EFFECT OF SEED-BORNE FUNGI ASSOCIATED WITH SEEDS OF
SILVER OAK (Grevillea robusta) ON SEEDLING GROWTH AND
DEVELOPMENT OF DIEBACK AND STEM CANKER IN
SOUTHEASTERN ETHIOPIA
MSc THESIS
URGESSA MERGA GARDEFA
NOVEMBER 2015
HARAMAYA UNIVERSITY, HARAMAYA
Effect of Seed-Borne Fungi Associated with Seeds of Silver Oak (Grevillea
robusta) on Seedling Growth and Development of Dieback and Stem Canker
in Southeastern Ethiopia
A Thesis Submitted to the Postgraduate Program Directorate
(School of Plant Sciences)
HARAMAYA UNIVERSITY
In Partial Fulfillment of the Requirements for the Degree of
MASTER OF SCIENCE IN AGRICULTURE
(PLANT PATHOLOGY)
By
Urgessa Merga Gardefa
November 2015
Haramaya University, Haramaya
HARAMAYA UNIVERSITY
POSTGRADUATE PROGRAM DIRECTORATE
I hereby certify that I have read and evaluated this MSc Thesis entitled “Effect of Seed-Borne
Fungi Associated with Seeds of Silver Oak (Grevillea robusta) on Seedling Growth and
Development of Dieback and Stem Canker in Southeastern Ethiopia’’ prepared under my
guidance by Urgessa Merga Gardefa. I recommend that it be submitted as fulfilling the Thesis
requirements.
Abdella Gure (PhD)
Major Advisor
_________________
Signature
Mashilla Dejene (PhD)
Co-advisor
_________________
Signature
_______________
Date
_______________
Date
As a member of the Board of Examiners of the MSc Thesis Open Defense Examination, I
certify that I have read and evaluated the Thesis prepared by Urgessa Merga Gardefa and
examined the candidate. I recommend that the Thesis be accepted as fulfilling the Thesis
requirements for the degree of Master of Science in Agriculture (Plant Pathology).
_____________________
Chairman
__________________
Signature
________________
Date
_____________________
Internal Examiner
__________________
Signature
________________
Date
_____________________
External Examiner
__________________
Signature
_______________
Date
Final approval and acceptance of the Thesis is contingent upon its final copy to the Council of
Graduate Studies (CGS) through the candidate’s department or school of graduate committee
(DGC or SGC).
ii
DEDICATION
This work is dedicated to my father Mr Merga Gardefa, mother Mrs Aberru Tullu and wife Mrs
Wanofi Nuguse.
iii
STATEMENT OF THE AUTHOR
By my signature below, I declare and affirm that this Thesis is my own work. I have followed
all ethical and technical principles of scholarship in the preparation, data collection, data
analysis and compilation of this Thesis. Any scholarly matter that is included in the Thesis has
been given recognition through citation.
This Thesis is submitted in partial fulfillment of the requirements for a MSc degree at the
Haramaya University. The Thesis is deposited in the Haramaya University Library and is made
available to borrowers under the rules of the Library. I solemnly declare that this Thesis is not
submitted to any other institution anywhere for the award of any academic degree, diploma or
certificate.
Brief quotations from this Thesis may be made without special permission provided that
accurate and complete acknowledgment of source is made. Requests for permission for
extended quotation from or reproduction of this manuscript in whole or in part may be granted
by the Head of the School when in his or her judgment the proposed use of the material is in the
interests of scholarship. In all other instances, however, permission must be obtained from the
author of the Thesis.
Name: Urgessa Merga Gardefa
Signature: __________________
Date of Submission: _______________________
School/Department: _______________________
iv
LIST OF ACRONYMS AND ABBREVIATIONS
ANOVA
Analysis of Variance
AUDPC
Area Under Disease Progress Curve
CRD
Completely Randomized Design
CV
Coefficient of Variation
DAI
Days After Inoculation
DPC
Disease Progress Curve
DPI
Days Post Inoculation
DPR
Disease Progress Rate
DPS
Days Post Sowing
FAO
Food and Agriculture Organization of the United Nations
FI
Frequency of Infection
H2O2
Hydrogen Peroxide
LSD
Least Significant Difference
m.a.s.l.
meters above sea level
MEA
Malt Extract Agar
MLD
Mycosphaerella Leaf Diseases
MOE
Ministry of Education
NaOCl
Sodium Hypochlorite
PDA
Potato Dextrose Agar
RF
Relative Frequency
SAS
Statistical Analysis System
SNNPRs
Southern Nations, Nationalities and Peoples’ Regional State
WA
Water Agar
WGCF-NR
Wondo Genet College of Forestry and Natural Resources
v
BIOGRAPHICAL SKETCH
The author was born in Ilu Woreda in Southwest Shoa Zone, Oromia Regional State, on the 14th
of January 1988. He attended primary and junior education in Ilu Woreda at Asgori Primary
and Junior School. He attended and completed his secondary school education at Sebeta
Comprehensive Secondary School during 2004-2007.
In 2008, he joined Wondo Genet College of Forestry and Natural Resources, Hawassa
University, and graduated with BSc Degree in Forestry in 2010. After his graduation, he was
employed as a Graduate Assistant by Wondo Genet College of Forestry and Natural Resources
at Hawassa University.
After three years of service at Hawassa University, he joined the School of Graduate Studies at
Haramaya University in 2012 to pursue a study leading to MSc Degree in Agriculture (Plant
Pathology).
vi
ACKNOWLEDGEMENTS
First and foremost, I would like to thank the Almighty God for giving me strength and patience
to conduct and complete the whole research work successfully.
My special thanks are extended to my major advisor, Dr. Abdella Gure, and my co-advisor Dr.
Mashilla Dejene, for their wholehearted assistance from the commencement to the completion
of this thesis work.
My special thanks also go to Mr Mengistab Mateos, a Coordinator of WGCF-NR’s
Laboratories, for his moral support and appreciation during my research work. I would also like
to thank laboratory technicians of WGCF-NR’s, including Mr Adugna Boru, Mrs Woyinishet
Afework and Sanayit Desalegn, for their moral and technical support throughout my thesis
work. My sincerest gratitude also goes to all staff members of WGCF-NR, especially to Mr
Adem Esimo, Ararsa Derese, Begashow Marinie, Denebo Billo, Mrs Genet Negash, Getachew
Birhanu, Getachew Deme, Tola Bayisa, Yedeta Teshome and Mrs Workinesh Tekele for their
valuable and moral support during my research work. I am also thankful to all my friends and
relatives for their cooperation with me throughout my study period.
My special thanks are to Haramaya University and Ministry of Education for their corporate
financial support for my study. I would also like to extend my gratitude to Wondo Genet
College of Forestry and Natural Resources for allowing me to collect Grevillea robusta seed
samples, and access to facilitated Forest Pathology Laboratory for my study. I am also thankful
to Arsi Forest Enterprise and its staff members for the provision of facilities during seed
samples collection.
Last but not least, I am particularly indebted to my wife Mrs Wanofi Nuguse for her moral
support and patience during my study.
vii
TABLE OF CONTENTS
DEDICATION
iii
STATEMENT OF THE AUTHOR
iv
LIST OF ACRONYMS AND ABBREVIATIONS
v
BIOGRAPHICAL SKETCH
vi
ACKNOWLEDGEMENTS
vii
TABLE OF CONTENTS
viii
LIST OF TABLES
x
LIST OF FIGURES
xi
LIST OF TABLES IN THE APPENDIX
xii
LIST OF FIGURES IN THE APPENDIX
xiii
ABSTRACT
xiv
1. INTRODUCTION
1
2. LITERATURE REVIEW
4
2.1. Biology of Silver oak (Grevillea robusta)
4
2.2. The Nature of Forest Tree Seeds
5
2.3. Seed-borne Fungi of Forest Trees
5
2.4. Forest Tree Diseases in Ethiopia
2.4.1. Root diseases
2.4.2. Needle blight
2.4.3. Foliage diseases
2.4.4. Stem canker and dieback diseases
2.4. 5. Disease of Grevillea robusta tree
8
9
9
10
10
12
3. MATERIALS AND METHODS
14
3.1. Description of the Study Area
14
3.2. Collection of Grevillea robusta Seed Samples
16
3.3. Isolation of Seed-borne Fungi
18
3.4. Identification of Seed-borne Fungi
18
3.5. Morphological characterization of Botryosphaeria species
19
Continues…
viii
3.6. Pathogenicity Test
3.6.1. Pathogenicity test on seeds of G. robusta
3.6.2. Pathogenicity test on seedlings of G. robusta
20
20
21
3.7. Data Collection
22
3.8. Data Analysis
3.8.1. Area under the disease progress curve (AUDPC)
3.8.2. Disease progress rate (DPR)
22
22
23
4. RESULTS AND DISCUSSION
24
4.1. Isolation and Identification of Seed-borne Fungi
24
4.2. Characterization of Fungal Isolates from G. robusta Seeds
27
4.3. Pathogenicity Test on Seeds of G. robusta
4.4. Pathogenicity Test on Seedlings of G. robusta
29
31
4.5. Area under Disease Progress Curve (AUDPC)
35
4.6. Disease Progress Rate (DPR)
36
5. SUMMARY AND CONCLUSION
38
5.1. Recommendations
41
6. REFERENCES
42
7. APPENDICES
51
ix
LIST OF TABLES
Table
Page
1. Relative frequency (RF%) of fungal isolates obtained from seed samples collected from
symptomatic and asymptomatic G. robusta stands at Gambo, Shashemene and WGCF-NR
sites
27
2. Effect of predominately isolated test fungi on seedlings emergence of G. robusta tree in the
laboratory
31
3. Mean disease severity (%) recorded on G. robusta seedlings after inoculation with different
fungal species
35
4. Mean AUDPC values calculated for each fungal species inoculated on G. robusta
seedlings
36
5. Initial and final disease progress rates (DPR) estimated on G. robusta seedlings inoculated
with different fungal species
37
x
LIST OF FIGURES
Figure
Page
1. Map showing fruit samples collection sites
15
2. Symptoms of stem cankers and dieback on G. robusta stand
17
3. Fruit samples collected from G. robusta stands
17
xi
LIST OF TABLES IN THE APPENDIX
Appendix Table
Page
1. Number of fungal isolates obtained from seed samples collected from symptomatic and
asymptomatic stands of G. robusta at Gambo, Shashemene and WGCF-NR sites
51
2. Number of seeds that yielded one or more fungal isolates out of 100 seeds incubated, and the
frequency infection of seed samples collected from symptomatic and asymptomatic stands of
G. robusta at Gambo, Shashemene and WGCF-NR sites
51
3. Disease progress rates (DPR) recorded on G. robusta seedlings inoculated with different
fungal species
52
xii
LIST OF FIGURES IN THE APPENDIX
Appendix Figure
Page
1. Colony and conidial morphological characteristics Botryosphaeria sp.1
53
2. Colony and conidial morphological characteristics Botryosphaeria sp.2
53
3. Colony and conidial morphological characteristics of Fusarium sp.1
53
4. Colony and conidial morphological characteristics of Fusarium sp.2
54
5. Colony and conidial morphological characteristics of Lasiodiplodia sp.
54
6. Seedlings of G. robusta inoculated with different fungal species and disease symptom
observed
55
xiii
EFFECT OF SEED-BORNE FUNGI ASSOCIATED WITH SEEDS OF
SILVER OAK (Grevillea robusta) ON SEEDLING GROWTH AND
DEVELOPMENT OF DIEBACK AND STEM CANKER IN
SOUTHEASTERN ETHIOPIA
ABSTRACT
Silver oak (Grevillea robusta A. Cunn ex R.Br.) is an important multipurpose tree species that
provides various goods and services. The species has been widely grown for timber production
by smallholder farmers as well as by forest enterprises. Grevillea robusta is being widely
grown for shade and aesthetic value in most urban centers in Ethiopia. Nevertheless, this tree
species is being severely affected by dieback disease and stem canker in Ethiopia and
elsewhere in Africa. The objective of the current study was to assess the impact of fungi
associated with G. robusta seeds on seed germination and seedling growth. Grevillea robusta
seed samples were collected from standing trees at three localities, namely Gambo,
Shashemene and WGCF-NR. Seed-borne fungi associated with G. robusta seeds were isolated
from seed samples collected from healthy and diseased-looking trees of each locality. A total of
12 fungal species were morphologically identified. The most commonly isolated and selected
fungi for pathogenicity test on G. robusta seeds and seedlings were Botryosphaeria sp.1,
Botryosphaeria sp.2, Fusarium sp.1, Fusarium sp.2 and Lasiodiplodia sp. Spore suspensions
and mycelium plug cut of each test fungi were used for seed and seedling inoculation test,
respectively. The in vitro seed and seedling inoculation test were conducted at WGCF-NR’
laboratory and greenhouse, respectively. The experiments were laid out in a completely
randomized design (CRD) with four replications. The data on seedling emergence were
collected by counting the number of seedlings emerged per treatment. Similarly, disease
severity and its progress on the inoculated G. robusta seedlings over time were recorded using
0-7 disease scale; and subjected to ANOVA after converted into percentage severity index
(PSI). The results of in vitro seed and seedling inoculation tests revealed that all test fungi had
variable effects on both seedling emergence, and seedling growth, of which Fusarium sp.1 and
Lasiodiplodia sp. had higher significant effect on seedling emergence and seedling growth than
the other species. The highest (613%-days) and lowest (233%-days) AUDPC values were
recorded on seedlings inoculated with Fusarium sp.1 and Botryosphaeria sp.1, respectively.
Similarly, on seedlings inoculated with Fusarium sp.1 and Botryosphaeria sp.1, there were the
highest (0.01465 units day-1) and lowest (0.00665 units day-1) disease progress rates recorded
at the final day of disease assessment (49th day after inoculation), respectively. A combination
of both disease progress rate and AUDPC values showed that Fusarium sp.1 was the most
pathogenic fungal species, whereas Botryosphaeria sp.1 was the least pathogenic of all the
other tested fungal species. Therefore, the finding of this study showed that seed is considered
to be the sources of inoculum for the incidence of dieback canker and stem disease of G.
robusta trees. It is, therefore, suggested to use pathogen-free seeds and planting materials.
Keywords: AUDPC, Botryosphaeria, Dieback disease, Disease progress rate, Fusarium, G.
robusta, Lasiodiplodia, Pathogenicity, Seed-borne fungi, Stem canker
xiv
1. INTRODUCTION
Forests are the most important components of the ecosystems and play significant role in
sustaining life on earth (Campbell et al., 2003). Forests are valuable and rich resources for
various ecological, social and economical requirements. They play a vital role in buffering
against global climate change, stabilizing soil conditions, agro-forestry development, and
manipulating waste products. Forests also offer recreational opportunities, serve as a sanctuary
for wildlife, soil conservation, provide organic matter, fuel wood, lumber, pulp and paper,
medicines and other valuable extracts (Spanos et al., 1999; Eyles et al., 2003; Noël et al.,
2005).
Forests are generally categorized as natural forests and plantation forests. Natural forests are
types of forests that are naturally-regenerated woody species diversity, while plantation forests
are defined as even‐aged forest stands established by man through seeding and/or planting in
the process of afforestation or reforestation (FAO, 2001). Plantation forests can be established
using either exotic or native tree species. There is a growing worldwide trend towards the
establishment of plantation of exotic tree species, especially in the tropics and subtropics
(Persson, 1995). The largest introduced plantation forest countries are Australia, Brazil, Chile,
Indonesia, New Zealand and South Africa (Vercoe, 1995). In these countries, the most
commonly planted exotic tree species include Acacia spp., Euclyptus spp., Grevillea robusta
and Pinus patula, and those tree species are used mainly for sawn timber, paper and pulp
industries.
Many countries in Africa also grow large areas of exotic plantations of Acacia spp., Cupressus
lusitanica, Eucalyptus spp., Grevillea robusta and Pinus patula to provide fuel and timber as
well as for production of paper and pulp (Vercoe, 1995). Particularly, the establishment exotic
tree species in Ethiopia also commenced with the introduction of Eucalyptus species from
Australia in 1894-1895 (Pohjonen and Pukkala, 1990). Since then, the tree species, like C.
lusitanica, G. robusta and P. patula are among the other exotic tree species that have been
introduced and widely planted in different parts of the country (Eshetu, 2002; Bekele, 2006).
2
Grevillea robusta is native to eastern Australia and it has been introduced to tropical and
subtropical highlands and warm temperate regions around the world commencing in the mid
to late 19th century (FAO, 2001). In this regard, the tree species is widely planted in Central
and South America, China, India, Indonesia, Sri Lanka, Vietnam and many countries in Africa
(Booth and Jovanovic, 2002)). These countries have the climatic and edaphic conditions
regarded as optimal for the growth of the tree species (Booth and Jovanovic, 2002); thus, it has
gained a widespread popularity in these regions originally as a shade tree for tea and coffee,
and more recently as an agro-forestry tree for small subsistence farms. Similarly, this tree
species is grown in different parts of Ethiopia as an agro-forestry, where in most cases it is
planted near and around homestead together with fruit trees, coffee (Coffea arabica), khat
(Catha edulis), enset (Ensete ventricosum), banana (Musa spp.) and some other agricultural
crops.
G. robusta is one of a fast-growing tree species with multipurpose uses that provide various
goods and services, including construction material, electric power transmission pole, timber,
fuel wood, shade, wind break, fodder and soil fertility improvement (Muchiri et al., 2002). In
Australia, and in vast areas of other regions where it is cultivated, G. robusta is also valued as
an ornamental or aesthetic tree for private and public gardens, promenades, street borders and
parks (Muchiri et al., 2005; Holding et al., 2006).
Forest tree species die at different stages of growth for various reasons of which diseases and
insect pests are the major factors that often lead to abnormal growth or development of tree
species (Wingfield, 1990; Mireku and Simson, 2001; Schroeder et al., 2002). In Ethiopia,
plantation forests of several tree species have been suffering from varying degrees of attack by
several disease-causing agents (Alemu et al., 2003). Particularly exotic plantation forests are
among the forests that have been subjected to attack by various diseases in recent years. As far
as G. robusta is concerned, the most recent and notable case is the occurrence of stem canker
and dieback disease. Disease symptoms of stem canker and die-back of G. robusta include
dieback of shoots and branches, formation of lesions and canker on the stems, yellow-to-red
exudates on stems and branches, formation of clusters of small and deformed leaves, and
sapling and tree mortality. The disease seemed to develop from actively growing tissues in
3
young shoots and inflorescences, and progressed into the branches and stems. Disease
progression from young infected shoot tissues could also develop further into the stem
(Njuguna, 2003; Njuguna et al., 2011).
The dieback and stem canker disease of this tree species is known to be caused by the fungal
species of the family Botryosphaeriaceae (Njuguna, 2003; Toljander et al., 2007; Njuguna et
al., 2011). However, so far, the source of inoculum has not been clearly identified. Thus, the
current study was directed to assess the effect of fungi associated with G. robusta seeds on
seed germination and seedling growth. In this aspect, this study was proposed to generate
research data that can contribute to the development of management strategies aimed at
reducing the incidence of seed-borne diseases within and outside plantations. It clued that
dieback and stem canker disease were incited by seed borne pathogenic fungi. To this effect,
proper collection of fruits, storage, extraction techniques and seed treatments are the
preliminary means of reducing the incidence of the diseases.
The general objective of the study was to assess the charactestics and effects of seed-borne
fungi associated with G. robusta seeds, on seed germination, seedling growth and their
development.
The specific objectives of the study were to:
1. Identify fungi associated with seeds of G. robusta;
2. Investigate the effect of seed-borne fungi on seed germination; and
3. Study the effect of seed-borne fungi on the seedling growth and development of dieback and
stem canker of G. robusta seedlings.
4
2. LITERATURE REVIEW
2.1. Biology of Silver oak (Grevillea robusta)
Silver oak (Grevillea robusta A. Cunn ex R.Br.) is an evergreen tree species belonging to the
family Proteaceae. It is an erect single-stemmed tree typically reaching an adult size of 25-35
m in height and 80 cm in diameter in its natural range, and is exemplified by its conical crown
and dense branches projecting upwards (Orwa et al., 2009). Leaves are silver-grey below and
pale green above, fern-like. Mature fruits are dark-brown capsules with a slender beak,
splitting to release two winged and light seeds. This tree species is characterized by its
proteoid root system (cluster of roots that grow in low fertility soils), and hence believed to
compete less for minerals with food crops (Akycampong et al., 1999). Furthermore, G.
robusta does not form symbiotic associations with soil rhizobacteria or mycorrhizal fungi
(Skene et al., 1996); thus, it is believed that this tree species develops under conditions of low
phosphorus availability.
G. robusta first flowers when about 6 years old. Flowers are in spikes, yellow-orange. In its
natural range, flowering occurs over a few weeks in October-November, but when planted in
equatorial latitudes, flowering is sporadic throughout the year or absent (Kalinganire et al.,
2001). The flowers are bisexual, and pollen is shed before the stigma becomes receptive.
Pollinating agents include honeybees, birds and arboreal marsupials, which collect nectar and
pollen from flowers (Kalinganire et al., 2001). The period from fertilization to fruit maturity is
about 2 months. Fruit opens during hot, dry weather, releasing the seeds, which can be carried
considerable distances by wind. G. robusta has mature fruit from September to January (Lott
et al., 2000).
According to Orwa et al. (2009) seed storage behavior of G. robusta is orthodox; whole seed
have 28.5% mc; 60-70% germination following 2 years of hermetic storage at -7 oC with 10%
mc; 35% germination following 12 months of open storage (Albrecht, 1993). Seeds were
maintained for 4 years in commercial storage conditions; viability was maintained for 2 years
5
in hermetic air-dry storage at 3 oC. G. robusta seeds germinate within 8-20 days, and the
expected germination rate is between 50 and 70% (Orwa et al., 2009).
2.2. The Nature of Forest Tree Seeds
Seeds are essential biological commodities in the regeneration of forests. They are the primary
means by which forest trees reproduce, maintain genetic variability, and become established
on appropriate sites (Fenner and Thompson, 2005). One of the main features of forest tree
seeds is their great diversity in size, shape and texture. The size and texture of tree seeds range
from small and hard, as the seeds of Eucalyptus spp., to relatively large and fleshy acorns of
some pine oak (Quercus spp) (Lusk and Kelly, 2003). The seeds of G. robusta are light and
medium sized. The longevity of tree seeds varies from a few days to many years. Mature tree
seeds generally have higher tolerance to low moisture stress levels or temperatures than many
fresh fruits, vegetables or flowers (Hong et al., 1996).
A constant and reliable supply of healthy and high quality seeds may be difficult to achieve for
a number of reasons (Morpeth and Hall, 2000). Firstly, seed production by forest trees is so
variable that for some species there may be no annual production of seeds, or production may
be very small or of poor quality. As far as G. robusta seeds concerned, there are 70,000100,000 seed per kilogram depending on the provenance and the climatic conditions of the
ripening year (Orwa et al., 2009). Secondly, forest tree seeds, like all other seeds, are also
exposed to different biotic and abiotic factors that can affect seed germination and the normal
developmental processes (Morpeth and Hall, 2000). Among biotic factors, forest tree seeds get
infected or contaminated by propagules of various microorganisms, especially seed-borne
fungi (Mamatha et al., 2000).
2.3. Seed-borne Fungi of Forest Trees
Forests tree species are known to be infected by various plant pathogens in forest nurseries,
plantations and also in natural forests (Mireku and Simson, 2001). Diseases of forest trees are
incited by different causal agents, such as fungi, bacteria and viruses. Among these, the impact
6
of fungi is considerable, and many of them are serious pathogens of maturing seeds that
reduce the yield and impair seed quality (Sutherland et al., 2002). A number of studies have
shown that several kinds of fungi can be isolated from forest tree seeds with a range of impacts
on the seeds and seedlings. The majority of the associated fungi recorded from seeds of forest
trees so far belong to the conidial (anamorphic, imperfect) states of the phylum Ascomycota
(Mittal et al., 1990). Hence, important seed-borne fungal diseases of different forest trees are
caused by species of Alternaria, Aspergillus, Botrytis, Cephalosporium, Chaetomium,
Cladosporium, Colletotrichum, Curvularia, Fusarium, Penicillium, Phoma and Phomopsis
(Khalid et al., 2002).
The seeds of forest trees are vehicles, victims and responsible for long or great distance spread
of numerous pathogens or diseases. To this effect, the term ‘seed-borne pathogen’ describes
the state of any pathogen being carried with, on or in the seed, which may or may not have the
potential of causing disease of seed or the subsequent plant (Agarwal et al., 1997). The
survivals of seed-borne pathogens depend upon the amount of inocula per seed, the location of
inocula in the seeds, the type of survival propagules and the seed storage environmental
conditions (Agarwal et al., 1997).
On the contrary, the term ‘seed-transmission’ refers to the act of infection of the seedlings
from seed-borne inocula (Berjak, 2000). The agents that are transmitted and cause diseases are
seed-transmitted pathogens, whereas pathogens that are associated with seeds, but do not play
a role in disease development are non-seed-transmitted pathogens (Mittal et al., 2003). The
main effects of seed-transmitted fungi are the diseases they cause, and to some extent, also the
seed viability reduction they pose (Dos-Santos et al., 2001). Seed transmission can be
influenced by the type, virulence, amount and location of inocula in seed. Similarly, the rate of
seed-borne pathogen transmission depends upon the host, pathogen race and virulence,
environment, vectors, and their interactions over time (Agarwal et al., 1997).
Seed-borne fungi of forest trees include all types of fungi contaminating the seed surface or
infecting its inner tissues (Dhingra et al., 2002). Seed-borne pathogenic fungi can greatly
affect seed quality and cause diseases that have negative impact on seed germination and
7
seedling establishment in nurseries (Mamatha et al., 2000; Burgess and Wingfield, 2002). The
presence of certain fungi on seeds is often significant because it may indicate problems with
the quality of the seed lot due to improper handling and storage of both cones and seeds
(Sutherland et al., 2002). In this case, the sources of contamination can be from improperly
cleaned seed lots containing bits of pathogen- contaminated needles, leaves, cones or other
debris, and infected seeds. Fungal propagules can gain access to the seed tissues at any time
from flowering to the post-shedding phases (Kabeere et al., 1997). This can happen while the
fruits and seeds are still on a tree, after falling onto the ground, during collection and
processing, and during transit or in seed storage (Dhingra et al., 2003).
According to some studies, various seed-borne fungi are commonly associated with seeds of
many tree species, and these include pathogens and saprophytes (Vujanovic et al., 2000). A
number of fungal species that are generally considered to be saprophytes do behave as parasite
under certain favorable conditions for those saprophytes (Vujanovic et al., 2000). Such
conditions include injury to the seed or seed coat, moisture and temperature conditions that
favor fungal growth and increase physiological and physical vulnerability of tree cones/fruits,
seeds, or seedlings to infection (Singh et al., 1999a). Moreover, seed-borne fungi can weaken
and predispose seeds and seedlings to a variety of other soil-borne pathogens (Mamatha et al.,
2000).
Pathogenic fungi can infect seeds internally through the stigma-style continuum during
flowering (Agarwal et al., 1997; Mathur and Kongsdal, 2003) and destroy the
endosperm/cotyledon and even the embryo, or contaminate the seed surfaces or mix with
seeds and affect seedling germination and their development. Fungi gain access to seeds
through direct penetration via ovary wall, floral parts, and systematic via vascular tissue
(Agarwal et al., 1997). Depending upon the presence of fungi either on seed-coat or inside the
seed, seed-borne fungi are further categorized as externally seed-borne fungi and internally
seed-borne fungi (Singh et al., 1999b). The former group includes species of the genera
Botrytis, Fusarium, Mucor, Phialophora, Rhizopus and Trichothecium. They are not usually
host-specific and may involve more than one species. Similarly, some of the well-known
internally seed-borne fungi include species of the genera Alternaria, Aspergillus, Botrytis,
8
Botryodiplodia, Caloscypha, Cephalosporium, Fusarium, Phoma, Schizophyllum and
Sirococcus. These may cause deterioration of seed quality and pre- or post-emergence
mortality of seedlings. In this regard, symptoms of seed-borne diseases are usually divided
into pre-and post-emergence damping off (Denman et al., 2003; Abdella, 2004). The former
consists of reduced emergence and death of the radicle just emerged from the seed coat; while
the latter is subdivided into root rot, cotyledon rot, and basal stem rot after the seedlings
emerged from the soil (Denman et al., 2003).
Moreover, based on their ecological requirements, the fungi found associated with seeds are
classified into ‘field fungi’ and ‘storage fungi’ (Agarwal et al., 1997). This grouping of fungi
is based on where the association begins. Field fungi are known to invade seeds as they are
developing on the plants in the field or after they have matured, but before they are harvested
and are favored by high relative humidity or high seed moisture content, while storage fungi
are found associated with seeds during storage (Agrios, 2005). The storage fungi of seeds
comprise mainly species of Aspergillus and Penicillum, and members of these fungal groups
normally do not infect seed prior to storage, but invade under conditions prevailing in storage
at a relatively low equilibrium relative humidity or low seed moisture content (Huang and
Kuhlman, 1990).
2.4. Forest Tree Diseases in Ethiopia
Microorganisms can have a mutualistic, saprophytic and parasitic/pathogenic association with
forest trees, among which the parasitic association often causes damage on several valuable
tree species (Spanos et al., 1999; Coetzee et al., 2001). Pathogens influence the survival of
regenerating seedlings and hence influence the occurrence and abundance of plant species. A
number of studies conducted worldwide indicated that pathogens causing forest tree diseases
have considerable impacts on forest tree species distribution, forest structure and composition,
succession and biodiversity. For example, Cryphnectria parasitica, a pathogen of American
chestnut, eliminated chestnut trees from the forest community and caused change in species
composition and structure (Coetzee et al., 2001; Venter et al., 2001; Myers and Bazely, 2003;
Irshad et al., 2007). Some studies have also been carried out on the prevalence of forest tree
9
diseases and losses they cause in forests. According to Alemu (2004), Armillaria root rot, stem
canker, tree dieback, wood rot, needle blight, foliage disease and damping-off were observed
under different instances.
2.4.1. Root diseases
Armillaria is one of the genera that affect the roots of several native and exotic woody plants
throughout the world (Alemu et al., 2003). Many of the species in this genus are serious
pathogens of a wide range of native and planted conifers, hardwood trees and shrubs in forests,
orchards and gardens (Coetzee et al., 2001). These fungal species were also recorded from
some important indigenous and exotic tree species in Ethiopia. It was reported that Armillaria
species were found on recently cleared and planted sites and where shade trees had been
removed (Otta et al., 2000). In this regard, the prevalence of symptoms of Armillaria root rot
was found in association with Pinus patula at Wondo Genet, Belete, Bedele and Jimma, on
Acacia abyssinica trees at Wondo Genet and Bedele, on stumps of Juniperus excelsa, at
Wondo Genet, on Cordia alliodora and Cedrela odorata trees in research plots at Aman and
on Grevillea robusta at Wondo Genet (Alemu et al., 2003).
A recent population study on Armillaria spp. in Ethiopia indicated that two Armillaria species,
namely A. mellea and A. fuscipes are involved in causing Armillaria root rot. A. mellea is
responsible for root rot on hardwood trees in the Jima and Kerita areas (Otta et al., 2000). On
the other hand, A. fuscipes affects Cedrela odorata, Cordia alliodora and P. patula trees. It
was also found that this fungus is associated with Acacia abyssinica and Juniperus excels
(Alemu et al., 2004).
2.4.2. Needle blight
Dothistroma needle blight caused by Dothistroma septospora is a serious disease in many
countries where Pinus radiata is grown. In some African countries, the severe defoliation
caused by Dothistroma needle blight had led to abandonment or restriction planting of the fast
growing P. radiata and in most cases it has been substituted with a slightly slow growing
10
Pinus patula (Ciesla et al., 1995; Alemu et al., 2003; Alemu et al., 2006). The occurrence of
Dothistroma needle blight was reported on Pinus radiata around Addis Ababa (Alemu et al.,
2003; Alemu et al., 2006).
2.4.3. Foliage diseases
Mycosphaerella leaf diseases (MLD) were reported to be associated with juvenile foliage of
Eucalyptus globulus (Alemu et al., 2003; Alemu et al., 2006). Symptoms of these leaf diseases
were recorded from samples obtained from Wondo Genet, Hossana, Endibir, Bedele,
Menagesha, Holetta and Addis Alem (Alemu et al., 2003; Alemu et al., 2006). Shoot dieback
and leaf blotch are the common symptoms of MLD (Alemu et al., 2006). It causes premature
defoliation, retarded growth and in severe case it may cause total abandonment of planting
susceptible species. In several cases, nearly 100% of the juvenile leaves and leaf surfaces were
affected by MLD.
According to Alemu et al. (2006) three different Mycosphaerella species namely, M. nubilosa,
M. marksii and M. parva were identified from Eucalyptus globulus trees planted in different
parts of Ethiopia. Mycosphaerella marksii was isolated only from leaf samples collected near
Hossana. However, M. parva was found on leaf samples obtained from Addis Alem, Endibir
and Hossana. Alemu et al. (2006) reported that Ethiopia is the third country to report the
occurrence of M. Parva. The occurrence of this species at different localities indicates the
importance of fungus and it might play significant role in MLD outbreak in Ethiopia as well.
On the other hand, Mycosphaerella leaf disease caused by M. nubilosa was found around
Endibir, Holetta, Hossana and Bedele (Alemu et al., 2006). This species commonly affects
juvenile leaves of E. globulus.
2.4.4. Stem canker and dieback diseases
Stem canker is the cracking of bark and sapwood caused by some fungal species (Agrios,
2005). Most canker-causing fungi overwinter in dead or infected bark tissue in which fungal
fruiting bodies, spores or mycelia are present. Many fungi cause canker diseases on a variety
11
of forest trees and shrubs. With this respect, some of the most common stem canker-causing
fungal genera include Botrysphaeria, Cytospora, Nectria, Ceratocystis, Hypoxylon and
Cryphonectria (Roux et al., 2001).
A serious stem canker and dieback disease of forest trees is caused by Botryosphaeria spp.
(Alemu et al., 2003; Alves et al., 2004; Slippers et al., 2004a). The Botryosphaeria spp. are
widely distributed in the tropical and temperate climates worldwide and recognized to have
multiple hosts with the ability to move between native and introduced host forest trees
(Slippers and Wingfield, 2007). Many literatures indicated that various diseases of forest tree
species are caused by these fungal species (Alemu et al., 2004; Copes and Hendrix, 2004;
Phillips et al., 2006; Bester et al., 2007). With this respect, infection caused by these fungal
species takes place either through wounds, or directly through the stomata and other natural
openings (Kim et al., 2002; Burgess et al., 2005). These fungi are associated with a wide
range of disease symptoms, such as shoot blight, dieback, stem canker, fruit rots and even
death of many woody plant hosts (Swart et al., 2000; Roux et al., 2001; Alemu et al., 2003).
A particularly dangerous feature of these species of fungi is that they can live in plant tissues
as endophytes, saprophytes and latent or opportunistic pathogens, without exhibiting clear
symptoms (Smith et al., 1996; Slippers et al., 2007). These fungi, which reside in the hosts
without manifesting any disease symptoms for a more or less extended period within the plant
tissues are able to attack their hosts when grown under stress conditions, and the onset of
disease from the latent infection is linked to stressful environmental conditions, such as
drought, water logging and winds that reduce the growth vigor of the host plants (Burgess et
al., 2006; Desprez-Loustau et al., 2006).
Botryosphaeria spp. are among the most common fungi that cause diseases of various
commercially grown forest trees species. Among the commonly infected tree species are
Acacia mearnsii, Eucalyptus spp., Grevillea robusta, Pinus spp., Podocarpus falcatus and
Prunus africana. It has been previously suggested that the introduction of exotic tree species
can also introduce pathogens into new areas via planting materials or seeds (Wingfield et al.,
2001). Evidence of the presence of pathogenic fungi, such as species of Botryosphaeria, on
12
seeds has been documented on Eucalyptus spp. and other hosts (Lupo et al., 2001; Slippers et
al., 2004c; Abdella et al., 2005). Abdella (2004) reported that Botryosphaeria spp. on native
forest tees in Africa is the most important pathogens affecting the dwindling forest resources.
The importance of stress-related pathogens, such as B. dothidea, is the most evident scenario
under consideration (Denman et al., 2000; Smith et al., 2000; Slippers et al., 2004b). This
fungal species has a wide host range amongst woody plants and has been recorded from all
Eucalyptus species commonly propagated in South Africa (Slippers et al., 2007).
Moreover, a number of stem canker disease-causing Botryosphaeria spp. in Ethiopia have also
been reported from Eucalyptus spp. (Alemu et al., 2003). A particular fungal species involved
in causing stem canker on Eucalyptus species was identified as Botryosphaeria parva. It was
recorded from several tree species, including E. globulus, E. saligna, E. grandis and E.
citrodora planted at Munessa, Shashemene, Wondo Genet and Menagesha areas. This fungal
pathogen is commonly found on both coppice stems and first generation stands irrespective of
the ages of the stands. Abdella (2004) also reported four other Botryosphaeria species that
were associated with seeds of Podocarpus falcatus and Prunus africana. Of these four species,
three of them were new records. The one which was recorded from seeds of Podocarpus was
B. parva, while the species from seeds of P. africana was reported to be new and the pathogen
was named as Diplodia rosulata (Abdella, 2004).
2.4. 5. Disease of Grevillea robusta tree
Grevillea robusta is a multipurpose tree that provides various goods and services. However,
this tree species is being severely affected by stem canker and dieback disease in several
African countries, including Ethiopia. Observation made under field conditions indicated that
stem canker and dieback disease could contribute to the decline of the population and area
coverage of G. robusta in different parts Ethiopia, particularly in Wondo Genet College of
Forestry and Natural Resources (WGCF-NR), and Arsi Forest Enterprise. It is one of the
limiting factors of commercial production of G. robusta. For a long time G. robusta was
regarded as having “no diseases of economic importance worldwide” (FAO, 2001). Due to
that fact, no isolations of pathogens had been done, and the species continued to be regarded
13
as disease-free. In Kenya, however, stem canker and dieback symptoms were first reported on
G. robusta in 1960 (Smith, 1960) and later in the 1980s (Milimo, 1988).
An occurrence of a canker and dieback disease of this tree species was observed for the first
time in Uganda in October 2001 (Toljander et al., 2007). According to the report of Toljander
et al. (2007) the incidence of canker and dieback disease symptom of G. robusta was also
reported in Ethiopia for the first time in February 2006. This canker and dieback disease seems
to be widespread in some parts the East African region since typical symptoms have been
observed on G. robusta in Kenya and Ethiopia. In response to this, nowadays stem canker and
dieback disease is being very severe in these countries.
Njuguna et al. (2011) stated that reports of canker and dieback symptoms in semi-arid areas
together with results from monitoring of on-farm experiments in Kenya showed that the
incidence of canker and dieback symptoms on G. robusta increased from 17 to 65% and
mortality of the trees also increased from 2 to 18% between 2001 and 2003. Infected trees
were characterized by poor growth, cracks on stems or branches resulted in rupturing of the
bark, followed by resin exudation. Njuguna et al. (2011) reported that as cankers increased,
girdling of young stems, branches and shoots led to dieback of shoots, branches and death of
trees. Cankers varied in size from small lesions of few millimeters to large open wounds
sometimes extending over 1 m along the stem on severely infected trees (dying or dead trees).
14
3. MATERIALS AND METHODS
3.1. Description of the Study Area
The present study was carried out by collecting samples of matured fruits of Grevillea robusta
from the forest plantation districts of the Arsi Forest Enterprise; namely, Gambo and
Shashemene Forest Districts, and Wondo Genet College of Forestry and Natural Resources
(WGCF-NR), in 2013/14. The experiments were conducted at the Forest Pathology
Laboratory of WGCF-NR. WGCF-NR is located in Southern Nations’, Nationalities’ and
Peoples’ Regional State (SNNPRs) at 263 km South of Addis Ababa. It is geographically
situated at 07o 06' 16"N and 38o 37' 41"E. The altitude of the area ranges from 1800-2500
m.a.s.l. with mean annual rainfall of 1,160 mm. The main rainfall period is from June to
September, while the period from December to February is relatively dry. The mean monthly
temperature ranges from 19 oC in August to 25 oC in March, April, May and September. It is
categorized under Weina dega ecological zone.
The WGCF-NR locality is characterized by both natural and plantation forests. The common
forest tree species found in WGCF-NR include Acacia spp., Cordia africana, Croton
macrostachyus, Cupressus lusitanica, Eucalyptus spp., Grevillea robusta, Pinus patula and
Podocarpus falcatus. Wondo Genet is endowed with fertile soil, water, forest and wildlife, to
mention some of its many natural features and resources. Sandy-loam soil is the dominant soil
type that covers the largest parts of the area. Reddish clay soil is also found in some parts of
the area.
15
Figure 1. Map showing fruit samples collection sites
Arsi Forest Enterprise is situated at about 240 km south of Addis Ababa, and geographically
located at 07° 21' N and 38° 42' E along the eastern escarpment of the Central Rift Valley. The
altitude of this area ranges from 2100-2450 m.a.s.l. with mean annual rainfall of 1,250 mm.
The main rainy season of the area extends from end of June to September, and the short rainy
season is from February to April. The average maximum temperature of the area is 25 oC and
the minimum is 7 oC, which occurs in November (Lüttge, et al., 2003). The total concession
area of the Enterprise is estimated to be 21,384 ha, of which 6,230 ha is occupied by plantation
forests and the rest (15,154 ha) is covered with natural forests (Kedir, 2009).
The entire forest area of the Enterprise is divided into three forest districts, namely Munessa,
Gambo and Shashemene. Of these, the present study was conducted in the plantation forest of
the two forest districts, specifically Gambo and Shashemene. The Gambo site is
geographically located at 07o 19' 40" N and 38o 49' 17"E with altitudinal range of 2100-2700
m.a.s.l. and mean annual rainfall of 1250 mm and mean annual temperature of 15-20 ºC
16
(Demel and Granström, 1995), while the Shashemene site is specifically and geographically
situated at 07o 08' 20" N and 38o 39' 33"E with altitude ranging from 1700 to 2600 m.a.s.l. The
mean annual rainfall and temperature of the area are 700-1000 mm and 12-19 ºC, respectively.
These forest districts are characterized by both natural and plantation forests. At present, the
main plantation species of the districts are exotics, such as Cupressus lusitanica, Eucalyptus
globulus, E. saligna, E. grandis. G. robusta and P. patula.
3.2. Collection of Grevillea robusta Seed Samples
For the present study, samples of matured fruits were collected from G. robusta stands in Arsi
Forest Enterprise sites, namely Gambo and Shashemene (Sole) forest Districts, and WGCFNR between September 2013 and January 2014. In each study site, four sampling points
(compartments) of standing G. robusta trees were randomly considered. At the beginning, a
preliminary disease survey was conducted at each sampling point (compartment) of the three
seed samples collection sites in order to have an overview of the incidence of the stem canker
and dieback disease on G. robusta trees in the stands. The survey was based on recognition of
diseased-looking trees bearing disease symptoms such as stem cankers, flow of yellowish to
reddish ooze from the infected tree stems as well as top death of the leading shoots and
branches, and those without these symptoms as healthy-looking trees (Figure 2). This was
followed by purposive selection of symptomatic and asymptomatic mother trees for fruit
collection.
Accordingly, 20 asymptomatic and 20 symptomatic trees were selected at each of the fruit
collection sites, namely Gambo, Shashemene (Sole) and WGCF-NR. Thus, a total of 120 trees
(60 diseased and 60 healthy) looking trees were selected for fruit collection. In all cases,
mature fruits were separately collected in plastic bags from each of the healthy and diseasedlooking trees by climbing. The fruit samples were labeled, and transported to the Forest
Pathology Laboratory at WGCF-NR for further processing. After transporting to the
laboratory, fruits collected from healthy-looking trees of respective site were mixed and
processed as one composite sample, while those from different sites were kept separately, and
so were those from diseased-looking trees.
17
The collected fruits were spread on clean polythene sheets for drying for four days (Figure
3A). After proper drying, seeds of G. robusta were manually extracted from the fruits (Figure
3B). Then, the extracted seeds were washed with tap water and air-dried by spreading them on
clean polythene sheets kept under shade, and stored in sealed paper bags for further analysis.
Figure 2. Symptoms of stem cankers and dieback on G. robusta stand: (A) Stem cracks and small
cankers; (B) Oozing from the cankered stem (trunk); (C) Severely affected tree with stem canker; (D)
Asymptomatic G. robusta stand; (E) Shoot and branch dieback; (F) Trees with severe dieback of shoots and
branches (Photos taken from Gambo, Shashemene and WGCF sites)
Figure 3. Fruit samples collected from G. robusta stands: (A) Matured fruits of G. robusta; (B) Seeds
extracted from fruits
18
3.3. Isolation of Seed-borne Fungi
Isolation of seed-borne fungi associated with seeds of G. robusta collected from the three sites
was undertaken separately from seed samples collected from healthy and diseased-looking
trees. In total, 200 seed samples (100 seeds each from symptomatic and asymptomatic) mother
tree were drawn for isolation at each locality. Seed-borne fungi were isolated from surfacesterilized seeds. Surface sterilization of the seeds was carried out using 3-33% (v/v) hydrogen
peroxide (H2O2) (Sutherland et al., 2002). The seeds were sterilized by immersing them in the
hydrogen peroxide solution for two minutes, followed by three consecutive rinsing thoroughly
with sterilized water. Another set of G. robusta seeds, to be used as a control, were surface
washed with sterile water only, i.e. without surface-sterilization with hydrogen peroxide
(H2O2, 33% v/v). In both cases (surface-sterilized and unsterilized seeds), the rinsed seeds
were allowed to dry by blotting on sterile filter paper. Then, five seeds per plate were
aseptically placed equidistantly onto 9 cm diameter of Petri-dish containing potato dextrose
agar (PDA) medium using a sterilized pair of forceps. Then, the plates were incubated at 22-25
o
C for 7 days under 12 hours alternating cycles of light and darkness. Finally, three to seven
days after incubation, the fungi emerging from seeds were monitored, and isolated onto
separate fresh culture medium as pure cultures; sealed, labeled and assigned with identification
numbers for further examination.
3.4. Identification of Seed-borne Fungi
The preliminary identification of seed-borne fungi growing out from seeds plated onto the
PDA medium was undertaken on the basis of the cultural or colonial features developed on the
agar medium and the morphological (conidial) characteristics of the individual fungal isolates
studied under a compound microscope (Barnett and Hunter, 1998). The cultural features used
to characterize fungal colonies were color of the obverse (upper or front) and reverse sides of
the cultures, shape of colony, margin of colony and growth pattern of mycelium (such as
fluffy aerial hyphae, appresssed or submerged hyphae). Morphological characteristics,
including shape and color of the conidia/spores, presence or absence of branching and
septation of hyphae, were also used to characterize the fungal colony. On the basis of the
19
above characteristics, fungal isolates found growing out from the seeds were identified, and
their percentages of relative frequencies were calculated by applying the following formula
(Ebele, 2011):
Relative frequency (%) = Number of fungal colonies of each species appeared x 100
Total number of colonies of all fungal species appeared
Frequency of infection (%) was also calculated as the total number of seeds infected by fungi
divided by the total number of seeds incubated (El-Awadi, 1993). A frequency of infection
was used to compare the degree of infection by seed-borne fungi of seed samples collected
from G. robusta stands found at three localities, namely Gambo, Shashemene and WGCF-NR.
Frequency of infection (%) = Number of seeds infected by fungi x 100
Total number of seeds incubated
3.5. Morphological characterization of Botryosphaeria species
Isolates of Botryosphaeria spp. from seeds of G. robusta were characterized based on cultural
characteristics and conidial morphology. Morphological identification of Botryosphaeria
isolates was based on conidial morphology from cultures grown on 2 % water agar (Luque et
al., 2005). In order to induce sporulation, autoclaved pine needles were placed onto the plates
containing 2% water agar (WA) (Slippers et al., 2004a; Luque et al., 2005). Then, the cultures
were inoculated onto sterilized pine needles paced on 2% water agar (WA) and incubated at
22-25 oC. Cultures releasing conidia from pycnidia formed on pine needles were transferred
on PDA, and the plates were incubated at 22-25 oC. Mounts of pycnidia were prepared, and a
morphological observation was made under light microscope (Phillips, 2002; De Wet et al.,
2003).
20
3.6. Pathogenicity Test
Inoculum for pathogenicity test was prepared from 10-day-old pure cultures of each test
fungus. It was prepared as described by Xue et al. (2004) that 10 milliliters of sterile distilled
water was added to each Petri dish containing mycelial mat, and the culture was agitated to
dislodge spores using a sterile scalpel into an electric blender. After blending for five minutes,
it was diluted in 200 ml of sterile distilled water to make a spore suspension for inoculation of
seeds (Xue et al., 2004). The resulting spore suspension was filtered through two layers of
muslin cloth, and the concentration of spores was estimated using a haematocytometer for
inoculation (Xue et al., 2004). In this connection, each fungal species used for pathogenicity
were adjusted to the specific concentration of spore per milliliter. With this regard, the
concentration of spore suspension of Botryosphaeria sp.1, Botryosphaeria sp.2, Fusarium
sp.1, Fusarium sp.2 and Lasiodiplodia sp. was 2.2×105, 2.4×105, 1x106, 2.6x105 and 2.5×105
spore/ml, respectively.
3.6.1. Pathogenicity test on seeds of G. robusta
Seed-borne fungal species that were most frequently isolated from each locality were tested
for their specific effects on G. robusta seed germination under regulated laboratory conditions.
In this case, seed samples collected from healthy-looking trees were surface-disinfected with
hydrogen peroxide (H2O2 33% v/v) for two minutes, followed by rinsing three times in sterile
water. The rinsed seeds were dried by blotting on sterile filter paper under a laminar flow
hood. Then, seeds were transferred and soaked in flooded and homogenized mycelium
suspension of 10-day-old cultures of each test fungus that included Botryosphaeria sp.1,
Botryosphaeria sp.2, Fusarium sp.1, Fusarium sp.2 and Lasiodiplodia sp. separately for 12
hours, and air-dried. After that, seeds treated with each test fungus were sown at a rate of 10
seeds per plate containing 2% water agar medium, and laid out in completely randomized
design (CRD) with four replications each in a laboratory. Seeds soaked in sterile water devoid
of any mycelium suspension of each test fungus served as a control or check plate.
21
Next to this inoculation process, the emergence of seedlings in each plate was monitored on
the 30th day post-sowing. Finally, the emerged seedlings were counted and emergence
percentage was calculated as indicated below:
Seedling emergence (%) = Number of seedlings emerged x 100
Total number of seeds sown
3.6.2. Pathogenicity test on seedlings of G. robusta
For pathogenicity test on seedlings, seeds collected from healthy-looking trees were surfacesterilized as descried before, and followed by rinsing thrice in sterile water. The rinsed seeds
were dried by blotting on sterile filter paper. Then, a seed was sown in each 10 cm diameter
pot containing sterilized mixed soil medium (5:3:2 v/v/v: forest soil, compost and sand). Each
pot containing sown seed was subsequently laid out in a green house, and was watered as
required with sterilized tap water.
Three months after emergence of seedlings, stem of a seedling per pot was properly washed
using sterile water, and the surface of the sapwood was wounded with sterile scalpel. Five mm
mycelium plug was aseptically cut with a sterile scalpel from actively growing margins of 10day-old culture of each test fungus, including Botryosphaeria sp.1, Botryosphaeria sp.2,
Fusarium sp.1, Fusarium sp.2 and Lasiodiplodia sp. Then, a mycelium plug cut was
immediately inserted into each wounded seedling with four replications. The inoculated
seedlings were sealed with parafilm to protect them from drying and possible contamination
by other microorganisms, and the parafilm was removed seven days after inoculation
(Pethybridge et al., 2004). Other healthy-looking seedlings grown under the same conditions
were wounded and agar plug cuts without test fungi were inserted into the wounds as controls
to compare the impacts of the inoculum on the seedlings (Dakin et al., 2010). Then, the
inoculated seedlings were sealed in the same manner.
22
3.7. Data Collection
On the 30th day post-sowing (DPS), the germination and emergence of seedlings in each plate
were monitored. Then, data on seedlings emergence were collected by counting the number of
seedlings emerged per treatments. Similarly, seven days after inoculation of seedlings, the
development of disease symptoms was assessed on each individual seedling at a seven-day
interval. The data on disease severity and its progress on the inoculated seedlings over time
were recorded using 0-7 disease scale described by Asad et al. (2010), where: 0= no disease
symptom, 1= 1-10% area of infection over total area observed, 2= 11-20%, 3= 21-30%, 4= 3140%, 5= 41-50%, 6= 51-60% and 7= 61% to onwards regarded as maximum severity.Then,
the disease severity scores were converted into percentage severity index (PSI) for analysis
using the following stated formula (Wheeler, 1969):
PSI =
`
Sum of all numerical ratings X 100
Total number of observations X maximal disease index
3.8. Data Analysis
The recorded data were subjected to analysis of variance (ANOVA) using SAS version 9.2
software package, and treatment mean values were compared using the least significant
difference (LSD) at 5% probability level (Gomez and Gomez, 1984).
3.8.1. Area under the disease progress curve (AUDPC)
Area under the disease progress curve (AUDPC) values were calculated for each treatment
from the raw data to each observation with respect to the replication using the following
formula (Shaner and Finney, 1977; Campbell and Madden 1990).
𝑛−1
AUDPC = ∑ 0.5(𝑥𝑖+1 + 𝑥𝑖 )(𝑡𝑖+1 − 𝑡𝑖 )
𝑖−1
Where,
23
xi= the percentage of disease at the ith assessment
ti = is the time of the ith assessment in days from the first assessment date
n = total number of disease assessments
Disease severity was expressed in percent and time (t) in days, thus, AUDPC values were
expressed in %-days (Campbell and Madden, 1990). Then after, the calculated AUDPC values
were subjected to analysis of variance to compare amount of disease with different treatments.
Means were separated using LSD 5% where ever it was appropriate.
3.8.2. Disease progress rate (DPR)
The disease progress rate was also calculated from the transformed raw data according to the
logistic model linearization ln [(Y/1-Y)], (Van-der Plank, 1963). The data resulting from the
transformation was used to determine the disease progress rate. Then, the calculated values
were used in analysis of variance to compare the disease progress among the treatments.
Means were separated using LSD 5% where ever it was appropriate.
24
4. RESULTS AND DISCUSSION
4.1. Isolation and Identification of Seed-borne Fungi
The result of the present study revealed that a total of 280 isolates of seed-associated fungi
were obtained from the collected seeds samples of Grevillea robusta stands found at three
sites. Based on the cultural features, fungal isolates were provisionally categorized into 15
distinct groups. With the help of morphological characteristics, these distinct groups of fungal
isolates were, in turn categorized into twelve different fungal species belonging to eight
genera. The identified seed-borne fungi were Alternaria sp., Aspergillus sp.1, Aspergillus sp.2,
Botryosphaeria sp.1, Botryosphaeria sp.2, Botryosphaeria sp.3, Curvularia sp., Fusarium
sp.1, Fusarium sp.2, Lasiodiplodia sp., Pestalotiopsis sp. and Phoma sp. Most of these fungal
species have been recorded from seeds of many different trees in different countries as
endophytes, saprobes or pathogens causing damages to seeds and seedlings (Mamatha et al.,
2000; Dhingra et al., 2003; Alemu et al., 2004).
In a previous study, Njuguna et al. (2011) also isolated about 40 different fungal species
associated with different diseased and healthy parts (stem, shoot, leaves and branches) of G.
robusta, of which 32 species were identified as new occurrence on G. robusta in Kenya. This
implied that G. robusta played a host to a wide range of fungal species, some of which are
known to cause diseases in other hosts that are of economic importance to agriculture
(Njuguna, 2003; Njuguna et al., 2011). In the present study, the results on the fungal isolates
(a total of 280) obtained from the collected seed samples of three localities showed that the
most common fungal species associated with the seeds were Botryosphaeria sp.1,
Botryosphaeria sp.2, Fusarium sp.1, Fusarium sp.2 and Lasiodiplodia sp. Of these seed-borne
fungi, Botryosphaeria sp.1 with (18.93%) was the most abundant fungus, followed by
Lasiodiplodia sp. with (16.07%), Botryosphaeria sp.2 with (14.28%), Fusarium sp.1 with
(13.21%) and Fusarium sp.2 with tallies (11.07%) (Table 1). In earlier studies it was also
indicated that some Botryosphaeria spp. were frequently isolated from seeds of P. falcatus and
P. africana (Abdella, 2004). Alemu et al. (2004) also isolated Botryosphaeria spp. from
Eucalyptus plantations in Ethiopia.
25
The distribution of fungal isolates among three localities showed that seed samples collected
from Wondo Genet site harbored the highest number (119) of fungal isolates and closely
followed by Shashemene site (101), whereas the least number (60) of fungal isolates was
recovered from seeds collected from Gambo site (Appendix Table 1). Within each locality,
varying numbers of fungal isolates were recovered from seeds sampled from symptomatic and
asymptomatic G. robusta stands. In this connection, more fungal isolates were detected from
seed samples collected from symptomatic (diseased-looking) than asymptomatic (healthylooking) G. robusta stands at three sites (Gambo, Shashemene and WGCF-NR). This result
was in agreement with the findings of Njuguna et al. (2011) which indicated that the leaves
and branches sampled from diseased standing G. robusta trees harbored more species of fungi
than that of healthy trees in Kenya. In the current study, a total of (23.57%), (21.42%) and
(11.78%) numbers of fungal isolates were recorded from seed samples collected from
diseased-looking G. robusta stands at WGCF-NR, Shashemene (Sole) and Gambo localities,
respectively (Table 1). However, seed samples collected from healthy-looking standing trees
at WGCF-NR, Shashemene and Gambo sites harbored (18.93%), (14.63%) and (9.63%)
fungal isolates, respectively.
The result of the current study further revealed that at WGCF-NR site, Lasiodiplodia sp. was
the most frequently isolated fungal species with (8.93%), followed by Botryosphaeria sp.1
with (8.57%) and Fusarium sp.2 with (7.14%), while the least abundant fungus was
Aspergillus sp.2 with (0.72 %) isolates (Table 1). However, the most abundantly isolated
fungus from seed samples collected from Shashemene (Sole) site was Botryosphaeria sp.1
with (6.79%), followed by Alternaria sp. with (5.71%) and Botryosphaeria sp.2 with (5.00%)
isolates, whereas Pestalotiopsis sp. with (1.07%) was the least frequently isolated fungus.
Similarly, the most predominantly isolated fungal species at Gambo site was Fusarium sp.1
with (4.28%), followed by Botryosphaeria sp.1 with (3.57%) and Botryospheria sp.2 with
(3.21%) isolates, while the least occurrence percentage of fungus was Fusarium sp.2 with
(1.07%) isolates (Table 1).
The result of the present study showed that Botryosphaeria sp.1 was the most frequently
detected fungal species from seed samples collected from diseased-looking standing trees at
26
WGCF-NR and Shashemene sites (Table 1). This fungal species was also identified
predominantly from seed samples collected from healthy-looking standing G. robusta trees at
Shashemene (Sole) site. However, Fusarium sp.1 was the first ranked isolated fungus from
seed samples of diseased-looking stands of G. robusta at Gambo site. In the current study, it
was revealed that the most abundantly isolated fungus from seed samples of healthy-looking
standing trees at WGCF-NR was Lasiodiplodia sp., whereas Botryosphaeria sp.2 was isolated
as predominant fungi from seed samples collected from healthy-looking trees of G. robusta at
Gambo site, with the least identified fungus being Phoma sp. (Table 1).
The present study also showed that the least detected fungal species from seed samples of
diseased-looking and healthy-looking of G. robusta stands at WGCF-NR was Aspergillus sp.2.
In contrast, Botryosphaeria sp.3 and Pestalotiopsis sp. were the least isolated fungal species
with equal proportion from seed samples collected from diseased-looking stands of G. robusta
at Shashemene, whereas fungal species, including Pestalotiopsis sp. and Phoma sp. were
found as the least frequently occurred fungi with equal proportion from healthy-looking stands
of G. robusta at Shashemene site (Table 1).
The result of the present study indicated that there was variation among three study sites in the
fungal abundance and species diversity they harbored. These differences in isolate frequencies
among three localities seemed to be due to some degree variation in environmental conditions
and geographic locations. Ecological factors also might have played some roles in the
disparity in the type and number of fungal species from those sites (Collado et al., 1999). A
previous study by (Taylor et al., 2000) indicated that differences in climate and the degree of
disturbance of habitats in which the host grows could influence the diversity of microfungi.
The result of the current study further showed that higher frequency of infection by seed-borne
fungi was observed on seed samples collected from symptomatic than asymptomatic G.
robusta stands at each locality. With this respect, seed samples collected from diseasedlooking mother trees at WGCF-NR site were found with the highest frequency of infection
(56%), and closely followed by Shashemene site (50%), whereas seed samples collected from
diseased-looking mother trees at Gambo site showed the least frequency of infection (29%)
27
(Appendix Table 2). Similarly, seed samples collected from healthy-looking G. robusta stands
at WGCF-NR site showed the highest frequency of infection (43%) (Appendix Table 2). Seed
samples collected from healthy-looking G. robusta mother tree at Shashemene and Gambo
sites were also found with the frequency of infection (36%) and (25%), respectively
(Appendix Table 2).
Table 1. Relative frequency (RF%) of fungal isolates obtained from seed samples collected
from symptomatic and asymptomatic G. robusta stands at Gambo, Shashemene and
WGCF-NR sites
No
Fungal species
1 Alternaria sp.
2 Aspergillus sp.1
3 Aspergillus sp.2
4 Botryosphaeria sp.1
5 Botryosphaeria sp.2
6 Botryosphaeria sp.3
7 Curvularia sp.
8 Fusarium sp.1
9 Fusarium sp.2
10 Lasiodiplodia sp.
11 Pestalotiopsis sp.
12 Phoma sp.
Total RF (%)
WGCF-NR
Shashemene
Gambo
Symp Asym
p
Symp
Asym
p
Symp
Asym
p
2.14
0.36
5.00
3.57
2.86
3.93
4.64
1.07
23.57
3.21
1.07
3.93
2.86
0.71
1.43
2.14
1.79
2.50
0.71
1.07
21.42
2.50
0.71
2.86
2.14
0.71
0.71
1.43
1.07
1.78
0.36
0.36
14.63
1.43
2.14
1.07
1.07
2.50
1.07
1.43
1.07
11.78
1.07
1.43
2.14
1.07
1.78
1.43
0.71
9.63
1.43
0.36
3.57
2.50
2.50
3.21
4.29
1.07
18.93
Total
RF
(%)
5.71
6.07
2.50
18.93
14.28
3.56
2.14
13.21
11.07
16.07
1.07
5.35
99.96
Keys:
- indicates no fungal isolates obtained
Symp - Seed samples collected from symptomatic mother trees
Asymp - Seed samples collected from asymptomatic mother trees
4.2. Characterization of Fungal Isolates from G. robusta Seeds
Based on cultural and morphological studies, the most commonly obtained Botryosphaeria
isolates considered in the present study could be grouped into two distinct species as
Botryosphaeria sp.1 and Botryosphaeria sp.2. The colony color of Botryosphaeria sp.1 turned
from white through greenish white to blackish brown on the upper surface some 10 days after
incubation, fluffy with slightly raised centre with aggregating vertical hyphae (Appendix
28
Figure 1A). The reverse side of this fungal species became faint yellow in color, which
persisted for some time and finally turned bluish-black. This fungal species was also
characterized morphologically by hyaline and septate conidia with ellipsoid shape.
Botryosphaeria spp. have a wide host range and geographical distribution (Alves et al., 2004;
Slippers et al., 2004a). They are known to infect many woody fruits, trees, and herbaceous
plants. These fungi are largely considered drought-stress opportunistic pathogens living as
saprophytes or endophytes most of the time (Swart et al., 2000; Mila et al., 2005; Slippers et
al., 2007). Difficulties distinguishing Botryosphaeria species are common because the group
of fungal organisms has many taxonomic and nomenclatural ambiguities (Slippers et al.,
2004a). Species identification has been based on characteristics such as colony and conidial
morphology (Denman et al., 2000; Smith et al., 2000; Slippers et al., 2004a). Differentiation
based on conidial characteristics is difficult because characters vary with age and type of
media (Phillips et al., 2006).
On the other hand, the cultures on PDA of Botryospheria sp.2 isolates showed on the upper
surface, initially whitish appearance with abundant aerial mycelium, gradually becoming grey
to dark grey (Appendix Figure 2A), while the reverse side of the culture was at first whitish
and finally turned black. Conidial morphology of Botryosphaeria sp.2 was characterized by
hyaline and narrowly or irregularly fusiform shape septate conidia (Appendix Figure 2B).
Fusarium spp. have been widely distributed on plants and in the soil (Lori et al., 2004). The
fungus Fusarium comprises cosmopolitan common pathogenic species to forest trees and
agricultural crops (Satou et al., 2001). Morphological characteristics are fundamental for the
identification of Fusarium spp. In the present study, according to macroscopic and
microscopic feature, Fusarium species those were identified from seeds of G. robusta were
grouped into Fusarium sp.1 and Fusarium sp.2.
Fusarium sp.1 showed white but usually with light purple mycelium color (Appendix Figure
3A). This fungal species was also characterized morphologically by clear/non-pigmented
hyphae with septate (showing divisions or walls within the hyphae). Macro- and micro-conidia
29
were formed frequently. Macro-conidia are produced from phialides on unbranched or
branched conidiophores. Macro-conidia are fusiform in shape, and have a slightly pointed
apical tip. The fusiform macro-conidia are also somewhat curved making it appear sickleshaped. They usually contain three to five divisions within the macro-conidium (Appendix
Figure 3B). In contrast, micro-conidia are non-septate, ellipsoidal and are straight or slightly
curved in shape as they are abundantly produced from the tip of these phialides.
On the other hand, Fusarium sp.2 was described both by its colony and morphological
characteristics. It was a woolly to cottony with cream to white aerial mycelium and a cream
reverse culture (Appendix Figure 4A). It was also characterized morphologically by septate
and hyaline hyphae, abundant ovoid micro-conidia and slightly curved macro-conidia with
zero to one septum and five to six septa (Appendix Figure 4B), respectively.
The mycelium color of Lasiodiplodia sp. appeared at first white and then turned to black five
days after incubation (Appendix Figure 5A). This fungal species was also characterized
morphologically by hyaline, aseptate and ovoid conidia in immature and dark brown septate
with ovoid conidia in mature state (Appendix Figure 5B).
4.3. Pathogenicity Test on Seeds of G. robusta
In the present study, the results from the in vitro seed inoculation tests showed that the
selected test fungal species, including Botryosphaeria sp.1, Botryosphaeria sp.2, Fusarium
sp.1, Fusarium sp.2 and Lasiodiplodia sp. exhibited pathogenic effects on seedlings
emergence of G. robusta. To this effect, 30 days after sowing (DAS), seeds inoculated with
Fusarium sp.1 were found with the lowest seedling emergence percentage (47.5%) compared
to seeds treated with other test fungi (Table 2). The remained seeds lots were found rotten.
This implied that this fungal species was found to be more pathogenic and caused significant
reduction in seedling emergence as compared to other fungal treatments.
According to reports of some authors, evaluations of seed inoculation tests indicated that
Fusarium spp. such as F. oxysporum and F. solani are major seed-borne pathogens that cause
30
mortality to seeds and newly germinated seedlings of forest trees (Dick and Dobbie, 2002;
Khalid et al., 2002; Lori and Salerno, 2002; Shailendra et al., 2004; Pathan et al., 2007). The
findings from in vitro seeds inoculation test done by Abdella (2004) also showed that
Fusarium spp., particularly F. oxysporum was strongly pathogenic to seeds and caused severe
loss of seed germination.
The result of the current study further revealed that seeds inoculated with Lasiodiplodia sp.
had lower seedlings emergence percentage with (55%), followed by Fusarium sp.2 with
(60%). There was no significant difference (p > 0.05) observed between these two fungal
species (Lasiodiplodia sp. and Fusarium sp.2) on seedlings emergence of G. robusta.
Lasiodiplodia sp. had also not showed a significant difference (p > 0.05) from the seeds
inoculated with Fusarium sp.1 on seedlings emergence of G. robusta (Table 2). In earlier
study, it was also reported that Lasiodiplodia spp. for instance, L. theobromae in seeds of
forest trees has shown discoloration and reduction in germination (Joshi et al., 2005).
Botryosphaeria sp.2 was found with seedlings emergence percentage (77.5%) (Table 2). On
the other hand, seeds treated with Botryosphaeria sp.1 showed the highest percentage seedling
emergence (90%) as compared to seeds inoculated with the other fungal species, while seeds
soaked in sterile water devoid of mycelium suspension of the test fungus (control treatment)
showed 100% seedlings emergence, and the control showed a significant difference from the
seeds inoculated with all the test fungi except those inoculated with Botryosphaeria sp.1
(Table 2). The result of the current study is in agreement with the reports of Abdella et al.
(2005) that Botryosphaeria spp., particularly B. parva tested on seeds of Podocarpus falcatus
had shown a little effect on seed germination.
Based on the results from in vitro seed inoculation tests, Abdella (2004) grouped fungi into
five categories, namely I) isolates that were pathogenic only to seeds and had no obvious
impacts on the germlings; II) isolates that were pathogenic only to the germlings; III) isolates
that were pathogenic both to seeds and the emerging germlings; IV) isolates that were more or
less harmless both to seeds and seedlings; and V) isolates that were germination promoters.
The result of both seed and seedling inoculation test in the current study indicated that all
31
fungal treatments selected for pathogenicity test were pathogenic to both seeds and seedlings.
Thus, the results of seeds and seedlings inoculation tests in this study matched with the third
pathogenic level category grouped by Abdella (2004) that fungal isolates were pathogenic both
to seeds and emerging germlings of P. falcatus.
Table 2. Effect of predominately isolated test fungi on seedlings emergence of G. robusta
tree in the laboratory
No Treatments
(Fungal species)
1
Botryosphaeria sp.1
2
Botryosphaeria sp.2
3
Fusarium sp.1
4
Fusarium sp.2
5
Lasiodiplodia sp.
6
Untreated (control)
CV (%)
LSD (0.05)
Emergence (%)
90.00d
77.50c
47.50a
60.00b
55.00ab
100.00d
11.29
12.20
Mean values with the same letter within a column are not significantly different from each other at p  0.05; CV
= Coefficient of variation; LSD = Least significant difference
4.4. Pathogenicity Test on Seedlings of G. robusta
Based on the results from in vitro seedlings inoculation tests, all fungal species selected for
pathogenicity test had variable effects on the tested seedlings. All the inoculated seedlings
with different treatments showed disease symptoms; however, no disease symptom was
detected on seedlings treated with agar plug cuts without test fungi (control). In this regard,
seedlings inoculated with Fusarium sp.1, Lasiodiplodia sp., Fusarium sp.2 and
Botryosphaeria sp.2 showed early development of disease symptoms seven days after
inoculation. However, seedlings treated with Botryosphaeria sp.1 were delayed almost by 14
days to show disease symptoms as compared to seedlings inoculated with other treatments
(Table 3). Earlier studies also indicated that Botryosphaeria spp. can live in plant tissues as
endophytes without exhibiting clear symptoms (the process known as latent infection) (Smith
et al., 1996; Pavlic et al., 2007). Many authors have also shown that diseases caused by the
Botryosphaeria fungi are usually linked to environmental stress factors acting on the host
32
species (Slippers et al., 2007; Úrbez-Torres and Gubler, 2009). The finding of the current
study is also consistent with the findings of other researchers (Njuguna, 2003; Njuguna et al.,
2011) that the development of disease symptoms (canker and dieback) was slower on
seedlings of G. robusta inoculated with Botryosphaeria sp. in Kenya.
In the present study, the result of pathogenicity test with the different test fungi showed varied
disease symptoms seven days post-inoculation (Appendix Figure 6). For instance, seedlings
inoculated with Fusarium sp.1 exhibited disease symptoms, such as formation of exudates at
the inoculation point, yellowing and wilting of leaves (Appendix Figure 6C). This result is in
agreement with the finding of Anderson et al. (2002) which reported that seedlings of Acacia
koa inoculated with Fusarium spp., particularly F. oxysporium, showed similar disease
symptoms with the symptoms observed in the current study. However, seedlings inoculated
with Fusarium sp.2 showed symptoms that included stunted growth and chlorosis of leaves as
compared to the control seedlings. On the other hand, seedlings treated with Lasiodiplodia sp.
showed small necrotic lesions at the inoculation point, followed by typical shoots dieback
symptoms (Appendix Figure 6E).
Many authors have also reported in their findings that Lasiodiplodia spp., particularly
Lasiodiplodia theobromae have been found as causal agents for the canker and dieback
diseases in most trees species, including G. robusta (Roux et al., 2001; Denman et al., 2003;
Toljander et al., 2007). According to Njuguna et al. (2011), pathogenicity tests further showed
that L. theobromae was highly pathogenic on young seedlings of G. robusta in Kenya. On the
contrary, disease symptoms observed on seedlings inoculated with Botryosphaeria sp.1 were
small necrotic spots on the leaves and dieback of shoots. This observation is in agreement with
the finding of Toljander et al. (2007) and Njuguna et al. (2011) which reported that
Botryosphaeria spp., like B. parva were pathogenic, and produced top dying of shoots on G.
robusta seedlings in Kenya. Conversely, top dying of shoot and leaf blight were the disease
symptoms observed on G. robusta seedlings inoculated with Botryosphaeria sp.2 in the
current study (Appendix Figure 6B).
33
The result of pathogenicity test in the current study revealed that the test fungi, such as
Botryosphaeria sp.1, Botryosphaeria sp.2 and Lasiodiplodia sp. caused more or less similar
typical shoot dieback symptoms on inoculated seedlings. In a previous study, Njuguna et al.
(2011) reported that the disease caused by Botryosphaeria species seemed to develop from
actively growing tissues in young shoots, and progressed into the branches and stems. In the
current study, the same condition was also observed on the seedlings inoculated with
Botryosphaeria species that dying back of the tissue started from actively growing shoots.
Disease progress from young infected shoot tissues (that is the most recent growth ring) could
also develop further into the stem and to older growth rings and, in this way, could have
provided links between infected young tissues and stem cankers (Toljander et al., 2007; Úrbez
et al., 2008; Njuguna et al., 2011).
In the current study, the results of the disease severity score on the seedlings inoculated with
different test fungi and recorded for 49 days at every seven days intervals showed that the
degree of damage (severity) increased progressively as the duration after inoculation
increased. In line with this, the highest (6.82%) mean disease severity score was observed on
seedlings inoculated with Fusarium sp.1, followed by Lasiodiplodia sp. with 3.65%, Fusarium
sp.2 with 3.25% and Botryosphaeria sp.2 with 2.37% severity at 7th day after inoculation
(Table 3). There was significant difference (p ≤ 0.05) among Fusarium sp.1, Lasiodiplodia sp.,
Fusarium sp.2 and Botryosphaeria sp.2 on the 7th day after inoculation, whereas no disease
severity score was recorded on seedlings inoculated with Botryosphaeria sp.1 and the control
at 7th day of disease assessment, and there was no significant difference (p > 0.05) observed
between these two treatments.
The result of the current study also showed that inoculation with all test fungal species
increased disease severity with increase in time after inoculation. It was indicated that fungal
treatments showed significant (p ≤ 0.05) difference from each other in disease severity at 14th
day of disease severity evaluation. To this effect, seedlings inoculated with Fusarium sp.1
showed the highest (12.325%) mean disease severity score at 14th day after inoculation,
followed by Lasiodiplodia sp. with (7.625%). G. robusta seedlings treated with Fusarium sp
2, Botryosphaeria sp.2 and Botryosphaeria sp.1 also attained mean disease severity scores of
34
6.00, 4.75 and 2.20%, respectively. In contrast, seedlings treated with agar plug cut without
test fungi (control) had zero (0.00%) disease severity score at all disease assessment periods
recorded for 49 days at seven-day interval.
The finding of the present study further revealed that at every seven-day interval of disease
severity assessment, there was an increase in disease severity score observed on seedlings of
G. robusta treated with different test fungi included in this study. To this effect, disease
assessment undertaken at 21st and 28th days after inoculation showed that seedlings inoculated
with Fusarium sp.1 had the highest disease severity score, followed by the effect of
inoculation with Lasiodiplodia sp., Fusarium sp.2 and Botryosphaeria sp.2 (Table 3). In
contrast to this, seedlings treated with Botryosphaeria sp.1 showed the lowest disease severity
recorded at the respective period of disease assessment (Table 3). There was significant (p ≤
0.05) difference observed among test fungi at 21st and 28th days of disease evaluation.
On the other hand, there was no significant (p > 0.05) difference exhibited between
Botryosphaeria sp.2 and Fusarium sp.2 in disease severity score at 35th day disease
assessment. At 42nd and 49th day of disease evaluation, all test fungi also showed higher
disease severity than the disease severity recorded at 7th to 35th day disease assessment. The
disease severities due to inoculation of seedlings with Fusarium sp.1 were 36.62 and 39.50%
at 42nd and 49th day of disease evaluation, respectively (Table 3). The mean disease severities
on G. robusta seedlings inoculated with Lasiodiplodia sp., Botryosphaeria sp.2, Fusarium
sp.2 and Botryosphaeria sp.1 were 28.62, 25.17, 22.57 and 16.75%, respectively, at 42nd day
of disease evaluation (Table 3). Furthermore, mean disease severities of 36.23, 31.40, 25.25
and 22.30% were recorded on G. robusta seedlings treated with Lasiodiplodia sp.,
Botryosphaeria sp.2, Fusarium sp.2 and Botryosphaeria sp.1, respectively, at the final day of
disease assessment (49th day after inoculation). There was significant (p ≤ 0.05) difference
observed among the test fungi at the final day of disease assessment.
35
Table 3. Mean disease severity (%) recorded on G. robusta seedlings after inoculation with
different fungal species
Treatments
(Fungal species)
Botryospheria sp.1
Botryospheria sp.2
Fusarium sp.1
Fusarium sp.2
Lasiodiplodia sp.
Untreated (control)
CV (%)
LSD (0.05)
7 DAI
0.00e
2.37d
6.82a
3.25c
3.65b
0.00e
6.95
0.28
14 DAI
21 DAI
28 DAI
35 DAI
42 DAI
49 DAI
2.20e
4.75d
12.32a
6.00c
7.62b
0.00f
6.32
0.52
5.00e
9.40d
19.42a
10.12c
11.85b
0.00f
5.16
0.72
8.30e
14.07d
27.17a
15.65c
16.67b
0.00f
4.53
0.93
12.10d
19.35c
33.27a
19.50c
22.25b
0.00e
3.79
1.02
16.75e
25.17c
36.62a
22.57d
28.62b
0.00f
2.63
0.86
22.30e
31.40c
39.50a
25.25d
36.22b
0.00f
2.36
0.92
Mean values with the same letter within a column are not significantly different from each other at p  0.05; CV
= Coefficient of variation; LSD = Least significant difference
4.5. Area under Disease Progress Curve (AUDPC)
Area under disease progress curve exhibited highly significant difference among test fungal
species in inciting infection on G. robusta seedlings. In this regard, the highest (613%-days)
AUDPC value was recorded on seedlings inoculated with Fusarium sp.1, followed by AUDPC
value due to Lasiodiplodia sp. (444%-days), Botryosphaeria sp.2 (372%-days) and Fusarium
sp.2 (358%-days) as compared to the control (Table 4), whereas G. robusta seedlings
inoculated with Botryosphaeria sp.1 were attained the lowest (233%-days) AUDPC value
(Table 4). On the contrary, seedlings treated with agar plug without test fungi (control) had
zero (0.00%-day) AUDPC value in comparison with other treatments. According to Campbell
and Madden (1990), the amount of disease developed with the highest and lowest AUDPC
values corresponded to susceptible and resistant varieties, respectively. Furthermore, the
amount of disease with the highest AUDPC value revealed that the disease-inciting agents
(pathogens) were relatively more pathogenic. Pursuant to this observation, the present study
revealed that Fusarium sp.1 was the most pathogenic fungal species and this was ascribed to
its highest AUDPC value (613%-days) recorded on G. robusta seedlings as compared to the
values due to other fungal species tested. Similarly, Lasiodiplodia sp. was found to be the
second highly pathogenic fungal species causing infection on G. robusta seedlings, followed
by Botryosphaeria sp.2 and Fusarium sp.2 obtained from their respective calculated AUDPC
36
values (Table 4). However, G. robusta seedlings inoculated with Botryosphaeria sp.1 showed
the lowest (233%-days) calculated AUDPC value, and it was considered to be relatively the
least pathogenic fungal species among the test fungi in the current study.
Table 4. Mean AUDPC values calculated for each fungal species inoculated on G. robusta
seedlings
No
1
2
3
4
5
6
CV (%)
LSD (0.05)
Treatments
(Fungal species)
Botryosphaeria sp.1
Botryosphaeria sp.2
Fusarium sp.1
Fusarium sp.2
Lasiodiplodia sp.
Untreated (control)
AUDPC
(%-days)
233e
372c
613a
358d
444b
0.00f
0.66
3.37
Treatment mean values with the same letter within a column are not significantly different from each other at p 
0.05; CV = Coefficient of variation; LSD = Least significant difference; AUDPC = Area under disease progress
curve
4.6. Disease Progress Rate (DPR)
Logistic model is considered to be the most appropriate for temporal analysis of disease
development because of wide application and goodness of fit for describing many epidemics
(Campbell and Madden, 1990). The result of the present study indicated that fungal
treatments, including Botryosphaeria sp.1, Botryosphaeria sp.2, Fusarium sp.1, Fusarium sp.2
and Lasiodiplodia sp. caused different infection rates. To this effect, the recorded infection
rates incited due to inoculation with Botryosphaeria sp.1, Botryosphaeria sp.2, Fusarium sp.1,
Fusarium sp.2 and Lasiodiplodia sp. on G. robusta seedling were 0.00321, 0.00364, 0.00962,
0.00431 and 0.00638 units-day-1, respectively, at initial day of disease assessment (7th day
after inoculation) (Table 5). Out of these fungal treatments used in the present study, Fusarium
sp.1 caused the highest (0.00962 units-day-1) disease progress rate, followed by Lasiodiplodia
sp. with 0.00638 units-day-1 as compared to inoculation with other fungal species. The result
of disease assessment taken on G. robusta seedlings at different periods showed that disease
infection due to inoculation with Fusarium sp.1 progressed at the fastest rate in contrast to the
37
rate of infection incited by other test fungi on the same host seedlings, whereas fungal
treatments, like inoculation with Botryosphaeria sp.1 showed the lowest (0.00321 units-day-1)
infection rate. Conversely, there was no recorded disease progress rate over time in the case of
the control at the respective disease assessment periods. Also, there was no significant (p >
0.05) difference in infection rates observed among fungal treatments, such as Botryosphaeria
sp.1, Botryosphaeria sp.2 and Fusarium sp.2 at the initial disease assessment.
On the other hand, disease progress rates estimated at the final day of disease assessment (49 th
day after inoculation) indicated that fungal treatments, including inoculation with
Lasiodiplodia sp., Botryosphaeria sp.2 and Botryosphaeria sp.1, increased infection at a rates
of 0.01214, 0.01025 and 0.00665 units-day-1, respectively (Table 5). However, retarded
infection rates were estimated at final day of disease assessment as compared to disease
assessment recorded at 7th to 42nd days on G. robusta seedlings inoculated with Fusarium sp.1
and Fusarium sp.2 (Appendix Table 3). The analysis of variance of the final disease
assessment also revealed that there was significant difference in disease progress rates among
all fungal treatments. In contrast, the lowest (0.00665 units-day-1) infection rate estimated at
final day of disease assessment was observed on seedlings inoculated with Botryosphaeria
sp.1. On the other hand, zero (0.00) units-day-1 infection rate was recorded at the respective
day of disease assessment on seedlings treated with agar plug cut devoid of fungal mycelium
(control).
Table 5. Initial and final disease progress rates (DPR) estimated on G. robusta seedlings
inoculated with different fungal species
No
1
2
3
4
5
6
Treatments
(Fungal species)
Botryosphaeria sp.1
Botryosphaeria sp.2
Fusarium sp.1
Fusarium sp.2
Lasiodiplodia sp.
Untreated (control)
CV (%)
LSD (0.05)
Initial Rate
(units day-1)
0.00321c
0.00364c
0.00962a
0.00431c
0.00638b
0.00f
18
0.001
Final rate
(units day-1)
0.00665e
0.01025c
0.01465a
0.00764d
0.01214b
0.00f
1.88
0.002
38
5. SUMMARY AND CONCLUSION
Silver oak (Grevillea robusta A. Cunn ex R.Br.) is an important multipurpose tree species that
provides various goods and services, including timber production, construction material,
electric power transmission pole, fuel wood, shade, wind break, fodder, soil fertility
improvement and aesthetic value. However, this tree species is being severely affected by stem
canker and dieback disease in several African countries, including Ethiopia. The current study
was directed to assess the impact of fungi associated with G. robusta seeds on seed
germination and seedlings growth.
Seed samples were collected from G. robusta standing trees found at three localities, namely
Gambo, Shashemene and Wondo Genet College of Forestry and Natural Resources (WGCFNR). Seed-borne fungi associated with G. robusta seeds were isolated separately from seed
samples collected from healthy and diseased-looking trees of each locality. The spore
suspension and mycelium plug cut of the most commonly isolated fungal species were used
for seed and seedling inoculation test, respectively. The layout of the trial was a completely
randomized design (CRD) with four replications. The data on seedlings emergence were
collected by counting the number of seedlings emerged per treatments. Similarly, disease
severity and its progress on the inoculated G. robusta seedlings over time were recorded using
0-7 disease scale, and subjected to ANOVA after converted into percentage severity index
(PSI).
Grevillea robusta seed-associated fungal isolates were identified on the basis of
morphological characteristics and categorized into twelve different fungal species, including
Alternaria sp., Aspergillus sp.1, Aspergillus sp.2, Botryosphaeria sp.1, Botryosphaeria sp.2,
Botryosphaeria sp.3, Curvularia sp., Fusarium sp.1, Fusarium sp.2, Lasiodiplodia sp.,
Pestalotiopsis sp. and Phoma sp. The most commonly isolated fungal species from the three
sites were Botryosphaeria sp.1, Botryosphaeria sp.2, Fusarium sp.1, Fusarium sp.2 and
Lasiodiplodia sp. Further, this study revealed that Lasiodiplodia sp. was the most frequently
isolated fungal species, while the least frequent fungus was Aspergillus sp.2 that occurred at
WGCF-NR site. However, the most abundant fungal isolates at Shashemene (Sole) and
39
Gambo sites were Botryosphaeria sp.1 and Fusarium sp.1, respectively, whereas
Pestalotiopsis sp. and Fusarium sp.2 were the least identified fungal isolates at Shashemene
and Gambo sites, respectively.
The most commonly and frequently identified fungal species, including Botryosphaeria sp.1,
Botryosphaeria sp.2, Fusarium sp.1, Fusarium sp.2 and Lasiodiplodia sp. were selected and
tested for their specific effects against G. robusta seed germination, and seedling growth. It
was found out that the fungal species had variable effects on both seed germination and
seedling growth. In vitro seed inoculation tests showed that seeds treated with Fusarium sp.1
exhibited the lowest seedling emergence percentage (47.50%), followed by Lasiodiplodia sp.
(55.00%) as compared to the control, in which all seedlings fully emerged (100%). Hence,
some fungal species showed significant and negative effect on seedling emergence. However,
there was no significant (p > 0.05) difference observed between Lasiodiplodia sp. and
Fusarium sp.1 on G. robusta seedling emergence. Lasiodiplodia sp. had also not showed a
significant difference (p > 0.05) from the seeds inoculated with Fusarium sp.1 on seedlings
emergence of G. robusta. On the contrary, seeds treated with Botryosphaeria sp.1 had the
highest (90.00%) seedling emergence percentage compared to inoculation with other fungal
species.
In vitro seedling inoculation test indicated that all fungal species selected for pathogenicity test
showed disease symptoms on the tested G. robusta seedlings. However, no disease symptom
was observed on seedlings inoculated with agar plug without any test fungus (control).
Disease symptoms, such as formation of exudates at the inoculation point, yellowing and
wilting of leaves were observed on seedlings inoculated with Fusarium sp.1. There was also
chlorosis disease symptom observed on seedling leaves of the respective host inoculated with
Fusarium sp.2. The test fungi, such as Botryosphaeria sp.1, Botryosphaeria sp.2, and
Lasiodiplodia sp. caused more or less similar typical shoot dieback symptoms on the
inoculated G. robusta seedlings.
Disease severity scores recorded 49 days after inoculation on seedlings inoculated with
different test fungi revealed that the degree of damage (disease severity) increased
40
progressively with increase in time after inoculation. At every seven-day interval of disease
assessment, the highest mean disease severity score was recorded on seedlings inoculated with
Fusarium sp.1, followed by severity due to inoculation with Lasiodiplodia sp., whereas the
least disease severity score was recorded on seedlings inoculated with Botryosphaeria sp.1 as
compared to seedlings treated with other test fungi. On the other hand, seedlings inoculated
with agar plug devoid of mycelium (control) showed zero disease severity score at every
seven-day interval of disease assessment.
Area under disease progress curve values exhibited significant difference among test fungal
species in inciting infection on G. robusta seedlings. To this effect, the highest (613%-days)
and lowest (233%-days) AUDPC values were recorded on seedlings inoculated with Fusarium
sp.1 and Botryosphaeria sp.1, respectively. The highest (0.01465 units day-1) disease progress
rate was also observed on seedlings inoculated with Fusarium sp.1 at the final day of disease
assessment (49th day after inoculation). In contrast to this, the lowest (0.00665 units day-1)
disease progress rate was recorded on G. robusta seedlings treated with Botryosphaeria sp.1 at
the respective duration of disease evaluation. Generally, a combination of both disease
progress rate and AUDPC values showed that Fusarium sp.1 was the most pathogenic fungal
species, whereas Botryosphaeria sp.1 was the least pathogenic of all the other tested fungal
species.
The results of the current study showed that a diverse group of fungal species were associated
with G. robusta seeds. To find out the source of inoculum for the occurrence of stem canker
and dieback of G. robusta, the most frequently isolated seed-borne fungal species were tested
for their specific effects against G. robusta seed germination and seedling growth. The
selected and tested fungal species had pathogenic effects on both seedling emergence, and
seedling growth. Therefore, the finding of this study concluded that seed is considered to be
the sources of inoculum for the incidence of dieback and stem canker disease of G. robusta
trees.
41
5.1. Recommendations
Both in vitro seed and seedling inoculation tests were only conducted under laboratory and
greenhouse conditions, respectively. As a result, further investigations are required to find out
how these fungal species behave under nursery or field conditions.
Chemical (fungicide) efficacy of these species of fungi should also be tested to develop
management strategy of stem canker and dieback disease causing pathogenic fungi.
It is also commendable to use disease/pathogen-free seeds and planting materials.
42
6. REFERENCES
Abdella Gure. 2004. Seed-borne fungi of the afro-montane tree species Podocarpus falcatus
and Prunus africana in Ethiopia. Doctoral Dissertation, Swedish University of
Agricultural Sciences, Uppsala. Acta Universitatis Agriculturae Sueciae, Silvestria 334.
Abdella Gure, Slippers, B. and Stenlid, J. 2005. Seed-borne Botryosphaeria spp. from native
Prunus and Podocarpus trees in Ethiopia with a description of the anamorph Diplodia
rosulata sp. Mycological Research, 109: 1005-1014.
Agarwal, V.K. and Sinclair, J.B. 1997. Principles of Seed Pathology, 2nd edition. CRC Press,
Inc. Boca Raton, FL.
Agrios, G.N. 2005. Plant Pathology, 5th edition. Elsevier Academic Press, Amsterdam.
Akycampong, E., Hitimana, E., Torquebiaus, E. and Munyemana, P.C. 1999. Multistrata
agroforestry with beans, bananas and Grevillea robusta in the highlands of Burundi.
Environmental Agriculture, 35:357-369.
Albrecht, J. ed. 1993. Tree seed hand book of Kenya. GTZ Forestry Seed Center Muguga,
Nairobi, Kenya.
Alemu Gezahegne, Roux, J. and Wingfield, M.J. 2003. Diseases of exotic plantation
Eucalyptus and Pinus species in Ethiopia. South African Journal of Science, 99: 29-33.
Alemu Gezahegne, Roux, J., Slippers, B. and Wingfield, M.J. 2004. Identification of the
causal agent of Botryosphaeria stem canker in Ethiopia Eucalyptus plantations. South
African Journal of Botany, 70: 241-248.
Alemu Gezahegne, Roux, J, Hunter, G.C. and Wingfield, M.J. 2006. Mycospharella Species
Associated with leaf disease of Eucalyptus globulus in Ethiopia. Forest Pathology, 36:
253-263.
Alves, A., Correia, A., Luque, J. and Phillips, A. 2004. Botryosphaeria corticola, sp. nov. on
Quercus species, with notes and description of Botryosphaeria stevensii and its anamorph,
Diplodia mutila. Mycologia, 96: 598-613.
Anderson, R.C., Gardner, D.E., Daehler, C.C. and Meinzer, F.C. 2002. Dieback of Acacia koa
in Hawaii: Ecological and pathological characteristics of affected stands. Forest Ecology
and Management, 162: 273-286.
Asad, M., Shafqat S., Naeem I., Muhammad T. M. and Munawer R. K. 2010. Methodology
for the Evaluation of Symptoms Severity of Mango Sudden Death Syndrome in Pakistan.
Pakistan Journal of Botany, 42(2): 1289-1299.
43
Barnett, H.L. and Hunter, B.B. 1998. Illustrated Genera of Imperfect Fungi, 4th edition. APS
Press, St. Paul, MN, USA.
Bekele Lemma. 2006. Impact of exotic tree plantations on carbon and nutrient dynamics in
abandoned farmland soils of southwestern Ethiopia. Doctoral Dissertation, Swedish
University of Agricultural Sciences, Uppsala. Acta Universitatis Agriculturae Sueciae.
Berjak, P. 2000. The effects of micofloral infection on the viability and ultrastructure of wetstored recalcitrant seeds of Avicennia marina (Forssk.) Vierh. Seed Science Research, 10:
341-353.
Bester, W., Crous, P.W. and Fourie, P.H. 2007. Evaluation of fungicides as potential
grapevine pruning wound protectants against Botryosphaeria species. Australasian Plant
Pathology, 36: 73-77.
Booth, T.H. and Jovanovic, T. 2002. Identifying climatically suitable areas for growing
particular trees in Africa: An example using Grevillea robusta. Agroforesrty Systems, 54:
41-49.
Burgess, T.I., and Wingfield, M.J. 2002. Quarantine is important in restricting the spread of
exotic seed-borne pathogens in the southern hemisphere. International Forestry Review,
4: 56-65.
Burgess, T.I., Taylor, A., Hardy, G. and Wood, P. 2005. Identification and pathogenicity of
Botryosphaeria species associated with grapevine decline in Western Australia.
Australasian Plant Pathology, 34: 187-195.
Burgess, T.I., Barber, P.A., Mohali, S., Pegg, G., De Beer, W. and Wingfield, M.J. 2006.
Three new Lasiodiplodia spp. from the tropics recognized based on DNA sequence
comparisons and morphology. Mycologia, 98: 423-435.
Campbell, C.L., Madden, V.L. 1990. Introduction to plant disease epidemiology. New York,
John Wiley and Sons, Inc.
Campbell, M.A., Medd, R.W. and Brown, J.B. 2003. Optimizing conditions for growth and
sporulation of Pyrenophora semeniperda. Plant Pathology, 52: 448-454.
Ciesla, W.M., Mbugua, D.K. and Ward, J.D. 1995. Ensuring forest health and productivity; A
perspective from Kenya. Journal of Forestry, 93: 36-39.
Coetzee, M.P., Wingfield, B.D., Harrington, T.C., Steimel, J., Coutinho, T.A. and Wingfield,
M.J. 2001. The root rot fungus Armillaria mellea introduced into South Africa by early
Dutch settlers. Molecular Ecology, 10: 387-396.
Collado, J., Platas, G., Gonzalez, I. and Pela’ez, F. 1999. Geographical and seasonal
influences on the distribution of fungal endophytes in Quercus ilex. New phytopathology,
144: 525-532.
44
Copes, W.E. and Hendrix, E.F. 2004. Effect of temperature on sporulation of Botryosphaeria
dothidea, B. obtusa, and B. rhodina. Plant Disease, 88: 292-296.
Dakin, N.D., White, G.E., Hardy, S.J. and Burgess, T.I. 2010. The opportunistic pathogen,
Neofusicoccum australe, is responsible for crown dieback of peppermint (Agonis
flexuosa) in western Australia. Australasian Plant Pathology, 39: 202-206.
De Wet, J., Burgess, T., Slippers, B., Preisig, O., Wingfield, B.D. and Wingfield, M.J. 2003.
Multiple gene genealogies and microsatellite markers reflect relationships between
morphotypes of Sphaeropsis sapinea and distinguish a new species of Diplodia.
Mycological Research, 107: 557-566.
Demel, T. and Granström, A. 1995. Soil seed banks in dry afro-montane forests of Ethiopia.
Journal of Vegetation Science, 6: 777-786.
Denman, S., Crous, P.W., Taylor, J., Kang, J., Pasco, I. and Wingfield, M. 2000. An overview
of the taxonomic history of Botryosphaeria and a re-evaluation of its anamorphs based on
morphology and ITS rDNA phylogeny. Studies in Mycology, 45: 129-140.
Denman, S., Crous, P.W., Groenwald, J.Z., Slippers, B., Wingfield, B.D. and Wingfield, M.J.
2003. Circumscription of Botryosphaeria species associated with Proteaceae based on
morphology and DNA sequence data. Mycologia, 95: 294-307.
Desprez-Loustau, M.L., Marçais, B., Nageleisen, M.L., Piou, D. and Vannini, A. 2006.
Interactive effects of drought and pathogens in forest trees. Annals of Forest Science, 63:
597-612.
Dhingra, O.D., Lustosa, D.C., Maia, C.B. and Mesquita, J.B. 2002. Seed-borne pathogenic
fungi that affect seedling quality of red angico (Anadenanthera macrocarpa) trees in
Brazil. Journal of Phytopathology, 150: 451-455.
Dhingra, O.D., Lustosa, D.C., Maia, C.B. and Mesquita, J.B. 2003. Seed-borne fungal
pathogens of jacaranda (Dalbergia nigra) tree. Seed Science and Technology, 31: 341349.
Dick, M.A. and Dobbie, K. 2002. Species of Fusarium on Pinus radiata in New Zealand. New
Zealand Plant Protection, 55: 58-62.
Dos-Santor, F.E., Sobrosa, R.D., Costa, I.F. and Corder, M.P. 2001. Pathogenic Fungi
Detection in Seed of Black Wattle (Acacia mearnsii De Wild). Ceincia-Florestal, 11: 1320.
Ebele, M.I. 2011. Evaluation of aqueous plant extracts to control rot fungi in paw. Journal of
Applied Biosciences, 37: 2419-2424.
45
El-Awadi, F.A, 1993. Sources and mechanism of resistance to root rot and wilt disease
complex in chickpea at sandy soil. PhD thesis, Faculty of Agriculture, Suez Canal
University. pp. 41-42.
Eshetu Yirday. 2002. Restoration of the native woody-species diversity using plantation
species as foster trees in the degraded highlands of Ethiopia. Doctoral Dissertation,
University of Helsinki. Viikki Tropical Resources Institute, Department of Forest
Ecology, Helsinki.
Eyles, A., Davies, N.W., Yuan, Z.Q. and Mohammed, C. 2003. Host responses to natural
infection by Cytonaema sp. in the aerial bark of Eucalyptus globulus. Forest Pathology,
33: 317-331.
FAO (Food and Agriculture Organization of the United Nations). 2001. Protecting plantation
from pests and diseases: Report based on the work of W.M. Ceisla. Forest Plantation
Thematic Papers, Working Paper 10. Forest Resource Development Service, Forest
Resource Division. FAO, Rome.
Fenner, M. and Thompson, K. 2005. The Ecology of Seeds. Cambridge University Press, New
York.
Gomez, K.A. and Gomez, A.A. 1984. Statistical procedures for agricultural research, 2nd
edition. John Wiley and Sons Inc., New York, Singapore.
Holding, C., Carsan, S. and Njuguna, P. 2006. Smallholder timber and firewood marketing in
the coffee and cotton/tobacco zones of Eastern Mount Kenya. pp. 18-23. In: Proceedings
of the IUFRO Conference hosted Galway-Mayo Institute of Technology, Galway, Ireland.
Hong, T.D., Linington, S. and Ellis, R.H. 1996. Seed Storage Behaviour: A Compendium.
Handbooks for Genebanks: No. 4. International Plant Genetic Resources Institute, Rome.
Huang, J.W. and Kuhlman, E.G. 1990. Fungi associated with damping-off of slash pine
seedlings in Georgia. Plant Disease, 74: 27-30.
Irshad, G., Bhutta, A.R. and Mukhtar, T. 2007. Effect of fungi on germination of forest tree
and their control. Pakistan Journal of Phytopathology, 19(1): 69-75.
Joshi, G., Thapliyal, R.C., Nayal, J.S. and Phartyal, S.S. 2005. Relation of seed viability and
vigour with nursery emergence in Dalbergia sissoo. Indian Forester, 131(6): 835-40.
Kabeere, F., Hampton, J.G. and Hill, M.J. 1997. Transmission of Fusarium graminearum
Schwabe from maize seeds to seedlings. Seed Science and Technology, 25: 245-252.
Kalinganire, A., Harwood, C.E., Slee, M.U. and Simons, A.J. 2001. Pollination and fruit-set of
Grevillea robusta in western Kenya. Austral Ecology, 26:637-648.
46
Kedir Nino. 2009. Isolation and identification of fungi associated with needle blight and death
of Pinus patula trees in forest plantations of Arsi Forest Enterprise. MSc Thesis, Hawassa
University, Wondo Genet College of Forestry. Ethiopia.
Khalid, N., Anwar, S.A., Haque, M.I. and Riaz, A. 2002. Study on occurrence of seed-borne
fungi and their impact on seed germination of five forest trees. Pakistan Journal of
Phytopathology, 13: 47-50.
Kim, D.H. and Uhm, J.Y. 2002. Effect of application timing of ergosterol biosynthesis
inhibiting fungicides on the suppression of disease and latent infection of apple white rot
cause by Botrosphaeria dothidea. Journal of General Plant Pathology, 68: 237-245.
Lori, G. and Salerno, M.I. 2002. Fusarium population associated with Douglas-fir and
Ponderosa pine seeds in Argentina. Seed Science and Technology, 30: 559-566.
Lori, G., Edel-Hermann, V., Gautheron, N. and Alabouvette, C. 2004. Genetic diversity of
pathogenic and nonpathogenic populations of Fusarium oxysporum isolated from
carnation fields in Argentina. Phytopathology, 94: 661-668.
Lott, J.E., Howard, S.B., Black, C.R.and Ong, C.K. 2000. Allometric estimation of aboveground biomass and leaf area in managed Grevillea robusta agroforestry systems:
Agroforestry Systems, 49(1):1-15.
Lupo, S., Tiscornia, S. and Bettuci, L. 2001. Endophytic fungi from flowers, capsules and
seeds of Eucalyptus globules. Revista Iberoam Mycology, 18: 38-41.
Luque, J., Martos, S., Phillips, A. 2005. Botryosphaeria viticola sp. nov. on grapevines: a new
species with a Dothiorella anamorph. Mycologia, 97: 1111-1121.
Lusk, C.H. and Kelly, C.K. 2003. Interspecific variation in seed size and safe sites in a
temperate rain forest. New Phytologist, 158: 535-41.
Luttge, U., Berg, A., Masresha Fetene, Nauke, P., Peter, D. and Beck, E. 2003. Comparative
characterization of photosynthetic performance and water relations of native trees and
exotic plantation trees in Ethiopian Forest Tree, 17: 40-50.
Mamatha, T., Lokesh, S. and Rai, V.R. 2000. Impact of seed mycoflora of forest tree seeds on
seed quality and their management. Seed Research, 28: 59-67.
Mathur, S.B. and Kongsdal, O. 2003. Common laboratory seed health testing methods for
detecting fungi, 1st edition. Published by International Seed Testing Association (ISTA),
Bassersdorf, Ch- Switzerland.
Milimo, P.B. 1988. Growth and utilization of Grevillea robusta in Kenya: Paper presented at a
workshop on the use of Australian trees in China Guangzhou, China.
47
Mila, A.L., Driever, G.F., Morgan, D.P. and Michailides, T.J. 2005. Effects of latent infection,
temperature, precipitation, and irrigation on panicle and shoot blight of pistachio in
California. Phytopathology, 95: 929-932.
Mireku, E. and Simson, J.A. 2001. Fungal and nematode threats to Australian forests and
amenity trees from importation of wood and wood products. Canadian Journal of Plant
Patholology, 24: 117-124.
Mittal, R.K., Anderson, R.L. and Mathur, S.B. 1990. Mico-organisms associated with tree
seeds, world checklist. Petawawa National Forestry Institute, PI-X-96E.
Mittal, R.K., Mathur, S.B. and Thomsen, K. 2003. Fungi of stoked Khaya anthoteca seeds and
their effect on germination. Journal of Tropical Forest Science, 15(4): 539-45.
Morpeth, D.R. and Hall, A.M. 2000. Microbial enhancement of seed germination in Rosa
corymbifera ‘Laxa’. Seed Science Research, 10: 489-94.
Muchiri, C.W., Ong, C.K., Black, C.R., Ngumi, V.W. and Mati, B.M. 2005. Tree and crop
productivity in Grevillea, Alnus and Paulownia based agroforestry system in semi-arid
Kenya. Forest Ecology and Management, 212: 23-39.
Muchiri, M., Pukkala, T. and Miina, J. 2002. Modeling trees effect on maize in the Grevillea
robusta and maize system in Central Kenya. Agroforestry System, 55: 113-123.
Myers, J.H. and Bazely, D. 2003. Ecology and Control of Introduced Plants. Cambridge
University Press, United Kingdom.
Njuguna, J.W. 2003. Wilt and stem cankers of multipurpose trees in Eastern Province: Kenya
Forestry Research Institute (KEFRI). Annual Report, 2003–2004. Nairobi, Kenya.
Njuguna, J.W., Barklund, P., Ihrmark, K. and Stenlid, J. 2011. A canker and dieback disease
threatening the cultivation of Grevillea robusta on small-scale farms in Kenya. African
Journal of Agricultural Research, 6(3): 748-756.
Noël, A., Levasseur, C., Le, V.Q. and Séguin, A. 2005. Enhanced resistance to fungal
pathogens in forest trees by genetic transformation of black spruce and hybrid poplar with
a Trichoderma harzianum endochitinase gene. Physiological and Molecular Plant
Pathology, 67: 92-99.
Orwa, C., Mutua, A., Kindt, R., Jamnadass, R. and Simons, A. 2009. Agroforestry Database:
A tree reference and selection guide version 4.0.
Otta, Y., Hattori, T. and Intini, M. 2000. Genetic Characterization of heterothalic non
heterothallic Armillaria mellea sensu Stricto. Mycological Research, 104: 1046-1054.
48
Pathan, M.A., Rajput, N.A., Jiskani, M.M. and Wagan, K.H. 2007. Studies on intensity of
shisham dieback in Sindh and impact of seed-borne fungi on seed germination. Pakistan
Journal of Agriculture, 23: 12-17.
Pavlic, D., Slippers, B., Coutinho, T.A. and Wingfield, M.J. 2007. Botryosphaeriaceae
occurring on native Syzygium cordatum in South Africa and their potential threat to
Eucalyptus. Plant Pathology, 56: 624-636.
Persson, A. 1995. Exotic prospects and risks from European and African viewpoint. Buvisindi
Agricultural Science, 9: 47-62.
Pethybridge, S.J., Hay, F.S. and Wilson, C.R. 2004. Short research notes: Pathogenicity of
fungi commonly isolated from foliar disease in Tasmanian pyrethrum crops. Australasian
Plant Pathology, 33: 441-444.
Phillips, A.J. 2002. Botryosphaeria species associated with diseases of grapevines in Portugal.
Phytopathology Mediterranea, 41:3-18.
Phillips, A.J., Alves, A., Correia, A. and Luque, J. 2006. Two new species of Botryosphaeria
with brown, 1-septate ascospores and Dothiorella anamorphs. Mycologia, 97: 513-519.
Pohjonen, V. and Pukkala, T. 1990. Eucalyptus globulus in Ethiopian forestry. Forest Ecology
and Management, 36: 19-31.
Roux, J., Byabashaija, D.M., Coutinho, T.A. and Wingfield, M.J. 2001. Diseases of plantation
Eucalyptus in Uganda. South African journal of science, 97: 16-18.
Satou, M., Ichinoe, M., Fukumoto, F., Tezuka, N. and Horiuchi, S. 2001. Fusarium blight of
Kangaroo Pay (Anigozanthos spp.) caused by Fusarium chlamydosporum and Fusarium
semitectum. Journal of Phytopathology, 149: 203-206.
Schroeder, T., Kehr, R. and Hüttermann, A. 2002. First report of the seed-pathogen
Geniculodendron pyriforme, the imperfect state of the ascomycete Caloscypha fulgens, on
imported conifer seeds in Germany. Forest Pathology, 32: 225-230.
Shailendra, K., Surinder, K., Kapoor, K.S., Ranjeet, S. and Chakrabarti, S. 2004. Mortality of
Dalbergia sissoo Roxb. (Shisham) in Subathu Forest Range of Solan, Himachal Pardesh:
a case study. Indian Forester, 130: 349-350.
Shaner, G. and Finney, R.E. 1977. The effect of nitrogen fertilization in the expression of
slow-mildewing resistance in Knox wheat. Phytopathology, 67: 1051-1056.
Singh, P.M., Mehrotra, D. and Singh, P. 1999a. Seed-borne fungi of some forest trees and
their control. Indian Journal of Forestry, 22(4): 320-24.
Singh, P.M., Khan, S.N. and Singh, P. 1999b. Assessment of seed-borne fungi of some forest
trees and their management. Indian Journal of Forestry, 22(3): 281-84.
49
Skene, K.R., Kierans, M., Sprent, J.I. and Raven, J.A. 1996. Structural aspects of cluster root
development and their possible significance for nutrient acquisition in Grevillea robusta
(Proteaceae). Annals of Botany, 77: 443-451.
Slippers, B., Crous, P.W., Denman, S., Countinho, T.A., Wingfield, B.D. and Wingfield, M.J.
2004a. Combined multiple gene genealogies and phenotypic characters differentiate
several species previously identified as Botryosphaeria dothidea. Mycologia, 96: 83-101.
Slippers, B., Crous, P.W., Coutinho, T.A., Wingfield, B.D. and Wingfield, M.J. 2004b.
Multiple gene sequences delimit Botryosphaeria australis sp. nov. from B. lutea.
Mycologia, 96: 1028-1039.
Slippers, B., Fourie, G., Crous, P.W., Coutinho, T.A., Wingfield, B.D., Carnegie, A. and
Wingfield, M.J. 2004c. Speciation and distributionof Botryosphaeria spp. on native and
introduced Eucalyptus trees. Studies in Mycology, 50: 343-358.
Slippers, B. and Wingfield, M.J. 2007. Botryosphaeriaceae as endophytes and latent pathogens
of woody plants: Diversity, ecology and impact. Fungal Biology Reviews, 21: 90-106.
Smith, A.N 1960. Boron Deficiency in Grevillea robusta Nature, London, 186: 4729-987.
Smith, D.R. and Stanosz, G.R. 2000. Molecular and morphological differentiation of
Botryosphaeria dothidea (anamorph Fusicoccum aesculi) from some other fungi with
Fusicoccum anamorphs. Mycologia, 93: 505-515.
Smith, H., Wingfield, M.J. and Petrini, O. 1996. Botryosphaeria dothidea endophytic in
Eucalyptus grandis and Eucalyptus nitens in South Africa. Forest ecology and
management, 89: 189-195.
Spanos, K.A., Pirrie, A., Woodward, S. and Xenopoulos, S. 1999. Responses in the bark of
Cupressus sempervirens clones artificially inoculated with Seiridium cardinale under
field conditions. European Journal of Forest Pathology, 29: 135-142.
Sutherland, J.R., Diekmann, M. and Berjak, P. 2002. Forest Tree Seed Health. IPGRI
Technical Bulletin No. 6. International Plant Genetic Resources Institute, Rome, Italy.
Swart, L., Crous, P.W., Petrini, O. and Taylor, J.E. 2000. Fungal endophytes of Proteaceae,
with particular emphasis on Botryosphaeria proteae. Mycoscience, 41: 123-127.
Taylor, J.E., Hyde, K.D. and Jones, E.B. 2000. The biogeographical distribution of microfungi
associated with three palm species from tropical and temperate habitats. Journal of
Biogeography, 27: 297-310.
Toljander, Y.K., Nyeko, P., Stenström, E., Ihrmark, K. and Barklund, P. 2007. First Report of
Canker and Dieback Disease of Grevillea robusta in East Africa Caused by
Botryosphaeria spp. Plant Disease, 91: 773.
50
Úrbez-Torres, J.R., Guerrero, J.C., Guevara, J. and Gubler, W.D. 2008. Identification and
Pathogenicity of Lasiodiplodia theobromae and Diplodia seriata, the Causal Agents of
Bot Canker Disease of Grapevines in Mexico. Plant Disease, 92: 519-529.
Úrbez-Torres, J.R. and Gubler, W.D. 2009. Pathogenicity of Botryosphaeriaceae species
isolated from grapevine cankers in California. Plant Disease, 93: 584-592.
Vander Plank, J.E. 1963. Epidemiology of Plant Disease. New York and London Academic
publishers.
Venter, M., Wingfield, M.J., Coutinho, T.A. and Wingfield, B.D. 2001. Molecular
characterization of Endothia gyrosa isolates from Eucalyptus in South Africa and
Australia. Plant Pathology, 50: 211-217.
Vercoe, T.K. 1995. International, social and economic importance of Australian Eucalyptus.
pp. 14-20. In: Diekmann, M. and Ball, J.B. (eds.), Eucalyptus pests and diseases.
International Plant Genetic Resource Institute (IPGRI). Bangkok, Thailand.
Vujanovic, V., St-Arnaud, M. and Neumann, P.J. 2000. Susceptibility of cones and seeds to
fungal infection in a pine (Pinus spp.) collection. Forest Pathology, 30: 305-320.
Wheeler, B.E. 1969. An introduction to plant diseases. John Wiley and Sons, New York.
Wingfield, M.J. 1990. Current status and future prospects of forest pathology in South Africa.
South African Journal of Science, 86: 60-62.
Wingfield, M.J., Slippers, B., Roux, J. and Wingfield, B.D. 2001. Worldwide movement of
exotic forest fungi, especially in the tropics and the southern hemisphere. Bioscience, 51:
134-140.
Xue, A.G., Armstrong, K.C., Voldeng, H.D., Fedak, G. and Babcock, C. 2004. Comparative
aggressiveness of isolates of Fusarium spp. causing head blight on wheat in Canada.
Canadian Journal Plant Pathology, 26: 81-88.
Zhou, S.G. and Stanosz, G.R. 2001. Relationships among Botryosphaeria species and
associated anamorphic fungi inferred from the analyses of ITS and 5.8S rDNA sequences.
Mycologia, 93: 516-527.
51
7. APPENDICES
Appendix Table 1. Number of fungal isolates obtained from seed samples collected from
symptomatic and asymptomatic stands of G. robusta at Gambo, Shashemene and
WGCF-NR sites
No Fungal species
WGCF-NR
Shashemene
Gambo
Symp Asym
p
6
4
1
1
14
10
10
7
8
7
11
9
13
12
3
3
66
53
Symp Asym
p
9
7
3
2
11
8
8
6
2
2
4
2
6
4
5
3
7
5
2
1
3
1
60
41
Symp Asym
p
4
3
6
4
3
6
3
3
7
5
3
4
4
3
2
33
27
Total
1 Alternaria sp.
2 Aspergillus sp.1
3 Aspergillus sp.2
4 Botryosphaeria sp.1
5 Botryosphaeria sp.2
6 Botryosphaeria sp.3
7 Curvularia sp.
8 Fusarium sp.1
9 Fusarium sp.2
10 Lasiodiplodia sp.
11 Pestalotiopsis sp.
12 Phoma sp.
Total
16
17
7
53
40
10
6
37
31
45
3
15
280
Keys:
- indicates no fungal isolates obtained
Symp–Seed samples collected from symptomatic mother trees
Asymp– Seed samples collected from asymptomatic mother trees
Appendix Table 2. Number of seeds that yielded one or more fungal isolates out of 100 seeds
incubated, and the frequency of infection of seed samples collected from symptomatic
and asymptomatic stands of G. robusta at Gambo, Shashemene and WGCF-NR sites
Source of seed samples
Gambo - Symp
Gambo - Asymp
Shashemene - Symp
Shasheshemen - Asymp
WGCF - Symp
WGCF- Asymp
Keys:
No of seeds that yielded one Frequency of infection (%)
or more fungal isolates
29
29
25
25
50
50
36
36
56
56
43
43
Symp - Seed samples collected from symptomatic mother trees
Asymp - Seed samples collected from asymptomatic mother trees
52
Appendix Table 3. Disease progress rates (DPR) recorded on G. robusta seedlings inoculated
with different fungal species
Treatments
(Fungal species)
Botryosphaeria sp.1
Botryosphaeria sp.2
Fusarium sp.1
Fusarium sp.2
Lasiodiplodia sp.
Untreated(Control)
CV (%)
LSD (0.05)
Rate1
0.00321c
0.00364c
0.00962a
0.00431c
0.00638b
0.00d
18
0.001
Rate2
Rate3
0.00375d 0.00431d
0.00567c 0.00674c
0.01199a 0.01429a
0.00564c 0.00721bc
0.00689b 0.00769b
0.00e
0.00e
8.091
4.917
0.007
0.005
Rate4
Rate5
Rate6
0.00490d
0.00779c
0.01547a
0.00770c
0.00877b
0.00e
3.48
0.004
0.00567e
0.00894c
0.01522a
0.00774d
0.01017b
0.00f
2.021
0.002
0.00665e
0.01025c
0.01465a
0.00764d
0.01214b
0.00f
1.88
0.002
Mean values having the same letter within a column are not significantly different from each other at p  0.05
level; CV = Coefficient of variation; LSD = Least significant difference
53
Appendix Figure 1. Colony and conidial morphological characteristics of Botryosphaeria
sp.1: (A) Colony of Botryosphaeria sp.1 on PDA after 10 days of incubation at 25 oC; (B) Conidial
morphology of Botryosphaeria sp.1
Appendix Figure 2. Colony and conidial morphological characteristics of Botryosphaeria
sp.2: (A) Colony of Botryosphaeria sp.2 on PDA after 10 days of incubation at 25 oC; (B) Conidial
morphology of Botryosphaeria sp.2
Appendix Figure 3. Colony and conidial morphological characteristics of Fusarium sp.1: (A)
Colony of Fusarium sp.1 on PDA after 10 days of incubation at 25 oC; (B) Macro-conidia with three to
five septa and non-septate micro-conidia of Fusarium sp.1
54
Appendix Figure 4. Colony and conidial morphological characteristics of Fusarium sp.2: (A)
Colony of Fusarium sp.2 on PDA after 10 days of incubation at 25 oC; (B) Slightly curved with five to
six septate macro-conidia of Fusarium sp.2
Appendix Figure 5. Colony and conidial morphological characteristics of Lasiodiplodia sp.:
(A) Colony of Lasiodiplodia sp. on PDA after 10 days of incubation at 25 oC; (B) Aseptate and ovoid
conidia in immature and dark brown septate with ovoid conidia in mature state of Lasiodiplodia sp.
55
Appendix Figure 6. Seedlings of G. robusta inoculated with different fungal species and
disease symptoms observed: (A) Necrotic spots and shoot dieback disease symptom observed on seedling
inoculated with Botryosphaeria sp.1; (B) Top dying of shoot and leaf blight disease symptom observed on
seedling inoculated with Botryosphaeria sp.2; (C) Yellowing and wilting of leaves disease symptom observed on
seedling inoculated with Fusarium sp.1; (D) Chlorosis of leaves disease symptom observed on seedling
inoculated with Fusarium sp.2; (E) Typical shoot dieback disease symptom observed on seedling inoculated with
Lasiodiplodia sp.; (F) No disease symptoms on seedling inoculated with agar plug devoid of mycelium (Photos
taken from greenhouse of WGCF)