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Chapter 2 Botryosphaeriaceae associated with Terminalia catappa in Cameroon, South Africa and Madagascar
ABSTRACT
Species in the Botryosphaeriaceae represent some of the most important fungal pathogens of woody plants. Although these fungi have been relatively well studied on economically important crops, hardly anything is known regarding their taxonomy or ecology on native or non-commercial tree species. The aim of this study was to compare the diversity and distribution of the Botryosphaeriaceae on Terminalia catappa, a tropical tree of Asian origin planted as an ornamental in Cameroon, Madagascar and South Africa. A total of 83 trees were sampled, yielding 79 Botryosphaeriaceae isolates. Isolates were initially grouped based on morphology of cultures and conidia. Representatives of the different morphological groups were then further characterized using sequence data for the ITS, tef 1-a, rpb2, BOTF15 and þ– tub gene regions. Five species of the Botryosphaeriaceae were identified, including Neofusicoccum parvum, N. batangarum sp. nov., Lasiodiplodia pseudotheobromae, L. theobromae and L. mahajangana sp. nov. Lasiodiplodia pseudotheobromae and L. theobromae, were the most commonly isolated species (62%), and were found at all the sites. Neofusicoccum parvum and N. batangarum were found in South Africa and Cameroon respectively, whereas L. mahajangana was found only in Madagascar. Greenhouse inoculation trials performed on young T. catappa trees showed variation among isolates tested, with L. pseudotheobromae being the most pathogenic. The Botryosphaeriaceae infecting T. catappa appear to be dominated by generalist species that also occur on various other hosts in tropical and sub-tropical climates.
INTRODUCTION
The Botryosphaeriaceae is a diverse group of fungi that accommodates numerous species spread over many anamorph genera, the best known of which are Diplodia, Lasiodiplodia, Neofusicoccum, Pseudofusicoccum, Dothiorella and Sphaeropsis (Crous et al. 2006). Members of the Botryosphaeriaceae have a worldwide distribution and occur on a large variety of plant hosts including monocotyledons, dicotyledons, gymnosperms and angiosperms, on which they are found as saprophytes, parasites and endophytes (Slippers and Wingfield 2007; von Arx 1987).
It has long been recognized that species of the Botryosphaeriaceae are important pathogens of several plants (Von Arx 1987). Infected plants can exhibit a multiplicity of symptoms such as die-back, canker, blight and rot on all above ground plant organs (Punithalingham 1980; Slippers et al. 2007). A particularly dangerous feature of these fungi is that they can live as endophytes in plant organs, in a latent phase, without producing clear symptoms, and diseases only emerge following the onset of unfavourable conditions to the tree (Smith et al. 1996). This implies that they can easily, and unobtrusively, be moved around the world with seeds, cuttings and even fruit.
Extensive studies have been conducted on diseases of economically important species of fruit (e.g. Lazzizera et al. 2008; Phillips 1998; Slippers et al. 2007; van Niekerk et al. 2004) and timber trees (e.g. Mohali et al. 2007; Sanchez et al. 2003) caused by fungi in the Botryosphaeriaceae. Much less is known about the Botryosphaeriaceae on plants with no large-scale international commercial value (Denman et al. 2003; Pavlic et al. 2008), such as Terminalia catappa, but which have social and environmental significance (Gure et al. 2005). Without knowledge of the Botryosphaeriaceae on hosts with limited or no commercial value, and hosts in their native environments, the impact and biology of the important pathogens in this group will never be fully understood.
Terminalia catappa, frequently referred to as “tropical almond”, belongs to the Combretaceae and originates from Southern India to coastal South-East Asia (Smith 1971). These trees are widely cultivated in tropical and subtropical coastal areas and utilised by local communities for a number of household uses. The multitude of non-wood products and services pertaining to this tree species make it an important component, especially for coastal communities. The tree is planted for shade and ornamental purposes in urban environments, the timber is converted into decorative tools, furniture and many other applications, leaves and bark are commonly used in traditional medicine and its fruits contain edible kernels from which high energy oil is extracted and which can also be admixed into diesel fuel (Chen et al. 2000; Hayward 1990; Kinoshita et al. 2007).
The diversity and spatial distribution of the Botryosphaeriaceae, associated with a specific host, is important. Whether it accommodates similar or different fungal assemblages depending on the environment, is useful in understanding the ecology and host-pathogen relationships of these fungi. This knowledge in turn can be applied where recommendations for disease management strategies are required. Several studies have compared assemblages of fungal endophytes in different geographic regions (Fisher et al. 1994; Gallery et al. 2007; Gilbert et al. 2007; Taylor et al. 1999). However, such studies dealing with a specific endophytic group of fungi are limited. Similarly, very few studies have compared the assemblages of Botryosphaeriaceae from a specific host at a regional level (Taylor et al. 2005; Urbez-Torrez et al. 2006).
Among all the species of Terminalia present on the African continent, T. catappa is one of the few species planted widely in West, Central, East and Southern Africa. As part of a larger project in which we explore diseases of Terminalia spp. in Africa, the broad distribution of this species over the continent made it an ideal candidate to characterise endophytic species of the Botryosphaeriaceae under variable geographic and climatic conditions. The aims of this study were, therefore, to investigate the diversity of the Botryosphaeriaceae occurring on introduced T. catappa and to analyse the patterns of their distribution in three African countries. Pathogenicity trials were also undertaken to assess the ecological significance of the Botryosphaeriaceae collected from T. catappa.
MATERIALS AND METHODS
Isolates
Collections were made from T. catappa trees in Cameroon, Madagascar and South Africa. In Cameroon, samples were collected along the beach front of Kribi, a seaside town within the tropical forest and bordering the Atlantic Ocean (N2 58.064, E9 54.904, 7 m asl). The climate in this area is characterized by high humidity, precipitation up to 4000 mm per annum and relatively high temperatures, averaging 26 ˚C. In South Africa, sampling was done in Richardsbay (S28 46.886 S, E32 03.816, 0 m asl), a harbour city on the Indian Ocean where catappa trees are planted to provide shade in open spaces and in parking areas. Climatic conditions in this area are typically subtropical to tropical. The average temperature in summer is 28 °C and 22 °C in the winter. The humidity levels tend to be very high in summer and the annual rainfall is ~1200 mm. In Madagascar, samples were collected from the towns of Morondava (S20 17.923, E44 17.926, 3 m asl) and Mahajanga (S15 43.084, E46 19.073, 0 m asl), both located on the west coast of the country. In these areas, the climate is between semi-arid and tropical humid with mean annual temperatures of 23.5 °C and average rainfall between 400 and 1200 mm per annum.
Samples were collected from 83 T. catappa trees in all three countries in 2007. Forty trees were randomly sampled in Kribi, 15 in Richardsbay, 20 and eight in Morondava and Mahajanga, respectively. Except for the trees in Richardsbay, that were showing symptoms of die-back at the time of collection, those at all the other sites were healthy. One branch (~0.5 – 1 cm diameter) per tree was cut and all the samples placed in paper bags and taken to the laboratory where they were processed after one day.
From each branch, two segments (1 cm in length each) were cut and split vertically into four halves. Samples were surface sterilized by dipping the wood pieces in 96 % ethanol for 1 min, followed by 1 min in undiluted 3.5 % sodium hypochlorite and 1 min in 70 % ethanol, before rinsing in sterile distilled water and allowing them to dry under sterile conditions. The four disinfected branch pieces from each tree were plated on 2 % malt extract agar (MEA) (2 % malt extract, 1.5 % agar; Biolab, Midrand, Johannesburg, S.A.) supplemented with 1 mg ml-1 streptomycin (Sigma, St Louis, MO, USA) to suppress bacterial growth. The Petri dishes were sealed with Parafilm and incubated at 20 °C under continuous near-Ultra Violet (UV) light. One week later, filamentous fungi growing out from the plant tissues and resembling the Botryosphaeriaceae were transferred to new Petri dishes containing fresh MEA.
All cultures are maintained in the Culture Collection (CMW) of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa. Representatives of all species have also been deposited at the Centaalbureau voor Schimmelcultures (CBS, Utrecht, Netherlands). Herbarium materials for previously underscribed species have been deposited at the National Fungal Collection (PREM), Pretoria, South Africa.
Morphology and cultural characteristics
Fungal isolates were grown on plates containing 1.5 % water agar (Biolab, S.A.) overlaid with three double-sterilized pine needles and incubated at 25 °C under near UV-light for two to six weeks to induce the formation of fruiting bodies (pycnydia and/or pseudothecia). Morphological features of the resultant fruiting bodies were observed using a HRc Axiocam and accompanying Axiovision 3.1 camera (Carl Zeiss Ltd., München, Germany). For previously undescribed species, sections of fruiting bodies were made with a Leica CM1100 cryostat (Setpoint Technologies, Johannesburg, South Africa) and mounted on microscope slides in 85 % lactic acid. For the undescribed species 50 measurements of all relevant morphological characters were made for the isolate selected as the holotype and 30 measurements were made for the remaining isolates. These measurements are presented as the extremes in brackets and the range calculated as the mean of the overall measurements plus or minus the standard deviation.
The morphology of fungal colonies growing on 2 % MEA at 25 °C under near UV-light for two weeks was described and colony colours (upper and reverse surfaces) of the isolates were recorded using the colour notations of Rayner (1970). Growth rates of cultures on 2 % MEA in the dark was determined at 5 °C intervals from 10 to 35 °C. For growth rates, evaluations of five plates were used for each isolate at each temperature. Two measurements, perpendicular to each other, were made after three days for each plate resulting in 10 measurements for each isolate at each temperature. The experiment was repeated once.
DNA extraction
Mycelium was scraped from 10-day-old cultures representing different morphological groups, using a sterile scalpel and transferred to 1.5 µl Eppendorf tubes for freeze-drying. The freeze- dried mycelium was mechanically ground to a fine powder by shaking for 2 min at 30.0 1s-1 frequency in a Retsch cell disrupter (Retsch Gmbh, Germany) using 2 mm-diameter metal beads. Total genomic DNA was extracted using the method described by Möller et al. (1992). The concentration of the resulting DNA was determined using a ND-1000 uv/Vis Spectrometer (NanoDrop Technologies, Wilmington, DE USA) version 3.1.0.
PCR amplification
were respectively used to amplify and sequence the internal transcribed spacer regions (ITS), including the complete 5.8S gene, the translation elongation factor 1-a gene (tef 1-a), partial sequence of the þ-tubulin gene (þ– tub), part of the second largest subunit of RNA polymerase II gene (rbp2) and an unknown locus (BotF15) containing microsatellite repeats. A “hot start” polymerase chain reaction (PCR) protocol was used on an Icycler thermal cycler (BIO-RAD, Hercules, CA, USA). The 25 µl PCR reaction mixtures for the ITS, BT and RPB2 regions contained 0.5 µl of each primer (10 mM) (Integrated DNA Technology, Leuven, Belgium), 2.5 µl DNTPs (10 mM), 4 µl of a 10 mM MgCl2 (Roche Diagnostics GmbH, Mannheim, Germany), 2.5 µl of 10 mM reaction buffer (25 mM) (Roche Diagnostics GmbH, Mannheim, Germany), 1 U of Taq polymerase (Roche Diagnostics GmbH, Mannheim, Germany), between 60-100 ng/µl of DNA and 13.5 µl of sterile distilled water (SABAX water, Adcock Ingram Ltd, Bryanston, S.A.). The amplification conditions were as follows: an initial denaturation step at 96 °C for 1 min, followed by 35 cycles of 30 seconds at 94 °C, annealing for 1min at 54 °C, extension for 90 seconds at 72 °C and a final elongation step of 10 min at 72 °C. To amplify the tef 1-a gene region, the 25 µl PCR reaction mixture contained 0.5 µl of each primer (10 mM), 2.5 µl DNTPs (10 mM), 2.5 µl of 10 mM reaction buffer with MgCl2 (25 mM) (Roche Diagnostics GmbH), 1 U of Taq polymerase, between 2-10 ng/µl of DNA and 17 µ l of sterile SABAX water. The amplification conditions used were similar to those of Al-Subhi et al. (2006) and the conditions used to amplify the BotF15 locus were the same as those of Pavlic et al. (2009a). The PCR amplification products were separated by electrophoresis on 2 % agarose gels stained with ethidium bromide in a 1x TAE buffer and visualized under UV light.
Acknowledgements
Preface
Chapter 1 Literature Review: Terminalia spp in Africa with special reference to its health status
ABSTRACT
1. INTRODUCTION
2. THE GENUS TERMINALIA
2.1. Origin and distribution
2.2. Botanical description and ecology
2.3. Propagation and management
2.3.1. Seed propagation
2.3.2. Vegetative propagation
2.3.3. Tending of trees
2.4. Functional uses of Terminalia trees
2.5. Pests and diseases
2.5.1. Insects
2.5.1.1. Fruit Borers
2.5.1.2. Stem Borers
2.5.1.3. Defoliators
2.5.1.4. Termites
2.5.2. Wildlife
2.5.3. Diseases
2.5.3.1. Root diseases
2.5.3.1. Stem diseases
2.5.3.3. Leaf diseases
2.5.3.4. Stain diseases
3. CONCLUSIONS
4. REFERENCES
Chapter 2 Botryosphaeriaceae associated with Terminalia catappa in Cameroon, South Africa and Madagascar
ABSTRACT
1. INTRODUCTION
2. MATERIALS AND METHODS
2.1. Isolates
2.2. Morphology and cultural characteristics
2.3. DNA extraction
2.4. PCR amplification
2.5. DNA Sequencing
2.6. DNA Sequence Analyses
2.7. Pathogenicity
3. RESULTS
3.1. Isolates
3.2. Morphologic characterization
3.3. DNA extraction and PCR amplification
3.4. DNA sequence analyses
3.5. Taxonomy
3.6. Distribution of the Botryosphaeriaceae
3.7. Pathogenicity
4. DISCUSSION
5. REFERENCES
Chapter 3 Botryosphaeriaceous fungi as endophytes on Terminalia species in Cameroon
ABSTRACT
1. INTRODUCTION
2. MATERIALS AND METHODS
2.1. Sample collection and fungal isolation
2.2. Morphology and cultural characteristics
2.3. DNA extraction, PCR reactions and DNA sequencing
2.4. Sequence Analyses
2.5. Pathogenicity
3. RESULTS
3.1. Isolates and morphology
3.2. DNA extraction and PCR amplification
3.3. Phylogenetic analyses
3.4. Pathogenicity
4. DISCUSSION
5. REFERENCES
Chapter 4 Phenotypic and molecular characterization of the Botryosphaeriaceae associated with
native Terminalia spp. of Southern Africa
ABSTRACT
1. INTRODUCTION
2. MATERIALS AND METHODS
2.1. Sample collection and fungal isolation
2.2. Morphology and culture characteristics
2.3. DNA extraction, PCR reactions and DNA sequencing
2.4. DNA Sequence Analyses
2.5. Pathogenicity
3. RESULTS
3.1. Isolation, morphology and culture characteristics
3.2. DNA extraction and PCR amplification
3.3. Phylogenetic analyses
3.4. Taxonomy
3.5. Pathogenicity
4. DISCUSSION
5. REFERENCES
Chapter 5 Genetic structure of Lasiodiplodia theobromae and L. pseudotheobromae from native
and non-native hosts in Cameroon
ABSTRACT
1. INTRODUCTION
2. MATERIALS AND METHODS
2.1. Fungal isolates
2.2. DNA extraction, PCR reactions and DNA sequencing
2.3. Simple sequence repeat (SSR)-PCR and GENESCAN analyses
2.4. Statistical analyses
2.4.1. Bayesian clustering analyses
2.4.2. Gene and genotypic diversity
2.4.3. Genetic differentiation and gene flow
2.4.4. Linkage disequilibrium
3. RESULTS
3.1. Fungal isolates
3.2. Microsatellite PCR amplification
3.3. Statistical analyses
3.3.1. Bayesian clustering analyses
3.3.2. Gene diversity
3.3.3. Genotypic diversity
3.3.4. Genetic differentiation and gene flow
3.3.5. Linkage disequilibrium
4. DISCUSSION
5. REFERENCES
Chapter 6 Aurifilum, a new fungal genus in the Cryphonectriaceae from Terminalia species in
Cameroon
ABSTRACT
1. INTRODUCTION
2. MATERIALS AND METHODS
2.1. Survey and specimen collection
2.2. DNA extraction and sequence comparisons
2.3. Morphology
2.4. Pathogenicity
3. RESULTS
3.1. Survey and specimen collection
3.2. DNA sequence comparisons
3.3. Morphology
3.4. Taxonomy
3.5. Pathogenicity
4. DISCUSSION
5. REFERENCES
Summary
