLES
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Plate 1. A section o f the nursery showing the arrangement o f seedling and budlings in 28
beds. 28
Plate 2. Characteristic symptoms of brown leaf spot on the variety o f rough lemon. 43
Plate 3. Muriform spores of Alternciria citri scrapped directly from the diseased lesion. 45 
Plate 4. Muriform spores of Alternaria citri entangled in mass o f  mycelia on P.D.A. ' 45 
Plate 5. Cultural characteristics o f Alternaria citri on Potato Dextrose Agar. 46
Plate 6. Brown leaf spot caused by Alternaria citri on the variety o f rough lemon test
seedlings. Note the absence of disease lesions on the control. 46
Plate 7. Characteristic symptom of citrus scab in the nursery. 48
Plate 8 Characteristic symptoms of greasy spot-like disease in the nursery. 52
Plate 9. Characteristic symptom o f the first stage of citrus canker. 55
Plate 10 Characteristic symptoms of the second stage o f the citrus canker disease. 55
Plate 11. Characteristic symptoms of the last stage o f citrus canker. 56
Plate 12. Cultural characteristics o f Xanthomonas citri isolated from the citrus canker 61
disease in the nursery. 61
Plate 13 Development o f young canker disease symptoms on inoculated test plants. 61
Plate 14. Citrus leaves showing young canker spots following inoculation with the 64
bacterium using the blunt syringe method (Arrowed). 64
Plate 15. Citrus leaves showing young canker spots (Arrowed) following inoculation 64
with the bacterium using the spraying method 64
Plate 16. Citrus leaf attacked by citrus leaf miner (Phyllocnistis citrella) in the nursery. 67
LIST OF PLATES
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ABSTRACT
A research work was carried out at the Agricultural Research Station to determine the 
disease status of the seedlings and budlings in the nursery. Field surveys were carried out 
on three separate occasions between October and December 2003 during the dry season
and repeated in March to May 2004 in the rainy season.
Four diseases were detected. These were citrus scab, citrus canker, greasy spot-like 
disease and brown leaf spot o f rough lemon. The causal agent for brown leaf spot was 
confirmed to be Altemaria citri, and that for citrus canker was confirmed to be 
Xanthomonas campestris pv. citri, a bacterium that has not been previously reported 
in  Ghana. Pathogens for scab and greasy spot-like disease could not be isolated and 
identified.
Brown leaf spot and scab were found to be restricted to rough lemon, which is used as 
rootstock in the nursery and therefore could not be transferred to the field. 
Comparatively, citrus canker and greasy spot -like  disease were found to have a wide 
host range and have infected the following citrus varieties in the nursery: late Valencia 
(sweet orange), waiters (grapefruit) Cleopatra mandarin, satsuma mandarin and ponkan 
(mandarins), king disemis and ortanique (hybrids); and the rough lemon. The two 
diseases have a potential o f being transferred to the field as they infect the scion as well.
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Both the incidence and severity of the diseases in all the species and varieties of the citrus 
studied were higher in the rainy season than in the dry season.
The use o f blunt syringe in inoculation studies o f the-canker disease gives quick results as 
compared to the spraying method.
Studies on the effect o f the diseases on yield, to complete their importance were not 
feasible within the period o f  the study.
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CHAPTER ONE 
INTRODUCTION
The different species o f cultivated citrus constitute major fruits o f  subtropical regions 
(Rice et al, 1986) and is the most important fruit crop in West Africa (Tweneboah, 2000). 
Primary species of cultivated citrus are the sweet orange (C. sinensis), lemon (C. limon), 
grape fruit (C. parcidisi), lime (C. ciurantifolia) and mandarin (C. reticulata). It also 
includes hybrids such as tangor and tangelo (Rice et al., 1986). They belong to the order, 
Geraniales and to the family, Rutacea. They are evergreen trees or small shrubs and vary 
in size from 3-5m tall (Rice et al., 1986; Albrigo and Davies, 1994; Tweneboah 2000).
The crop is used mainly as human food either in the raw state or in the processed state. 
The crop serves as an important source o f vitamin C and was used extensively in the past 
to prevent the occurrence o f scurvy in ships (Albrigo, and Davies, 1994). In the 
processing industry the crop, especially sour orange is processed into orange marmalade 
and other products. By- products o f the juice extraction are important in soft drink 
production where the oils and juice are used for flavours, the juice pulp cloud and the 
peel and rag extracts are used for pectin production (Albrigo and Davies, 1994). Large 
amounts o f oils in the peels are extracted for the making o f perfumes (Tweneboah, 2000), 
sections o f the fruit are canned and exported from most producing countries to other 
countries, while dried peel pellets are used in the processing o f feed for cattle (Albrigo 
and Davies, 1994). In India, the dry peel pellets are milled and used in cleaning metals 
(Morton, 1987).
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In Ghana, citrus is cultivated in most parts o f the country and is gradually gaining 
popularity among farmers in certain areas in Ashanti, Central and Eastern regions. In 
some o f these areas the crop is gradually replacing cocoa as the main cash crop o f the 
people (Ofosu-Budu; Personal communication). The crop also serves as source o f income 
to the farmers. Earnings from the crop amounted to Two hundred and fifty thousand 
dollars to Ghana in 2002 (Ghana Export Promotion Council, 2002).
Despite the growing popularity of the crop in Ghana, its production is hampered by the 
incidence o f diseases. Currently not less than fifty different diseases o f  the crop have 
been reported attacking the crop in Ghana (Oduro, 2000). These diseases affect all the 
parts o f the crop, including roots, leaves and fruits (Clerk, 1974). Almost all the major 
pathogens o f plants, including fungi, bacteria and viruses, have been reported attacking 
the crop in Ghana, predominated by fungi diseases (Clerk, 1974; Oduro, 2000).
Though citrus can reproduce sexually through seeds, most commercial plantings in 
Ghana are done vegetatively. This is partly due to the fact that the crop exhibits 
polyembryology and as such desirable characteristics o f  parent trees would not be 
exhibited by sexually propagated offsprings (Tweneboah, 2000). Furthermore there are 
some advantages offered by certain rootstocks such as the ability to withstand certain 
diseases endemic in an area, which are good for the establishment o f  the crop in the field 
(Clerk, 1974)
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The commonest propagation method is by grafting or budding o f nursery plants before 
they are planted on the field (Tweneboah, 2000). At the nursery, budlings showing 
undesirable characteristics are destroyed to ensure that plants on the field are only the 
desirable ones.
The nursery is therefore very important in the cultivation o f  citrus in Ghana. Commercial 
growers o f the crop either depend on people specialized in the raising o f seedlings for 
sale or have their own nursery where seedlings are raised for propagation (Tweneboah, 
2000). Others with large plantations have their own nurseries where planting materials 
are raised for the orchards and at the same time to be supplied to other farmers. One o f 
the last group o f cultivators of the crop is the Agricultural Research Station o f the 
University o f  Ghana.
The farm is located at Kade in the Eastern Region o f Ghana and carries out research into 
the citrus crop. The station has large plantations o f  the crop and a large nursery that feed 
the orchards with planting materials and also serve as source o f planting materials for 
most commercial farmers o f the crop in the country.
For a very long time, very few leaf spot diseases had been recorded in the nursery. These 
diseases had been managed successfully with cultural practices such as proper 
fertilization and general farm sanitation. Also when deemed necessary, the use o f  copper 
based fungicides been have also been successful in controlling these diseases Due to this, 
the outbreak o f diseases at epidemic levels were rarely encountered in the nursery. Also
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most o f  these diseases were restricted to rough lemon, which was used mainly as a 
rootstock, and as such, its spread to the farmer’s field was not much o f a problem. Effect 
o f  diseases on foliage o f  scion, which could result in poor quality planting materials were 
rarely encountered, hence incidence o f  foliage diseases in the nursery was not o f  much 
concern to the station. (Brentu; Personal comm.)
In recent times however, there appear to be an upsurge o f foliage diseases in the nursery. 
Presently all the different varieties o f the crop in the nursery, including both the scions 
and rootstocks are diseased. The alarming rate of spread o f these diseases in the nursery 
and the rapid destruction of the foliages o f the seedlings has become a great concern to 
the station. The most disturbing observation about these diseases is that the routine 
disease control measures in the nursery appear to be ineffective against these diseases. 
With the exception o f funguran, which appear to suppress these diseases, all the other 
fungicides have proved ineffective against these diseases (Brentu; personal comm.). With 
the diseases affecting foliage o f scions, there is high risk o f disseminating these diseases 
to the field o f farmers, who depend on the station for planting materials. This would 
ultimately prove detrimental to' the citrus industry in the country as a whole, if  these 
diseases prove to be destructive in the farmer’s field.
Since the station carries out a lot o f research into the crop, different varieties and planting 
materials of the crop from different places are taken there for study. In such a case, 
accidental introduction of new and destructive diseases to the nursery may be
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encountered. With the important role played by leaves in the survival o f  trees especially, 
o f  budded materials, urgent steps need to be taken to control destruction o f  the foliages o f 
the young trees in the nursery. This therefore calls for urgent steps aimed at identifying 
the various diseases in the nursery, with the view to ensuring that highly destructive ones, 
which may be present, are quickly eradicated from the nursery and also to ensure that 
measures are put in place to prevent the occurrence o f such diseases in future.
Therefore a study in the nursery to identify these diseases and their causal agents would 
yield very important information, such as, which disease or diseases are most damaging 
in the nursery and therefore require urgent attention. It would also determine whether 
some o f the cultural practices and other disease controlling measures in the nursery need 
to be changed completely or modified.
The objectives o f  this project work were to identify the various diseases and their causal 
agents in the nursery. The relative incidence and severities o f  the diseases were also 
ascertained.
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CHAPTER TWO  
LITERATURE REVIEW
2.1 Origin and spread of citrus.
Cultivated species of citrus are believed to be indigenous to south-east Asia (Tweneboah, 
2000), particularly the subtropical regions (Rice et al„ 1986). Recent evidence suggests 
that, the Yunmen province in south central China, may be an important source o f origin 
o f the crop due to the diversity o f the citrus species found there and the system o f rivers 
that could have provided dispersal o f the fruit to the south (Gmitter and Hu, 1990). Many 
types o f  the citrus species are believed to have moved to various Arabian areas such as 
Oman, Persia, Macedonia and Palestine long before Christ (Tolkowsky, 1938) and had 
been playing important role in the Jewish religion as early as 50 AD-150 AD (Webber et 
al., 1967).
Limes apparently originated in the East Achipelago and were probably brought across the 
sea o f  Oman by Arabian sailors and subsequently to Egypt and Europe (Albrigo and 
Davies, 1994). Sweet orange was introduced to Europe during the last half o f the 15th 
century by Arab traders and finally into West Africa by the Portuguese and Europeans 
(Tweneboah, 2000).
Citrus was believed to be cultivated in their natural habitat before introduction into 
Europe by the Portuguese (Tweneboah, 2000). Currently the crop is cultivated primarily 
between latitude 40° N and 40° S. More northern and southern locations o f  commercial 
production exist where temperatures are moderated by ocean winds (Albrigo and Davies,
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1994). The main centers of production in the world are southern Africa, Israel, The 
United States o f America, Brazil, Spain, Japan and Italy, with the United States being the 
largest producer. In Africa, large-scale production occurs in the Mediterranean coast and 
Zimbabwe, Mozambique, South Africa and Swaziland. In the other areas cultivation of 
the crop is on small scale and mainly for auto-consumption (Albrigo and Davies, 1994: 
Tweneboah 2000; Rice et a l ,  1986).
2.2 Importance of nurseries in the propagation of citrus.
Citrus can be propagated sexually through seeds, but commercial growers o f the crop 
prefer to use vegetative propagation. The use o f vegetative propagation offers many 
advantages to the grower. It enables the grower to enjoy the benefits o f  a particular citrus 
cultivar, such as abundant fruiting (Tweneboah, 2000). The method also widens the 
choice o f site for the establishment o f orchards, as cultivars, which would normally not 
perform well in a particular environment when grown sexually, can be grown profitably 
in the same environment with the choice o f the right type o f  rootstock. Another 
advantage o f this method is the early fruiting due to reduction in juvenile period of 
growth.
Before transplanting the budded materials, they are nursed in the nursery to ensure that 
the union o f the stock and scion had been obtained. Secondly the young trees are 
observed carefully and diseased or weak trees are destroyed. This ensures that only good 
and healthy planting materials are transplanted on the field (Albrigo and Davies, 1994), 
because the availability o f healthy planting materials is essential if  the grower is to have
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any surety o f  the continued health and productivity o f the orchard (Fraser, 1967). The 
importance o f nursery in the cultivation of the crop cannot be overemphasized and the 
success or failure o f an orchard is largely dependent on how well the propagating and 
growing o f  the crop has been done in the nursery.
2.3 Diseases as constraints to citrus production.
Worldwide, the most serious limitation to the profitable production o f  citrus in otherwise 
suitable environment is the diseases caused by bacteria, mycoplasma, viruses and fungi 
(Albrigo and Davies, 1994). As with other diseases, symptoms observed on leaves can 
either be as a result of direct attack on the foliage or as an infection on other parts o f  the 
plant (Agrios, 1997).
Diseases that affect leaves o f plants can be very devastating due to their ability to reduce 
drastically, the photosynthetic ability o f leaves, which could reach as far as 40% 
reduction. (Wood et al., 1988). This could be very devastating when it occurs in the 
nursery, where proper development o f the foliage is very important for the success o f  the 
propagation o f the crop. Currently, pathogens recorded causing diseases o f  citrus leaves 
include fungi, bacteria and other graft transmissible pathogens (Agrios, 1997).
Symptoms induced by these pathogens are varied. These include leaf distortion (Clerk, 
1974), destruction of leaf chlorophyll (Albrigo and Davies, 1994), presence o f materials 
that mask the leaf surface (Wood et a l ,  1988), and puncturing o f  holes in the leaves. In 
other cases there is premature defoliation and the plant loses almost all the diseased
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leaves (Amador, 2003). Though these symptoms vary, their end effect is always inimical 
to the growth o f the plant as they hamper the leaves ability to photosynthesise (Albrigo 
and Davies, 1994).
2.4 Importance and aetiology of some foliage disease of citrus
2.4.1 Greasy spot.
This disease was first reported in Cuba and Florida in 1915. Currently the disease has 
spread to Central and South America, The Carribeans and some Asian countries (Timmer 
eta l., 1988).
The disease is reported to be o f economic importance in areas of citrus cultivation with 
relative humidity almost up to 100%, which occurs simultaneously with high 
temperatures (Timmer et al., 1988). Morton (1987) has reported that high humidity in 
excess o f 90%, which occur after heavy infestation o f the rust mite, can also lead to the 
rapid development of the disease.
The most serious consequences o f the disease are defoliation. Losses up to 25% in fruit 
yield in sweet orange and about 45% in grapefruit have been recorded in Florida (Timmer 
et al., 1988). It has been reported to cause yield loss in humid climates in America and 
Asia (Albrigo and Davies, 1994). The disease is responsible for premature leaf drop, 
which usually occur at the end o f the first growing season. When leaves drop 
prematurely, it results in shortage of carbohydrates and their redistribution in the tree to
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stimulate leaf replacement at the expense of fruit development, culminating in low yields 
(Albrigo and Davies, 1994).
Greasy spot is a fungal disease caused by Mycosphaerella citri and its anamorph, 
Cercospora citri-grisea (Fisher) (Whiteside, 1972). The organism does not produce any 
fruiting structures on living leaves. However after leaves have fallen and decomposed, 
ascocarps containing ascospores are produced in abundance (Timmer et al, 1980).
Symptoms o f the disease first appear as a mottle on the upper side o f the leaf with a 
slightly raised pale orange to yellowish brown blister on the lower leaf surface. Later 
affected areas o f the leaf become darker brown or black and greasy in appearance, and 
much o f the chlorosis disappear. Leaves often drop before the lesions develop the dark 
greasy symptom that gives the disease its name (Timmer et al., 1988).
Certain diseases with similar symptoms as the greasy spot have been recorded in many 
parts o f  the world. The aetiological agents o f these diseases are not known though certain 
fungi have been implicated. They are believed to be fungal disease mainly because they 
could be controlled by copper fungicides (Timmer et a l ,  1988). An example o f  such a 
disease is reported in Japan and is known as psuedo-greasy spot (Koizumi, 1986). 
Another one has been reported in Australia (Timmer et al., 1988), and in Argentina 
(Marco and Whiteside, 1986).
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2.4.2. Melanose.
Melanose is reported o f being present in most citrus growing countries in the world, but 
important only where inoculum is abundant and rainfall occurs during the period of early 
fruit development (Albrigo and Davies, 1994).
The disease is reported to be of economic importance in the eastern and central parts o f 
the Texas valley where humidity is high, and attacks all the varieties o f  citrus (Albrigo 
and Davies, 1994). Injury caused to fruit rind is superficial and may not be important if 
the crop is to be processed (Timmer et al., 1988).
Symptoms appear as small, brown, discrete or confluent sunken spots, which later 
develop into pustules. The pustules on the leaves are at first surrounded by a yellow halo, 
which later regreens, leaving only the corky pustules. Pustules are also produced on fruits 
and can be confused with citrus rust mite injury, though in the case o f  the rust mite 
injury, the pustules are not raised (Timmer et al., 1988). Infections o f leaves occur after 
severe stress (Albrigo and Davies, 1994).
2.4.3 Citrus scab
The disease referred to as scab occur in three forms. These include, citrus scab, the 
commonest form; sweet orange scab and Tryons scab (Timmer et al., 1988). The disease 
is reported to be a problem in raising rootstock in nurseries and seedbeds, particularly on 
rough lemon and sour orange. Sweet orange scab is also reported as causing infections on 
fruits as well resulting in reduction in fresh fruit production (Timmer et al., 1988).
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The causal agent attacks both sides o f the leaf, but more frequently, the under surface o f 
the leaf producing numerous corky pustules (Clerk, 1974). Symptoms appear first as 
translucent dots that later becomes pustules. As the disease progresses, the pustules turn 
into warts consisting o f a mass o f corky tissues, pale tan in colour. The leaves become 
twisted and distorted (Amador, 2003). When the disease appears at a time o f leaf 
resistance pustules become smaller and little or no leaf distortion occurs ( Timmer et al., 
1988).
The three different forms o f disease, namely citrus scab, sweet orange scab and Tryons 
scab are caused by Elsinoe fawcetti (Bitancourt and Jenkins) anamorph, Sphaceloma 
fawcetti (Jenkins), Elsinoe australis (Bitancourt and Jenkins) anamorph, Sphaceloma 
fawcetti (Bitancourt and Jenkins) and Sphaceloma fawcetti var Scabiosa  (MacAlp and 
Tryon), respectively (Timmer et al., 1988). The form o f the disease caused by Elsinoe 
fawcetti is a leaf spot disease and citrus species are only susceptible to the disease when 
young and is thus confined to new growth and may attack all citrus species except sweet 
and sour orange (Timmer et al., 1988).
Elsinoe fawcetti produces nonseptate or one to two septate conidiophores that are closely 
compacted. Conidia are hyaline one celled and ellipsoidal. Being a slow growing 
organism, its isolation in the laboratory becomes difficult as it is easily overshadowed by 
other organisms present as contaminants (Timmer et al.. 1988).
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2.4.3 Lime anthracnose
This disease, also known as wither tip, is reported of being prevalent in areas where 
rainfall becomes frequent during shoot emergence, bloom or early stages o f fruit 
development. It has been reported only on key limes and never on any other citrus species 
(Wheeler, 1963). The disease can be a serious limiting factor to the production o f the key 
limes and has been reported of being particularly troublesome in the wetter climates o f 
the Caribbean (Timmer et al., 1988). It was reported to be the cause o f  great destruction 
o f limes in Florida (Amador, 2003).
The disease is characterized by localized necrosis o f different shapes and sizes. As the 
disease progresses the necrotic areas dry up and fall out, forming what is known as shot 
holes (Albrigo and Davies, 1994). Only young tissues are susceptible to attack. Severe 
infection can cause the tips o f affected shoot to shrivel for several centimeters, hence the 
name, wither tip. The disease also causes blossom blight and affected fruits may shed. 
Infected fruits may later develop corky lesions, which may be confused with those o f 
canker, but unlike canker they have no yellowish halo (Timmer et al., 1988).
Lime anthracnose is caused by the fungus Gloesporium limetticola Clausen. The 
organism produces conidia that are small but numerous and salmon pink coloured. The 
mycelium in potato dextrose agar is white and the medium becomes orange, without any 
setae on the acervuli (Timmer et al., 1988).
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2.4.4 Alternaria brown leaf spot of mandarins
The disease was first described in 1903 on emperor mandarin in Australia, causing 
blemishes on both the leaves and fruits (Pegg, 1966). It appeared in Florida where 
currently is a major problem on Dancy tangerine and M inneola tangelo (Whiteside, 
1976).
This disease is characterized by very large, round, spots on the leaf surfaces, causing loss 
o f chlorophyll. The spots usually occur in concentric rings with a characteristic target 
appearance (Albrigo and Davies, 1994). The extension o f necrosis into veins is a 
characteristic o f  the disease (Timmer et al., 1988). Symptoms also develop on fruit as 
small slightly depressed black spots. Older fruit when infected may drop. They form 
periderm, creating corky eruptions on the rind surface similar to those caused by 
melanose. However these pustules can be much larger than those caused by melanose 
(Timmer et al., 1988).
The causal organism of the disease is Alternaria citri, a pathogen bearing the same name 
as the one causing Alternaria  leaf spot o f  rough lemon but differ pathologically, in that 
toxins produced by the former is not effective on rough lemon. (Timmer et al., 1988). 
Spores produced by the organism are easily .recognized by the presence o f both cross and 
longitudinal septa (Barnett and Hunter, 1972).
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2.4.5 Altemaria lea f  spot of rough lemon
The disease was first described in South Africa in 1929 (Timmer et al., 1988). It has also 
been reported in many areas including Florida where it was erroneously referred to as 
anthracnose. It can be a major problem in the raising o f rough lemon seedlings for 
rootstock (Timmer et al., 1988).
Lesions vary from large, necrotic, blighted areas to small, circular spots. Spores o f 
Altemaria citri, are often present. Necrotic areas are usually surrounded by an extensive 
chlorotic halo. Lesions tend to extend out along leaf veins. Stem infection and defoliation 
that commonly follows infection o f the leaf blade cause die back o f  the shoot apices. 
Severe attacks lead to the development o f plants with small intemodes and excessive 
branching, which are difficult to bud (Timmer et al., 1988).
The causal organism is the fungi, Altemaria citri, a pathogen similar to the one causing 
Altemaria brown rot o f mandarin, but differing from it, pathologically. Toxins produced 
by this causal agent are only pathogenic to rough lemon and rangpur lime, but not on 
mandarins (Whiteside, 1978).
2.4.6 Sooty mould and sooty blotch
The causal organisms o f  these diseases produce a black velvety membranous fungal 
coating which appear on the surfaces of the leaves. This limits light interception and
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consequently, photosynthesis. This greatly hampers the formation o f sugars and thereby 
resulting in retarded growth and flowering (Tweneboah, 2000).
These diseases are not regarded as true diseases because the fungi associated with them 
do not feed on the tissues o f the citrus leaves, but rather on the rich honeydew, secreted 
by insects, such as aphids. However, the high economic loss, as high as 40% reduction in 
photosynthesis, associated with these diseases, justifies their inclusion among disease o f  
citrus (Wood et al., 1988). Sooty mould is caused by the fungi, Capnodium citri 
(Tweneboah, 2000), while sooty blotch a more intense form o f sooty mould is caused by 
Gloeodes pomigena (Amador, 2003)
2.4.7 Areolate Leaf Spot
This disease occurs in the humid, tropical areas o f South America. The causal organism is 
the fungus, Pellicularia filamento'sa. The disease can become serious in seedbeds and 
nurseries, particularly on sour orange seedlings (Stahel, 1940).
The disease is characterized by light coloured areas with dark concentric rings on the 
leaves. Lesions cease to expand under unfavourable conditions and hence vary 
considerably in size. Chlorotic halos form around the lesions and severely affected leaves 
drop off (Whiteside et al., 1988).
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2.4.8. Cercospora Leaf Spot
This disease is caused by Cercospora angolensis, and seems to be restricted to sub- 
Saharan Africa. Most varieties o f citrus are susceptible to the disease (Timmer et al., 
1988).
On leaves, the fungus causes circular, mostly solitary spots, up to 4mm in diameter. The 
centers o f these spots are usually grayish brown and shriveled and are surrounded by a 
prominent yellow halo. When the disease becomes severe a major part o f the leaf blade 
may be chlorotic. Leaf abscission is also common. Sometimes, the necrotic area falls out 
creating a shot-hole effect. Lesions resemble those o f canker, but differ in being flat or 
shrunken (Timmer et al., 1988).
2.4.9 Bacterial blast
The disease occurs under cool wet conditions and may affect twigs as well. Grape fruit 
and oranges are the most susceptible varieties o f  citrus. Symptoms o f the disease 
commonly appear on wings and petioles o f leaves as water soaked lesion or reddish 
brown to black lesions, which quickly move in both directions, up the mid vein o f the 
leaves and down to twigs. Once the phloem in the petiole is seriously damaged, the 
leaves wither, curl, and eventually drop Infection occurs through wounds created during 
windstorms and heavy rains (Timmer et al., 1988).
The causal organism is the bacterium, Pseudomonas syringae (Albirigo and Davies, 
1994). The bacteria is a normal inhabitant o f citrus leaves, becoming abundant on leaf 
surfaces only after conditions o f prolonged wetting by rains at relatively low
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temperatures. The disease rarely progresses when temperature is above 20° C (Timmer et 
al., 1988).
2.4.10 Citrus Canker
Citrus canker has been described as the most serious bacteria disease o f citrus (Civerelo, 
1981; Graham et al., 1992;). The disease causes necrotic lesions on fruit, leaves and 
twigs. Losses caused by the disease are reduced fruit quality and quantity and premature 
fruit drop (Agrios, 1997).
The disease is reported as having a devastating socio-economic and political impact 
because o f the market standards for fresh fruits and the perception o f possible inoculum 
transfer on fresh fruit product (Gottwald et al. , 2002).
Citrus canker can be endemic (Gottwald et al., 2002) and hence has a greater tendency to 
limit greatly, a grower’s choice o f cultivars that have been grown profitably in other parts 
o f the world. In certain parts o f the world, the cultivation o f certain cultivars o f the crop 
that are highly susceptible to the disease has been abandoned. For example, the 
cultivation of grape fruit (citrus paradisi), has been stopped in Japan (Albrigo and 
Davies, 1994). Gottwald et al., (2002) also reported that the cultivation o f  the same 
cultivar in Florida is almost non-profitable because o f requirement for multiple 
bactericidal sprays per year along with other canker management schemes to maintain 
yield and quality. The control o f the disease is also very expensive and cost as much as
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70 million dollars and destruction of close to a quarter of million trees to control it in 
Florida, in the late 1980’s (Gabriel, 2002).
Citrus canker is reported to have originated from South East Asia (Peltier et al., 1926) 
and Japan (Agrios, 1997). Currently the disease has widened its geographical range and 
appears to be found worldwide, particularly in areas where rainfall and temperature 
increase simultaneously (Gabriel, 2002). The disease can be found in all G ulf States o f  
America (Stall and Seymour, 1983). It has been reported in the Middle East, Africa, the 
Americas (Gabriel, 2002) and Australia (Agrios, 1997).
Spread o f canker disease across geographical barriers has been attributed mainly to the 
movement o f diseased propagating materials such as budwood, rootstocks and seedlings 
or budded trees (Agrios, 1997).
Citrus canker is caused by different strains o f Xanthomonas campestris pathovar citri 
(Hasse) Dye (Civerolo, 1981). Currently the organism has been elevated to the species 
level and is now known as Xanthomonas citri ex Hasse (Gabriel et a l, 1989). Another 
organism known as Xanthomonas campestris pathovar aurantifolli has been discovered 
as another causal agent o f  the same disease (Gabriel, 2002). The two organisms have the 
same cultural characteristics. However the latter appears to grow well on most common 
media used in the laboratory, only after being cultured in a special media, while the 
former grows freely on most common media (Gabriel. 2002). Differentiating between the 
two has been reported as being possible using serological means (Bradbury, 1986),
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The two organisms are reported to be pathogenic to almost all cultivated species o f 
Rutacea, particularly, Citrus spp, Fortunella spp, and Poncirus spp. and though different 
types o f  the disease have been named including Asiatic canker, false citrus canker and 
Mexican lime cancrosis, based on the difference in strains that caused each disease, the 
symptoms elicited on susceptible host are the same in all cases o f  the disease (Gabriel, 
2002).
Symptoms on the leaf first appear as oily looking about 2 -10mm circular spots, usually 
on the abaxial leaf surfaces. Later both epidermal surfaces may become ruptured. On 
Leaves, stems and fruits, the circular lesions become raised and blister like, growing into 
white or yellow spongy pustules. These pustules then darken and thicken into a light tan 
to brown corky canker, which is rough to touch. Older lesions tend to have more elevated 
margins and are at times surrounded by yellow chlorotic halo, which may disappear later 
on, and a sunken center. Defoliation and premature abscission o f affected fruits mar 
occur on heavily affected trees (Gabriel, 2002). On affected leaves, the necrotic center 
may fall out leaving a scattering o f holes in the affected leaves (Anon, 1997).
In the past, certain diseases have been misdiagnosed as citrus canker due to the 
similarities in disease lesions. One of such diseases was mancha foliar, a disease caused 
by Alternaria limicolla (Lopez, 1988). This disease was found in Mexico in 1982 and 
misdiagnosed as citrus bacteriosis, a form o f citrus canker. In contrast to citrus canker 
experiments, results from experiments carried out on the disease show that the disease
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rather develops strongly in the dry season,, and that bacterial oozing was not observed 
when the diseased lesion was cut (Stapleton and Garza- lopez, 1988). Currently there are 
no Xanthomonas strains known to cause the disease known as citrus bacteriosis (Gabriel, 
2002).
In 1910, when the citrus scab disease was introduced into the Gulf area o f the United 
States on nursery stock from Japan, it was initially misidentified as citrus canker (Maloy, 
1993)
Another opportunistic leaf spotting diseased caused by Xanthomonas campestris 
pathovar citrumelo, known as citrus bacterial leaf spot or nursery leaf spot was found in 
Florida in 1984 and was misdiagnosed as citrus canker (Gabriel, 2002). In contrast to 
citrus canker older lesions o f  the disease did not show bacteria streaming, and matured 
trees in grooves were never affected by the disease (Gabriel, 2002). Currently, due to the 
tissue hyperplasia associated with the disease and the extensive damage associated with 
the disease in nurseries (Gottwald et al., 1991), most researchers treat the disease just like 
citrus canker (Mavriodieva et al. , 2004)
Two characteristic symptoms of the disease has been described as very reliable either 
separately or combined. The first one is the presence o f  water soaked margin around the 
diseased lesion (Timmer et al., 1988) and the second is the tissue hyperplasia that results 
in canker on the affected parts (Gabriel, 2002).
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The causal agent resides on leaf surfaces and can survive for sometime but the erupting 
pustules on the leaf surface are a major source o f  inoculum for infection. Infection o f 
fruits and leaves are associated with leaf injury and also stomatal opening (Graham et a l ,  
1992).
2.4.11 Tristeza
This is a foliage disease caused by the citrus tristeza virus. The virus is usually 
debilitating and is one o f the most destructive diseases o f citrus. It suppresses new growth 
in the affected plant, and causes stem pitting and death o f the whole plant.
Symptoms on the foliage include leaf deformities such as vein clearing, chlorosis and leaf 
cupping. In the advanced stages o f the disease, leaf bronzing or yellowing occur followed 
by gradual defoliation (Roistacher, 1991).
2.4.12 Citrus exocortis
This is caused by a viroid known as the citrus exocortis viroid. The disease is present in 
almost all the citrus growing areas in the world, although some plants may not show any 
symptoms (Roistacher, 1991).
Symptoms on the leaf include the presence o f brown, necrotic and cracked veins, 
especially in the mid vein. Leaf petiole may be severely wrinkled (Roistacher, 1991).
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2.5. Control of foliage diseases of citrus
Control o f  foliage diseases o f citrus is specific by diseases, but some general concepts are 
applicable (Albrigo and Davies, 1994). These concepts can be classified as regulatory, 
cultural, chemical or an integration of two or more methods (Agrios, 1997).
2.5.1 Regulatory
This refers to the control measure aimed at excluding a pathogen from a certain 
geographical area in which it was not previously known. The methods include the use o f  
quarantine measures, use o f pathogen-free planting materials and siting o f  farms far away 
from contaminated fields (Agrios, 1997). This method has been used to control the spread 
of many foliage diseases o f citrus, especially, those that are debilitating, and whose 
introduction into an area in which they were previously unknown, could cause 
devastating loss to the grower. These include those diseases induced by the graft 
transmissible pathogens, such as greening, tristeza and vein enation (Roistacher, 1991), 
diseases induced by bacteria, such as canker (Amador, 2003), and certain fungi that are 
difficult to control with chemicals (Timmer et al., 1988).
2.5.2 Cultural methods
These are methods that depend primarily on certain actions o f  the grower that creates an 
unfavourable conditions for the pathogen to survive, thereby, reducing the amount o f 
pathogens present in an area, a plant or plant parts (Agrios, 1997).
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One cultural method is the removal and burning of all infected plant parts that might 
harbour the pathogen. This method has been used to control bacterial canker in Florida 
(Agrios, 1997). This is also recommended for the control of mal secco disease, where 
diseased shoots and branches are pruned out and burned immediately (Salerno and 
Cutuli, 1977). Another cultural practice, the avoidance o f overhead irrigation, has been 
recommended for the control o f citrus scab (Timmer et al., 1988) while Salerno and 
Cutuli, (1977) reported that the same method was effective in the control o f  mal secco 
disease. General farm sanitation such as washing the soil o ff farm equipment before 
moving it from one field to the other can effectively help to control certain diseases such 
as bacterial canker (Agrios, 1997).
2.5.3. Chemical control
Chemical control is the most commonly known method o f controlling plant diseases, 
where chemicals that are toxic to the pathogen are used. Such chemicals either inhibit 
germination, growth and multiplication o f pathogen or are outright lethal to the pathogen 
(Agrios, 1997). Chemical control is a faster way o f effecting the control o f  plant diseases 
wherever plants are cultivated. The method is fairly easy to use and sometimes is the only 
alternative method o f disease control. Most o f these chemicals are copper based, with 
Bordeaux mixture, being the commonest one (Agrios, 1997). Other non- copper based 
fungicides that have been used to control foliage disease o f citrus include Bendamidazole 
fungicides, which have been used to control mal secco, a fungal disease (Salermo and 
Cutuli, 1977) and sulphur dust which has been used to control citrus powdery mildew
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(Timmer et al., 1988). In some cases, oil has been used to control some fungal diseases 
such as greasy spot o f citrus (Timmer et al., 1988).
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CHAPTER THREE 
MATERIALS AND METHODS
3.1 Characteristics of area of study
The survey and sampling o f diseases were done at the citrus nursery at the University o f 
Ghana Agricultural Research Station located at Okumaning near Kade, about 120km 
North West o f  Accra. An annual rainfall o f 1600mm, with a bimordial distribution 
pattern, characterizes the climate o f the area. The temperature ranges between 25-38°, 
with a humidity o f  between 60-90%. The peaks o f the rainy season occur in May/  June 
and September/October. A short dry spell occurs in July/August, while the major dry 
season stretches from November to February.
3.2 Area, location and some practices in the nursery.
The nursery covers an area o f about 3200m2 and is found closer to the main offices o f  the 
station. Both seedlings (raised from seeds) and budlings (raised from rootstock and buds) 
were found in the nursery during the period o f the study. The budlings and seedlings were 
raised in seedling bags and have been arranged in rectangular batches referred to as beds, 
with each bed made up o f seedlings or budlings o f the same variety o f  the crop. There 
was an average number o f  200 seedlings or budlings per bed. Ages o f the budlings and 
seedlings ranged from one to two years. These beds were scattered in the nursery such 
that beds o f the same variety could be found at different locations in the nursery (Plate I).
Budding o f the seedlings is carried out in the nursery using buds obtained from the citrus 
orchards o f the station. Overhead irrigation is the major form o f irrigation practiced in the 
nursery and is carried out at three days intervals. Proper fertilization, general farm
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sanitation and the occasional use o f  copper based fungicides constitute the major disease 
control measures in the nursery. There was no evidence o f tool sterilization o f any form 
by the workers in the nursery.
Apart from the main nursery where this study was carried out, three other smaller 
nurseries belonging to private individuals at the station were found to be present during 
the period o f this study.
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Plate 1. A section o f the nursery showing the arrangement o f seedling and budlings in 
beds. Mg. X 0 .7
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3.3 Field survey
3.3.1 Determination of field symptoms of the diseases
Field surveys were conducted on three separate in November 8th, 22nd and December 20th 
representing the dry season and another three occasions in May 8th, 22nd and June 19th 
representing the rainy season. Survey was done by random picking o f  beds. Seedlings 
and the budlings o f the different varieties o f  citrus were examined for characteristic citrus 
diseases and the different symptoms recorded and named according to Timmer et al, 
(1988). Samples o f  the diseased leaves were collected during field inspections. Samples 
were cut using scalpel that was sterilized by dipping into 1% sodium hypochlorite and 
stored in labeled polythene bags and sent to the Crop Science Department laboratory for 
the isolation o f the causal agents o f  the diseases.
3.3.2 Determination of disease incidence and severity
The determination o f disease incidence and assessment o f  severity were done during the 
last survey o f  each season. Three beds representing three replicates o f  each o f the eight 
varieties o f  the crop were selected at random. The number o f plants that were affected by 
a particular disease was expressed as percentage o f the total number o f  plants on the bed. 
The mean incidence from the three replicates was found and recorded as the incidence o f 
the disease on the variety. Where the same disease was found on different varieties, the 
means obtained were subjected to the analysis o f variance and separated by the Duncans 
Multiple Range Test at 5%.
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Severity o f diseases was assessed on twenty-four young plants per selected bed. The 
severity for the different diseases were found as follows:
a) Severity for greasy spot-like disease was found by determining the percentage o f the 
leaf area affected by the disease. This was done by comparing the diseased leaf to 
standard leaf area diagrams developed by Gottwald (2002). The scale was modified by 
assigning the following indices to the various percentages;
0=No spot
l=Traces to 5% o f leaf area covered 
2=6% to 10% o f leaf area covered 
3=11% to 20% o f leaf area covered.
4=>20% o f leaf area covered.
In the rainy season, the mean severity figures for the different varieties were subjected to 
the Analysis o f Variance and separated by the Duncans Multiple Range test at 5%.
b) Severity for citrus canker was determined by counting the number o f spots developed 
on the surface o f the leaf. Therefore the severity was calculated as the average number o f 
spots per leaf. In the rainy season, the mean severity figures for the different varieties 
were subjected to the Analysis o f  Variance and separated by the Duncans Multiple Range 
test at 5%.
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c) Severity for citrus scab and brown leaf spot on rough lemon were estimated by 
determining the area of the diseased leaf covered by the diseased lesions and expressing it 
as a percentage o f the total leaf area, using the formula;
s=t/T, where s=severity, t=area o f diseased leaf covered by the lesion and T=total area of 
the leaf.
3.3.3 Effect o f diseases on matured citrus trees in the nursery
During the period o f  the study 18 matured citrus plants, which had grown out o f seedlings 
and budlings that were not transplanted on the field, were also inspected to determine the 
types o f diseases on them. Samples of diseased leaves from these trees were taken to the 
laboratory and the causal agents isolated.
3.3.4 Incidence of leaf miners
Leaves o f some o f the plants showing extensive curling and damages were inspected to 
identify the pest responsible for the damage. Two hundred plants in the nursery were 
picked at random and the percentage o f plants with such leaves was recorded.
3.3.5 Incidence of foliage diseases in the other nurseries at the station
After the surveys in the main nursery were completed, it was extended to the other 
nurseries belonging to private individuals working at the station. The foliages o f the 
seedlings and budlings in those nurseries were inspected and the types o f diseases
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observed were recorded. Samples of the diseased materials were also taken to the 
laboratory to determine their causal agents.
3.4 Laboratory work
3.4.1 Isolation and confirmation of causal agents of diseased leaves 
Two methods o f isolation were used. These were direct isolation and induced mycelial 
growth and possible sporulation (Tuite, 1969). In the case where the two methods did not 
yield enough results for diagnosis, other alternative methods used by other workers for 
isolation of causal agents o f diseases similar to what was observed in the nursery, were 
also used.
a) Direct isolation
In this method, samples o f  the diseased leaves were taken and a sterile razor blade was 
used to scrape the surface of the diseased lesion onto slides. The scrapping was mounted 
in a drop o f  water and observed under the compound microscope to determine signs o f 
the pathogen.
b) Induced mycelia growth and possible sporulation.
The isolation of fungi from the diseased symptoms was done first on water agar (WA) 
and then on potato dextrose agar (PDA). Agar agar and dehydrated PDA (Oxoid) were 
purchased commercially and prepared separately at the rate o f 20 g/L and 39 g/L, 
respectively using clean distilled water. The mixtures were autoclaved At a temperature 
of 121°C for 15 minutes. These were poured into clean and heat sterilized plates. Tissue
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segments from the advancing margins of the disease lesions o f the leaves were excised 
with a flamed scalpel; surface sterilized in 1% sodium hypochlorite solution for 45 
seconds and plated on water agar plates and covered (3 pieces per plate). The covered 
plates were enclosed in clean polythene bags and incubated under ambient conditions on 
a bench in the laboratory. Fungi, which grew out o f the platted tissues, were sub- cultured 
on plates o f potato dextrose agar (PDA), using flamed inoculating pins and cockborer. 
The plates were incubated in the laboratory until sufficient growth was observed.
Morphological features o f the organism were observed by mounting mycelia bits and 
spores in water and observing with a compound microscope. Identification o f the fungal 
isolates was based on the following culture characteristics on PDA: morphology o f 
mycelia; growth rate and nature o f spores as described by Whiteside et al., (1988)
The checklist o f plant diseases in Ghana (Oduro, 2001) and America (United States 
Department of Agriculture, 1970) were used to ascertain the status o f the pathogen in 
Ghana and elsewhere.
3.4.2 Alternative methods for the isolation of causal agents of the diseases
a) Isolation of causal agent of scab.
The alternative method used for the isolation o f the organism o f citrus scab was that o f 
Timmer et al., (1988). A sterile razor blade was used to scrape some o f the dried pustular 
growth on the diseased sample and cultured on water agar. These were incubated under 
ambient conditions on benches in the laboratory. Three days after incubation, growths
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that were observed on the water agar were sub-cultured onto potato dextrose agar in 
plates and incubated until sufficient growth was observed. The morphological features 
were then observed under the compound microscope to identify the organism.
b) Isolation of the causal agent of greasy spot-like disease.
The alternative methods employed for the isolation were those o f Timmer et al., (2003). 
Samples o f detached diseased leaves were moistened for 60 min. with water and 
incubated at a temperature o f 28°C, for one week. The diseased specimen was soaked in 
water for 60 minutes every other day, during the incubation period. After the seventh day, 
growths on the leaves were scrapped onto a slide, fixed with lactophenol and observed 
under the compound microscope to examine the pathogen.
The second alternative method involved the scrapping o f the surface o f fallen and rotten 
diseased leaves onto a slide, and examining the scrapings for the conidia o f the pathogen.
c) Isolation of causal agent of canker.
The method employed for the isolation o f the causal agent o f citrus canker was that o f 
Gabriel (2002). Bacteria infection was first confirmed by placing a cut water- soaked 
margin of the disease lesion in sterile distilled water in the depression o f a concave slide 
and covering it with a cover slip. The preparation was observed under the compound 
microscope using oil immersion to determine the oozing o f bacterium cells.
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The bacterium isolation on media was done by first taking samples of the diseased leaves 
washing them in running sterile distilled water. Then lesions with water soaked margins 
were excised using sterile scalpel and sterilized in 10% dilution of household bleach. 
Using a sterile razor blade, the water soaked margins were carefully removed, sectioned 
and the pieces dragged on the surface o f yeast nutrient agar contained in plates. The shiny 
yellowish bacterium colonies that grew were re-streaked onto nutrient media to obtain 
pure cultures. The isolated bacterium was preserved on slants for pathogenicity test and 
characterization.
3.4.3 Pathogenicity test of isolates
Suspensions o f fungal isolates were prepared by suspending the mycelium and spores 
from PDA, in sterile distilled water. These were used to spray three (3) months old 
healthy seedlings o f the citrus varieties from which they were isolated. The seedlings 
were then covered with clean polythene bags to create a humid condition around them, 
and arranged in Completely Randomised Design (C.R.D), with three replicates tests of 
each isolate. Control was a seedling sprayed with sterile distilled water, which was also 
tied in polythene bags. An isolate was confirmed pathogenic to the plant if  it caused the 
disease as observed on the original diseased leaf from the field.
Pathogenicity test o f isolated bacteria was done using the inoculation method described 
by Gabriel (2002), called the blunt syringe method. In this method, a one-week-old 
preserved culture o f the organism was cultured in glucose nutrient broth for two days and 
the bacterium together with the medium was drawn into a syringe without a needle. The
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syringe containing the inoculum was then pressed gently, but firmly to the lower leaf 
surface and the slurry forced into the leaf through the stomata until about 2 cm 2 o f the leaf 
became water congested. Then some few minutes after the congestion had cleared, the 
seedlings were arranged in a completely randomized design in the green house. The 
treatment was replicated three times with a control made up o f seedling inoculated with 
the nutrient broth without the bacteria. The bacterium was confirmed as the causal agent 
of the disease when it was able to cause the disease in the test plants.
3.4.4 Characterisation of isolated bacteria
a) Cultural growth on glucose media.
A glucose nutrient agar media was prepared by combining 2.5 g o f  glucose with 23 g of 
nutrient agar and autoclaved at 121° C for ,15 minutes and allowed to cool. The molten 
media was then poured into sterile plates and allowed to set. Using a sterile loop, cells o f 
the preserved bacteria were streaked onto the surface o f the media in the plates, inverted 
and incubated for two days under ambient conditions in the laboratory and the cultural 
characteristics o f the bacterium recorded to aid in its characterisation.
b) Gram staining.
The Gram stain reaction (Bradbury, 1970) was done using freshly prepared reagents.
A thinly spread film of the bacteria was made on a clean slide, air-dried and the lower 
side o f the slide lightly flamed to fix the bacteria on the slide. The smear was then 
flooded with crystal violet solution for 1 minute. This was then washed in tap water for 10 
seconds and excess water drained off. It was carefully blotted dry using a paper towel and
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flooded with iodine solution for lm im ., washed in tap water for 15 sec. and blotted dry. 
The smear was then decolourised with ethyl alcohol for 30 sec. and rinsed in tap water 
for 2 sec. Safranin solution was then used to counterstain for 10 sec., washed briefly in 
water and blotted dry. The  smear was then observed under oil immersion under the 
compound microscope. The  resul ts were recorded to aid in the characterization of the 
bacteria. A  culture of Escherichia coli obtained f r om the Noguchi Memorial Institute for 
Medical Research served as the reference bacterium and was given the same treatment as 
the test bacterium.
c) Motility test
The motility test was conducted to examine whether the isolated bacterium was 
flagellated. A thin layer o f vaseline was applied around the edge o f the well on a concave 
slide. A loopful o f  the isolated bacteria suspension was placed in the center o f  a cover 
slip and the concave slide was inverted and placed on the slip so the drop o f the bacteria 
suspension, was enclosed within the concave depression. The preparation was then turned 
right up again and observed under the microscope under oil immersion.
d) Catalase test.
Yeast nutrient agar was prepared by combining 5 g of yeast extract with 23 g o f nutrient 
agar. The mixture was dissolved in 1L of distilled water, dispensed into test tubes and 
autoclaved at 121 °C for 15 minutes and allowed to set as slants. Three o f the slants were 
inoculated with the bacteria and one, which served, as a control was not inoculated with 
the bacteria. The slants were incubated for two days under ambient conditions in the
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laboratory. Few drops o f 3% hydrogen peroxide was poured gently along the walls o f the 
tube onto the culture and the effect o f the chemical on the organism was recorded. The 
slants that were not inoculated with the bacterium also received the same treatment.
e) Starch hydrolysis test
The method employed was that o f Lelliot and Stead (1987). The media for the test was 
prepared by combining 28 g o f nutrient agar and 2 g of soluble starch The mixture was 
dissolved in I litre o f distilled water and autoclaved at 121°C for 15 minutes. The media 
was poured into sterilized plates and allowed to set. An inoculating pin was used to stab 
the media in the plates, with a two-day-old culture o f the bacteria. Each plate was stabbed 
at four different points in the plate. The plates were incubated in the laboratory for seven 
days. After the incubation, the plates were flooded with Lugols iodine solution and the 
colour development was recorded.
3.4.5. Further studies on citrus canker 
Investigating the inoculation method on number o f days to disease expression and 
the stages of disease development.
Results from the pathogenicity test indicated that aetiology o f  the canker diseased 
condition could be due to the bacterial that causes citrus canker. Consequently some more 
experiments were designed to further conduct some detailed studies on the disease and 
the causal organism
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A suspension o f the bacterium was prepared by streaking colonies o f the bacterium taken 
from a two week old culture. This was first streaked onto nutrient agar and after two 
days, the colonies were suspended in sterile tap water (Gabriel, 2002).
Two methods o f  inoculation were evaluated in the study. These were the blunt syringe 
method as described in section 3.3.3 and the spraying method. Each inoculation method 
was tested on a sweet orange seedling with the inoculum prepared. The controls were 
sweet orange seedlings inoculated with sterile tap water using each o f  the two inoculating 
procedures. Each treatment was replicated ten times and the number o f days to first 
disease expression were recorded and subjected to the ‘t ’ test at 5%
Temperature and humidity were monitored during the period o f the experiment in the 
screen house in both the first and repeated inoculation trials, using the Tiny plus 
electronic data logger.
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CHAPTER FOUR  
RESULTS
4.1 Varieties of citrus grown in the nursery
Eight different citrus varieties were used for this study (Table 1). Six o f  these varieties 
were from four different species o f  the crop while the other two were hybrids made up o f 
the combination o f different genotypes from different species o f  the citrus crop. Six o f 
them were budlings while the other two namely Cleopatra mandarin and a variety of 
rough lemon, were seedlings. These two varieties were used mainly as rootstocks. The 
scions on the budded materials were obtained from the orchards on the station.
Table 1. Varieties of citrus identified in the nursery.
Variety Species Genetics Use Seedling/Budling
Late Valencia Sweet orange (C. sinensis) pure line scion Budlings
Walters Grapefruit (C.paradisi) pure line scion Budlings
Satsuma Tangerine (C. reticulata) pure line scion Budlings
Ponkan Tangerine (C. reticulata) pure line scion Budlings
Cleopatra Mandarin (C. reticulata) pure line rootstock Seedlings
King disemis Rough lemonx Tangerine hybrid scion Budlings
Ortanique Sweet, orangex Tangerine hybrid scion Budlings
Rough lemon Rough lemon (C. limon) Pure line rootstock Seedlings
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4.2 Diseases identified in the nursery
4.2.1 Brown leaf spot
a) Disease symptoms:
This was a leaf spot disease made up o f large, round, and mostly, solitary spots. The spots 
were dark brown to black in colour and appeared in concentric rings with a pinhead 
center, giving a characteristic target appearance. Most o f  these spots appeared at leaf 
margins. Black dots, which were spores o f  Alternaria sp. were present in concentric rings 
in most o f  the diseased lesions (Plate 2).
b) Host range, incidence and severity.
The disease was found to be restricted to rough lemon. (Table 2). Disease incidence and 
severity on the rough lemon variety appeared to be higher in the dry season than in the 
rainy season_(Table 2). In the rainy season an incidence o f 89% was recorded on the 
variety compared to 45% in the dry season. Similarly a severity o f 16% was recorded in 
the wet season as compared to 6.3% in the dry season.
c) Identification of the causal agent.
Alternaria citri was isolated. The signs o f the organism were observed when the 
scrapings from the surface of the diseased lesions were examined under the microscope. 
Conidia observed were muriform with vertical septations that ranged from four to six in 
number, with slight constrictions at the septa. They were also divided by one or two 
horizontal septa. Some of the conidia were catenulated and they vary greatly in shape and
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Cultural characteristics o f the organism on potato dextrose agar showed a loosely 
interwoven, slender mycelium. The organism grew and filled the entire plate within ten 
days after culturing on potato dextrose agar (Plate 4). When the organism was observed 
under the microscopes, the spores o f the organism as described above were found 
scattered among the mycelium (Plate 5).
The pathogen had been listed in the checklist of plant diseases both in the United States 
and that o f Ghana as the causal agent o f  the disease, thereby confirming the organism as 
the causal agent o f the disease. The pathogen was able to induce the disease when it was 
inoculated onto the healthy rough lemon seedlings further confirming it as the causal 
agent o f  the disease (Plate 6 ).
size. Most o f  them were short, clavate, and oblong and appeared dark olive brown (Plate.
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Plate 2. Characteristic symptoms of brown leaf spot on the variety of rough lemon. 
Mg. X 0.7
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Table 2. Host range, incidence and severity o f brown leaf spot in the nursery.
Variety
Dry season Rainy season
Host Incidence/% Severity/% Host Incidence/% Severity/%
Cleopatra 0 0 0 0
Walters - 0 0 . “ 0 0
King disemis - 0 0 - 0 0
Ortanique - 0 0 0 0
Ponkan “ 0 0 0 0
Satwuma " 0 0 - 0 0
Late Valencia 0 0 - 0 0
of Rough 
lemon
+ 45 6.3 ■+ 89 16
- = absent. + present
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Plate 3. Muriform spores o f Alternaria citri scrapped directly from the diseased lesio 
Note the presence o f  catenulated spores (arrowed) at the lower left o f the figure.
Mg. X 800
Plate 4. Muriform spores of Alternaria citri entangled in mass of mycelia on P.D.A. 
Mg. X 800
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Plate 5. Cultural characteristics o f Altemaria citri on Potato Dextrose Agar. Mg. X 0.8
Plate 6 . Brown leaf spot caused by A ltemaria citri on the variety of rough lemon test 
seedlings. Note the absence of disease lesions on the control. Mg. X 0.7
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4.2.2 C itrus scab
a) Disease symptoms.
The disease was characterized by protuberances present at the lower surface o f the 
affected leaves, with a corresponding depression on the opposite side o f the leaf. The 
depressions were surrounded by large yellow halo. The protuberances were brown in 
colour and in most cases appeared as pustules. Scrapings from the pustules revealed the 
presence o f pieces o f mycelia o f  the pathogen under the microscope. The pustules in most 
cases were spread on the surface o f the affected leaf, resulting in distorted leaves (Plate 
7).
b) Host range, incidence and severity
The scab disease was found restricted to the variety o f  the rough lemon in the nursery 
(Table 3). The disease was also found in two out o f the three other nurseries inspected.
In the rainy season, an incidence o f 78% was recorded on the variety as compared to 48% 
in the dry season. Similarly, severity of 55% was recorded on the variety in the wet 
season as compared to 25% in the dry season, an indication that the disease was more in 
the rainy season than in the dry season (Table 3).
c) Identification of the causal agent.
All the attempts at isolating Elsinoe fawcetti and its anarmorph, Spaceloma fawcetti 
proved futile. Instead Fusarium sp. was consistently isolated when the isolation method 
proposed by both Tiute (1969) and Timmer et al.. (1988) were used.
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Plate 7. Characteristic symptom o f citrus scab in the nursery. Mg. X 0.7
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Table 3. Host range, incidence and severity o f citrus scab in the nursery
Variety/species
Dry season Rainy season
Host Incidence/% Severity/% Host Incidence/% Severity/%
Cleopatra 0 0 0 0
Walters - 0 0 0 0
King disemis - 0 0 0 0
Ortanique - 0 0 0 0
Ponkan - 0 0 0 0
Satsuma - 0 0 0 0
Late Valencia - 0 0 0 0
Rough lemon + 48 25 + 78 55
- = disease is absent. + = disease is present.
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4.2.3 Greasy spot
a) Disease symptoms
The disease symptom was characterized by the presence of yellow mottle on the upper 
part o f  some o f the affected leaf surfaces, with a matching, slightly raised, pale orange to 
yellowish brown blisters on the lower surface. On some o f the leaves, affected areas of 
the leaves appeared darker brown and greasy in appearance, with a yellow halo 
surrounding some o f the spots on the upper surface (Plate 8 ).
b) Host range, incidence and severity.
Greasy spot was found to have a wide host range, affecting all the varieties o f the crop in 
the nursery. In the surveys conducted during the dry season, the disease was found to 
have affected only sweet orange and Cleopatra mandarin. During this period, the disease 
was also recorded on 10 out o f  the 18 mature citrus trees in the nursery. The disease was 
not found in the other nurseries in the station and hence confined to the main nursery. In 
the rainy season the disease was found on all the varieties o f  the crop in the nursery.
Disease incidence on the different varieties o f  the crop in the rainy season varies 
significantly, ranging from 37%, which was recorded on waiters (C. paradisi) to 6.7 on 
the variety o f rough lemon (C. limon). Severity figures also varied among the various 
varieties with the highest of 3.8, recorded on late Valencia (C. sinensis) and the lowest of 
1.7 recorded on waiters (C. paradisi), king disemis, ortanique, ponkan (C. reticulata) and 
satwuma mandarin (C  reticulata).
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c) Identification of the causal agent.
Mycosphaerella citri, the causal organism o f the disease known as greasy spot in other 
citrus growing area was not isolated in this study. The repeated wetting and incubation 
method used did not result in the production o f the ascospores o f the organism. Also 
scrapings from fallen and diseased leaves did not show any sign of the organism. The 
anamorphic stage o f the organism was also not isolated on media.
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Plate 8  Characteristic symptoms o f greasy spot-like disease in the nursery. Mg. X 0.7
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Table 4. Host range, incidence and severity o f greasy spot-like disease in the nursery.
Variety Dry season Rainy season
Host Incidence/% Severity Host Incidence/% Severity
Cleopatra + 27 2.9 + 35.7t>c 3 7bc
Walters 0 0 . 0 + 6.7b 1.7a
King disemis - 0 0 . 0 + 5.0a 1.7a
Ortanique - 0 o.o- + 37.3°° 1.7a
Ponkan 0 0 . 0 + 34.3bc 1.7a
Of R. lemon “ 0 0 .0 + 1 0 .0 b 2.7b
Satsuma 0 0 .0 + 31.7bc 1.7a
Late Valencia + 35 3.3 + 37.0bc 3.8bc
-= disease is absent, + disease is present
Means followed by the same alphabets in a column are the same.
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4.2.4 Citrus canker
a) Disease symptoms.
The disease symptom was a complex one made up o f three stages based on the stage of 
development o f the spots, with each stage showing distinguishing features, but retaining 
some of the features of the stages preceding it.
i) In the first stage, the spots appeared as very tiny but visible spots or as translucent dots 
surrounded by enlarged, yellow chlorotic areas. The translucent dots appear visible only 
when held against a source o f light (Plate. 9).
ii) The second stage as seen in Plate 10, was made up o f clearly visible spots, which were 
brown and sunken on the upper surface o f the affected leaves and were surrounded by 
yellow chlorotic areas. At the lower surfaces o f  the leaves, the spots appeared as round, 
raised corky growths, surrounded by yellow halo. Some of the larger spots appeared 
irregular in shape as dry warty pustules that were sunken at the upper surface o f  the 
leaves. Tissues found at the center o f the spots appeared papery and overstretched. On the 
lower surface o f the leaves, the warty growth appear raised, rough to touch and were 
bordered by a water soaked area.
iii) Plate 11 showed the final stage o f the disease which was a. ‘shot’ hole effect produced 
on the affected leaves, with the internal margins o f the holes showing traces o f  the 
overstretched tissues associated with the second stage.
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Plate 9. Characteristic symptom o f the first stage o f  citrus canker.
Note the pinpoint spots surrounded by yellow chlorotic halo. Mg. X 0.7
Plate 10 Characteristic symptoms of the second stage o f the citrus canker disease. 
Note the shapes of the yellow chlorotic halo surrounding the spots. Mg. X 0.7
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Plate 11. Characteristic symptoms of the last stage o f citrus canker. 
Note the presence o f shot holes. Mg. X 0.7
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Citrus canker was found to have a wide host range, affecting all the varieties o f the crop 
in the nursery. In the dry season survey, the disease was found on only sweet orange and 
Cleopatra. However all the varieties o f  citrus were found infected with the disease within 
the rainy season survey (Table 5). Three out o f the 18 matured citrus trees found in the 
nursery were found with the disease in the wet season. The disease was also present in all 
the nurseries on the station.
Incidence of the canker disease on the citrus varieties of the crop ranged from 38.3% on 
rough lemon to 100% on grapefruit in the rainy season. With the exception o f ponkan, all 
the budlings showed more than 50% incidence in the rainy season. In the dry season, only 
two of the varieties, namely late Valencia and Cleopatra mandarin were diseased. Late 
Valencia showed a high incidence o f  80% while Cleopatra showed an incidence o f  6 8 % 
(Table 5).
Results from the severity determination tests showed higher severity o f  the disease on the 
budlings as compared to the seedlings. Severity score o f 18.2 in late Valencia was highest 
followed by waiters, with a severity figure o f 8.5 (Table 5).
b) Host range, incidence and severity.
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Table 5. Host range, incidence and severity o f citrus canker disease in the nursery.
Variety Dry season Rainy season
Host Incidence/% Severity Host Incidence/% Severity
Cleopatra + 6 8 3.5 + 92 3  bed 5.6
Walters - 0 0 . 0 + 1 0 0 bcd 8.5
King disemis - 0 0 . 0 + 47.7s 6 . 2
Ortanique - 0 0 . 0 + 38.3bac 6.4
Ponkan - 0 0 . 0 + 83.3bc 6 . 8
Of R. lemon - 0 0 . 0 + 9 4  0bcd 3.9
Satsuma - 0 0 . 0 + 83.0bc 2 . 1
Late Valencia + 80 6.5 + 94.0bcd 18.2
-= disease is absent, + disease is present
Means followed by the  sam e alphabets in a column are the same.
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c) Identification of the causal agent.
Xanthomonas campestris p v  citri was isolated from both young and old lesions o f the 
disease. The bacterium cells were observed to be oozing out when pieces of the water 
soaked margins o f the disease were put into a drop o f water. On glucose nutrient agar, the 
colonies o f  the bacterium appeared shiny yellow, mucoid and convex (Plate 12). In the 
motility test, the bacterium cells were observed to be motile. In the Gram stain test, the 
organism retained the red colour indicating that it was a Gram-negative bacterium. The 
stained bacterium cells were short rods. The bacterium was also catalase positive 
producing gas bubbles in few seconds after the introduction o f the hydrogen peroxide. In 
the starch hydrolysis test, the area immediately surrounding the bacterium cleared while 
the other area showed the black colouration, showing the bacterium to be a starch- 
hydrolyzing organism.
Xanthomona scampestris pv. citri has not been listed as a causal agent o f  the disease in 
Ghana. The organism induced similar characteristics symptoms o f citrus canker when it 
was inoculated onto healthy seedlings of sweet orange.
The first symptom o f the disease was evident seven days after inoculation. It consisted of 
a yellow area on upper leaf surface. Then fifty (50) days after inoculation, scab like 
outgrowth, characteristic of the disease started to appear at the lower part o f the 
inoculated leaves, surrounded by large yellow chlorotic area. Small solitary canker spots 
with raised margins, which turned slightly warty, developed on the upper leaf surface
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(Plate 13). Further expansion o f the disease lesion was not observed until leaves began to 
senence.
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Plate 12. Cultural characteristics o f Xanthomonas citri isolated from the citrus canker 
disease in the nursery. Mg. X 0.7
Plate 13 Development of young canker disease symptoms on inoculated test plants. 
Mg. X 0.7
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4.3 The influence of method of inoculation on the development of citrus canker 
disease on seedlings
Results from the inoculation showed that number o f days for the first stage o f  the canker 
symptoms to develop varied significantly between the two inoculation methods. Whereas 
symptoms developed within an average of 7.8 days following inoculation, it took 52.6 
days after inoculation for the disease to develop using the spraying method (Table 6 ).
b) Stages of disease development
i) Using the blunt syringe inoculation method.
First disease symptoms on the plants inoculated with the inoculum was evident seven 
days after inoculation. It consisted of a yellow area on upper leaf surface with a slight 
bulged area on the upper surface with the presence o f black spots at lower side o f the leaf 
(zone o f  inoculation).
The second stage o f the disease, characterized by the presence o f scab like outgrowth, 
started to appear at the lower part o f the disease leaves, twenty-one days after the 
emergence o f the first stage o f the disease (Plate 14). On the upper surface o f the affected 
leaves, small, solitary canker spots with raised rough warty margins developed. There 
was a gradual expansion o f  the lesions until the leaves begin to senence.
ii) Using the spraying method.
The first stage o f the disease was observed about fifty three (53) days after inoculation. 
The symptoms first appeared as tiny solitary spots surrounded by yellow halos. These
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spots underwent very little expansion and the scab like growths surrounded by water 
soaked margins on the lower surface also begins to form (Plate 15).
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Plate 14. Citrus leaves showing young canker spots following inoculation with the 
bacterium using the blunt syringe method (Arrowed). Mg. X 0.7
Plate 15. Citrus leaves showing young canker spots (Arrowed) following inoculation 
with the bacterium using the spraying method. Mg. X 0.7
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Table 6 . Influence o f method o f ino'culation on canker disease development.
Method o f inoculation Mean number o f days
Blunt syringe 7.8
Spraying 52.6
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4.4 Incidence of leaf m iners at the nursery
Leaf miners were found to cause the curling and leaf destruction of citrus seedlings 
observed in the nursery. The pest was identified by the presence of meandering 
serpentine larva mines, which were present vertically on the ventral side o f  affected 
leaves (Plate 16). The larvae appeared as very minute and translucent greenish yellowish 
worms, located inside the leaf mines. The mines were covered with a silvery film o f the 
leaf epidermis. A high incidence of the pest infestation was recorded on 125 o f the 200 
trees inspected being affected, representing an incidence of 62.5%.
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Plate 16. Citrus leaf attacked by citrus leaf miner (Phyllocnistis citrella) in the nursery. 
Note the serpentine nature of the mines. Mg. X 0.7
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CHAPTER FIVE 
DISCUSSIONS
Four different kinds o f disease were found in the nursery. These were citrus canker, 
brown leaf spot o f rough lemon, citrus scab and greasy- spot like disease o f  citrus. Citrus 
canker was characterized by water soaked margins and a yellow halo surrounding the 
disease lesion which has been found as a reliable diagnostic symptom (Gabriel, 2000; 
Stall, 1988). Xanthomonas campestris pv. citri. which was isolated and confirmed in this 
study has also been reported as the causal agent of the disease (Civerolo, 1981). The 
disease has also been reported in many citrus nurseries (Agrios. 1997).
The second disease, brown leaf spot has also been reported as very prevalent in citrus 
nurseries. The target appearance of the disease lesion and the presence o f  the spores of 
the causal agent on the surface of the disease lesion as well as the restriction o f the 
disease to the rough lemon variety of the citrus crop have been reported as being 
diagnostic features of the disease (Timmer et al.. 1988). The causal agent o f the disease. 
Alternatia citri was also isolated and confirmed in this study.
The third disease found in the nursery was the greasy spot like disease, with the 
characteristic greasy nature of the disease lesion (Timmer et al.. 1988). Mycosphaerella 
citri and its anamorph, Cercospora citri. reported to be the causal agents could not be 
isolated
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The fourth disease [bund in the nursery was  the ci t rus scab.  I'he cocky,  and pustular  
nature o f  the spots and the distort ion o f  d iseased lea l 'had  been repor ted as a character ist ic 
symp tom  o f  the cit rus scab (Clerk.  1974). FJsinoc fawcetti. the causal  agent  o f  scab could 
not be isolated.
Not all the four diseases were o f equal importance. Alternaria leaf spot and citrus scab 
were found not be of much importance to the station. Excessive branching and shorter 
nodes o f affected trees, associated with the brown leaf spot on rough lemon that makes 
the plants difficult to bud, was not observed in this study. Similarly, stunted growth of 
affected trees associated with scab disease was also not observed. Also, disease severity 
in both the dry season and the wet season were not very high. Secondly, the two diseases 
were found to be restricted to the rough lemon variety, which was utilized mainly in the 
nursery as a rootstock. This means there is a less likelihood o f the diseases being 
transferred into the field, when proper pruning of the budded material is carried out 
before transplanting. This also makes the diseases o f less importance to the nursery.
On the other hand, greasy spot like disease and citrus canker could be important diseases 
at the station. All the varieties of the crop in the nursery were found to be susceptible to 
these two diseases and this could make the control of such diseases very difficult and 
expensive. Being found on the foliages of the scions as well, these diseases have a 
potential of being transferred onto the farmers field and the orchards o f the station. The 
infection of matured trees in the nursery is an indication that these diseases may not be 
restricted to nurseries only but can establish well  on matured trees in the field as well.
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Between the two diseases, greasy spot- like disease may not be the most threatening 
disease in the nursery. Unlike the citrus canker, greasy spot -like disease showed very 
low incidence figures in both the wet and dry seasons. Also with the exception o f late 
Valencia, all the budlings had low severity figures. Cleopatra mandarin and rough lemon, 
which had high severity figures were used mainly as rootstock and therefore might not 
affect the quality o f planting materials produced.
Determination o f  the effects o f the diseases on yields to complete the studies on their 
importance was not feasible because the seedlings and budlings in the nursery had not 
reached the stage o f  fruit bearing during the period in which the study was carried out.
Citrus canker was found to be a very devastating disease in the nursery. The wide host 
range, high incidences and the physical destruction o f foliage o f the seedlings and 
budlings make it a disease that demands rapid attention. The disease remained the only 
one with a 100% incidence on a waiters (C. paradisi) within the period o f  the experiment. 
The damage caused to the foliage was rapid resulting in production o f poor quality 
planting material. The high severities showed on the hybrids, namely ortanique and king 
disemis, indicates that combination o f different genotypes to control the disease may not 
be possible, and thus confirms the assertion by Lelliot and Stead (1987), that all hybrids 
of the citrus crop are susceptible to the disease. The high incidence and severity on 
waiters (C. paradisi) indicate that the raising o f planting materials o f the variety could be 
very unprofitable in the nursery.
The cultural practices being adopted in the nursery could favour the development o f the 
disease. Overhead irrigation, just like rainfall, wet the leaf surfaces and thereby induces
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the oozing out o f bacterium cells from diseased lesion, thereby aiding the spread o f the 
disease among the seedlings and budlings in the nursery ..Secondly, budding and pruning 
of leaves with unsterilised tools could contribute to the spread of the bacterium in the 
nursery. Also the arrangements o f the seedling and_budlings in crowded rows and 
columns can facilitate the spread o f the disease in the nursery.
From the on going discussions it could be inferred that with the emergence o f citrus 
canker in the nursery there is the need for a modification o f certain practices in the 
nursery. For example the station needs to explore other alternative methods o f irrigation, 
since the practice o f overhead irrigation would continue to accelerate the development of 
the disease. Secondly, tools sterilisation must be taken as one o f the disease prevention 
measures in the nursery to prevent further spread o f not only the canker, but also any 
other disease that could spread through the use of infected working tools and equipment. 
Also with the disease being found in all the. nurseries at the station, any control measure 
instituted against the disease must not be restricted to the main nursery alone, but must be 
extended to the other nurseries as well to ensure its total eradication from the station.
Apart from cultural practices, the climate in the area is favourable for the development o f 
the disease. Frequent rainfalls and high temperatures that characterise the climate o f the 
area are known to exacerbate the disease leading to serious damage and tree death 
(Anon., 1997; Gottwald et a l, 2000; Roistacher, 1988) and this may account for the high 
incidence o f the disease recorded in the rainy season.
Another possible factor contributing to the high incidence o f the disease was the 
incidence of the Asian leaf miner ( Phyllocnistics citrella ) in the nursery. The association
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o f the pest with citrus species and related Rutacea species has been reported (Kalshoven, 
1981). Its presence in West Africa has long been suspected (Heppner, 1998). The larvae 
mines observed on the affected leaf surfaces in the nursery might serve as entering points 
of the bacterium in the nursery (Hill, 1918; Ando et al., 1985). In its burrowing activities, 
the larva o f the pest may bore holes into the canker spots, which may result in the spread 
of the bacterium (Mavrodieva et al., 2004). Though the pest was highly suspected as a 
contributory factor in the spread o f  the disease in the nursery this was not investigated in 
this study and therefore further studies in this area is recommended.
Most o f the budded materials recorded higher severity figures. This observation could be 
attributed to the type o f rootstocks used in the nursery. From the survey it was realised 
that the commonest rootstocks used in the nursery were rough lemon (C. limori), 
cleopatra mandarin (C. reticulata) and Citrus volkameriana. These three rootstocks are 
known to promote vigourous growth in scions resulting in rapid emergence o f  young 
growths on the plant (Hearn and Hutchinson, 1977; Castle, 1988) and hence predisposing 
the scion to the canker disease, which affects young growths more than matured tissues 
(Albrigo and Davies, 1994). This observation was not investigated and therefore further 
work in this area is recommended.
Results from the screen house experiment in which different inoculation methods were 
used further confirmed the disease as citrus canker. The various stages o f the disease as 
recorded in this study have been reported by earlier researchers (Gabriel, 2000; Gottwald 
et al., 2000) and thus confirm the disease as canker. The result also showed that factors
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that accelerate the entrance of the bacterium in to the leaves could greatly accelerate the 
development o f  the disease, since the syringe places the bacterium directly into the 
leaves. Thus in pathogenicity studies on the disease, the blunt syringe method is highly 
recommended as compared to the spraying method.
Canker disease is a threat not only to the station, but also to the citrus industry in the 
country as a whole. The spread o f the disease in Ghana can have a very devastating effect 
on the citrus industry in the country. Currently citrus is gaining popularity in Ashanti, 
Eastern and Western regions, which have high humidities and temperatures and frequent 
rainfalls in most parts o f the year. Such environmental characteristics have been reported 
as very favourable for the disease (Whiteside et al., 1988: Anon., 1997). Serious damage 
and death o f trees affected by the diseases have also been reported as being accelerated 
by severe rains and winds and so with occurrence o f the disease in such areas its effect 
would be very devastating.
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REFERENCE
Agrios, G., N. (1997). Plant Pathology. Academic Press. New York. 635 pp.
Albrigo, L. G., and Davies F.S., (1994). Crop Production Science in Horticulture. CAB 
International U.K. 224 pp.
Amador, J. M. (2003). Diseases Affecting Localised Parts o f Trees. http://aggie- 
horticulture.tamu.edu/citrus 12315 .htm
Ando, T„ T aguchi, K.Y.,Uchiyama, ML, Ujiye, T„ Kuroko, H. (1985). (7Z-11Z), 11- 
hexadecadienal sex attractants o f the citrus leaf miner moth Phyllocnistics citrella 
Staiton. (Lepidoptera, Phyllocnistidae). Agriculture, Biology, Chemistry. Tokyo 49:3633- 
3653.
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APPENDIX
ANOVA for incidence of greasy spot-like disease in the nursery
s v DF SS MS V. r. Fpr.
Treatment 7 4530.50
(2835.87)
647.21
(404.84)
28.29
(19.17)
<0.001
Residual 16 3660.00
(22.88)
Total 23 4896.50
(3171.84)
Transformed data in brackets.
ANOVA for severity o f  greasy spot-like disease in the nursery
SV DF SS MS V. r. Fpr.
Treatment 7 18.374062 2.624866 335.98 <0 . 0 0 1
Residual 16 0.125000 0.007812
Total 23 18.499062
Transformed data in brackets.
ANOVA for severity o f  canker disea.se in the nursery
SV DF SS MS V .r. Fpr.
Treatment 7 12249.96
(6786.118)
1749.99
(969.445)
147.57
(115.63)
<0 . 0 0 1
Residual 16 190.00
(134.147)
Total 23 12439.96
(6920.265)
Transformed data in brackets.
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ANOVA for severity o f  canker disease in the nursery
s v DF SS MS V. r. Fpr.
Treatment 7 490.49167
(12.994762)
70.07024
(1.85639)
6468.12
(4013.83)
<0.001
Residual 16 0.17333
(0.0074000)
0.1083
(0.0004625)
Total 23 490.66500
(13.002162)
Transformed data in brackets.
83
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