Diseases of exotic plantation forestry trees in Ethiopia

Alemu gezahgne

Boswell papyrifera is a tree species which is found in Amhara, Tigray, and Benshangul Gumuze region and used for the production of frankincense. Frequent tapping at different rounds and at different position of the tree is made to produce frankincense. A study aiming at evaluating the health status of tapped Boswellia papyrifera was carried out on samples obtained from Metema and Humera. Galls were commonly observed on most tapped Boswellia trees and a black wood discoloration was found on the wood beneath these galls. Isolation made from the discolored part of the wood resulted in growth of fungal colonies with a white to gray fluffy aerial mycelia which later changed into blackish mycelial growth as it matures. The spore of the fungus has hyaline, ellipsoid to ovoid shaped and it has one septa. The spore has a rounded base and truncate apex. The color, the shape of the colony and the characteristics of the conidia is found to be identical with description of Lasiodiplodia theobro...

Trees with stem bark lesions are frequently observed in Eucalyptus globulus Labill. plantations, particularly in the central west region of Uruguay. These lesions constitute a problem for trunk decortications at harvest and they also facilitate the access of fungi that could cause wood decay. Seven, three and oneyear-old plantations, located at three sites in close proximity were selected. Four types of trunk lesions were present in trees regardless the age of plantation and more than one type was found in each plantation. The aim of this study was to investigate the fungal composition associated with these lesions and compare them to healthy tissues and try to find out the origin of these symptoms. Another purpose was to elucidate the real role of the fungi considered pathogens by means of experimental inoculations. Segments from lesions and healthy tissues yielded 897 fungal isolates belonging to 32 taxa, 681 isolates from bark lesions and 216 from healthy tissues. Both healthy an...

15 Several species in the Botryosphaeriaceae family cause wood stain, cankers and dieback 16 of trunks and branches in a wide range of forest tree species. The aim of this study was to 17 characterize the botryosphaeriaceous fungi associated with decline symptoms observed in 18 Acacia mangium and Pinus caribaea var. hondurensis, two economically important forest 19 tree species grown in commercial plantations in Venezuela. Fungi isolated from 20 symptomatic samples collected from both hosts in commercial sites were identified based 21 on their morphology and DNA sequences of the internal transcribed spacer region (ITS1-22 5.8S-ITS2) and part of the β-tubulin and translation elongation factor 1-α genes. 23

Diseases of exotic plantation forestry trees in Ethiopia by Alemu Gezahgne M. Phil. (University College of North Wales, Bangor) Submitted in fulfilment of the requirements for the degree Doctor of Philosophy In the faculty of Natural and Agricultural Sciences, Department of Microbiology and Plant Pathology University of Pretoria Pretoria, South Africa March 2003 Promoter Co-promoters Dr. J. Roux Prof. M. J. Wingfield Prof. B. D. Wingfield © University of Pretoria Diseases of exotic plantation forestry trees in Ethiopia by Alemu Gezahgne M. Phil. (University College of North Wales, Bangor) Submitted in fulfilment of the requirements for the degree Doctor of Philosophy In the faculty of Natural and Agricultural Sciences, Department of Microbiology and Plant Pathology University of Pretoria Pretoria, South Africa March 2003 Promoter Co-promoters Dr. J. Roux Prof. M. J. Wingfield Prof. B. D. Wingfield Dedicated to my late younger sister, Getenesh Gebre Tsadik. TABLE OF CONTENTS Acknowledgments ........................................................................... .1 Preface ................................. :................................................JV Chapter One: Impact and Importance of Diseases in Exotic Plantations in Ethiopia and Other African Countries-Literature ........................................................................... 1 Review Introduction .................................................... .. ................... .. 2 Nursery diseases ............................................................ ... ... 4 Diseases of plantation Pinus spp. in Africa ....................................... 5 Root diseases ........... ......................................................... 5 Stem diseaseslcanker ......... ................... ............................. 8 Needle diseases ......................... ... ..................................... 9 Diseases of plantation Eucalyptus spp. in Africa ........................... .. 11 Root diseases ............................... .. ................................ 11 Stem diseaseslcanker ....................... ................................ .13 Leafdiseases ..................................... .................... .. ...... 16 Wilt diseases ................................................................. 19 Diseases of plantation Cupressus spp. in Africa .............................20 .............................21 Diseases of plantation Acacia spp. in Africa Root diseases ................................................... .... .......... 21 Stem diseases/canker ........ ;... ............................................ 22 Leaf diseases ............................................. .. .................. 23 Wilt disease ................................................................. 24 ......................................24 Exotic plantation Forestry in Ethiopia Conclusions ....................... " .................................... . ............26 References ........................... , . .............................................27 Chapter Two: Diseases of Exotic Plantation Eucalyptus and Pinus Species in Ethiopia .............. ..... ............................................ .49 Abstract ...... . ...................................................................50 Introduction ............................................... ... ........................51 Materials and Methods .............................................................. 53 Survey areas and sample collection ............ ... .......................53 Isolation techn(ques ........................................................ 53 Results ..........................................................................54 Root diseases ................................................................. 54 Stem cankers ...... ............... ....... ......... .... ........................ 55 Leaf disease ...................... ........................................... 56 Other fungi .............................................. ............ .......57 Discussion References .................................... .. ........ ............................57 ... . ... '" ................................................................60 Chapter Three: Identification ·of the Armillaria Root Rot Pathogen in Ethiopian Plantations ................ ....................................... 70 Abstract .......... .. ..............................................................71 Introduction ....... . ..................................................................72 Materials and Methods .... .. ................ . .................................74 ...................................... 74 Sample collection and isolation Basidiocarp morphology ..................... ......... ... .............. 75 DNA Extraction ............................................ ,........... 75 DNA amplification ........................................................ 76 Restriction enzyme digestion .... ... ........................................ 76 Cloning ......... . ........ ........ ....................................... 77 DNA sequencing ........................................................ 77 Analysis ofDNA sequence data ...................................... 78 Results ................................................................... . ...... 78 ...................................... 78 Sample collection and isolation Basidiocarp morphology .................................. .... ......... 79 DNA amplification ........................................................ 79 Restriction enzyme digestion .................................... ........... 79 DNA sequencing .......................................... .. ..... .. ..... 80 Analysis ofDNA sequence data ............. . ....... ................. 80 Discussion ............................. ................. .. ................ . ......... 80 .................................................. .. ......................83 References Chapter Four: Characterisation of the Coniothyrium Stem Canker Pathogen on Eucalyptus camaldulensis in Ethiopia .. ......... ...... . 102 Abstract ......... . ............................................................. .103 Introduction .............. .......................................................... 104 Materials and Methods ... . .......... .................. .. . ...... .. ........... 105 Sample collection and isolation .. ......................................... 105 DNA extraction .............................................. . ....... 106 PCR amplification .................................................... .. 107 DNA sequencing ........................................ .. ............ 107 Sequence analysis ........................ .............................. 108 ......... , ........ ......................... ... ...... .. 108 Pathogenicity test Results ........................................................................ 109 Sample collection and isolation . ........ .. ....... ... ............... 109 PCR amplification and analysis ofsequence data ............. .. ... 109 Pathogenicity test .... .... .................................. ............ 111 Discussion ... ,................ . .. '" ... ....... . .................. . ................ 111 References .... . ............................ . ...................................... 113 Chapter Five: Identification of the Causal Agent of Botryosphaeria Stem Canker in Ethiopian Eucalyptus Plantations .. ................ 128 Abstract . ... ................................. . ........................ ... ....... 129 Introduction .................................. .. .................................... 130 Materials and Methods .......'.............. ............................. .... 132 Symptoms, sample collection andfungal isolation .................. 132 .................................... 133 Morphological characterisation ........ ............ ....................... ........... 133 DNA extraction peR amplification ......................................... ............. 134 Restriction Fragment Length Polymorphysims (RFLP) ...... ... 134 DNA sequencing ............. ..... .............. " .................... 135 ...................................................... 135 Sequence analysis Pathogenicity tests ...................................................... 135 Results .. .. .......... ........... . .. ............................................ 137 Symptoms, sample collection andfungal isolation ...... ............ 137 .......... .. ........................ 137 Morphological characterisation peR amplification ...................................................... 138 .................. 138 Restriction Fragment Length Plymorphisms DNA sequencing and analysis ........................ .. .......... 138 Pathogenicity tests ................................................. .. ... 139 Discussion ................................ ... . ............... . ....... ............. 140 ..................... .. ...................................... . .......... 142 References Chapter Six: Characterisation of Diplodia pinea and First Report of Botryosphaeria parva From Pinus patula in Ethiopia ......... 170 Abstract ........... ... ................ ; . ....................... ... .............. 171 Introduction .................... : - ~' .; ...-; .. . ..... . ................................... 172 Materials and Methods .......... .- ........................................... 174 Fungal isolation and morphological characterisation ......... 174 DNA extraction .. .. .......................................................... 174 peR amplification ...................................................... 175 Morphotype determination .................. .... ........ ............... 176 DNA sequencing ...................................................... 176 Sequence analysis .................................... .. ................ 176 ...................................................... 177 Pathogenicity test .............................. .. ...... .. ........................... . .... 178 Results Fungal isolation and morphological characterisation ......... 178 peR amplification and DNA sequencing ........................... 178 Morphotype determination ... .............. .... .................. ... .. . 179 Pathogenicity trial ...................................................... 180 Discussion ........................................................................ 180 References . .................... ...... ............................................. 182 Chapter Seven: Mycosphaerella Species Associated With Leaf Blotch Disease of Eucalyptus globulus in Ethiopia .. .... ..................... 215 Abstract ......................................... ................... ............216 Introduction .... .. .................. .. ....................................... ....... 217 ......................................................219 Materials and Methods ....................... .. .. ......... 219 Sample collection and isolations ................................ .... 220 Morphological characterisation ...................................................... 221 DNA extraction peR amplification .... .... .............................................. 221 DNA sequencing andphylogenetic analysis .. ................. ........ 222 Results ..................... .................. . ................. . ... ...........222 ....... ...................... .. ..... 222 Sample collection and isolation llforphological characterisation .................................... 223 DNA sequencing and phylogenetic analysis . . ......................... 224 Discussion ...... ......... .. ......... .... ..... .......... .......... .. ......... .. .... 224 References .............. .. ........................................................ 226 Summary ................................................................................. 247 ACKNOWLEDGEMENTS I am very grateful to God for guiding me and for giving me the strength and patience to accomplish this work and for cherishing me ih times of joy and sorrow. I would like to extend my gratitude and SIncere appreciation to the following institutions and people who have made significant contributions to the completion of this study. Without your assistance this study would not have been possible. My sincere thanks go to Dr. Jolanda Roux for her unqualified moral support and technical guidance from the inception to the finalisation of this project. Your guidance, comments and above all your friendship were instrumental to my accomplishment. I have learnt a lot from your wealth of knowledge on forest pathology. I am also very much indebted to Professor Michael J. Wingfield, not only for accepting me to be part of F ABI but also for his guidance, comments and support, as well as for his moral encouragement whenever I needed it. I thank Professor Brenda D. Wingfield for her assistance in the area of molecular biology and for her constructive comments and suggestions on the content of this study. The wonderful people in the Forestry and Agricultural Biotechnology Insti tute (FABI) deserve special thanks for their extended support during my study at the institution. I have learnt a lot from each one of you. I thank you all. I am really proud of being a member of this highly renowned institution. I would also like to thank Bernard Slippers, Martin Coetzee, Mauricio Marin, Gavin Hunter and Juanita de Wet for their tireless assistance in specific chapters of this thesis. My wife Yeshi Ketema and my daughter Rekik Alemu deserve special thanks for their understanding and patience in my absence from home. Yeshi, without your moral support, encouragement, endurance and love, it would have been difficult to accomplish this work. I very much appreciate your determination to bear alone the responsibility of bringing up our daughter in the three years of my absence. I am proud of you and I love you very much. I do not have enough words to express my gratitude to my mother Shewaye Demessie. Your determination, dedication and all round support have been the comer stone of all my achievements. You are a wonderful mother. I honour you and love you very much. I also thank my aunt Lackech Demessie for being with me and supporting me in difficult times. I really appreciate this support. I thank my brothers Getachew Shale, Fitawek Gebre Tsadick, Afework Gebre Tsadick, Lakew Bekele, my sisters Tezeru Ayele, Haregua Zewde as well as Betelihame Girma, Tizita Girma, Eden Girma, Mulumbet Girma, Firegenet Alemu and Etaferu Getachew for their continuous moral support and encouragement as well as for the support you gave to my wife Yeshi and for taking care of my daughter Rekik. Many, many thanks go to my mother-in-law Desta Wolde Gebrael, my father-in-law Ketma Meshesha as well as my brother-in-law Binyam Ketma and sister-in-law, Jerusalem Ketema for closely attending my family in my absence. It is a blessing to have had you on my side at such a critical time. Mesfin Woje, Belay, and Kedir also deserve special thanks for taking care of my family while I was away. Dr. Pia Barklund deserves special honour for her all-rounded assistance from the inception up to the completion of this study. I also thank my colleague Dagne Duguma for his unqualified support during the fieldwork and thereafter. I thank the Agricultural Research and Training Project (ARTP) of the Ethiopian Agricultural Research Organisation (EARO) for the financing of my study. I also thank the Forestry Research Centre and its staff for facilitating and supporting the fieldwork . If Wondo Genet Forestry College, Munesa Shashemene Forest Development and Marketing Enterprise and the lima Zonal Agricultural Bureau of Oromia region are aclmowledged for allowing me to do field work in their forests and for their assistance during the field work. Lastly, but not least the University of Pretoria, FABI and the Tree Pathology Cooperative Programme are thanked for providing the infrastructure and equipment for the lab work, as well as for funding. III PREFACE In several parts of Africa, exotic tree species are planted mainly in agroforestry development programs, for reduction of soil erosion, run-off control to combat desertification and rehabilitation of degraded land. They also contribute to the production of fuelwood, sawn timber and in some cases for pulp and paper production. In Ethiopia, planting exotic species commenced 110 years ago with the introduction of Eucalyptus globulus. Currently, several exotic species including those of Eucalyptus, Pinus, Acacia and Cupressus have been planted in Ethiopia. The government, the community and individual small-scale farmers own these plantations. These plantations contribute to the production of round wood for sawn timber, poles and posts and to meet wood requirements for local use, such as for construction material and for wood fuel. Eucalyptus spp. are the preferred planting stock, especially for fuel wood owing to their rapid growth and immediate economic return. Pinus spp. and Cupressus spp. are mostly planted in state owned forest areas. Plantations of exotic speCIes are successful in most areas where they have been planted. The success of these plantations is ascribed, at least in part, to the separation of the trees from their natural enemies. Despite this, pathogenic fungi, including native and introduced organisms pose serious threats to the development of exotic plantations. In Ethiopia, even though plantations of exotic species commenced over a century ago, little attention has been afforded to diseases of plantation trees. Currently, very little knowledge is available on the status of diseases in these plantations. The aim of studies making up this thesis has, therefore, been to increase the knowledge base pertaining to diseases in Ethiopian plantations. The studies in this thesis focus mainly on identifying and recording the major diseases found in plantations of Eucalyptus and Pinus species. The thesis is comprised of seven separate chapters and each should be seen as an independent unit. Except for chapter one, the remaining six chapters were structured based on results of disease surveys conducted in 2000 and 2001. They have been presented as separate manuscripts and, therefore, some redundancy in introduction and methodologies used, could not be avoided. IV Chapter one of the thesis presents a review of the impact and importance of diseases recorded on the most commonly planted exotic forestry species in Africa. The review discusses briefly, diseases recorded on Eucalyptus, Pinus, Acacia and Cupressus species. Root diseases, stem diseases, foliage diseases and wilt diseases recorded on plantations of exotic species in Africa have been included in the review. Knowledge pertaining to the disease situation on trees in Ethiopia was also evaluated and the lack of information on this aspect of forestry in the country is highlighted. In order to manage forests and plantations effectively and to obtain maximum returns from them, it is essential to obtain information on the prevalence of diseases and their importance. In 2000 and 200 1, disease surveys were conducted in major plantation areas situated in South, South Western and Western Ethiopia. Chapter two of the thesis deals with the results of these disease surveys, focusing on plantations of Eucalyptus and Pinus spp. This chapter presents the first record of major diseases in Ethiopian plantation forests. Armillaria root rot was one ofthe most common diseases found associated with Pinus spp. It was, however, also found on some indigenous and exotic species. Although Armillaria root rot had previously been recorded from Ethiopia, there was uncertainty regarding the identity of the fungus found in the country. Chapter three discusses the results of the survey on the distribution and host range of this pathogen in plantations of Ethiopia. Also included is a taxonomic study, identifying the Armillaria sp. responsible for the disease in areas that we have considered. Eucalyptus camaldulensis is one of the most widely planted Eucalyptus species in Ethiopia. A serious stem canker was frequently observed on E. camaldulensis planted in South and South Western Ethiopia. Preliminary investigations suggested that the disease was Coniothyrium stem canker. Chapter four presents the results of a study aimed at identifying this stem canker pathogen. Chapter five of this thesis deals with Botryosphaeria stem canker of Eucalyptus spp. Botryosphaeria spp. are well known as wound and stress related opportunistic pathogens. Symptoms of Botryosphaeria stem canker, including production of brown v SUMMARY In Ethiopia, the planting of exotic species commenced with the introduction of Eucalyptus globulus approximately 110 years ago. Today several different Eucalyptus, Pinus, Cupressus and Australian Acacia species are planted to provide wood for fuel/energy and raw material for furniture and construction. In many areas, people are dependent solely on wood to provide for their basic fuel and construction needs. Despite this, little attention has been given to improve the silvicultural and management practices of plantations in Ethiopia. In particular, disease surveillance and management has never received due attention. The aim of the studies that make up this thesis have been to address the issue of diseases of plantation trees in Ethiopia. Studies have thus focused on the prevalence, identity and importance of major diseases of especially Eucalyptus and Pinus spp. As a background to this thesis, available information on diseases of exotic tree species in Africa has been reviewed and this is presented in the first chapter. In the review, diseases of the major exotic plantation species including Eucalyptus, Pinus, Cupressus and Acacia species have been considered. A section was also devoted to highlight tree diseases reported from Ethiopia. The review shows clearly that there is a great lack of information on diseases of exotic plantation species in most African countries, with the exception of South Africa. This suggests the need for more pathology studies in African plantations. The review also highlights the importance of diseases in plantation forests. In Ethiopia, little information is available on tree diseases in plantation forests. To partially address this problem, disease surveys were conducted in 2000 and 2001 in Eucalyptus and Pinus plantations in South and South Western Ethiopia. The results of this survey showed that a number of pathogens, known from other countries, including Armillaria root rot, stem canker and foliage diseases are found in plantations of Ethiopia. The major diseases discovered during the survey are discussed in Chapter two of this thesis and an indication is given of their impact and distribution. During the disease survey, Armillaria root rot was found to be associated with both exotic and native tree species. Morphological and molecular identification techniques revealed that the Armillaria sp. collected in this study is A. jitscipes. This is discussed in chapter three, where I also provide preliminary data regarding the host range and distribution of Armillaria root rot in Ethiopia. Prior to this study it was suggested that A. mellea is responsible for Armillaria root rot of 247 hard woods in Ethiopia. The current study, however, showed that at least two Armillaria spp., A. mellea and A. Jusicpes are causing Annillaria root rot in the country. Of significance is the fact that A. jilscipes was isolated from two indigenous tree species, A. abyssinica and J excelsa. Chapter four of this thesis deals with the identity of the fungus causing stem canker on Eucalyptus camaldulensis. Disease symptoms identical to those caused by Coniothyrium zuluense were commonly found on E. camaldulensis in restricted areas in Western Ethiopia. The causative agent was detennined based on DNA sequence analysis of the ITS 1, ITS 2 and 5.8S gene region and 0- tubulin genes. According to the phylogenetic tree generated for these sequence data, the Ethiopian Coniothyrium isolates seem to be closely related to C. zuluense, however, the Ethiopian isolates fonned a separate group. This may suggest that C. zuluense represents a species complex, but this needs further investigation. Coniothyrium canker is considered to be one of the most serious diseases of Eucalyptus spp. especially to the sawn timber and construction industry as it weakens and flaws the timber. It occurrence in Ethiopia is, therefore, of great importance. Disease symptoms similar to those of Botryosphaeria canker on Eucalyptus were commonly observed in all the areas where surveys were conducted. Botryosphaeria spp. are known as opportunistic stress related and endophytic pathogens on a wide range of woody plants, worldwide. In Ethiopia, symptoms similar to those associated with Botryosphaeria infection elsewhere, were found in almost all plantations surveyed. The disease was found on several Eucalyptus spp. including E. globulus, E. saligna, E. grandis and E. citriodora. Both morphological and molecular identification techniques were used to detennine the identity of the fungus and the results are presented in chapter five. It was shown that B. parva is responsible for Botryosphaeria stem canker of Eucalyptus spp. in Ethiopia and the pathogenicity of Ethiopian isolates was also tested. This pathogen can have a serious effect on Eucalyptus in Ethiopia, as growing conditions in the country are often harsh and many people rely on coppicing to reproduce their stands. All these factors are conducive to stress and thus to Botryosphaeria infection. Diplodia pinea is a fungus that commonly resides in the cones of Pinus spp. and it tends to move from these sites to infect stems, when trees are under stress. Therefore, isolations were made from Pinus patula cones to detennine whether D. pinea was present in these structures in Ethiopia. Chapter 6 of the thesis provides results of this study. It was expected that D. pinea would be the most common inhabitant of the cones. Contrary to this, a Fusicoccum sp. was found more 248 frequently than D. pinea. The results presented in this chapter show clearly that the A morphotype of D. pinea is found in cones of P. patula in Ethiopia. The Fusicoccum sp. found associated with P. patula cones is most closely related to B. parva. Results of greenhouse inoculation studies showed that both these fungi are pathogenic to Pinus tadea, with D. pinea being the more pathogenic. Serious leaf spot and shoot die-back symptoms were observed on leaves of E. globulus at several localities. The leaf blotch symptoms closely resemble those caused by Mycosphaerella spp. Even though 30 different Mycospha erella spp. are known to be associated with Eucalyptus species world-wide, the cause of Mycosphaerella leaf blotch on E. globulus in Ethiopia is not known. Morphological and DNA based comparisons were used to determine the identity of the species found in Ethiopia and the results are provided in chapter seven. I was thus able to show that three Mycosphaerella spp. namely, M marksii, M. grandis and M nubi/osa are involved in causing Mycosphaerella leaf disease of E. globulus in Ethiopia. This is the first report of these species from Ethiopia and the first report of M grandis from a country other than Australia. The results presented in the various chapters making up this thesis provide the first detailed studies on diseases of plantation trees in Ethiopia. Most tree diseases discussed in the thesis are first reports for the country. The thesis provides information on the identity of the pathogens and their significance in plantation development in Ethiopia. It also highlights the need for adequate management and silvicultural practices, as well as the need for selecting disease tolerant provenances and/or individuals. The information presented in the thesis also expands the host range and geographic distribution of all the pathogens included in the study, giving the study international significance. 249 exudate as well as stern cracking, were commonly found on several Eucalyptus spp. in all areas surveyed. This chapter discusses the results of the morphological and DNA based comparisons conducted to determine the identity of the Botryosphaeria spp. in Ethiopia. Chapter six investigates the occurrence of species of Botryosphaeria sensu lato in Pinus patula cones in Ethiopia. Diplodia pinea is a common endophyte and stress related pathogen in Pinus spp. Interestingly, the most common inhabitant of P. patula cones in Ethiopia proved to not be D. pinea. In this chapter I discuss these findings, the identification of the two Botryosphaeria type fungi present, as well as their relative pathogenicity to Pinus patula. Serious leaf spotting and shoot die-back typical of Mycosphaerella leaf blotch disease was observed on E. globulus, widely planted in cooler areas of Ethiopia. The last chapter of the thesis deals with the identification of the causal agents of MLD in Ethiopia. Ascospore germination patterns, cultural morphology and DNA sequence data were used to determine the identity of the fungus. The research presented in this thesis represents the first comprehensive senes of studies on diseases of plantation trees in Ethiopia. From an Ethiopian point of view, the information contained in this thesis will hopefully create due awareness among forest managers regarding the importance of diseases in plantation development. I also hope that they will form a foundation and pave the way for further studies on these and other tree diseases in Ethiopia in the future. VI INTRODUCTION The demand for forest products has increased considerably over the past 100 years. The result has been the near depletion of indigenous wood sources. This increasing demand for timber and fuel energy has necessitated the establishment of large areas of plantation forests. Plantation forests can be developed using either exotic or native tree species. However, there is a growing worldwide trend towards the establishment of plantations of exotic tree species, especially in the tropics and sUbtropics (Evans 1984, Turnbull 1991, Persson 1995). These provide a source of energy, paper and pulp and wood extracts such as tannins. Plantations also playa substantial role in agroforestry development, the reduction of soil erosion, run-off control, combating desertification, rehabilitation of degraded land, fodder and they provide shade and shelter (Evans 1984). The establishment of exotic plantation forestry has grown substantially worldwide. It is estimated that approximately 78 million hectares of exotic plantations exist today (Vercoe 1995). The most commonly planted species are Pinus radiata D. Don, P. patula Schiede & Deppe, Eucalyptus grandis Hill ex Maid and other Eucalyptus hybrids and species, Australian Acacia spp. and Tectana spp. Plantations of Eucalyptus spp. alone covers about 10 million ha worldwide (Eldridge et al. 1997). The largest exotic plantation forestry countries are Chile, Brazil, Indonesia, South Africa, Australia and New Zealand (Vercoe 1995). In these countries, plantations are utilized mainly for the paper and pulp industries and for sawn timber, forming multi-billion dollar industries. Many countries in Africa grow large areas of exotic plantations of Eucalyptus, Pinus, Cupressus and Acacia spp., to provide fuel and timber as well as for the production of paper and pulp for the local and especially for the export markets (Evans 1984, Vercoe 1995). Exotic plantations in South Africa for example cover approximately 1.5 million ha, of which E. grandis and P. patula are the dominant species (Denison & Kietzka 1993, Anonymous 1998). In Kenya, almost all wood required for fuel-wood and industrial purposes are obtained from plantations of exotic species, which include P. patula, Cupressus lusitanica Mill. and different Eucalyptus spp. (Ciesla, Mbugua & Ward 1995). Exotic trees are commonly used as plantation species in Ethiopia. The establishment of exotic plantation forestry in this country commenced with the introduction of Eucalyptus species, approximately 110 years ago, around 1890 (Persson 1995). Since then, a number of other exotic 2 tree species including Pinus spp., Acacia spp., and Cupressus spp. have been introduced and planted in different parts of the country. Plantations of exotic tree species cover more than 200 000 ha ofland (Anonymous 1994, Vercoe 1995). To meet the increasing demands for wood and wood products, both natural forests and plantation forests have to be protected. Threats against these wood sources include fire, indiscriminate cutting, encroachment, pests and diseases. The occurrence of pathogens on woody plants is a common phenomenon on trees growing in natural forests, plantations and ornamentals and has resulted in serious losses to forestry programmes worldwide (Manion 1981, Wingfield 1990). In many countries exotic plantation forestry has been highly successful, largely because these trees have been removed from their natural enemies, found in their areas of onein , This, might aCC01.mt specifically to the success of Eucalyptus spp. in Africa (Wingfield 1990, Persson 1995, Bright 1998). Exotic plantation species, though separated form their natural enemies, have on the other hand, been planted in a new environment. They can thus be exposed to potentially new pests and diseases, to which they do not have a natural resistance (Wingfield 1990). This situation may increase the disease risk associated with exotics (Wingfield 1987a). Despite being isolated from their natural enemies, a number of serious disease problems have emerged on exotic species in most countries where they have been planted (Wingfield 1990). Both native pathogens originating from endemic hosts as well as accidentally introduced pathogens cause damage in plantations (Wingfield, Swart & Kemp 1991). A large number of new and emerging tree disease have been recorded in the last decade. This may be attributed to the increased movement of people and plant material between countries (Palm 1999, Wingfield & Roux 2000a, Wingfield et al. 2001, Allen & Humble 2002). Pathogenic fungi pose an enormous threat to trees planted in monoculture, due to the uniform age of the trees and the reduced diversity of plantations (Hodges 1979, Wingfield 1990). It is generally believed that mono cultures are more vulnerable to disease and pest damage. The damage to monocultures could be serious because risk is not spread widely. Monocultures are also believed to facilitate the emergence of new and more virulent forms of pathogens (Hodges 1979, Heybroek 1980, Libby 1982, Leakey 1987, Roberds, Namkoong & Skf0ppa 1990, Persson 1995). On the other hand, it has been shown that the susceptibility of monoculture to disease has been very much generalised and unbalanced. Choice of species and provenances and site selection has more influence than effect of stand composition on incidence of disease in plantations (Chou 1981). 3 Similarly, pathogens with wide host ranges for example Armillaria spp. (Raabe 1962) and Phytophthora cinnamomi Rands (Newhook & Podger 1972) could have a devastating effect even in mixed stands. Fungal tree diseases, especially, have had a major impact on woody hosts world-wide. This includes those caused by both introduced and native pathogens. Chestnut, which was one of the major forest species in North America, has been nearly eliminated by the Chestnut blight fungus, Cryphonectria parasitica (Murr.) Barr (Hepting 1974, Anagnostakis 1987, 1988). Similarly, Dutch elm disease caused by Ophiostoma ulmi (Busim.) Nannf. and Ophiostoma novo-ulmi Brasier has devastated native elm trees in Europe and North America (Gibbs 1978, Brasier 1990). Cronartium ribicola Fischer, the cause of White pine blister rust has had a dramatic impact on White pines in America (Ziller 1974). Similarly, Dothistroma needle blight caused by Dothistroma pini Hulbury (syn. = D. septospora), which was initially identified in the United States of America, occurs in eastern and southern Africa, Chile, Australia and New Zealand and causes devastating damage on exotic P. radiata plantations in these countries (Ivory 1968, Gibson 1972, Lundquist & Roux 1984). Pathogens of native plants have also developed the capacity to infect exotic plantation species. For example, rust caused by Puccinia psidii G. Winter, a pathogen on native Mytraceae in South and Central America, suddenly appeared on various exotic Eucalyptus spp. planted in this region (Coutinho et al. 1998). This review focuses on the importance and impact of pathogens on exotic plantations in some African countries. Special consideration is given to the disease situation in plantations of Ethiopia. Hence, in the following sections, some of the most important diseases of the major exotic plantation species in Africa are discussed. Special emphasis is given to species of Eucalyptus, Pinus, Ctpresses and Australian Acacia, because they are dominant in the plantations of Ethiopia and other African countries. NURSERY DISEASES As a result of the use of non-sterilised growth medium and water in many African nurseries, diseases cause significant losses in production. Nursery diseases reduce germination, cause seedling death and enhance malformation and stunting. These result in seedling rejection and lower field survival rates. The most commonly observed disease symptoms include damping-off, root rot, 4 blight, stem cankers and leaf spots (Darvas, Scott & Kotze 1978, Shanna, Mohanan & Florence 1984). Nursery plants are predisposed to nursery diseases by environmental and management factors. Control of nursery diseases relies heavily on sound sanitation and management. Nursery diseases are commonly spread by infected seed, water and planting media. Mechanical and insect wounds are other factors increasing disease risks. Watering and weeding regimes, spacement and nutrition all impact on nursery health (Bloomberg 1981, Anderson, Belcher & Miller 1984, Dwinell, Barrows-Broaddus & Kuhlman 1985). Available reports of diseases in African nurseries have been summarised in Table 1 and will not be discussed individually. DISEASES OF PLANTATION PINUS SPP. IN AFRICA Plantations of Pinus spp. have been established especially in South, East and Central Africa (Gibson 1979). The most commonly planted species are P. patula, P. radiata, P. caribea L., P. elliottii Englam and P. taedea L. (Gibson 1975, Evans 1984). These trees are grown mostly for industrial purposes, sawtimber, pulpwood and plywood veneers (Evans 1984, Ivory 1987). Root diseases Armillaria spp. include some of the most prominent causes of death and decay of coniferous trees and shrubs in natural forests, native and exotic plantations, and gardens world-wide (Wargo & Shaw 1985, Ivory 1987, Shaw & Kile 1991). Armillaria spp. have a wide host range and occur in both tropical and temperate areas of the world (Ivory 1987, Shaw & Kile 1991). Reports indicate that Armillaria spp. are dominant in deciduous forests as secondary pathogens attacking trees weakened by biotic or abiotic stress. Such stress includes defoliation by insects, frost, drought, foliage diseases, stem cankers, water logging, soil compaction and air pollution (Wargo & Shaw 1985). Armillaria spp. can, however, also be aggressive primary pathogens, frequently killing healthy trees of all ages (Wargo & Shaw 1985). A typical sign of Armillaria root rot in forests and plantations are the concentration of dead and dying trees in circular patches. Infections of trees take place through the roots, resulting in root rot and eventual tree death. Root death results in yellowing and wilting of crowns, resin exudation 5 around the root collar and basal areas of stems and well developed white mycelial mats under the bark of infected trees (Bottomley 1937, Liickhoff 1964, Ivory 1987). The wood beneath the fungal mycelium has a soft, wet, stringy, white rot. Under favourable conditions rhizomorphs will fonn (I vory 1987, Shaw & K.i1e 1991). It has been reported that Armillaria spp. colonise the stumps of indigenous trees and serve as a source of ino'culum for the infection of exotic tree species especially, Pinus spp. (Kotze 1935, Bottomely 1937, Liickhoff 1964, Gibson 1970, Ivory 1987). Armillaria mellea (Vahl:Fr.) Kummer sensu lato and A. heimii Pegler are names commonly used for the causal agents of Armillaria root rot in Africa (Ivory 1987, Coetzee 1997). Mohammed (1994) assumed that A. mellea is the cause of Armillaria root rot in temperate regions of Africa. However, recent reports have indicated that A. heimii, A. meflea sensu stricto, A. mellea (Vahl.:Fr.) P. Kumm. sub sp. aJricana Mohammed, A. mellea (Vahl.:Fr.) P. Kumm. var. cameruensis Henn. and A. juscipes Petch occur in African plantations (Gibson 1975, Pegler 1977, Ivory 1987, Masuka 1989, Mwangi, Lin & Hubbes 1989, Abomo-Ndongo & Guillaumin 1997, Coetzee et al. 2000). Armillaria root rot has been reported in P. patula, P. elliottii, P. oocarpa Shchiete and P. radiata plantations in Zimbabwe, Uganda, Kenya, Tanzania, Malawi and South Africa (Lee 1970, Gibson 1975, Ivory 1987, Masuka 1989, Mwangi etal. 1989, Abomo-Ndongo & Guillaumin 1997, Coetzee et al. 2000). In South Africa, Armillaria root rot is known in plantations of several pine species including P. patula, P. caribea and P. elliottii. (Kotze 1935, Bottomley 1937, Liickhoff 1964). Despite this, the identity of Armillaria found in South Africa was not detennined until recently. Initially it was arbitrarily called A. mellea (Lundquist 1987) and later the pathogen was assumed to be A. heimii (Wingfield & Knox-Davies 1980a). Coetzee et al. (2000), however, showed that the isolates obtained from plantations in South Africa are clearly different from both A. mellea and A. heimii and rather represents A.juscipes. In Zimbabwe, Armillaria root rot was considered to be one of the most serious threats to Pinus plantations in that country. Affected species included P. elliotii and P. oocarpa (Masuka 1989). Mwenje & Ride (1996) characterized the Armillaria isolates from Zimbabwe into three morphological groups, namely groups I, II, and III. Coetzee et al. (2000) showed that most Armillaria isolates from Zimbabwe (Harare area) resemble neither A. Juscipes that is found in South Africa, nor A. mellea, but represents A. heimii. 6 In Kenya Armillaria root rot is widely spread in plantations, indigenous forests and cash crops (Mwangi et aI. 1994). It was isolated from P. patuIa, P. eIliottii and P. radiata (Abomo-Ndongo & Guillaumin 1997). Mewnje and Ride (1996) showed that both A. mellea and A. heimii are found in Kenya. Sphaeropsis sapinea (Fr.:Fr.) Dyko and Sutton (syn. DipIodia pinea (Desm.) Kickx ) has a worldwide distribution and is associated with a wide range of disease symptoms (Punithalingam & Waterston 1970, Gibson 1979, Swart, Knox-Davies & Wingfield 1985, Swart, Wingfield & KnoxDavies 1987). It was reported to cause serious root disease on P. taeda and P. eIliotii in South Africa and Swaziland (Wingfield & Knox-Davies 1980a, Swart et aI. 1985, Swart et aI. 1987). The above ground symptoms of S. sapinea infection include discoloration of the root collar, foliage chlorosis, needle fal1, exudation of resin and death of tree tops. It also causes dark blue radial lesions in young roots, which extend into the lateral root and even up to the trunks of trees (Wingfield & Knox-Davies 1980a, Swart et aI. 1985). This root disease is associated with stress such as overstocking, drought and poor site conditions (Wingfield & Knox-Davies 1980a, Swart et aI. 1987). Rhizina undulata Fr. (= R. inflata (Schaff.) Krast) is a fire associated pathogen that causes root rot of Pinus and other conifers (Morgan & Driver 1972, Lundquist 1984a, Wingfield, Swart & Von Broembsen 1988). It is found in Europe, North America, Africa and Asia (Gibson 1979). According to Germishuizen (1984), Rhizina root rot caused serious losses to Pinus plantations in South Africa. It was also reported to occur in Swaziland (Germishuizen 1979). Studies indicated that slash burning, which was conducted to remove logging debris, induced infection by R. unduIata (Morgan & Driver 1972, Germishuizen 1984, Ivory 1987). Phytophthora spp. are known to cause serious damage to woody plants in many parts of the world and have a wide host range (Zentmyer 1980, Tidball & Linderman 1990, Strouts & Winter 1994, Erwin & Ribeiro 1996). Phytophthora spp. are important pathogens in forestry nurseries (Von Broembsen & Donald 1981) and in sites that are wet or with poor soil nutrients (Erwin & Ribeiro 1996). In South Africa, P. cinnamomi is associated with root rot and death of several Pinus spp., especially P. patuIa, both in nurseries and plantations (Von Broembsen 1984, Wingfield & KnoxDavies 1980b, Linde, Kemp & Wingfield 1994a). Other Pinus spp. affected includes P. pinea L., P. radiata, P. elliottii and P. patuIa (Wingfield & Knox-Davies 1980b, Von Broembsen 1984, Ivory 1987). 7 Pythium spp. are not considered as important pathogens of mature pine trees (Markes & Kassaby 1974). Yet, some Pythium spp. cause death of young Pinus seedlings planted out in the field (Linde, Kemp & Wingfield 1994b). Some reports from South Africa indicate that a number of Pythium spp. are associated with death of P. patula established on previously cultivated lands. P. irregulare Buisman was consistently isolated from dying plants as well as from the soil in Pinus plantations and is considered as a major cause for the failure of P. patula planted on previously cultivated land (Linde, Kemp & Wingfield 1994c). Helicobasidium compactum Boedijn causes purple root rot and is found in Asia, Europe, Australia and Africa. It was reported from several Central and Southern Africa countries (Gibson 1979). It infects the roots of a wide range of plants including both hardwoods and conifers (Browne 1968). H. compactum is associated with root and collar rot of Pinus spp. (Bottomley 1937, Gibson 1979) in Zimbabwe, Nigeria, Kenya, Malawi, Tanzania (Browne 1968, Gibson 1975) and South Africa (Bottomley 1937). It causes stunting of terminal shoots, yellowing of needles, wilting and death of trees (Bottomley 1937). Infection by Helicobasidium is also associated with collar constriction. It is possible to find purplish brown fungal growth at the base of the tree and at the lowest parts of the branches (Bottomley 1937). Pseudophaeolus root and collar disease, caused by Pseudophaeolus baudonii (Pat.) Rev. (syn. Phaeolus manihotis Heim, Polyporus baudoni Pat.) is distributed throughout central and Southern Africa (Gibson 1979, Rattan & Pawsey 1981). This pathogen attacked Pinus spp. in Congo Brazzaville (Rattan & Pawsey 1981), Ghana (Ofosu-Asiedu 1975) and South Africa (Liickhoff 1955, 1964, Van Der Westhuizen 1973, Lundquist 1987, Wingfield 1987b). Infection spreads by means of root contact. The white to yellow mycelial fans found beneath the bark at the base of trees is used to distinguish the disease. It also produces large yellow fruiting structures on the roots near the base of infected trees (Van Der Westhuizen 1973, Ivory 1987, Wingfield 1987b). Stem diseaseslcanker Sp/1aeropsis sapinea is arguably the most common stem pathogen on Pinus spp. in Africa. Apart from causing root rot, S. sapinea is best known for the stem disease it causes on Pinus spp. It is known to be an opportunistic pathogen (Marks & Minko 1969, Swart et al. 1985) that subsists in cones and stems of healthy pine trees as endophytes (Smith et al. 1996a). Disease symptoms are 8 only expressed when trees are under stress (Zwolinski, Swart & Wingfield 1990a, Smith et al. 1996a). Sphaeropsis sapinea is known in pines wherever they are native and has been recorded from most countries where these trees are grown as exotics (Currie & Toes 1978, Gibson 1979, Palmer & Nicholls 1985). It has been associated with a wide range of disease symptoms, both in nurseries and on mature trees in the plantation. Plantation related symptoms include collar rot, shoot blight (Gibson 1979, Wingfield & Knox-Davies 1980b), blue stain, stem and branch cankers (Marks & Minko 1969, Wright & Marks 1970) and root disease (Punithalingam & Waterston 1970, Gibson 1979, Palmer, McRoberts & Nicholls 1988). In Africa, S. sapinea has been recorded as a pathogen of Pinus spp. in countries such as Malawi, South Africa, Swaziland, Zimbabwe and Tanzania (Gibson 1964, Lee 1970). Pinus radiata and P. patula are especially susceptible to infection (Swart et al. 1987, Wingfield 1990). In South Africa, a close association with hail damage has been shown (Swart et al. 1987). In summer rainfall areas of the country, where hail damage is frequent, the planting of P. radiata was abandoned (Wingfield 1990). Similarly, P. elliottii replaced P. patula in hail prone areas due to the susceptibility of P. patula to infection (Lundquist 1987). It was estimated in 1986 that infection by S. sapinea in South Africa has resulted in more than nine million Rand of loss per annum (Zwolinski et al. 1990a). Infection by S. sapinea not only results in death of trees, but reduces profit through the reduction of increment (Wright & Marks 1970), the loss of marketable volume (Currie & Toes 1978, Zwolinski et al. 1990a) and blue stain of timber (Laughton 1937, Da Costa 1955, Eldridge 1961). South African exports of saw logs often loose as much as half their marketable volume due to blue stain of timber (Zwolinski, Swart & Wingfield 1990b). Needle diseases Dothistroma needle blight, caused by Dothistroma pini Hulbury (=Dothistroma septospora Dorog. Morelet) is one of the most important needle diseases of exotic Pinus spp. Dothistroma needle blight was first recognized as a serious disease of ornamental, windbreak and Christmas trees in the United States of America. The disease was also reported from several other countries, including East and Southern Africa, Chile, Australia and New Zealand in exotic Pinus plantations (Ivory 9 1968, Gibson 1972, Peterson 1977, Lundquist & Roux 1984). According to Ivory (1968), three varieties of Dothistroma are found on needles of Pinus spp. in different parts of the world. These varieties are D. pini Hulbery var. keniensis Ivory, D. pini var. pini and D. pini Hulbery var. Iinearis Thyr & Shaw. Of these three varieties only D. pini var. keniensis has been reported from Africa, whereas D. pini var. pini is distributed in different parts of the world and D. pini var. linearis is mainly found in the United States of America and Canada. In Africa, Dothistroma needle blight was first observed in Tanzania (Gibson, Christensen & Munga 1964, Ivory 1968) and later in Kenya, Zimbabwe, Malawi and Uganda (Gibson 1964, Ivory 1968). The disease spread very fast in P. radiata plantations, causing defoliation and stunting of trees (Gibson et aI. 1964). Because of Dothistroma needle blight, the planting of P. radiata, has been mostly abandoned or severely restricted in African countries such as Kenya, Malawi and Zimbabwe (Ivory 1968, Lee 1970, Gibson 1972, Ciesla, Mbugua & Ward 1995). In many other countries, P. radiata has been substituted by P. panda. Lundquist & Roux (1984) reported the occurrence of D. pini in South Africa. It is, however, only found in a very small area of the country and is not considered to be economically important (Lundquist & Roux 1984, Wingfield & Roux 2000b). Dothistroma needle blight first appears on the lower branches of young trees (Gibson et aI. 1964, Wingfield & Roux 2000a). Early infection of needles produce yellow bands that later develop a reddish tint as the disease develops. Following this chlorosis, necrosis of the needles appears, first at the base of the tree, later spreading higher up the tree. In favourable conditions, the disease results in severe defoliation, to the extent that only the needles at the extremes of the branches remain (Gibson et al. 1964). Black fruiting bodies commonly appear on the dead epidermis in the · red bands (Gibson 1964, Wingfield & Roux 2000a). In severe cases infection may result in malformation and tree death. Dothistroma needle blight is more severe in younger trees less than ten years in age (Gibson 1972). In Tanzania, death of pine trees associated with Dothistroma needle blight was not experienced in areas where the rainfall was below 1500 mm/annum. In these areas only the diameter and height growth was reduced considerably (Christensen & Gibson 1964). A study conducted in Kenya on the effect of defoliation by D. pini on the increment rates of P. radiata, showed that diameter growth is considerably reduced. Nearly all growth of trees is inhibited when 75% of the foliage is affected (Christensen & Gibson 1964, Gibson et aI. 1964). 10 Cercospora needle blight is the other needle disease recorded on various Pinus spp. in several African countries (Gibson 1972, 1979, Ivory & Wingfield 1986, Ivory 1987) including Madagascar, East Africa, South Africa, Swaziland, Zambia and Central Africa (Gibson 1964, 1979, Ivory & Wingfield 1986, Ivory 1994). Cercospora needle blight is caused by Cercoseptoria pini- densiflorae (Hori. & Nambu) Deightn (Telemorph= Mycosphaerella gibsonii H. Evans). In South Africa it infects P. patula and P. radiata (Ivory & Wingfield 1986, Wingfield & Roux 2000a). The disease causes severe defoliation on young pine seedlings in nurseries and plantations. The fungus initially infects old needles and in severe cases, it also attacks young needles (Wingfield & Roux 2000a). Infection causes light green bands on the needles, which later change to yellow, brown and finally to a grey colour. Fruiting bodies are seen on dead needles and are "brush like" and grey in appearance (Wingfield & Roux 2000a). DISEASES OF PLANTATION EUCALYPTUS SPP. IN AFRICA In Africa, the production of Eucalyptus trees takes place either by the raising of seedlings from seed, the production of cuttings or by tissue culture (Leakey 1987, Denison & Kietzka 1993). In South Africa alone, approximately 500 000 ha are planted to a variety of Eucalyptus spp. and clones (Anonymous 1998). Other African countries also depend on Eucalyptus spp. for export income and importantly, as a substitute for indigenous trees. E. grandis, E. saligna Sm. , E. globulus Labil., E. camaldulensis Dhen., E. citriodora Hook and E. urophylla Blake are the most commonly planted species (Gibson 1975, Evans 1984). Several pathogens pose a threat to Eucalyptus planting on the continent. The most important ofthese are presented in the following section. Root diseases Phytophthora spp. are among the most common pathogens of Eucalyptus spp. (Marks & Kassaby 1974, Heather, Pratt & Chin 1977, Zentmyer 1980). In South Africa, P. cinnamomi results in death and stunting of E. Jastigata Deane and Maid., E. smithii R. T. Baker and E. fraxinoides Deane and Maid. (Wingfield & Knox-Davies 1980b). Hence, as Linde et al. (1994a) indicated, the susceptibility of these species has necessitated planting of other Eucalyptus spp. not prone to P. cinnamomi root disease. Recently P. nicotianae Breda de Haan has been isolated from diseased and dying E. nitens trees in the Kwazulu Natal Midlands of South Africa (Maseko et al. 2001). 11 Another Oomycetous root pathogen, Pythium splendens H. Braun, has been reported to cause mortality of young E. grandis in South Afiica (Linde, Wingfiled & Kemp 1994d). This fungus has caused a root and root collar disease on established E. grandis in the warmer sub-tropical areas of the country (Linde et al. 1994d). The disease is characterised by reddening of the leaves, rapid wilting as well as girdling of the roots and root conars and consequent death of trees. Lasiodiplodia theobromae (Pat.) Griff. and Maubl. (telemorph Botryosphaeria rhodina (Cooke) Von Arx.), has been reported from the Republic of Congo in association with rot of E. grandis roots (Raux et al. 2000a). The root collars and the stems above soil level exuded kino and developed small cankers. The disease spreads from the roots to the root collars and rest of the stem. The cankers associated with this disease caused a complete girdling, wilting and death of branches (Raux et al. 2000a). In Kenya, Armillaria root rot has been reported on E. microcorys F. Mull and E. saligna (Mwangi et al. 1989, Onsando, Wargo & Waudo 1997). In South Africa, Armillaria root rot has been recorded on Eucalyptus sp. planted on a site cleared of indigenous forest (Bottomley 1937, Kotze 1935, Liickhoff 1964), while in Malawi Armillaria root rot was found associated with E. saligna, E. microcorys and E. pilularis Sm. (Lee 1970). Ivory (1987) reported that Armillaria spp. also infects E. pilularis Sm. in Zimbabwe. Ganoderma species cause root and butt rot on several woody plants including Eucalyptus spp., worldwide (Browne 1968). In Zimbabwe E. grandis trees were infected by this pathogen (Masuka 1990, Masuka & Nyoka 1995). The causative agent was identified as G. sculptrutum Uyod (Masuka & Nyoka 1995). Infected trees showed longitudinal bark splitting, stem swelling and gummosis, with dark to black lesions on the affected roots. Affected trees developed epicormic shoots and trees died from the crown downwards (Masuka & Nyoka 1995). Basidiocarps of the fungus may be found at the base of the stems or attached to lateral roots (Masuka & Nyoka 1995). Infection causes death of trees in patches or death of trees in a line, with the most recently killed trees at the edges of the patches (Masuka & Nyoka 1995). Polyporus baudonii Pat. Ryv., also known as Pseudophaelus baudonii (Pat.) Ryv. and Phaeolus manihotis Heim is found in several African countries on many woody plants (Browne 1968, Gibson 1979, Ivory 1987). This fungus is known to cause root rot on Eucalyptus spp. in South Africa and Ghana (Van der Westhuizen 1973, Ofosu-Asiedu 1975, Wingfield & Raux 2000a). P. baudonii 12 attacks roots and root collars of susceptible trees. Leaf chlorosis, unseasonal leaf shedding as well as die-back of small branches at one side of the crown are characteristic symptoms of infection. The bark of the infected trees also changes colour and becomes cracked and charred as if it has been burnt by fire (Ofosu-Asiedu 1975). Stem diseaselcanker Cryphonectria canker caused by Cryphonectria cubensis (Burner) Hodges is considered to be one of the most serious canker diseases of Eucalyptus spp. in the tropics. It has been reported from different countries in Central and South America, Africa, Asia and Australia (Boerboom & Maas 1970, Hodges, Geary & Cordell 1979, Sharma, Mohanan & Florence 1985, Florence, Sharma & Mohanan 1986, Hodges, A1fenas & Ferreria 1986, Wingfield, Swart & Abear 1989, Conradie, Swart & Wingfield 1990). In Africa, Cryphonectria stem canker has been reported on Eucalyptus from Northern Africa (Gibson 1981), Cameroon (Sharma et al. 1985), South Africa (Wingfield et al. 1989, Conradie et al. 1990), and the Republic of Congo (Brazzavi1e) (Roux et al. 2000b). Cryphonecria canker has been reported on many different Eucalyptus spp. In Africa these include E. grandis in South Africa, E. grandis and E. urophylla in the Congo (Sharma et at. 1985, Conradie et al. 1990, Roux et al. 2000b). It is not known which species were affected in Cameroon and North Africa. Typical symptoms of Cryphonectria canker in South Africa differ from those found in other parts of Africa and the world. In South Africa, the disease is characterised by the formation of swollen, cracked, basal cankers and root/collar rot (Wingfield et al. 1989, Conradie et at. 1990). Young trees die readily from root and root collar infections, while older trees tend to become stunted (Wingfield et at. 1989, Conradie et al. 1990). In the Congo, Cryphonectria canker is characterised by the more typical target shaped stem cankers found on above grounds parts of the trees such as those described from other parts of the world (Roux et al. 2000b). These stem cankers commonly coalesce to girdle and kill and can occur over the length of the stem, often around branch knots (Sharma et al. 1985, Wingfield & Roux 2000a). Long-necked fruiting bodies, with orange spore masses, form abundantly in the cracks and the basal cankers (Wingfield & Roux 2000a). In South Africa the disease has lead to the abandonment of some clones in subtropical areas of the country (Wingfield 1990). Cryphonectria eucalypti Venter & MJ Wingfield, previously known as Endothia gyrosa (Schew) Fr. represents a newly described fungus, which is known to occur only in South Africa and l3 Australia (Venter et al. 2001). In South Africa it is commonly known as a pathogen of minor concern (Van der Westhuizen et al. 1993, Wingfield & Roux 2000a). Symptoms of infection are commonly characterised by the formation of superficial cracks in the bark of trees. These cankers commonly form in bands and may occur over the length of tree stems, although they are often most concentrated towards the bases of trees (Van der Westhuizen et al. 1993, Wingfield & Roux 2000a, Venter et al. 2001). Orange fruiting bodies are common between the cracks on infected stems. In some cases, these cracks provide entry sites for opportunistic pathogens such as Botryosphaeria spp. In some parts of South Africa, the pathogen has been associated with cankers and death of young stressed trees (Venter et al. 2001). Coniothyrium canker is a relatively newly discovered and important stem canker disease of Eucalyptus spp. This disease was first observed in 1988 in South Africa (Wingfield, Crous & Coutinho 1996). The causal agent was described as Coniothyrium zuluense Wingfield, Crous & Coutinho (Wingfield et al. 1996). The disease most typically damages E. grandis propagated from seed. It is also reported to affect several E. grandis clones and hybrids of E. grandis with E. urophylla and E. camaldulensis (Wingfield et al. 1996) in the warmer, humid areas of South Africa. The disease is characterised by discrete necrotic lesions on young green bark. At later stages, it forms large necrotic patches, which may spread over most of the stem. In severe cases, the trees produces epicormic shoots as a result of infection. In advanced stages of disease, infection may lead to top die-back and subsequent reduction in height growth (Wingfield et al. 1996, Wingfield & Roux 2000a). Botryosphaeria spp. and their anamorphs in the genera Fusicoccum, Lasiodiplodia, Sphaeropsis and Microphomopsis have one of the widest host ranges known for any group of pathogens. Amongst these hosts are many species of Eucalytpus. In Africa, confirmed reports of B. rhodina (anamorph: Lasiodiplodia theobromae) on Eucalytpus spp. have been made from South Africa (Smith, Kemp & Wingfield, 1994), the Republic of Congo (Roux et al. 2000b), Uganda (Roux et al. 2001a) and Malawi (Gibson 1964). Recently, a new Botryosphaeria sp., namely B. eucalyptorum Crous, H. Smith et M. J. Wingfield has been described on Eucalyptus in South Africa. This Botryosphaeria sp. is reported to be associated with cankers of the main stems of E. grandis and E. nitens (Deane Et maid.) Maid. (Smith et al. 2001) . Symptoms of infection by BotJyosphaeria spp. range from leaf spots, to stem cankers, tip and shoot blight and root rot (Davison & Tay 1983, Barnard et al. 1987, Shearer, Tippett & Bartle 1987, 14 Smith et al. 1994). Stern cankers are characterized by cracking of the bark and the exudation of resin (Smith et al. 1994). When the bark is removed, extensive resin formation is observed in the cambium and wood (Smith et al. 1994). Trees often recover from current infections, resulting in dead, discoloured heart wood (Smith et al. 1994, Roux et al. 2000b), and such trees continue to grow until the next infection cycle. Stress is known to be a major factor contributing to development of diseases caused by Botryosphaeria spp. on Eucalyptus spp. (Smith et al. 1994). According to Smith et al. (1994), plantations situated on marginal land are especially prone to infection by B. dothidea (Moug.) Ces. De Not. This pathogen is frequently associated with damage from hot and cold winds, late frost, drought, mechanical damage from hail, insect feeding or silvicultural practices (Schoeneweiss 1979, Ramos et al. 1991, Smith 1995, Wingfield & Roux 2000b). B. dothidea occurs as latent endophytic infections in several Eucalyptus spp. (Bettucci & Saravay 1993, Fisher et al. 1993, Smith et al. 1996a, Smith, Wingfield & petrini 1996b). In South Africa, B. dothidea was reported to be found as a latent pathogen in E. camaldulensis, E. grandis, E. nitens (Deane et Maid) Maid. and E. smithii R. T. Bak. (Smith et al. 1996a, 1996b). It causes asymptomatic endophytic infections in the leaves and in the xylem of E. grandis and E. nitens. Symptoms appear only when environmental conditions favour disease development (Bettucci & Saravay 1993). Cytospora spp. and their Valsa telomorphs are commonly isolated from Eucalyptus spp. in association with cankers. Cytospora australiae Speg, C. eucalypticola Van der Westhuizen and C. eucalyptina Speg., have been reported to cause cankers on Eucalyptus spp. in South Africa (Van der Westhuizen et al. 1993, Wingfield & Roux 2000a). Cytospora spp. were also reported in association with stern cankers of Eucalyptus spp. in Congo and Uganda (Roux et al. 2000b, Roux et al. 2001a). In Malawi, Cytospora cankers were observed on E. sa ligna , E. citriodora and E. maculata Hook (Lee 1970). These oppommistic pathogens are mainly isolated from trees under severe stress due to drought, or trees planted in wet swampy areas with poor drainage. Mechanical wounds are also common sites of infection (Shearer et al. 1987, Old, Yuan & Kobayashi 1991, Roux et al. 2001 a). It is assumed that these fungi are endophytes (Smith 1995, Wingfield & Roux 2000b) although this matter has not been comprehensively investigated . 15 In tropical areas of the world, woody plants including Eucalyptus and Acacia spp. suffer from a canker disesase commonly known as pink disease. In South Africa, E. macarthurrii Deane & Maiden is attacked by the pink disease pathogen, Erythricium salmonicolor (Berk & Broome) Burds. [syn. Corticium salmonicolor Berk. & Br., Phanerochaetea salmonicolor (Berk. & Br.) JulichJ (Wingfield & Roux 2000b, Roux et al. 200 1c). Infection by E. salmonicolor causes the inner tissue of the bark, mainly the phloem and the cambium to become brown and eventually die. Later, epicormic shoots develop just below the cankered region. These epicormic shoots also die due to downward spread of infection and a wilting of young shoots (Sharma et al. 1988). Apart from South Africa, pink disease has been reported from Eucalyptus spp. in Nigeria and the Democratic Republic of Congo (Gibson 1964). Leaf diseases Leaf diseases can have a serious impact on the growth of trees. Many fungi have been recorded on Eucalyptus leaves (Gibson 1975, Lundquist & Baxter 1985, Crous, Knox-Davies & Wingfield 1989a). In severe cases these fungi may result in complete defoliation of trees and seriously impact on tree growth. Cylindrocladium leaf blight, caused by several species of Cylindrocladium is one of the most devastating leaf diseases of Eucalyptus spp. Cylindrocladium leaf blight has been reported from several countries, especially in the tropics. Reports from Africa include those from South Africa (Crous, Phillips & Wingfield 1991) and the Republic of Congo (Brazzaville) (Roux et al. 2000b). Cylindrocladium leaf blight caused by C. theae (Petch) Subramanian was observed on Eucalyptus in the Congo in 1998 (Roux et al. 2000b). Lesions initially develop at the edges of leaves and gradually affect the entire leaf. The disease was also observed on twigs and branches. Leaves die on one or two branches and in severe cases the entire tree can be defoliated (Roux et al. 2000b). In South Africa, C. colhounii Peerally var. macroconidialis Crous, Wingfield & Alfenas var. nov. and C. pauciramosum C. L. Schoch et Crous have been recorded on Eucalyptus (Crous, Philips & Wingfield 1993, Schoch et al. 1999). Mycosphaerella leaf blotch disease (MLB) on Eucalyptus is associated with 30 specIes of }Ylycosphaerella Johnson (Crous 1998, Carnegie 2000, Hunter et at. 2002) and has a world-wide distribution including the tropics, subtropics, temperate and Meditenanean areas (Lundquist & Pumell 1987, Crous et at. 1989a, Camegie & Keane 1994). Infections of Eucalyptus spp. by 16 jVJycosphaerella spp. often show high host specificity (Crous & Wingfield 1996). For example, lV!. cryptica (Cooke) Hansf. is responsible for foliage damage on both juvenile and mature trees of E. nitens (Carnegie 1991), whereas lV!. molleriana Lindau. is the main cause of such foliage damage on E. globulus and E. maidenii F. Muel. (Lundquist & Purnell 1987). Leaf spot fungi in the genus lvJycosphaerella result in severe leaf necrosis, premature defoliation and loss of growth in Eucalyptus plantations (Crous et al. 1989a). In Africa, M molleriana is responsible for leaf spot and defoliation on both juvenile and mature trees of E. globulus (Dungey et al. 1995, Lundquist & Purnell 1987). M molleriana has resulted in an inability to establish E. globulus and E. maidenii in South Africa (Wingfield 1990). Similarly, planting of E. nitens, the most promising tree species for afforestation of frost prone areas is restricted because of attack by Mycosphaerella spp. (Lundquist 1985, Wingfield 1990). Mycosphaerella leaf blotch has also been reported from Uganda (Roux et al. 2001a), Zimbabwe and Kenya (Gibson 1964) as well as from Malawi (Lee 1970). Mycosphaerella nubilosa (Cooke) Hansf. (Syn. Sphaerella nubilosa Cooke) is another Mycosphaerella sp. found in South Africa and Zambia. This fungus is the cause of leaf spot and blight of E. globulus, E. maidenii, E. regnans F. Mueller, E. viminalis L. and several other Eucalyptus spp. (Gibson 1975, Park & Keane 1982, Lundquist & Baxter 1985, Lundquist & Purnell 1987). Recently five new Mycosphaerella spp., namely M. juvenis Crous et MJ. Wingf. (Anamorph: Uwebrauni juvenis Crous et MJ. Wingf.) , M africana Crous et M.J. Wingf., M ellipsoidea Crous et MJ. Wingf. (anamorph: Uwebrauni ellipsoidea), M crystallina Crous & M.J. Wingf., and M lateralis Crous et MJ. Wingf. (anamorph: Uwebrauni lateralis crous et MJ. Wingf.) were described in association with different Eucalyptus spp. in South Africa (Crous & Wingfield 1996). In South Africa, M marksii Carnegie & Keane has also been reported from E. grandis and E. nitens (Crous & Wingfield 1996). Phaeoseptoria eucalypti Hansf. Emand [syn=Kirramyces epicoccoides (Cooke & Massee)] J. Walker, B. Sutton & Pascoe, which is now more correctly known as Phaeophieospora epicoccoides (Cooke & Massee) Crous, F.A. Ferreira & Sutton, (Crous, Ferreira & Sutton 1997), has been recorded in Malawi, Zambia and South Africa (Chipompha 1987, Wingfield 1987a, Crous, KnoxDavies & Wingfield 1988, Shakacite 1991). It causes discrete leaf spots on several Eucalyptus spp. (Crous, Knox-Davies & Wingfield 1989b). High levels of infection have been reported on E. camalduiensis, E. globulus, E. saiigna, E. grandis and E. tereticornis Sm. (Chipompha 1987, Crous 17 et al. 1988), whereas the infection levels recorded on E. grandis have tended to be low. Infection is commonly observed on the older leaves and lower branches of trees (Crous et al. 1989b). Infected leaves initially develop numerous minute spots with purple margins. As infection develops the spots enlarge slightly. Black masses of spores are found on the under surfaces of the spots. In severe cases, infection leads to defoliation (Shanna et · al. 1984, Chipompha 1987, Crous et al. 1988). According to these authors, the disease also affects seedlings in nurseries. Nichol, Wingfield & Swart (1992) indicated that plantation establishment conditions such as site preparation and fertilisation influence the susceptibility of Eucalyptus spp. to infection by P. eucalypti. Two Pestalotiopsis spp. have been reported from Eucalyptus leaves in African countries. Pestalotiopsis disseminata (Thurn.) Steyaert is reported to cause brown leaf blight on E. citriodora, while P. Junerea (Desm.) Steyaert (syn.=Pestalotia fimerea Desm.) has been reported to cause leaf spots on E. globulus (Doidge et al. 1953, Lundquist & Baxter 1985). Aulographina eucalypti (Cooke & Massee) Von Arx and Muller [anamorph: Thyrinula eucalypti (Cooke & Mass.) Swart] was recorded in South Africa on several Eucalyptus spp. including E. globulus, E. grandis and E. nitens (Crous et al. 1989b). It causes circular necrotic lesions on the upper or lower leaf surfaces (Doidge et al. 1953, Lundquist & Baxter 1985) and may result in extensive defoliation (Crous et al. 1989b). This disease is also known as corky leaf spot because of the distinct raised, corky spots, often with concentric rings. Pseudocercospora eucalyptorum Crous, Wingfield, Marasas and Sutton (Crous et al. 1989c), Coniothyrium ovatum Swart (Crous et al. 1988), Fairmanieffa leprosa (Fainn.) Petrak and Syd. (Crous, Knox-Davis & Wingfield 1989d) and Harknessia eucalypti Cke. Apud Cke. & Hark. (Crous et al. 1989d) are among the other leaf pathogens recorded on several Eucalyptus spp. in South Africa. Harknessia eucalypti, Pestalotiopsis sp., Botrytis sp. and Melanconium eucalypticola Hansf. were reported from Zimbabwe in association with Eucalyptus spp. (Gibson 1964). Harknessia sp. and Cryptosporiopsis sp. have also been recorded on Eucalyptus spp. in Uganda (Roux et al. 2001a). ConnieffaJragariae (Oud.) Sutton was reported as the cause of leaf spot on Eucalyptus in Congo (Roux et al. 2000b). These diseases were not considered to be of great economic importance. 18 Wilt diseases Wingfield (1990) indicated that the number of diseases affecting forest trees can be expected to increase significantly in the future. A large number of pathogens that have as yet, not appeared in a country, could result in devastation. For example' Roux et al. (2000a) discovered an important new wilt and die-back disease of Eucalyptus in the Congo in 1998. The causal agent of this disease was identified as Ceratocystis fimbriata Ell. and HaIst (Roux et al. 2000a). Ceratocystis spp. are well known causal agents of wilt disease and are amongst the most serious pathogens of woody plants in the world (Kile 1993). They range from weak pathogens to aggressive primary pathogens (Kile 1993). The occurrence of C. fimbriata as a pathogen of Eucalyptus spp., in the Republic of the Congo, was the first record of a Ceratocystis sp. causing a vascular wilt on Eucalyptus spp. in the world. Infection by this pathogen led to a serious wilt disease of E. urophylla X E. pellita F. Muell (UP) and E. tereticornis Sm. X E. grandis (Roux et al. 2000a). Ceratocystis wilt has also recently been found in Uganda (Roux et al. 200 1a). Plantations of E. grandis trees showed the development of epicormic shoots, dead tops and tree death (Roux et al. 2001 a). Close examination of the main stems of the affected trees revealed extensive brown to blue streaking of the xylem. As was the case in the Congo, Ceratocystis wilt in Uganda resulted in high levels of mortality (Roux et al. 200la). The most typical symptoms of this disease is the irregular (streaks) dark brown discoloration of the xylem (Roux et al. 2001 a). Bacterial wilt of Eucalyptus spp. caused by Ralstonia solanacearum Yabuuchi et al. (syn.: Pseudomonas solanancearum) was first described from Brazil (Hayward 1964, Ciesla, Diekmann & Putter 1996). The first report of bacterial wilt of Eucalyptus in Africa was in the mid 1990' s, from South Africa (Coutinho et al. 2000). Its occurrence was also reported from the Republic of Congo and Uganda (Roux et al. 2000a, 200la). The isolates found in Congo belong to Biovar 3, similar to the bacteria found in South Africa (Roux et al. 2000a, Coutinho et al. 2000). Ralstonia solanacearum survives in soil or on plant debris. This bacterial pathogen affects several different Eucalyptus spp ., which include E. urophylla, E. camaldulensis, E. gran dis, and E. sa ligna. R. solanacearum causes root disease and cracking around the root collars of infected trees (Roux et al. 2000a). The disease also causes extensive xylem discoloration and black streaks are present in the discoloured xylem (Hayward 1964, Roux et at. 2000a). A creamy to white bacterial ooze appears on the surface of cut stems (Roux et al. 2000a). 19 DISEASES OF PLAl~TION CUPRESSUS SPP. IN AFRICA Trees in the Cupressaceae are widely planted in African countries as a source of sawn timber (Nsolomo, Madoffe & Maliondo 2000). In Africa, information on the diseases affecting the Cupressaceae is very limited, despite the fact tharmoitalities are commonly experienced. The best described disease of the Cupressaceae in Africa is Seiridium canker. This destructive canker disease is caused by Seiridium spp. and is described world wide as a devastating disease in plantations and on ornamental cypresses (Graniti 1986). S. cardinale (Wagener) Sutton, S. unicorni (eke and Ell.) Sutton & Gibson and S. cupressi (Guba) Boesewinkel are the three Seiridium spp. involved in causing stem canker on Cupressaceae (Boesewinkel 1983, Graniti 1986, Barnes et al. 200 1). All three species have been found in Africa, although the taxonomy of these fungi has been a matter of substantial debate. Seiridium canker has been reported from Kenya, North and South Africa (Rudd Jones 1953, Nattras Booth & Sutton 1963, Graniti 1986, Wingfield & du Toit 1986). The disease results in twig and branch cankers characterized by reddening or browning of the living bark. Infection results, in necrosis of tissue and gradually leads to girdling of the branches and stems of plants. This eventually results in death, first of the branches and then entire trees (Graniti 1986, 1998). Seiridium canker, caused by S. cupressi(syn= Rhynchosphaeria cupressi) has resulted in serious losses in Kenya (Rudd Jones 1953, 1954a, 1954b, Gibson 1964). In this country, damage to fast growing C. macrocarpa Hartw. has resulted in termination of the planting of this tree. It has been substituted by slow growing, but less susceptible, C. lusitanica Mill. (Gibson 1964). Apart from Kenya, Seiridium canker has also been reported from Malawi, South Africa, Tanzania and Uganda (Gibson 1964, Graniti 1986, Wingfield & du Toit 1986). Other diseases of Cupressaceae reported from Africa include stem gall, caused by Agrobacterium tumefasciens (Smith & Towsend 1907) Conn 1942 from Uganda and Kenya (Gibson 1964) and Rhizoctonia lamellifera Small. from C. lusitanica in Kenya (Gibson 1964). A serious stress-related disease caused by Sphaeropsis sapineaJ sp. cupressi SoleI et at. has also been reported from South Africa (Linde, Kemp & Wingfield 1998). In Kenya, Armillaria root rot is found in association with Cupressus lusitanica (Mwangi et al. 1989, Onsando et al. 1997). 20 DISEASES OF PLANTATION ACACL4 SPP. IN AFRICA A number of Australian Acacias, commonly known as wattles, have been introduced into Africa. A. tnearnsii de Wild., A. decurrens Wendl., A. mangium Wild. and A. auriculiformis A. Cunn. Ex Benth are the major species planted in Africa (Anonymous 1978, Kihiyo & Kowero 1986, Khasa, lallee & Bousquet 1994). Mostly, Acacias are used to extract tannin, for timber, pulp production, to promote biological nitrogen fixation and some are used for sand dune stabilization (Anonymous 1978, Kihiyo & Kowero 1986, Ngulube 1988, Khasa et al. 1994). Roux, Kemp & Wingfield (1995) provided an extensive review of the diseases of A. mearnsii in South Africa. The current treatment will, therefore, only briefly mention the major diseases of this tree in South Africa and will focus on diseases of other Acacia spp. and other African countries. Root diseases Phytophthora spp. are commonly associated with root diseases of Australian Acacia spp. Phytophthora nicotianiae (Dastur) Waterhouse [P. parasitica (Dastur)] was first reported from A. mearnsii in South Africa in the 1960's (Zeijlemaker 1971). The disease caused by P. nicotianiae, is commonly called black butt disease and results in collar rot of infected trees (Zeijlemaker 1971). The disease derives its common name from the resultant black discoloration and cracking of the bark at the bases of trees (Zeijlemaker 1971, Roux 2002). Infection may result in the death of trees, in a reduction of bark yield as well as a reduction in the quality of the thickest most valuable bark at the bases of trees (Sherry 1971, Haigh 1993, Roux & Wingfield 1997). More recently Roux & Wingfield (1997), isolated other Phytophthora spp. from black butt and root disease symptoms of A. mearnsii in South Africa. These included P. boehmeriae Sawada and P. meadii. McRae. In pathogenicity trials, both species were shown to be capable of causing lesions similar in size to those caused by P. nicotianae (Roux & Wingfield 1997). It has been suggested that black butt is a complex of diseases not caused by a single organism (Zeijlemaker 1968). This hypothesis was supported by results of a survey conducted by Roux and Wingfield (1997). Several other pathogens were isolated from disease symptoms. It is currently thought that Phytophthora spp. are the primary pathogens resulting in cracks and other wounds that become infected by secondary and opportunistic pathogens . Reports also state that P. nicotianae can only be isolated from the basal part of infected trees (Wingfield & Roux 2000a, Roux 2002). i l btO;l.'l Uu. ~ b \S94U4b3 21 This has, however, been shown to be inaccurate, with Phytophthora spp. being isolated from areas at breast height and also from the xylem of wilting trees (Roux, personal communication). In South Africa, three Ganoderma spp. are reported to be associated with root and collar rot of A. mearnsii (Gibson 1964, Liickhoff 1964). Ganoderma lucidum (Leyss.:Fr.) Karst causes white spongy rot (Bakshi 1976, Gorter 1977), G. applanatum (Pers. Wallr.) Pat. causes heart rot and G. rugosum Blume and Nees has been given as the cause of collar rot (Gibson 1964, Liickhoff 1964, Sherry 1971). It has been reported that Lasiodiplodia theobromae (Synonym= Botryodiplodia theobromae Pat., Diplodia natalensis Pole Evans) caused collar rot of A. mearnsii in South Africa (Sherry 1971, Gibson 1975, Roux, Wingfield & Morris 1997). The disease is reported to affect the whole root system and infection spreads up the stems to form black cankers. In South Africa, several other root pathogens have also been recorded on A. mearnsii. These include Macrophomina phaseolina (Gibson 1975, Bakshi 1976), Armillaria mellea sensu lato and Rhizoctonia sp. (Kotze 1935, Laughton 1937). In Kenya, Armillaria root rot is found in association with Acacia mearnsii, A. melanoxylon R. Br. and A. saligna (Labill.) HL Wendl. (Mwangi et al. 1989, Onsando et al. 1997). Armillaria root rot, which is broadly ascribed to A. mellea, and collar rot caused by Corticium spp. have also been recorded on A. mearnsii in Malawi (Lee 1970). Stem diseases/canker In recent years, severe mortality of A. mearnsii has been reported from South Africa, caused by Botryosphaeria dothidea (Roux et al. 1995, Roux et al. 1997, Roux 2002). Infection causes stem cankers, tip die-back, wilt and death of infected trees. The internal symptoms of Botryosphaeria canker and wilt include discoloration and death of the cambium and xylem, which is manifested as a visible dark brown ring in cross sections of infected trees (Roux 2002). The disease is also associated with frost and drought stress. Fusarium spp. are known to be pathogens on a wide range of hosts. Roux & Wingfield (1997) reported several Fusarium spp. from die-back and canker symptoms on A. mearnsii. Included were F. graminearium Schwabe, which was isolated from stem and branch cankers (Roux et al. 2001 b). Fusarium spp. have also been isolated from basal cankers associated with black butt disease (Zeijlemaker 1971), blister and mottle lesions associated with Ceratocystis wilt and mechanical 22 wounds on stems and branches (Zeijlemaker 1971, Roux et al. 2001 b). Fusarium graminearum was shown to be pathogenic to A. mearnsii in inoculation experiments (Roux et al. 2001 b). Pink disease caused by Erythricium salmonicolor affects several different Acacia spp. In Africa its occurrence was reported on A. mearnsii, A. auriculiformis and other Acacia spp. from Mauritius and South Africa (Sherry 1971, Gibson 1975, Bakshi 1976). Infection by E. salmonicolor resulted in death of branches and leaf cast due to the girdling of the branches. As the infected bark dies, patches of pink mycelium appear on the surface of the dying bark (Gibson 1975, Wingfield & Roux 2000a, Roux 2002). In South Africa Physalospora abdita (Berk & curt) N. E. Stevens (Bakshi 1976) and Sphaeropsis sp. (Roux & Wingfield 1997) have also been isolated from A. mearnsii. P. abdita caused stem and twig cankers on A. mearnsii and A. decurrens Wendl. (Browne 1968) while a Sphaeropsis sp. has been isolated from stem cankers (Roux & Wingfield 1997). In inoculation trials, the Sphaeropsis sp. showed high levels of pathogenicity (Roux & Wingfield 1997). Schizophyllum commune Fries is an opportunistic wound parasite that leads to the eventual death of trees. Pruning wounds are thought to be sites of infection for this pathogen (Ledeboer 1946). In Kenya, Phoma herbarum Westend. was reported to cause die-back on A. mearnsii and A. deccurrens (Olembo 1972). Infection by P. herbarum is also initiated through wounds (Olembo 1972, Gibson 1975). Leaf diseases Few foliage diseases have been reported on exotic Australian Acacia spp. They include leaf spot caused by Camptomeris albiziae (Petch) Mason (Sherry 1971, Wingfield & Roux 2000a) and C. verruculosa Syd. on A. mearnsii in South Africa (Bakshi 1976). The disease is associated with early leaf drop in autumn (Wingfield & Roux 2000a). A rust caused by Uromy cladium alpinum McAlp. has also been reported from A. mearnsii in South Africa (Morris, Wingfield & Walker 1988). Apart from these two diseases on A. mearnsii, the only recorded leaf disease of Australian Acacia spp. grown in plantations in Africa is leaf spot of A. longifolia caused by Cylindrocladium scoparium Morgan (Hagemann & Rose 1988). Wilt disease One of the most devastating diseases of A. mearnsii IS that caused by the wilt pathogen, Ceratocystis albofundus De Beer, Wingfield & Morris. This disease was first described from South Africa in 1988 (Morris, Wingfield & De Beer 1994). The most common symptom associated with C. albofundus infection is the rapid wilt and death of susceptible trees, the formation of swollen gum pockets in the stems, stem cankers, extensive oozing of gum and discoloration of the xylem (Morris et al. 1994, Raux et al. 1997, 2000a). Ceratocystis wilt, caused by C. albofundus, has been reported only from South Africa (Morris et al. 1994) and Uganda (Raux et al. 200Ia). Raux et al. (200Id) suggested that C. albofundus is native to South Africa. The only alternative hosts known for C. albofundus are South African Protea spp. (Gorter 1977) and A. decurrens (Raux et al. 2001 d). Ceratocystis spp. require wounds to initiate infection. Thus, C. albofundus is especially damaging where trees are affected by hail, insect or silvicultural practices (Raux 2002) that cause wounding of trees. EXOTIC PLANTATION FORESTRY IN ETHIOPIA In Ethiopia, wood plays a major role in meeting more than 85% of the energy requirements of the country. Mostly, this wood comes from the natural forests. For this reason, natural forest resources are diminishing rapidly. Estimates indicate that the natural forest cover has declined from 40% to 2.4% in the 1990's (Davidson 1988, Anonymous 1994). At present, the annual rate of forest exploitation is much higher than the annual replacement, both in terms of area and yield. If this trend continues, the remaining natural forests will not remain for long and it may not be possible to meet the demand for wood products. To overcome this problem, exotic tree planting was commenced and has been practiced for many years in different parts of Ethiopia. Generally speaking it is said that the planting of exotic tree species started with the introduction of E. globulus in the late 1890's. Currently, in Ethiopia, fast growmg exotic speCIes such as E. globulus, E. camaldulensis, E. saligna, E. grandis, and E. citriodora are widely planted in different parts of the country (Persson 1995, Negash 1997). C. lusitanica, P. patula, Grevillea robusta Cunn., A. mearnsii and A. decurrens are among other genera planted, both in plantations and around homesteads. Plantations of these exotic species cover a total area of about 200 000 ha (Anonymous 1994, Vercoe 1995). 24 This figure indicates only the areas of the plantations in National Forest Priority Areas, Peri-urban plantations and community woodlots. It does not include those trees planted around homesteads, farmlands or those planted for rehabilitation of degraded land. Many exotic plantation speCIes die at the seedling stage or at maturity for vanous reasons. However, no study has been conducted to investigate the cause of this death and it has been usually arbitrarily associated with poor species site matching and inadequate tending practices. The role of biotic factors in tree death is underestimated, poorly understood and has not received much attention in Ethiopia. Generally, little information is available on the damage pathogens cause to plantation trees in Ethiopia. A few records of tree diseases in Ethiopia can be found. Several of these deal with Armillaria spp. One such report mentions the infection of Armillaria spp. of pine trees (Mengistu 1992) and another indicates the occurrence of A. mellea in CojJea arabica L. plantations (Eshetu, Teame & Girma 2000). According to these reports Armillaria spp. were found on recently cleared and planted sites and where shade trees had been removed. Recently Ota, Intini & Hattori (2000) reported that the Armillaria sp. found on a hard wood species at Kerita and Jimma is A. meUea sensu stricto. Some records of tree pathogens from Ethiopia can be found in herbarium and survey reports ..-Eor example, Gibson (1972) mentioned that T. Middleton had found Dothistroma needle blight on P. radiata trees around Addis Ababa. Simillarly, based on unpublished records, erous et al. (1989c) mentioned that Pseudocerospora eucalyptorum, the cause of Eucalyptus leaf spot, has been recorded from Worota, North Ethiopia. Walker, Sutton & Pascoe (1992) also mentioned that they obtained a specimen of Phaeoseptoria epicocoides from Ethiopia that was collected from E. sa ligna and E. globulus at Gora and Gumuro. However, the importance of these fungi in causing leaf spot on Eucalyptus in Ethiopia is not clear. There are some reports that deal with diseases of native tree species in Ethiopia. The results of one of these studies reported the occurrence of Antrodia juniperina (Murrill) Niemela & Ryvarden on Juniperus exelsa Hochest. Ex. Endl. (Niemela & Ryvarden 1975). A. juniperina is reported to be parasitic and saprophytic on stems of J exelsa and to cause heart rot and necrosis of the butt. Infection causes intensive brown cubical rot of the wood (Niemela & Ryvarden 1975). In another report Niemela, Revenvall & Hjortstam (1998) recorded several decay fungi in natural stands of 25 Hagenia abyssinica (Bruce) J. F Omel. in East Africa, including Ethiopia. mentioned fungi involved in decaying H. abyssinica. They This report only included Hymenochaete ochromarginata Talbot. collected from living trunks and stumps. This fungus is considered to be the main decayer of living Hagenia trees. Other wood rot fungi included Phellinus ferruginosus (Schrad. Fr:) Bourdot and Oalizn and Trametes socotrana Cooke, collected from fallen branches and stems. Their role in disease of Hagenia can, however, be questioned as they are not generally considered to be primary pathogens. A number of Corticioid fungi have also recorded from H abyssinica branches and stems. They include Asterostroma medium Bres. , Cystidiodontia isabellina (Berk. & Broome) Hjortstam and Dichostereum kenyense Boidin & Lanq (Niemela et al. 1998). Recently, tree deaths of unknown causes have been experienced in many plantations in Ethiopia. Many different disease symptoms are associated with the dying trees. This situation necessitates a study of the pathological problems in plantations in order to develop strategies to reduce losses to this important enerprise. CONCLUSIONS In this review I have attempted to include information on as many of the diseases of exotic plantation trees in Africa as possible in the limited space available. However, the information available on forest diseases from African countries is scanty and often recorded in unpublished government reports. This limitation has meant that information has been gained form work done in only a few countries and often published only in the form of brief notes. Nevertheless, the information included in this review should help to provide insight into the impact of diseases in exotic plantations in Africa. Hopefully, it will also provide valuable foundation for future forest pathology studies in Africa. The rapidly growing demand for forest products will necessitate the expansion of exotic plantations in Africa. The introduction of exotic tree species into Ethiopia commenced a century ago. Up to now, not less than 160 exotic trees and shrubs have been introduced to the country, for different purposes. In a situation where exotic plantations substitute the native forests, an outbreak of disease could severely damage plantations. 26 Diseases have had a serious impact on exotic plantations in various parts of Africa. Various of these have been discussed in this review. It must be expected that other diseases will negatively impact of exotic plantation forestry in the future. Thus, every effort must be made to reduce this situation. It is clear from this review that the problems already experienced with tree diseases in Africa highlight the urgency of studying tree diseases in Ethiopia. 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Africa, Kenya, Viljoen, Wingfield & Seedling dieback Zimbabwe, Tanzania Crous 1992, Gibson 1964, fuckeliana de Bary) Colh totrichum acutatum References Browne 1968 P. radiata Necrosis (Shoot tip, terminal S. Africa, Kenya bud, & needles), stunting & Simmonds Gibson & Munga 1969, Lundquist 1984b stem malformation C. g:o eosporioides (Penz.) E. dives, E. grandis, E. Pem. & Sacco globulus, E. saligna Leaf spot, stem canker S. Africa Viljoen et al. 1992, Baxter, Van Westhuizen & Eicker 1983 Comella castaneicola (Ell. E. camaldulensis, E. Ev.) Sutton globulus Cylindrocladiella spp. Eucalyptus spp., Acacia Leaf spot S. Africa Viljoen et al. 1992 Dampingoff, seedling blight S. Africa Crous et al. 1993 Root rot, leaf spot S. Africa Crous et al. 1993 Root infection, seedling death S. Africa, Malawi Crous et al. 1991 spp., Pinus spp. C. cC!.melliae Eucalyptus spp., A. mearnsii Venkataramani & Venkata P. radiata, Eucalyptus spp. Ram C. parva Anderson 44 Pathogen Cylindrocarpon destructans Host Symptoms Distribution References P. radiata, P. roxburghii Root rot, Dampingoff South Africa Darvas et at. 1978 Eucalyptus spp., Acacia spp., Root rot, dampingoff, S. Africa, Kenya Lundquist & Baxter 1985, Crous Conifers leaf blight, stem canker (Zins) Scholten Cylindrocladium pauciramosum c.L. Schoch et at. 1991, Crous et at. 1993, Schoch et al. 1999, Roux 2001 * et Crous C. colhouni Peeraly E. grandis Leaf spot, root rot, wilt S. Africa Crous et al. 1993 C. clavatum Hodges & May Eucalyptus spp., P. radiata S. Africa Crous et al. 1993 Fusarium spp. Pinus spp., Eucalyptus spp., Dampingoff, seedling blight Dampingoff S. Africa, E. Africa, Browne 1968, Hocking 1968, Uganda Maiteki et al. 1999 S. Africa, E. Africa, Darvas et at. 1978, Viljoen, Uganda Wingfield & Crous 1992, 1994 S. Africa, E. Africa, Bakshi 1976, Viljoen et al. 1995, Uganda Roux et al. 2001 b Dampingoff S. Africa, E. Africa Zeijlemaker 1968 Dampingoff E. Africa, S. Africa Hocking 1968, Darvas et al. 1978 Acacia spp. F. oxysporum (Schlecht. Ex P. palustris, P. roxburghii, P. Fr. taeda, P. caribaea, P. khasya Dampingoff A. mearnsii F. solani (Mart.) Sacco P. patula, P. caribaea, P. khasya Dampingoff A. mearnsii F. moniliformis Sheld. P. radiata, P. roxburghii, P. caribaea, P. khasya F. equiseti (Corda) Sacco P. caribaea, P. khasya, P. radiata, P. roxburghii Roux et al 2001* personal communication 45 Pathogen F. sel11,itectum Berk. & Rav Host P. caribaea, P. khasya Symptoms Dampingoff Distribution E. Africa, S. Africa References Hocking 1968, Darvas et al. 1978 F. subglutinans P. patula Root rot, dampingoff S. Africa Viljoen et al. 1997a, 1997b Hainesia Iythri (Desm.) E. globulus, E. robusta, Leaf spotting, stunting, multiple S. Africa, Zambia Baxter et al. 1983, Lundquist Hohn E. saligna, E. grandis stems 1986, Lundquist & Foreman 1986, Crous et al. 1993 Harknessia hawaiiensis E. grandis, E. nitens Leaf spot S. Africa Crous et al. 1989b P. patula Root infection Tanzania Browne 1968 A . mearnsii Leaf deformation, leaf drop, S. Africa, Uganda Roux et al. 2001 a, Roux (Stevens & Young) Helicobasidium compactum (Boedijn) Oidium spp. stunting 2002 Macrophomina phaseolina A. mearnsii, A. Stunting, chlorosis, foliage death, Malawi, Zimbabwe, Gibson 1975, Bakshi 1976 (Tassi.) G. decurrens, Eucalyptus necrotic lesions on roots Tanzania, S. Africa Darvas et al. 1978 Leafblight, shoot dieback S. Africa Coutinho et al. 2001 spp. & Pinus spp. Pantoea ananatis Corrig. E. grandis, E. saligna, E. (syn= Erwinia ananas, E. dun ii, E. nitens, E. u redo vora smithii 46 Pathogen Symptoms Host Phaeoseptoria eucalypti E. bicostata, E. camaldulensis. Hansf. (Kirramyces E. cladocalyx, E. dunnii, E. epicoccoides ) globulus spp., E. saligna Phytophthora spp. Eucalyptus spp., Pinus spp. Leaf spot, defoliation Distribution References S. Africa, Malawi, Crous et al. 198ge, Chipompha, Uganda 1987, Roux etal. 2001a, Viljoen et al. 1992 Dampingoff, root rot S. Africa, Uganda Darvas et al. 1978, Maiteki et al. 1999 P. cinnamomi Rand E. citriodora. P. elliottii, P. Dampingoff, root rot S. Africa Darvas et al. 1978 Dampingoff, root rot S. Africa, E. Africa, Doidge 1950, Hocking 1968, Uganda Maiteki et al. 1999 halepensis. P. patula. P. pinaster. P.radiata Pythium spp. P. patula & Pinus spp. P. ultimum Trow E. grandis Root rot S. Africa, E. Africa, Gibson 1970, Darvas et al. 1978 P. irregularae Buisman Pinus spp., Eucalyptus spp. Dampingoff S. Africa Hocking 1968, Viljoen et al. 1992 P. pyrilobum Vaartaja Pinus spp., E. grandis Dampingoff S. Africa Linde et al. 1994a P. splendens H.Braun E. grandis Dampingoff S. Africa Linde et al. 1994b Pseudocercospora P. halepensis, P. patula, P. Needle blight S. Africa Viljoen et al. 1992 pinidensiflorae (Horri & radiata Nambu) Deighton 47 Pathogen Host Symptoms Distribution References Rhizoctonia solani Kuhn Pinus spp., Dampingoff, root rot, S. Africa, E. Africa, Darvas et al. 1978, Viljoen et [anam.=Thanetophorus cucumeries E. grandis collar rot, seedling Uganda al. 1992, Maiteki et al. 1999, (Frank) Don =Corticium solani blight Hocking 1968, Gibson & (Prill & Delacr.) Bourd. & Galz.] Hudson 1969, Gibson 1970 Pinus spp. Root infection Kenya, Tanzania Gibson 1964, Browne 1968 Sphaerotheca pannosa (Wallr.: Fr.) E. camaldulensis, E. Leaf spot, malformation S. Africa, Uganda, Crous et al. 198ge, 1989c, Lev. (syn= Aephitomorpha globulus, E. maidenii of leaves & shoots Kenya Chipompha 1987, Roux et al. Rosellinia necatrix (Hartig) Prill. [anam= Dematophora necatrix Hartig] 2001 a, Roux 2001* pannosa, Erysiphe pannosa, Oidium. leucocnium, Oidium eucalypti) Sphaeropsis sapinea P. patula Needle & shoot blight S. Africa Darvas et al. 1978 Sporothrix eucalypti Wingfield, E. grandis Leaf spot, defoliation, S. Africa Wingfield, Crous & Swart Crous & Swart sp. nov. shoot dieback 1993 * Personal communication. 48 ABSTRACT A survey of diseases in exotic plantations was undertaken in Southern and South Western Ethiopia during 2000 and 2001. The aim was to consider the occurrence and distribution of diseases of major plantation species in this country and to provide a foundation for further research. Samples were collected from plantations and trees planted around farms and homesteads in and around Wondo Genet, Munessa Shashemene, Jima, Bedele, Mizan and Menagesha and included those from roots, stems and leaves. Armillaria root rot was the most common disease, mainly associated with Pinus patula but was also found on Acacia abyssinica, Cordia alliodora and Cedrela odorata trees. Stem cankers associated with Botryosphaeria spp. were common on Eucalyptus globulus, E. saligna and E. citriodora. Stem canker disease associated with a Coniothyrium sp. was frequently observed on E. camaldulensis. Leaf blotch associated with Mycosphaerella spp. was common on E. globulus in most areas where this species is planted. In addition, Sphaeropsis sapinea on Pinus spp., cankers associated with Cytospora spp. and pink disease caused by Erythricium salmonicolor on Eucalyptus were also recorded in some plantations. This is the first general evaluation of plantation diseases in Ethiopia and it will provide a foundation for developing planting and disease management strategies, to ensure optimum production in plantations. 50 INTRODUCTION Establishment of exotic plantation forestry has been successful and profitable in many tropical and subtropical countries (Gibson 1979, Evans 1984). The timber derived from these plantations is commonly used to produce pulp and paper, viscose and rayon. It also provides a resource for construction and in developing countries, is an important source of fuel wood (Evans 1984, Turnbull 1991). In Ethiopia, wood provides 85% of the country's energy requirements and is used for construction purposes. However, the natural forest resource is diminishing rapidly (Anonymous 1994). This is while the demand for forest products is rapidly increasing, necessitating the establishment of plantations of rapidly growing trees. In Ethiopia, the introduction of fast growing exotic tree species took place a century ago, with the introduction of Eucalyptus globulus Labill. in the late 1890's (Persson 1995). Since then, several other Eucalyptus spp., as well as Cupressus, Pinus, Grevillea and Acacia species have been widely planted in plantations and around farms and homesteads (Evans 1984, Persson 1995). Plantations of these exotic species now occupy approximately 200 000 ha (Anonymous 1994). Plantations of exotic species have been highly successful in many countries (Wingfield 1990, Persson 1995). This is partially attributed to the separation of the trees from their natural enemies. However, these trees are established as monocultures in new environments and they are exposed to unique suites of pests and diseases. Thus, serious disease problems have emerged in most countries where they have been planted (Wingfield 1990). Diseases have had serious impacts on exotic plantation forestry and in some cases, have resulted in the abandonment, or restriction of species to specific localities. For example, the fastgrowing Pinus radiata D. Don. has been abandoned in several Eastern, Central and Southern African countries due to Dothistroma needle blight caused by Dothistroma septospora (Dorog.) Morelet (Gibson 1972, Ciesla, Mbugua & Ward 1995). In these 51 countries, P. radiata has been substituted by slower growing P. patula Schl. & Cham. but, in South Africa, severe losses to P. patula have subsequently occurred due to an interaction between hail damage and Sphaeropsis sapinea (Fr.:Fr.) Dyko & Sutton (Swart, Wingfield & KnoxDavies 1987). Similarly, planting Cupressus macrocarpa Hartw., which showed remarkable growth in Kenya and other East African countries, has been abandoned due to cypress stem canker caused by Seiridium cupressi (Guba) Boesew (Rudd Jones 1953, Gibson 1964). As a result, the slow growing C. lusitanica Mill. has been introduced as an alternative species (Rudd Jones 1953, Gibson 1964). Diseases have negatively affected the planting of Eucalyptus spp. for example, Mycosphaerella leaf blotch on E. globulus, E. nitens (Deane et Maid.) Maid. and E. maidenii F. Muel!. has resulted in reduction of planting these species (Lundquist & Purnell 1987). Likewise, E. jastigata Deane et Maid. and E. jraxinoides Deane et Maid., which initially perfonned well in frost prone areas of South Africa, have been abandoned due to root disease caused by Phytophthora cinnamomi Rands (Linde, Kemp & Wingfield 1994). Several diseases new to Eucalyptus, for example stem canker caused by Oyphonectria cubensis (Bruner) Hodges (Conradie, Swart & Wingfield 1990), Coniothyrium zuluense Wingfield, Crous & Coutinho (Wingfield, Crous & Coutinho 1996), and wilt caused by Ceratocystis jimbriata Ellis & HaIst. (Roux et al. 2000), have appeared in recent years. These pathogens not only damage the trees in their exotic habitat, but now also threaten Eucalyptus in their areas of origin (Wingfield 1990, 1999). Knowledge of plantation diseases in Ethiopia is limited. This is despite the fact that tree death is common in the country. These deaths are typically attributed to poor sitespecies matching, poor management and adverse climatic conditions. The role of biotic factors in tree death is underestimated, poorly understood and has received little attention. Thus, a survey of plantation tree species was conducted in some parts of South and South Western Ethiopia in 2000 and 2001. The objective was specifically to detennine the occurrence of diseases of exotic plantation species, to provide a basis for further study 52 and to establish a foundation for disease avoidance. This study provides the first detailed overview of plantation tree pathogens in Ethiopia. MATERIALS AND METHODS Survey areas and sample collection Surveys were conducted in April 2000 and in JuneSeptember 2001 in Southern and South Western Ethiopia. Collections were made in plantations and smallholdings around Munessa Shashemene, Wondo Genet, Jima, Mizan Teferi, Bedele, Menagesha and Addis Ababa (Figure 1, Table 1). Samples were ptimatily collected from Eucalyptus and Pinus plantations and included samples from roots, bark, stems, twigs and leaves. Isolation techniques Samples were collected from all trees showing symptoms of disease. Diseased plant tissue was collected and kept in paper bags for transport to the laboratory. Growth media used to isolate the fungi included 2% malt extract agar (MEA, Biolab) and MEA amended with 100 ppm streptomycin (MEAS) for the isolation of Ascomycetes and Coelomycetes. A selective medium containing benomyl was used for the isolation of Basidiomycetes (Harrington, Worall & Baker 1992). In the laboratory, several different techniques were used to isolate diseasecausing organisms. These included the transfer of pieces of mycelium or fruiting bodies from diseased plant tissue directly onto the growth medium; incubating symptomatic plant matetial in moist chambers; as well as inoculating segments of plant parts with disease symptoms onto growth media. All plates were incubated at 25°C to induce fungal growth. For the isolation of Mycosphaerella spp., discs of leaves with disease symptoms were attached beneath the cover of petti dishes with the pseudothecia facing downward, so that spores were released onto MEAS (Crous, Phillips & Wingfield 1991). After 24 hr, ascospore germination was checked under the microscope and single germinating 53 ascospores were transferred to MEA. Microscope slides were prepared of each isolate to determine the germination pattern of ascospores. Fungi isolated in this study were identified and representative isolates of the pathogens are maintained in the culture collection of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa. Specimens have also been deposited in the herbarium of the South African National Fungus Collection, Pretoria (PREM). RESULTS The results of this survey clearly demonstrate the prevalence of some important tree diseases in the exotic plantations examined. Diseases recorded during this survey included root diseases, stem cankers and leaf diseases. Root diseases Armillaria Root Rot Armillaria root rot was commonly found associated with the death of P. patula in Wondo Genet, Belete and Bedele. The causal fungus (PREM 57377 and 57378) was also isolated from dying Acacia abyssinica Hochst. trees growing in Pinus plantations. In addition, Annillaria root rot was found in association with dead and dying Cordia alliodora (Ruiz & Pav.) Oken. and Cedrella odorata L. trees in a research plot near Arnan. Symptoms were typical of those known for the disease and included death of trees in groups, wilting and yellowing of the crowns (Figure 2a), the occurrence of white mycelial fans between the bark and the wood of symptomatic trees (Figure 2b) as well as the occurrence of rhizomorphs on the bark of infected trees. Armillaria root rot was the major cause for the death of P. patula at Wondo Genet. The damage caused at other plantation sites appeared to be mild at the time of this survey. 54 Stem cankers Stem cankers were observed on several Eucalyptus spp. Disease symptoms included bark cracking, production of copious amounts of kino, stem discoloration and malfOlmation, as well as the production of kino pockets in the xylem (Figures 3, 4). Stem Canker associated with Botryosphaeria The most common disease observed on Eucalyptus spp. in Ethiopia was canker, from which a Botryosphaeria sp. (PREM 57379, 57380 and 57381) was isolated. At Wondo Genet these cankers were found on E. saligna Sm., E. gJ'andis Hill ex Maid., E. citriodora Hook and E. globulus. At Munessa Shashemene, they were observed on E. globulus, both on coppice and first generation stands, as well as on mature E. saligna. At these two sites, stem cracking and kino exudation was observed over the entire length of stems of affected trees. When the bark was removed from these trees, well developed kino pockets were visible in the cambium and xylem (Figure 3a). In the Jima area, similar disease symptoms were observed on E. citriodora and E. saligna. Here the damage was most severe on E. citriodora and not less than 50% of the trees in the plantation near Jima were symptomatic, but no death of trees were observed at the time of the survey. On this species, large basal cankers were observed. The disease was characterised by black discoloration and cracking of stems, starting at ground level up to approximately one metre height (Figure 3b). When the bark was removed, the cambium was completely discoloured and soaked with kino. Two or three layers of black lines were observed in the wood indicating different seasons of infection (Figure 3c). At Menagesha symptoms of stem canker were commonly found on coppice stems of E. globulus. At this site, several coppice stems were dead and wilting. A Botryosphaeria sp. was frequently isolated from symptomatic plant material collected from all sites. 55 Coniothyrium Stem Canker Stems of E. camaldulensis Dehnh. trees in Jiren plantation at Jima and on trees in wood lots between Jima and Woliso, as well as between Wolkite and Sodo, were seriously affected by a stem canker disease. The disease resulted in extensive stem malformation. Initial symptoms of the disease include small discrete lesions on young green bark. Patches of large necrotic lesions developed from these on the stems, branches and twigs (Figure 4a). A Coniothyrium sp. (PREM 57382) was consistently found sporulating on the surface of the lesions. Stems often showed a reddish colour due to the exudation of kino from the cracks. The wood of the affected stems showed the formation of pitted kino pockets (Figure 4b). Several of the infected trees produced epicormic shoots. It was estimated that at least 50% of the trees in a stand were affected by this pathogen. Pink disease Stem samples of diseased E. camaldulensis obtained from Pawe in the Benshangul Gumuz region, yielded structures typical of the pink disease pathogen, Erythricium salmonicolor (Berk. & Broome) Burds. (Syn. Corticium salmonicolor Berk. & Br.). Branch dieback, stem cankers, branch and stem girdling, production of epicormic shoots on the stems, death of trees as well as the production of pink mycelial growth on symptomatic plant parts are characteristic symptoms of pink disease. The fungus produced typical flat/resupinate fruiting structures on the surface of affected stems. Leafdisease Mycosphaerella leaf blotch Leaf spot and blight was commonly observed on E. globulus, wherever this species is grown. The symptoms observed on E. globulus are characteristic of those caused by Mycosphaerella spp. and in many cases, resulted in defoliation of young trees (Figure 5a, 5b). Isolations from leaves with leaf blotch symptoms consistently yielded 56 Mycosphaerella spp. (PREM 57386). From the examination of the germination patterns of ascospores, it is clear that more than one Mycosphaerella sp. is involved in causing leaf blotch on E. globulus leaves in Ethiopia. Other fungi Several other fungi, known to be associated with tree disease elsewhere in the world, were found in Pinus and Eucalyptus plantations, although they appeared to be relatively unimportant. For example, Sphaeropsis sapinea was isolated from pine cones collected from Wondo Genet and Munessa Shashemene. Species of Cytospora, Fusarium graminearum Schwabe and Cylindrocladium Morgan were also isolated from Eucalyptus branches collected from Wondo Genet, Wolkite and Menagesha. Phaeophleospora eucalypti (Cooke & Massee) Crous, F. A. Fen·eira & B. Sutton was common on E. camaldulensis and E. grandis leaves, in all areas examined. DISCUSSION Planting exotic speCies in plantations has been practised for more than a century in Ethiopia. The impact of diseases on plantation development has, however, received minimal attention. In recent years, tree deaths have been frequent but usually attributed to extreme climatic and poor site conditions. The results of this study have shown that several wellknown fungal pathogens are involved in causing considerable damage in exotic plantations. This study thus provides the first comprehensive documentation of plantation diseases in Ethiopia and provides a firm foundation for future study. Root rot caused by an Armillaria sp. was frequently found in P. patula plantations. Armillaria spp. are known to cause root rot on a wide range of tree species including both exotic as well as native trees and are known worldwide (Shaw & Kile 1991). The identification of Armillaria root rot from native A. abyssinica as well as from C odorata and C alliodora suggest that this disease could be important not only in P. patula, but also on other trees, including native species. Further study is essential to determine its 57 role in causing root rot in other localities not included in this survey. Previous studies have attributed Annillaria root rot in Ethiopia to A. mellea (Mengistu 1992, Eshetu, Teame & Girma 2000, Ott a, Intini & Hattori 2000). Fruiting bodies recovered from this survey, however, do not match the macromorphological characteristics of A. mellea. We are currently conducting further studies to identify the species isolated during the present surveys. Botryosphaeria spp. have a cosmopolitan distribution and are found on many different hosts including Eucalyptus spp. (Barnard et al. 1987, Smith, Kemp & Wingfield 1994) and it was not surprising to find them in this study. They are considered to be opportunistic wound and stress related pathogens (Pusey 1989, Smith et al. 1994). Environmental stress such as drought (Pusey 1989) and frost (Wene & Schoenesweiss 1980) especially, provide conducive conditions for disease development. Botlyosphaeria spp. are also known as endophytes and are found in healthy plant tissues (Smith, Wingfield & Petrini 1999). In some areas the presence of this pathogen seems to have resulted in poor growth of the coppice sprouts of E. globulus and it most likely contributed to the failure of coppice development. Regenerating Eucalyptus spp. by coppicing is widely practised in Ethiopia, and further investigation is needed to determine the association of the stem canker with poor growth and coppice failure. Currently studies are underway to determine which BOllyosphaeria spp. are involved in causing stem canker on Eucalyptus spp. in Ethiopia and thus to evaluate their relative importance. Stem canker associated with a fungus that closely resembles C. zuluense was the most common stem canker found affecting E. camaldulensis. This is the most widely planted Eucalyptus sp. in Ethiopia and given the importance of the disease on Eucalyptus spp., clones and hybrids in South Africa, Thailand and Mexico (Wingfield et al.1996, Van Zyl et al. 2002, Roux, Wingfield & Cibnin 2002), this disease is of considerable concern. Coniothyrium canker is considered to be one of the most important till·eats to Eucalyptus plantation forestIy in the world. This disease not only complicates debarking but it also affects the quality of sawn timber, growth and in severe cases may also result in death of trees (Wingfield et al. 1996, Roux et al. 2002, Van Zyl et al. 2002). At present little is 58 known regarding its occurrence in other E. camaldulensis growing areas of Ethiopia or whether it infects other Eucalyptus spp. It will, therefore, be important to conduct further surveys for this disease. A study is currently also in progress to confirm the identity of the Coniothyrium sp. found in Ethiopia and to determine whether it is the same fungus found in South Africa and elsewhere in the world. Pink disease caused by E. salmonicolor is common in the tropics, affecting a wide range of hosts including Eucalyptus spp., coffee, rubber, cacao, tea, Acacia and Podocarpus spp. (Gibson 1975, Roux et al. 200la) and its discovery on Eucalyptus in Ethiopia is considered important. In South Africa, pink disease has been reported on E. macarthurii Deane et Maid. and E. cloeziana F. Mueller in temperate areas of the country (Roux et al. 2001a). The damage caused by this disease is of concern for the development of E. camaldulensis in Ethiopia and studies including those relating to distribution and host range are required in Ethiopia. Leaf blotch caused by Mycosphaerella spp. is widely distributed and important on Eucalyptus spp. worldwide. It is especially wellknown for the defoliation it causes on E. globulus and E. nitens (Park & Keane 1982, Lundquist & Purnell 1987), and its occurrence on E. globulus in Ethiopia is significant. Elsewhere in Africa, MLB has been reported from South Africa (erous & Wingfield 1996), Uganda (Roux et al. 200lb), Malawi (Lee 1970), Zimbabwe and Kenya (Gibson 1964). Thirty Mycosphaerella spp. have been described in association with leaf blotch of Eucalyptus, of which 11 have been recorded on the African continent (Hunter et al. 2003). Nothing is known regarding the diversity, distribution or importance of Mycosphaerella spp. in Ethiopia and these questions deserve investigation. Results of this study include many new records of diseases of Pinus and Eucalyptus spp. in Ethiopia. They also provide a foundation on which to base future studies and to develop management strategies. In the past, tree deaths have been ascribed to factors such as adverse climatic conditions, poor species selection and inadequate postplanting management. This study has shown that the situation is more complicated and that 59 diseases play an important role. These findings suggest that management strategies to reduce the impact of diseases, and facilities to diagnose and monitor these problems should be instituted. In addition, most of the pathogens require more detailed taxonomic study and pathogenicity tests should be conducted to better understand their role in tree death. REFERENCES Anonymous (1994) Ethiopian Forestry Action Plan (EFAP). Final report, vol. I, pp 14 imd vol. II, pp 34. Addis Ababa, Ethiopia. Barnard, E. L., Geary, T., English, J. T. & Gilly, S. P. (1987) Basal cankers and coppice failure of Eucalyptus grandis in Florida. Plant Disease 71: 358361. Ciesla, W. M., Mbugua, D. K. & Ward, J. D. 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Observations on pathology, spread and possible origins of the disease. Annals ofApplied Biology 40: 323-343. Shaw, C. G. & Kile, G. A. (1991) Armillaria root disease. Agriculture Hand book No. 691. Forest Service, United States Department of Agriculture. Washington D.C. Smith, H., Kemp, O. H. J. & Wingfield, M. 1. (1994) Canker and die-back of Eucalyptus in South Africa caused by Botryosphaeria dothidea. Plant Pathology 43: 10311034. 62 Smith, H., Wingfield, M. J. & Petrini, o. (1999) Botryosphaeria dothidea endophytic in Eucalyptus grandis and Eucalyptus nitens in South Africa. Forest Ecology and Management 89: 189195. Swart, W. J., Wingfield, M. J. & KnoxDavies, P. S. (1987) Factors associated with Sphaeropsis sapinea infection of pine trees in South Africa. Phytophylactica 19: 505510. Turnbull, J. W. (1991) Future use of Eucalyptus. Opportunities and problems. In Proceedings of the IUFRO Symposium on Intensive Forestry: The role of Eucalyptus (A.P. G. Schonau ed): pp. 227. South African Institute of Forestry. Pretoria, South Africa. Van Zyl, L. M., Coutinho, T. A., Wingfield, M. J., Pongpanich, K. & Wingfield, B. D. (2002) Morphological and molecular relatedness of geographically diverse isolates of Coniothyrium zuluense from South Africa and Thailand. Mycological Research 106: 5159. Wene, E. G. & Schoeneweiss, D. F. (1980) Localised freezing predisposition to Botryosphaeria canker in differentially frozen wood stems. Canadian Journal of Botany 58: 14551458. Wingfield, M. J. (1990) Current status and future prospects of forest pathology in South Africa. South African Journal ofScience 86: 6062. Wingfield, M. J. (1999) Pathogens in exotic plantation forestry. International Forestry Review 1: 163168. Wingfield, M. J., Crous, P. W. & Coutinho, T. A. (1996) A serious canker disease of Eucalyptus in South Africa caused by a new species of Coniothyrium. Mycopathologia 136: 139145. 63 Table 1. Climatic conditions and altitude of survey areas Locality Mean Annual Temperature Mean Annual Rainfall Altitude (masl) °c (mm) Bedele 19 1 800 2010 Hossana 16 1 300 2320 Jima 20 1 500 1 750 Menagesha 14 1 017 2400 Mizan/Arnan 24 2200 1 350 Munessa Shashemene 19 1 200 2 1402600 Woliso 17 1 100 2 150 Wondo Genet 19 1 200 1 8002200 64 \ ~OUTI LakeTana Menagesha 0 g SOMALIA Addis Ahaha o Wolisso Bede1e = • Jima = Hossana o Munessa Shashemenne = Wondo Genete Turkana SOMALIA Figure 1. Map of Ethiopia showing the plantation areas where samples were conducted. 65 ABSTRACT Armillaria root rot is a well!mown disease on a wide range of plants, worldwide. In Ethiopia, the disease has previously been reported on Pinus spp., CojJea arabica and on various native hardwoods. The causal agent of the disease has been attributed to Armillaria meilea, a species now !mown to represent a complex of many different taxa. The aim of this study was to detelmine the extent of Atm ill ari a root rot and the identity of the Armillaria sp. in Ethiopian plantations. As part of a plantation disease survey in 2000 and 200 I, samples were collected in plantations at and around Munessa Shashemene, Wondo Genet, Jima, Mizan and Bedele, in South and South Western Ethiopia. Basidiocarps were collected and their morphology studied. Morphological identification was confilmed by sequencing the IGSl region of the ribosomal rRNA operon and comparing data with published sequences of Armillaria spp. Armillaria isolates were collected from Acacia abyssinica, Pinus patula, Cederela odorata and Cordia alliodora trees. Sporocarps were found on stumps of native Juniperus exelsa. Basidiocarp morphology suggested that the Armillaria sp. collected from J exelsa is A. Juscipes. This identification was confilmed for all isolates, based on sequence data. A. Juscipes is !mown to be common in Southern Africa. Its widespread occunence in Ethiopia suggests that it is also the major cause of Atmillaria root rot in that country. 71 INTRODUCTION Armillaria species cause root rot on a wide range of hosts, worldwide. These include many species such as Eucalyptus, Pinus, Acacia and Cupressus that are utilized in plantations (Wargo & Shaw 1985, Hood, Redfern & Kile 1991 , Kile, McDonald & Byler 1991). Armillaria spp. have been regarded as primary pathogens, stress induced secondary invaders and saprophytes (Wargo & Shaw 1985, Shaw & Kile 1991). Group death, wilting and yellowing of tree tops, resin exudation, as well as the occurrence of white mycelial fans under the bark of infected trees are common symptoms of Armillaria infections. In many cases, rhizomorphs are also found associated with Armillaria root rot and these structures facilitate spread of Armillaria through the soil (Morrison, Williams & Whitney 1991). The morphological characteristics of Armillaria spp. including the mycelium, rhizomorphs and basidiocarps have traditionally been the most impOl1ant criteria for identification. The mycelium and the rhizomorphs of many species of these fungi, however, exhibit limited variation, restricting their use in species identification (Watling, Kile & Gregory 1982, Garraway, HOttermann & Wargo 1991). In contrast, morphological characteristics of the basidiocarps, have provided useful taxonomic characters for species delimitation (Shaw & Kile 1991). However, the seasonal and irregular production of these structures, coupled with their scarcity, has complicated identification of Armillaria spp., based on morphology (Watling et al. 1982, Wargo & Shaw 1985, Mohammed et al. 1994). It is largely due to these limitations that Armillaria mellea (Vahl:Fr.) Kumer sensu lato was long considered to be a single variable species causing root rot, worldwide (Singer 1956). In recent years, mating studies (Korhonen 1978, Ulhich & Anderson 1978, Anderson & Ullrich 1979), biochemical comparisons (Morrison et al. 1985, Mwangi, Lin & Hubbes 1989, Mwenje & Ride 1996) and DNA based techniques (Anderson & Stasovski 1992, Coetzee et at. 2000) have been used to study the biology, taxonomy, and phylogeny of Armillaria spp. Currently, it is known that the Armillaria species complex, originally 72 treated as A. mellea sensu lato, consists of at least 36 different species (Wargo & Shaw 1985, Yolk & Burdsall 1995). DNAbased characterisation provides a useful tool to identify Armillaria spp. The intergenic spacer region (lGS1) of the rDNA operon is most commonly used to identify and study the relationship of Armillaria isolates (Anderson & Stasovski 1992, Coetzee et al. 2000). Restriction fragment length polymorphism (RFLP) patterns of this rDNA region are also commonly used to discriminate between Armillaria isolates (Harrington & Wingfield 1995). Armillaria root rot has been reported from several countries in South, East and Western Africa. In Africa, this disease has been found associated with both cash crop plants such as coffee and tea as well as on forest plantation species including those of Pinus, Eucalyptus, Acacia and Grevillea (Mwangi et al. 1989, Onsando, Wargo & Waud 1997). The disease has generally been ascribed to Armillaria mellea (Yahl.:Fr.) P. Kumm. and A. heimii Pegler (Pegler 1977, Ivory 1987, Mohammed, Guillaumin & Berthelay 1989). However, recent studies conducted on Armillaria isolates from Africa reported the occurrence of A. heimii, A. mellea sensu stricto (Mwangi et al. 1989, Augustian et al. 1994, Guillaumin, Mohammed & AbomoNdongo 1994, Mohammed et al. 1994, Mwangi et al. 1994, Mwenje & Ride 1996, AbomoNdongo & Guillaumin 1997), A. camerunensis (Henn.) Yolk & Burdsall [=A. camerunensis (Henn) = A. mellea (Yahl.:Fr.) P. Kumer var camerunsis Henn] (Singer 1986, Mohammed et al. 1989, Yolk & Burdsall 1995), A. mellea (Yahl.:Fr.) P. Kumm. sub species Africana (Mohammed et al. 1994, Yolk & Burdsall 1995) and A. fuscipes Petch (Coetzee et al. 2000). In Ethiopia, damage due to Armillaria root rot has been reported from Pinus patula Schiede & Deppe plantations at various sites (Mengistu 1992, Dagne 1998, Alemu, Roux & Wingfield 2003). Tree death in plantations due to this disease has been estimated to be between 520 % (Dagne 1998). Eshetu, Teame & Girma (2000) also noted that Armillaria root rot caused minor damage in coffee (Coffea arabica L.) plantations. Despite this, little attention has been given to the disease. It has generally been assumed 73 that Armillaria root rot is caused by A. mellea (Mengistu 1992, Eshetu et al. 2000) and no detailed study has been conducted to identify the Armillaria spp. found in Ethiopia. However, a recent study using somatic incompatibility, isozyme comparisons and Random Amplified Polymorphic DNA (RAP D) analyses has suggested the presence of A. mellea on hard woods in the Kerita and Jima areas of Ethiopia (Ota, Intini & Hattori 2000). During a survey of plantation forestry diseases in Ethiopia, conducted in 2000 and 2001, Armillaria root rot was identified as a common cause of tree mortality (AIemu et al. 2003). The species identity of the causal agent was, however, not known. The aim of this study was thus to identify the Armillaria isolates obtained from the surveys and to consider their phylogenetic relationships with other Armillaria species. To accomplish these objectives morphological characteristics of the basidiocarps and DNAbased compalisons including RFLP and DNA sequencing of the IGSl region of the rRNA operon, were used. MATERIALS AND METHODS Sample collection and isolation Surveys were conducted in forestry plantations at Munessa Shashemene, Jima, Bedele, AmanlMizan and Wondo Genet (Figure 1). Typical symptoms of Armillaria root rot were used to recognise centres of infection. Samples were collected from roots, stumps and stems of dead and dying trees. Small pieces of mycelium from the white mycelial fans, between the bark and the wood were transferred to a selective medium, containing benomyl and streptomycin (Harrington, Worall & Baker 1992). Cultures were incubated at 25°C in the dark for three weeks. Pieces of mycelium from the tips of the cultures were then transferred to 2% MEA (2% Biolab Malt Extract, 1.5% Biolab Agar) plates to multiply them for further use. Stock cultures of all the isolates used in this study are maintained on 2% MEA slants at 5 °c in the culture collection (CMW) of the Forestry 74 and AgIlcultural Biotechnology Institute (FABI), University of Pretoria, South Africa (Table 1). Basidiocarp morphology Basidiocarps collected from stumps of felled Juniperus exelsa Hochest. Ex. Endl. trees were used to study their morphology. Morphological characteristics of these structures were compared with those published for other species. Characters examined included the colour of the basidiocarps and size of the pileus and stipes. Rayner's (1970) colour cha11 was used to determine colors. DNA extraction Representative isolates (CMW5837, 5844, 5846, 8967, 8969, 8971) (Table 1) from different sites and hosts were grown in liquid MY medium (2% Biolab malt extract, 0.3% Biolab yeast extract agar) in the dark at 25°C, for approximately tlu'ee weeks. Mycelium was harvested from cultures by centrifugation (8000 g, 30 min) and freeze dried. The dried mycelial samples were ground to a fine powder in liquid nitrogen using a mortar and pestle. DNA was extracted using a modified version of the DNA extraction method of Raeder and Broda (1985). Extraction buffer (200 mM TrisHCl pH 8; 25 mM EDTA; 250 mM NaCI) (1000 ).!1) was added to about 0.5 g of powdered mycelium and incubated at 60°C for 30 min. This was followed by a phenol:chlorophorm extraction step. Cell debris was removed by centrifugation at 13000 g for one hour. FUl1her phenol:chlorophorm extractions were done on the aqueous phase until a clean interphase was obtained. Chlorophorm extractions were done to remove the traces of phenol. Sodium Acetate (3M NaAc) and absolute ethanol were added to precipitate the nucleic acids and they were collected by centrifugation at 13000 g. The nucleic acid pellet was washed with 70% ethanol, vacuum dried and dissolved in 50 ).!l sterile water. RNase A (0.01 mg/ml) (Roche) was added to the DNA and water suspensions to remove RNA and incubated overnight at 37°C in a water bath. The resulting DNA was visualised under 75 UV illumination after electrophoresis on a 1% agarose gel (Prom ega, Madison, Wisconsin) stained with ethidium bromide. DNA amplification The IGS1 reglOn of the ribosomal RNA (rRNA) operon was amplified usmg the polymerase chain reaction (PCR). This region was amplified with Primers Pl (5' TTG CAG ACG ACT TGA ATG G 3') (Hsiau 1996) and 5S2B (5' CAC CGC ATC CCG TCT GAT CTG CO 3 ') (Coetzee et al. 2000). The PCR mixtures used included dNTPs (20~M of each), MgCh (2.66mM), 10 x buffer containing MgCh (supplied with enzyme), 0.375 ~M of each primer, Taq polymerase (2.6 U) (Roche) and approximately 80 ng template DNA. The final reaction volume was adjusted to 50 ~l with H 20. The PCR programme consisted of an initial denaturation step at 96°C for 2 min. This was followed by 35 cycles of annealing at 58 °c for 30 s, elongation at 72 °c for 2 min., a ramp time of 1.5 s and another denaturation at 94°C for 30s. A final elongation step was allowed for 7 min at 72 °C. Prior to sequencing, the PCR products were purified using a QIAquick PCR Purification Kit (QIAGEN, Germany). The fragment sizes of the PCR products were determined after electrophoresis on a 1% agarose gel stained with ethidium bromide and visualised under UV illumination. A 100 bp molecular marker was used to determine the size of the PCR fragments. Restriction enzyme digestion Restriction Fragment Length Polymorphism (RFLP) profiles of isolates included in this study were obtained by digesting the IGS1 amplicons with the restriction endonuclease Alu I (Harrington & Wingfield 1995). IGS1 amplicons were digested by adding 3 units of Alu I restriction endonuclease to 18 ~l of unpurified PCR product. Digestion was allowed to occur overnight at 37°C. DNA fragments were separated on a 3% (w/v) agarose gel (Promega, Madison, Wisconsin) stained with ethidium bromide and profiles were visualised under UV illumination. A 100 base molecular weight marker was used to determine the fragment sizes. The absolute fragment sizes were determined using the 76 programme Gelreader 20.5 (National Center, Supercomputing Applications, University of Illinois at UrbanaChampaign, 1991). RFLP patterns and sizes oflGSl amplicons for the Ethiopian Armillaria isolates were compared with those of Armillaria spp. published by Coetzee et at. (2000). Cloning It was not possible to sequence the IGSl amp1icons directly and they were subsequently cloned to resolve this problem. Ligation of the PCR products was conducted using the PGEM® T Easy Vector System (Promega Corporation), 2X Rapid Ligation Buffer, T4 DNA Ligase, PCR products and deionized water according to the protocols outlined by the manufacturer. This reaction was incubated for one hour at room temperature. For · transformation, JMI09 High Efficiency Competent cells provided with the PGEM® T EASY Vector System II were used. Two III of the ligation reactions were transferred to 1.5 ml Eppendorf tubes and 25 III competent cell solution added to each Eppendorf tube. Isolation of recombinant plasmid DNA was accomplished using a standard plasmid miniprep procedure, using the instructions provided by the company. DNA sequencing Plasmid DNA was used as template to sequence the inserted IGS1 region of the Armillaria samples. DNA sequences were determined using an automated (ABI PRISMTM 3100) DNA sequencer. The inserted region was sequenced in both directions using primers T7 (5'ATT ATG CTG AGT GAT ATC CC- 3') and SP6 (5' ATT TAG GTG ACA CTA TAG AA3') (Promega 1999). The sequencing reactions were prepared using the Big Dye sequence system (ABI Advanced Biotechnology Institute, PerkinElmer) as recommended in the manufacturer's protocols. 77 Analysis ofDNA sequence data Sequence Navigator version 1.01 (ABI PRISMTM, Perkin Elmer) was used to manually align the sequence data by inserting gaps. Analysis of the sequence data was done using PAUP* version 4.0b2 (Swofford 1998). In the sequence data analysis, indels of more than 1 base were excluded and substituted with multistate characters and gaps treated as a 5th character. IGS1 DNA sequences obtained in this study were aligned against the data matrix published by Coetzee et al. (2000) and available on Tree Base (Table 1). Phylogenetic trees generated were rooted to A. heimii as the monophyletic sister group to the taxa. Analyses were done using NeighborJoining distance analysis and the total character difference was used to generate the tree. The confidence levels of the branching points were determined by 1000 bootstrap replicates. RESULTS Sample collection and isolation Symptoms of Armillaria root rot were found in plantations at Wondo Genet, Munessa Shashemene, Belete/Jima, Bedele and Arnan/Mizan. Armillaria root rot was identified on 1013 year old P. patula, Acacia abyssinica Hochest, Cordia alliodora (Ruiz & Pav) Oken and Cedrela odorata L. trees (Table l, Figure 1). The characteristic symptoms of infection included groups of dead trees (Figure 2a), wilting and chlorosis, as well as the occurrence of white mycelial fans (Figure 2b) under the bark of diseased trees. Masses of light brown rhizomorphs were found on diseased C. alliodora trees, in a research plot at Arnan (Figure 2c). The causal fungus was successfully isolated from symptomatic trees and grown on the selective medium. A total of 32 isolates were collected from the different hosts. In culture, the Armillaria isolates produced a flat whitish mycelium. Brown, cylindrical rhizomorphs were produced abundantly in cultures (Figure 2d). At the time of sample collection, the incidence of Armillaria root rot damage was most pronounced on P. patula at Wondo Genet. 78 Basidiocarp morphology Ten basidiocarps were collected from stumps of J exelsa trees, in a plantation at Wondo Genet (Figure 2e). These basidiocarps were used to partially identify the Armillaria sp. in this study. When the colour, the size of the stipe and the pileus of the basidiocarps were considered, the basidiocarps from Ethiopia showed close similarities with the basidiocarp morphology of A. juscipes (Coetzee et al. 2000) and differed from those of the much smaller A. heimii. The pileus of the fungus had an average diameter of 45 mm and the length of the stipes varied between 6087 mm. These measurements are more similar to those of the basidiocarps of A. juscipes (Pileus = 51 nun, Stipe = 6484 nun) than of A. heimii (Pileus = 30 mm, Stipe = 2545 mm) [Figure 2e]. DNA amplification The IGS regions of all Armillaria isolates from Ethiopia were successfully amplified with primers P1 and 5S2B. The PCR products of all Armillaria isolates used in this study yielded fragments of approximately 1200 base pairs (bp). This PCR fragment size is similar to that published for A. juscipes (Coetzee et al. 2000). Restriction enzyme digestion Alu I restriction digestion of PCR amplicons generated identical fragment patterns for all isolates. Three distinct bands with sizes of approximately 370, 250 and 95 bp were obtained (Figure 3). Comparison of RFLP profiles of the Ethiopian Armillaria isolates with published profiles for A. juscipes and A. heimii (Coetzee et al. 2000) showed that the RFLP patterns of Armillaria isolates from Ethiopia are different to both A. juscipes and A. heimii (Table 2). Furthermore, the Ethiopian RFLP profile did not match that of any other Armillaria sp. for which such profiles are available. 79 DNA sequencing The IGS sequence of the Armillaria isolates from Ethiopia, before alignment, varied between 1056 and 1100 bp. A Blast search using the IGSl and 5S gene sequences for these isolates against sequences in GenBank [National Centre for Biotechnology infonnation (NCB I), US National Institute of Health Bethesad, (http:/ www.ncbi.nlm. Nih.gov/BLAST)], indicated that the DNA sequences of Armillaria isolates from Ethiopia most closely match with the sequences of A. juscipes and A. heimii. Therefore, the DNA sequences of the Ethiopian Armillaria isolates were aligned with these two species (Coetzee et al. 2000). A total of 1247 characters were obtained for analysis after manual alignment. Analysis ofDNA sequence data The Armillaria isolates used in this study formed two main groups in a neighbourJoining tree (Figure 4). Sequences of Armillaria isolates from South Africa and La Reunion, which were previously identified as A. juscipes (Coetzee et al. 2000) grouped together with a bootstrap support of 90%. The Armillaria isolates from Ethiopia resided in a separate cluster with 74% bootstrap support. The Ethiopian Armillaria isolates grouped separately from A. heimii, showing the closest affinity to A. juscipes, although with some differences. The Ethiopian isolates differed from A. juscipes in having several indels. Isolate CMW8971 differed from A. juscipes with only 11 bp indels (of which 7 bps are deletions), while other Ethiopian isolates showed more variation. The most notable of these are isolates CMW5838 and CMW5846, which have 16 bp deletions, whereas isolates CMW5844, CMW8967 and CMW8969 have 33 bp deletions and contain one restriction site at position nine. Despite these differences, the Ethiopian isolates group with the A. juscipes clade with a bootstrap of 100 % and separately from A. heimii. DISCUSSION Recently, the importance of plantation forestry diseases in Ethiopia has been afforded considerable attention. Results from this study thus, form part of the first comprehensive 80 plantation disease survey conducted in the country (Alemu et at. 2003). This study furthennore presents results of the first extensive survey of Annillaria root rot in Ethiopian forest plantations. Our data clearly show that the dominant Armillaria sp. causing root rot and death in plantations is A. juscipes. This is the first report of A. filscipes from Ethiopia and also greatly extends its host range. Damage from Annillaria root rot has been observed in several African countries, where it has been attributed mainly to A. mellea and A. heimii (Pegler 1986, Ivory 1987). Armillaria juscipes was recently reported to be common in Southern Africa (Coetzee et at. 2000). Outside Africa, A. juscipes is known only from Sir Lanka, where it was first described and where Pegler (1986) suggested that it could have been introduced from Aflica. The taxonomic status of A. heimii and A. juscipes has, however, been confused for many years. It has thus been suggested that A. heimii is conspecific with A. filscipes and the latter name was retained (Pegler 1986, Ki1e & Watling 1988, Watling, Ki1e & Burdsall 1991). Recent studies have shown the existence of significant vmiation between A. heimii isolates from various Aflican countries (Augustain et at. 1994, Mohammed et at. 1994, Mwenje & Ride 1996). A DNA based study conducted on Southern African Armillaria isolates, thought to represent A. heimii showed that they are dissimilar to A. heimii from Zambia, Zimbabwe and Cameroon (Coetzee et at. 2000). In the study of Coetzee et at. (2000), Armillaria isolates from South Africa were shown to represent A. juscipes, and not A. heimii. Similarly, Armillaria isolates from La Reunion, believed to represent A. heimii were found to be identical to the South African Armillaria isolates and identified as A. filscipes (Coetzee et at. 2000). This study provided clear evidence that these two species represent distinct taxa. The results of the present study show that the Ethiopian Armillaria isolates represent A. juscipes, although some differences were observed in RFLP and lOS sequence data. Basidiocarp morphology has commonly been used to detennine the relationships of Armillaria spp. (Berube & Dessureault 1989, Watling et at. 1991). The macromorphological characters including colour and structures of the pileus, veil, annulus and stipe are reliable characters for this purpose (Berube & Dessureault 1989). Seasonal 81 availability of the basidiocarps, however, limits the use of basidiocarp morphology for species identification. In this study, very few fruiting structures were obtained and these were only from Wondo Genet. The macromorphological characters of these basidiocarps were different from those of A. heimii, having larger pileus and stipes, compared to the small basidiocarps of A. heimii (Kile & Watling 1988). The basidiocarps from Ethiopia were very similar to those from South Africa, known to represent A. juscipes. It was not possible to collect a culture linked to these basidiocarps but the proximity of the dying trees to others from which cultures and DNA sequences were obtained provides strong circumstantial evidence that the fungus is the same. Coetzee et al. (2000), showed that the 5S libosomal rRNA gene of African A. juscipes and A. heimii isolates are in opposite orientation in comparison to other Armillaria spp. Because of this, primers used to amplify the IGSl region of nonAfrican isolates failed to amplify the IGSI region of African Armillaria isolates (Coetzee et al. 2000). Primer 5S2B was, therefore, used to amplify the IGSl region of African Armillaria spp. The IGSl region of the Armillaria isolates from Ethiopia was successfully amplified with primers Pl and 5S2B indicating that the 5S gene of Ethiopian Armillaria isolates has the same orientation as that of other African A. jilscipes and A. heimii isolates. This provides further SUppOlt for our belief that the Ethiopian isolates represent A. juscipes. A recent population study on Armillaria spp. in Ethiopia reported that A. mellea is responsible for root rot on hardwood trees in the lima and Kerita areas (Ota et at. 2000). An isolate from symptomatic P. patula trees near lima in our study, produced the same RFLP profile as those of other Armillaria isolates that we have identified as A. juscipes. This suggests that the causative agent of Armillaria root rot of P. patula around lima is identical to other isolates included in our study and that it also represents A. jilscipes. The results of Ota et al. (2000) and this study, thus suggest that more than one Armillaria spp. might be involved in causing Annillaria root rot in Ethiopia. This emphasises the impoltance of conducting further and more comprehensive studies on the diversity, distribution, and host range of Annillaria root rot in Ethiopia. 82 RFLP patterns of all Ethiopian Armillaria isolates differed from those of A. juscipes and all other Armillaria spp. This difference in RFLP pattern was supported by DNA sequence data, which showed the deletion of indels within one of the restriction sites. Although the Ethiopian isolates grouped closely to A. fuscipes, they formed a separate subclade. This suggests that the Armillaria samples from Ethiopia could represent a distinct species, closely related to A. jitscipes. Macro and micromorphological comparison of the basidiocarps will be essential to understand the significance of this variation. Results of this study have shown that Armillaria root rot not only affects P. patula, but that it also kills Co. alliodora and C. odorata trees planted in research plots at Arnan, near Mizan. The fungus was also found on A. abyssinica and J excels a, species native to Ethiopia and growing in the Pinus plantations at Bedele and Wondo Genet. Most plantations in Ethiopia are made up of exotic species and these are planted on sites previously occupied by indigenous hardwoods. This suggests that stumps of the native hardwoods could be sources of the initial inoculum needed to infect exotic species. 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(1995) A nomenclatural study of Armillaria and Armillariella species. Synopsis Fungorum 8: 112l. Wargo, P. M. & Shaw, C. G. (1985) Annillaria root rot: the puzzle is being resolved. Plant Disease 69: 826832 Watling, R., Kile, G. A. & Gregory, N. M. (1982) The genus Armillaria-nomenclature, typification, the identity of Armillaria mellea and species differentiation. Transactions ofthe British Mycological Society 78: 271285. Watling, R., Kile, G. A. & Burdsall, H. Jr. (1991) Nomenclature, taxonomy and identification. In Armillaria root disease (C. G. Shaw & G. A. Kile eds). pp. 19. Agriculture Handbook No. 691. Forest Service, United States Department of Agriculture, Washington DC. 87 Table 1. Armillaria isolates used in this study Identity A. jilscipes b A. filscipes b A. filscipes b A. filscipes b A. filscipes b A. Juscipes b A. filscipes c A. filscipes A. filscipes c A. heimii A. heimii A. heimii A. heimii C C C C C Culture number a CMW5838 CMW5844 CMW5846 CMW8967 CMW8969 CMW8971 CMW27 17 CMW2740 CMW3l67 CMW3l52 CMW3l64 CMW3173 CMW3955 Host Origin Collector Pinus patula P. patula P. patula Cordia alliodora Acacia abyssinica P. patula P. elliottii P. patula P. elliottii Unknown Pelargonium asperum Tectona grandis Acacia xanthophloea Wondo Genet, Ethiopia Won do Genet, Ethiopia Won do Genet, Ethiopia AmanJMizan, Ethiopia Bedele, Ethiopia Belete/Jima, Ethiopia Sabie, South Africa Entabeni, South Africa Sabie, South Africa Western Province, Cameroon SaintDenis, La Reunion Dola Hill, Zambia Harare, Zimbabwe Alemu Gezahgne & Roux, J. Alemu Gezahgne & Roux, J. Alemu Gezahgne & Roux, 1. Alemu Gezahgne & Roux, J. Alemu Gezahgne & Roux, J. Alemu Gezahgne & Roux, 1. Wingfield, M. J. Wingfield, M. J. Ivory, M. Watling, R. F abergue, C. Ivory, M. Wingfield, M. J. & Coetzee, M. P. A. Accession No. AYl72029 AYl72032 AYl72030 AYl72031 AYl72034 AYl72033 AF204821 AF204822 AF204823 AF204826 AF204824 AF204825 AF204827 CMW numbers refer to the culture collection numbers of the Tree Pathology Cooperative Programme (TPCP), FABI, University of Pretoria. Pretoria, South Africa. b Isolates sequenced in this study. Sequence of Armillaria, in FABI database, identical to those submitted to GenBank (Coetzee et al. 2000). a C 88 Table 2. Comparison ofRFLP sizes of Armillaria isolates Armillaria from Ethiopia A. jilscipes a A. heimiz.(J A. mellei 370 365 530 215 250 245 220 175 95 135 175 150 a Data obtained from Coetzee et al. 2000 b Data obtained from Coetzee et al. 2001 89 , " \ LakeTana SOMALIA ]0 Bedele = • Jima Addis Ababa o Wolisso D MillleSSa Shashemenne = Wondo Genete SOMALIA Figure 1. Map of Ethiopia showing the plantation areas where surveys were conducted. 90 150 CMvJ3173 CMW3152 CMW3955 CMW5837 CMW5846 CMW8968 CMW5844 CMW 8967 CMW 8971 CMW2717 CMW2740 CMW3167 CMW3164 ZAMB IA CAM EROON ZIMBABW E ETHI OPIA ETHI OPIA ETHIOPIA ETHIOPIA ETHIOPIA ETHIOPIA SOUTH AFRICA SOUTH AFRI CA SOUT H AFRI CA LA REUNION CMW317 3 CMW315 2 CMW39 55 CMW5837 CMW5846 CMW8968 CMW5844 CMW8967 CMW8971 CMW2717 CMW2740 CMW3167 CMW3164 ZAMBIA CAM EROO N ZIMBABWE ETHIOPIA ETHI OP I A ETHI OP I A ETHI OPIA ETHIOPIA ETHIOPIA SOUTH AFRICA SOU TH AFRICA SOUTH AFRICA LA REUNION 160 170 1 80 190 200 2 10 AACAGCATGT TTAATGGA AGCCTATT GTGTATAATA TTGGTATATA CGGTGTACGG · ..... ... . · ....... ....... G · ......... · ......... · ......... · ......... · ......... · ......... · ......... · ......... · .......... · ......... · ......... · ....... ---------- . G ... . .. GG . G ...... GG .G ...... GG .G ...... GG . G ... ... GG .G ..... . GG . G ... . .. GG . G ...... GG . G ..... . GG .G ...... GG GGTATGGATC GGTATGGAT C GGTATGGAT C GGTATGGATC GGTATGGATC GG TATGGATC GG TAT GGATC GGTATGGATC GGTATGGATC GGTATGGATC CA ... G.... CA ... G.... CA ... G.... CA ... G.... CA ... G.... CA .. . G .... CA ... G.... CA ... G.... CA ... G.... CA ... G.... -------- ---------------------- --------------- --------------- --------------- · · · . . · · · · · ...... T. ...... T. ...... T. ...... T. . ..... T. . ..... T. ...... T. .. .... T. ...... T. ...... T. 280 250 270 220 230 240 260 AGTACGGGTA TACAGAAGAG TATAC AGTACAGTAG ACAGTATATA TATATATA TTATATA · ......... · ......... · ......... · ......... .. . G ... ... · ..... . ... .. . G ...... .. . G . . .... ... G...... .. . G ...... .. . G .. .... .. . G ...... .. . G .. .... .. . G ...... .. . G .. .... .......... · ......... · ........ . · ......... · ......... · ......... · ......... .......... .......... . . . . . . . . . . · ........ . . . . . . . . . . . . . . . . . . . · ......... ........ ...... . ..... AAGAG ..... .. . G ..... C .......... ........ TA TA .... G.A. AAGAG ..... .. . G .... . C ......... . ....... . TA .... G.A . AAGAG ..... .. . G ..... C .......... ........ TA TA .... G.A. AAGAG ..... . .. G... .. C .......... . ....... TA TA .... G.A. AAGAG ..... .. . G ..... C .......... . ....... TA TA .... G.A . AAGAG ..... .. . G ..... C · ......... ........ TA TA .... G.A . AAGAG ..... ... G..... C · ......... . ... . ... TA .... G.A. AAGAG ..... ... G..... C . . ........ ... .... . TA .... G.A . AAGAG ..... .. . G . .... C .. ........ ........ TA .... G.A . AAGAG ..... ... G.. .. . C .......... .. ..... . TA . ... G.A . 94 290 CMW317 3 CMW31 52 CMW3955 CMW5837 CMW5846 CMW8968 CMW5844 CMW8967 CMW8971 CMW2717 CMW2740 CMW3167 CMW3164 ZAMBIA CAM ER OON ZIMBABW E ET HI OP IA ETHI OP I A ETHIOPIA ETHI OP I A ETHI OP I A ETHIOPI A SOU TH AFRICA SOU TH AFR ICA SOU TH AFRICA LA REUNION CMW31 7 3 CMW3 15 2 CMW3955 CMW5837 CMW5846 CMW8968 CMW5844 CMW8967 CMW897 1 CMW2717 CMW2740 CMW3 1 67 CMW3 1 64 ZAMBIA CAMEROON ZI MBABWE ETHI OP I A ETHIOPIA ETH IOPIA ETHIOPIA ETHIOPIA ETHIOPIA SOU TH AFRICA SOU TH AFRICA SOUTH AFRICA LA REUNION 300 310 330 320 340 350 TCTATGAC TTG GAC TT GG ACTTGTACTT GGACTTGGAT CTT GGATCAC AATG CAAGTA AGGTAGTAGG . C. .... CAT ... .... CAT ... .... CAT ... .... CAT ... . . .. CAT .. . .... CAT ... .... CAT ... .... CAT .. . .... CAT .. . .... CAT ... · ......... · ......... · ......... · . .. ...... · ......... · ...... ... · .. . ...... · ......... · ..... . ... · . . . ...... · ......... · ..... ... . · .... ..... · ......... · .......... · ... .. . ... · ......... · ..... ... . · .. ....... · . ... ..... · ..... . ... · . . ....... · .......... · ..... . ... · ......... · ... .. ..... · ......... · ......... · ......... · ......... . .... G . .... G . .... G . .... G . .... G . .... G ..... G.. . .... G.. ..... G.. . .... G.. .T .T .T .T .T. A . AT .T. A . AT .T.A.AT .T.A.AT .T.A.AT .T.A.AT G... T .A.AT G... T . A .AT G ... T.A.AT G... T.A.AT 370 380 390 400 410 420 360 CAATGCAACG CAAGGCTAGT AGACAACGCA AGGCAATGCA AGGATAGTAG ACAATGCAAG GCAATGCAAG . . . . . . . . . . · ......... .A .. G . . CA . . CA . . CA. .CA. .CA. .CA. .CA. .CA. .CA. .CA. · · · · · · · · · · . A ....... .A ....... . A .. . .... .A . . . .... .A .... ... .A ....... .A ....... .A ....... .A ....... . A ....... · · · · · · · · · · ... A ... A ... A ... A . . . A ... A ... A ... A ... A ... A - 95 CMW315 2 CMW 39 55 CMW5 837 CMW5846 CMW8968 CMW5844 CMW896 7 CMW8 971 CMW27 1 7 CMW2 740 CMW3 1 67 CMW3164 ZAMB I A CAM EROON ZI MBABWE ETHIOP I A ETH IOP I A ET HI OPI A ET HI OPI A ETHIOP IA ETHIO PI A SOUT H AFRICA SOUT H AFRICA SOU TH AFRICA LA REUN I ON CMW3 1 73 CMW3152 CMW39 55 CMW58 37 CMW5846 CMW8968 CMW 5844 CMW8967 CMW89 71 CMW27 17 CMW2740 CMW3 1 67 CMW 3164 ZAMBI A CAMEROON ZI MBABWE ETHIOP I A ETH IOP I A ETHIOP IA ETHIOPI A ETH IOP I A ETHI OP I A SOUT H AFR I CA SOU TH AFR I CA SOU TH AFRICA LA REUNI ON CM~)317 470 430 440 450 460 4 80 4 90 GC TAGTAGAC AACGCAACGC AATGCAA GG CTAG TAGACA ACGCAAGGC AAG TAAGC T AGCAGGCAGA ---- - - - - - - ------- - - - · . . ....... · ..... .... · .... . . . . · ... .. .... · ... ...... · ......... · ......... · ..... . . . . · ........ . · . .. .. . ... · · · · · · . T .. . . G. . T . . . . G. . T .... G. . T ... . G. . T .... G. . T .... G. .... . . .. . G. · . . . . .. . .. . GA . G.C .. ..... . . . C ....... T · ....... . . . GA . G. C . . . ...... . C .. ..... T . GA .G.C . GA . G. C . GA.G.C .GA . G . C - -- - - - - - -- - -- - - - - - - - - - - - - - - - . GA.G . C - - - - - - - - - - - - - - --- --- - - - - - - - - · . T ....... . GA . G. C . . . T ....... .GA.G .C -- - - - - - - - - - - - - - - - - - - - - - - - - - - · .N . ..... . .GA . G . C o ••••••••• · . . .. . . .. . · . . . ...... · ... . ..... · ...... . .. . . ....... ... . ..... ......... .. . ... . .. . . .. ..... . .. . ..... . ........ ... . ..... . C . G. . C. . C. . C. . C. . C. . C. . C. 50 0 510 520 530 54C 55 0 560 CTTGTGAG TTGAGAGCTT GTACGCATGT CTTAG TTGGT GTGCA - - - - - - - - -- - - - - - - -- -. ......... · ... . ..... · ......... · ....... · . . . .... · .. . ..... . · .... ... .. · .. . . . ... . · . . . A .. . .... A . .. · .... .. . · .. ... . . · ....... · . ...... TC .. . ..... . . . ... .... TC ....... ... · ... .... TC ..... .... . · .... . .. TC ..... . .. . . ... ... . . TC . ... . ..... -- - - - - - - - - -- - - - - - - - - · ........ . T ........ C .C ... TAGAG TC TTT GGACT TGGGAC TT GG · ..... . .. . ...... . . . C . C ... TAGAG TCTT TGGAC T TGGGAC TT GG .. . .. C . .... C ..... C · ........ . .. .. ... . . C . .... . . . C. ..... .. . . C .... . .. . C. . . . . . . . . . C .... .. . . C. ... . . . ... C . . ... .. . C . . ... ..... C .C .. . TAGAG .C . .. TAGAG . C ... TAGAG .C ... TAGAG .C ... TAGAG .C ... TAGAG .C ... TAGAG . C ... TAGAG TCT TTGGACT TCTTT GGACT TCT TTGGACT TCTTT GGAC T TC TTT GGAC T TCTTTGGACT TCTTT GGAC T TCT TTGGAC T TGGGACT TGG TGGGAC TT GG TGGGACTT GG TGG GACT TGG TGG GACTT GG TGGGAC TT GG TGGGAC TT GG TGGGACTT GG 96 CMW3 1 73 CMW3 152 CMW3 955 CMW5 837 CMW5846 CMW8968 CMW5844 CMW8967 CMW8971 CMW2717 CMW2740 CMW3167 CMW3164 ZAMBIA CAM EROON ZIMBABWE ETHI OPIA ET HI OPIA ETHI OP I A ETHI OP I A ET HI OPIA ETHIOPIA SOUTH AFRI CA SOU TH AFR I CA SOU TH AFRI CA LA REUN I ON 570 58 0 590 61 0 600 TTGCGGAC TTGG G CAT TGAGGG ..... ... · .. . . . . . . ....... . · ..... . . . ACAGCCAACG GA ........ ... . ACAGAA TTGCAAGCT. · .. . . . . C . ACAGCCAACG GA ........ ... . ACAGAA TT GCAAGCT . · ..... . C. ACAGCCAACG GA ........ ... . ACAGAA TT GCAAGCT. · .... . .C . ACAGCCAACG GA .. ... .. . .. . . ACAGAA TTGCAAGCT. · ..... . C . ACAGCCAACG GA ........ .. . . ACAGAA TTGCAAGCT. · . . ... . C. ACAG CCAACG GA ........ . . . . ACAGAA TT GCAAGCT. · . . ... . C. ACACCCAATG GA ....... . ... . ACAGAA TTGCAAGCT . · ..... . C . ACACCCAATG GA .... .. .. . .. . ACAGAA TTGCAAGCT. · ..... . C. ACACCCAATG GA ..... ... .... ACAGAA TTG CAAGC T . ..... . A.C . ACACCCAATG GA ... ... .. ... . ACAGAA TT GCAAGCT. · ..... . C . 620 630 CTT GTATGCA CGCA CCT · ......... T . ... ... · . C ... C ... · .C ... C ... · . C ... C. .. · . C ... C. .. · . C .. . C ... · .C ... C ... · .C . .. C ... · .C ... C ... · . C ... C . .. .. C .. . C ... T ... TG .. T ... TG ... T ... TG ... T ... TG ... T ... TG ... T ... TG ... T ... TG ... T ... TG ... T ... TG ... T ... TG ... CMW3 1 73 Cj\1W3152 CMW3955 CMW5837 CMW5846 CMW8968 CMW5844 CMW8967 CMW8971 CMW2717 CMW2740 CMW3 1 67 CMW3164 ZAMB I A CAlvJEROON ZI MBABWE ETHIOPIA ETHIOPIA ETH IO PIA ETHIOPIA ET HI OPIA ETHI OP I A SO UTH AFR I CA SOU TH AFR I CA SOU TH AFRICA LA RE UNI ON 640 670 650 660 TAACGGACTT GGACATTGAG GTGTATGCAC G CTT · . ....... . . . . . . . . . . . . . . . . . . . . .GACA . . GAG · . - . . . . . . . . .... . . ... . .. . .. .... . CAC ... ACG · . CTT.T ... . C. .. C.. C .. C.. T .A... GCC · . CTT. T ... .C . .. C .. C .. C .. T . A... GC C .. CTT . T ... . C. . . C.. C .. C .. T . A ... GCC · . CTT. T .. . . C. .. C.. C .. C .. T .A . .. GCC · .CTT . T ... . C . .. C.. C .. C .. T . A... GCC · .CTT . T ... .C. .. C.. C . . C .. T .A... GCC · . CTT.T ... . C. .. C . . C . . C . . T .A ... GCC · . CT T .T ... . C. .. C .. C.. C .. T .A ... GC C · . CTT. T ... . C . .. C .. C.. C .. T . A... GC C · . CTT. T ... . C . .. C . . C .. C .. T . A ... GCC 690 GGACATTGA ......... ......... T. .. . . C. T. .... C. T. . .. . C. T. .... C . T . .... C. T. .... C . T . ...... T . . . .... T. . ... .. T. ...... 700 G - 680 GTGTATGCAC GACT T CTCAAGCAAA CTCAAGCAAA CTCAAGCAAA CTCAAGCAAA CTCAAGCAAA CTCAAGCAAA CTCAAGCAAA CTCAAGCAAA CTCAAGCAAA CTCAAGCAAA · .......... ATGCG TCGAC ATGCGTCGAC ATGCGTCGAC ATGC GT CGAC ATG CGT CGAC ATGCGTCGAC ATGCGTCGAC ATGCGTCGAC ATGCGTCGAC ATGCGTCGAC 97 CM~\137 CMW3152 CMW3955 CMW5837 CMW5846 CMW8968 CMW5844 CMW8967 CMW8971 CMW2717 CMW2740 CMW3167 CMW3164 CMW3 173 CMW3152 CMW3955 CMW5837 CMW5846 CMW8968 CMW5844 CM ftJ8 967 CMW8971 CMW2717 CMW 2740 CMW3167 CMW3164 ZAMBIA CAMEROON ZIMBABWE ETHI OP IA ETHIOPI A ETHIOPI A ETHIOPIA ETHIOPIA ETHI OP I A SOUTH AFRICA SOU TH AFRICA SOU TH AFRICA LA REUNION ZAMBIA CAMEROON ZIMBABWE ETHI OPIA ETHI OP IA ETHIOPIA ETHIOPIA ETHIOPIA ETHIOPIA SOUTH AFRI CA SOUTH - AFRICA SOUTH AFRICA LA REUNION 740 750 760 720 710 730 770 GTGT ATGCA CGGACAT TGAGG TGT AT ---------- ------ - ---------- ---------- ---------- ------ - ---------- ---------- ....... . ......... ....... . ......... TTGCAAGCTA TTGCAAGCTA TTGCAAGCTA TTGCAAGCTA TTGCAGGCTA TTGCAAGC TA TTG CAAGCTA TTGCAAGCTA TTGCAAGCTA TTGCAAGC TA .... TGCGCA .... TG CGCA .... TGCGCA .... TGCGCA .... TGCGCA .... TGCGCA .... TGCGCA .... TGCGCA .... TGCGCA .... TG CGCA TATT ..... T TATT ..... T T~ ..... T TATT ..... T TATT ..... T TATT ..... T TATT ..... T TATT ..... T TATT ..... T TATT ..... T GTCTTACTTG GTCTTACTTG GTCTTACTTG GTCTTACTTG GTCTTACTTG GTCTTACTTG GTCTTACTTG GTCTTACTTG GTC TTACTTG GTCTTACTTG CATTTCGCTA CATTTCGCTA CATTTCGCTA CATTTCGCTA CATTTCGCTA CATTTCGCTA CATTTCGCTA CATTTCGC TA CATTTCGC TA CATTTCGCTA GTTA.C .... GTTA.C .... GTTA.C .... GTTA.C .... GTTA.C .... GTTA.C .... GTTA.C .... GTTA.C .... GTTA. C .... GTTA.C .... .. . CT .. C.A ... CT .. C.A .. . CT .. C.A .. . CT .. C .A .. . CT .. C . A .. . CT .. C.A .. . CT .. C.A .. . CT .. C.A .. . CT .. C.A .. . CT .. C.A 780 790 800 810 820 830 840 GCACGCACCT TACG GAC ------------------------ - - - - - - - - - - - - - - - - - - - - - - - - - - - - - · ......... · ......... ------ · . --- .. AG. · .--- .. AG. · .--- .. AG . · . --- .. AG. · . --- .. AG. · . --- .. AG . · .--- .. AG . .. --- .. AG . · . --- .. AG. · . --- .. AG. .... CTAGTT · ... CTAG TT · ... CTAGTT .... CTAGT T · ... CTAGTT · ... CTAG TT · ... CTAGTT · ... CTAGTT · ... CTAGTT .... CTAGTT AGTTA ... AA AGTTA ... AA AGTTA ... AA AGTTA ... AA AGTTA ... AA AGTTA ... AA AG TTA ... AA AGTTA ... AA AGTTA ... AA AGTTA .. . AA ---------- ---------- ---------- ---------- GCTTGGTTTG GCT TGGTTTG GCTTGGTTTG GCTTGGTTTG GCTTGGTTTG GCTTGGTTTG CC TTGGTTTG CCTTGG TTTG CCTTGGTTTG CCTTGGTTTG ACTTTGGCAA ACTTTGGCAA ACTTTGGCAA ACTTTGGCAA ACTTTGGCAA ACTTTGGCAA ACTT TGGCAA ACTTTG GCAA ACTTTGGCAA ACTTTGGCAA ATGCGTTCAC ATGCGTTCAC ATGCGT TCAC ATGCGTTCAC ATGCGTTCAC ATGCGTTCAC ATGCGTTCAC ATGCGTTCAC ATGCGTTCAC ATGCGTTCAC TTGCAAGCTT TTGCAAGCTT TTG CAAG CTT TTG CAAGCTT TTGCAAG CTT TTGCAAGCTT TTGCAAGCTT TTGCAAGCTT TTGCAAGCTT TTGCAAGCTT 98 850 CMW3173 CMW3152 CMW3955 CMW5837 CMW 58 46 CMv.J8968 CMW5844 CMW 8967 CMW897 1 CMW2717 CMW2740 CMW 31 67 CMW3164 ZAMBI A CAMEROON ZIMBABWE ETHIOPIA ETHIOPIA ETHIOPIA ETHIOPIA ETHIOPIA ETHIOPIA SOUTH AFRICA SOUTH - AFR I CA SOUTH AF RI CA LA REU NION ZAMBIA CAME ROON ZIMBABWE ETHIOPIA ETHIOPIA ETHIOPIA ETHIOPIA ETHIOPIA ETHIOPIA SOUTH AFRICA SOUTH - AFRICA SOUTH - AFRI CA LA REUNION 870 TT GGACAT TGAGGGCTTG TA--------------.... .... . ...... ... -------........ . ......... AG . . . ... TA ... TTT.G .. C .TTGAAATA AG .. .... TA . .. TTT.G .. C.TTGAAATA AG . .. ... TA ... TTT. G .. C.TTGAAATA AG ...... TA . .. TTT.G .. C.TTGAAATA AG ...... TA ., .TTT.G .. C .TT GAAATA AG ...... TA ... TTT.G .. C.TTGAAATA AG .. . ... TA ... TTT.G .. C .TTGAAATA AG .. .... TA ... TTT.G .. C.TTGAAATA AG ...... TA .. . TTT.G .. C. TT GAAATA AG ...... TA .. . TTT . G .. C.TTGAAATA 920 CMW3173 CMW3152 CMW3955 CMW5837 CMW58 46 CMW8968 CMW5844 CMW8967 CMW8971 CMW 27 1 7 CMW2740 CMW31 67 CMW3 1 64 860 930 940 880 900 910 ---------- ---------- -------CGC ---------- ---------- ---------------- ---------- - -- ---CAAGT CAACA TGCTAGCTAG CACTTCAT .A CAAG TCAACA TGCTAGCTAG CACT TCA .. A CAAGTCAACA TGCTAGCTAG CACTTCA . . A CAAG TCAACA TGCTAGCTAG CACTTCA .. A CAAGTCAACA TGCTAGCTAG CACTTCA .. A CAAG TCAACA TGCTAGCTAG CACTTCA .. A CAAGTCAACA TGCTAGCTAG CACTTCA .. A CAAGTCAACA TGCTAGCTAG CACTTCA .. A CAAG TCAACA TGCTAGCTAG CACTTCA .. A CAAGTCAACA TGCTAGCTAG CAC TTCA .. A AC GCACCTTA 970 980 AT-AAAGACT TG CAAGCTAA · . - . . . . . . . · ......... · . - . ...... · ...... .. . . GC.TT .... · ......... · GC. TT .... · ..... .. .. · GC. TT .... · ......... . GC.TT .... · ........ . · GC. TT .. ' . · ......... · GC . TT .... · . ........ . GC. TT .... · ......... . GC. TT .... · ......... · GC. TT .... · ..... .. .. . GC. TT .... · .......... GC TT GATTGG 950 CTTT GTT GGC GCAA- ----- ---------- --------AA ------ -- - ------- -------. ....... . . .......... ------ ---------- -------G... A.A. -- - ... GTATGC CACCTATAGC CAAGTACG .. G... A .A.-- - ... GTAT GC CACC TATAGC CAAGTACG .. G.. . A. A .-- - ... GTATGC CAC CTATAGC CAAG TANG.. G... A . A .-- - ... GTATGC CACCTATAGC CAAGTACG .. G... A. A. -- - ... GTATGC CACCTATAGC CAAGTACG .. G... A.A .-- - ... GTATGC CACCTATAGC CAAGTACG .. G... A.A .-- - ... GTATGC CACCTATAGC CAAGTAC G.. G... A.A .-- - ... GTATGC CACCTATAGC CAAGTACG .. G... A.A.-- - . .. GTAT GC CACCTATAGC CAAGTACG .. G . .. A.A. -- - ... GTATGC CAC CTATAGC CAAG TACG .. 890 960 · ......... · ......... .T.G.A . .. G .T.G.A ... G .T.G.A ... G .T.G. A ... G .T. G.A ... G .T.G.A ... G .T.G.A ... G .T.G.A ... G .T.G. A . .. G .T. G.A ... G · ......... · .... .. ... . ... CG .. .. .... CG .... . . .. CG .... . ... CG .... . ... CG .... .... CG .... . .. . CG .... .... CG .... ., .. CG . . .. .... CG . ... 99 990 CMW3173 CMW3152 CMW3955 CMW5837 CMW5846 CMW8968 CMW5844 CMW8967 CMW8971 CMW2717 CMW2740 CMW3167 CMW3164 ZAMBIA CAMEROON ZIMBABWE ETHIOPIA ETHIOPIA ETHIOPIA ETHIOPIA ETHIOPIA ETHIOPIA SOUTH AFRICA SOUTH AFRICA SOUTH - AFRICA LA REUN I ON ZAMBIA CAMEROON ZIMBABWE ETHIOPIA ETHIOPIA ETHIOPIA ETHIOPIA ETHIOPIA ETHIOPIA SOUTH AFRICA SOUTH AFRICA SOUTH AFRICA LA REUNION 1020 ACT GGAGT ------ .T.CTCTATT AGTTACATCT ACTT ... C.A .T .CTCTATT AGTTACATCT ACTT ... C .A .T. CTCTATT AGTTACATCT ACTT ... C.A .T. CTCTATT AGTTACATCT ACTT ... C . A .T.CTCTATT AGTTACATCT ACTT ... C .A .T.CT CTATT AGTTACATCT ACTT ... C.A .T.CTCTATT AGTTACATCT ACTT ... C.A .T.CTCTATT AGTTACATCT ACTT ... C . A .T.CT CTATT AGTTACATCT ACTT ... C.A .T. CTCTATT AGTTACATC T ACTT ... C.A 1060 CMW3173 CMW3152 CMW3955 CMW5837 CMW5846 CMW8968 CMW5844 CMW8967 CMW8971 CMW2717 CMW2740 CMW3167 CMW3164 1010 1070 1080 1030 1040 1050 1060 CA TGGCTGACAC GCAAAAAGCA AAGGGGGA CTTGTTGG .. TGGCTGACAC GCAAAAAGCA AAGGGGGA CTTGTTGG .. TG GCTGACAC GCAAAAAGCA AAGGGGGGGA CTTGTTGG .. TGGCTGACAC GCAAAAAGCA AAGGGGGA CTTGTTGG .. TGGCTGACAC GCAAAAAGCA AAGGGGGA CTTGTTGG .. TGGCTGACAC GCAAAAAGCA AAGGGGGA CTTGTTGG .. TGGCTGACAG GCAAAAAGCA AAGGGGGA CTTGTTGG .. TGGCTGACAG GCAAAAAGCA AAGGGGGA CTTGTTGG .. TGGCTGACAG GCAAAAAGCA AAGGGGGA CTTGTTGG .. TGGCTGACAG GCAAAAAGCA AAGGGGGA CTTGTTGG .. 1090 1100 1110 1120 GACTTGA TATTCGT ACTTAAT GCTATCTTGC · ...... · .. .... · · · · · · · · · · .A .... ACT .A . ... ACT . A .. .. ACT .A .... ACT . A .... ACT . A .... ACT .A .... ACT .A .... ACT . A .... ACT .A .... ACT . ...... ....... TTT.C ..... TTT.C ..... TTT.C ..... TTT.C ..... TTT.C ..... TTT. C . .. .. TTT.C ..... TTT.C ..... TTT. C ..... TTT.C ..... TTACAGCGTG TTACAGCGTG TTACAGCGTG TTACAGCGTG TTACAGCGTG TTACAGCGTG TTACAGCGTG TTACAGCGTG TTACAGCGTG TTACAGCGTG CGCCGTGCGC CGCCGTGCGC CGCCGTGCGC CGCCGTGCGC CGCCGTGCGC CGCCGTGCGC CACCGTGTGC CACCGTGTGC CACCGTGTGC CACCGTGTGC CGTGCTGGGT CGTGCTGGGT CGTGCTGGGT CGTGCTGGGT CGTGCTGGGT CGTGCTGGGT CGTGCTGGGT CGTGCTGGGT CGTGCTGGGT CGTGCTGGGT . ...... · ......... . ...... · ......... CAG ....... CAG ....... CAG ....... CAG ..... .. CAG ....... CAG ....... CAG ....... CAG ....... CAG ....... CAG ....... · · · · · · · · · · .C .. G.. .C .. G.. . C .. G . . . C . . G .. . C .. G.. .C .. G.. . C .. G.. . C .. G.. . C .. G.. . C .. G.. - 100 1140 11 73 ZAMBIA 152 CAMEROON CM\rJ3955 IMBABWE 837 ETH CMW8 Cl'1W5 ETHIOPIA 1150 TATCTTACTA TCTT ACTATCAA AAACCACAGC ACCCAGGATT .......... . '" .. " '" · '" . ., " '" . . .... ACTATC TT . . . . . . . . · " .. .. '" . . . ,. . . '" . . '" " .. '" ......... · .. .. " . ,. ,. . . . " . ,. .. . . . . . . . . . . . .. .. . . . .. . T ... '" . " . .. . .. . " . .......... ~ ~ ~ ~ -~ 5 ZIMBABWE a .. .. .. .. " .. .. .. .. .. . " ~ ~ ~ .. .. .. ~ ~ • ., . ~ ~ ~ ~ .. .. .. • .. • a ~ • $ ~ .. .. .. " .. .. • .. ~ " • .. .. " ., • a .. ,. ...... ~ ~ ~ .. . ~ ~ . ~ . . . . . ................ -~ 1210 " ...... " .... . . . . .. • .... a .... " 0 GTGGTACTAA CTAGGCGGCA CTTTGNNNNN . .. .. . . " " . . · .. . . . . " . · .NNNNNNNN . . . . ., .......... · .G ....... ..... GTTA .. . . . . . .. NNNNNNNN NNNNNNNNNN . . . . . . . G .. G... . . . . .... ATTA · . . " . . . . · .G ....... .... . ATTA . . .. . . . . . .. . · .G . . ... ATTA ,. ............... .. G ...... ..... ATTA . . . . .. . . . . . · .G ..... · .... ATTA ......... " . · . . . . . . . . · .. .ATTA ....... . . '" .. . . " . · .... GATTA . . . . '" . " " .... · .G ....... . .... GATTA .. .. • .. .. + .. .. • · .G ....... · '" .ATTA ~ . 0" ·. ~ ·. ~ ~ ,. ~ ~ ~ ........ " .... . . .. . . . . .. . . . . .. . . . . ., . · " . " ....... · . ........... .. .. . . .. .. . . . . . . " . ........ ,. ..... .. . . " . -------- ~ CMWB CM\rJB CMW2717 AFRICA 740 SOUTH AFRICA CMW31 SOUTH AFRICA CMW31 4 LA REUNION .. ~ ~ 1200 CM\rJ31 a ~ ~ -~ ~ F,THIOPIA ETHIOPIA AFRICA 7 AFRICA CMW31 AFRICA CM\rJ3164 LA ION 1170 1160 ...... .. • a .. .. . .. 1 NNNNNNNNNN NNNNNNNNNN ACTGCGCAGA NNNNNNNNNN ACTGCGCAGA ACTGCGCAGA ACTGCGCAGA ACTGCGCAGA ACTGCGCAGA ACTGCGCAGA ACTGCGCAGA ACTGCGCAGA ACTGCGCAGA ... . " . ........ .......... " " . 12 1180 1190 GCCCACGTGG TCCCCCACC C. C .. l, . . • • • C .. GCG. .GCG. C .. GCG ... C .. GCG ... ... ... C. . .GCG .. C .. GCG ... C .. GCA ... C .. GCA. C .. GCA ... C .. GCA- 1247 NNNNNNNNNN NNNNNNN NNNNNNNNNN NNNNNNN TNNNNNNNNN NNNNNNN NNNNNNN TCAGACGGGA TGCGGNN TCAGACGGNN NNNNNNN TCAGACGGGA TGCGGNN TCAGACGGGA TGCGGNN TCAGACGGNN TCAGACGGGA TGCGGTG TCAGACGGGA TGCGGTG TCAGACGGGA TGCGGTG TCAGACGGGA TGCGGTG 101 ABSTRACT During a survey of Eucalyptus diseases in Ethiopia, a senous stem canker disease was discovered on E. camaldulensis trees, at several localities in the South and South Western parts of the country. The disease was characterised by the presence of discrete necrotic lesions, stem cankers, cracking of stems, production of kino pockets in the wood, as well as malformation of stems. These symptoms are similar to those caused by Coniothyrium zuluense in South Africa. The aim of this study was to identify the causal agent of the disease in Ethiopia. This was achieved by sequencing the ITS region of the rRNA operon and the ~ tubulin gene for a representative set of isolates. Sequences for the Ethiopian isolates were compared with those from authenticated isolates collected in South Africa, Thailand and Mexico, as well as with Coniothyrium-like isolates collected from diseased Eucalyptus trees in Uganda. Based on these data, the Coniothyrium isolates from Ethiopia and Uganda grouped together in a separate clade, but closely related to C. zuluense from South Africa, Mexico and Thailand. This study represents the first definitive report of C. zuluense and the disease caused by it in Ethiopia and Uganda. In Ethiopia, Coniothyrium canker is causing considerable losses in yield and quality of timber and it impacts negatively on the lives of the subsistence farmers. Research will thus be required to minimize these losses. 103 INTRODUCTION Eucalyptus species, which originate from Australia and nearby islands, have been introduced and planted in many tropical and subtropical countries. Estimates indicate that plantations of Eucalyptus spp. cover approximately 10 million hectares of land, worldwide (Eldridge et al. 1997). These plantations provide furniture, timber, distillates, tannins, essential oils, nectar, pollen and fibre for the production of paper, rayon and viscose. They are also a valuable source of fuel wood and construction timber (Poynton 1979, Turnbull 1991). In Ethiopia, the planting of exotic trees commenced with the introduction of Eucalyptus spp. in the 1890's. Eucalyptus globulus Lab ill, E. camaldulensis Dehn., E. saligna Sm., E. grandis Hill ex Maid and E. citriodora Hook are the most common species planted in Ethiopia. E. camaldulensis is widely planted, usually at lower elevation and warmer localities, while E. globulus is commonly planted in cooler areas. Plantations of Eucalyptus constitute the major proportion of exotic plantation species and cover about 100 000 ha of land. These Eucalyptus plantations provide wood for energy, construction material, transmission poles and fencing material (Pohjonen & Pukkala 1990, Persson 1995). Eucalyptus spp. have showed great promise in most areas where they have been planted as exotics. However, diseases pose a serious threat to these economically important plantation specles. A number of important diseases have been recorded on different Eucalyptus species and clones. These diseases infect stems, roots and leaves. Cryphonectria canker caused by Cryphonectria cubensis (Bruner) Hodges (Hodges, Alfenas & Ferreria 1986, Wingfield, Swart & Abear 1989, Conradie, Swart & Wingfield 1990), canker and dieback caused by Botryosphaeria spp. (Smith, Kemp & Wingfield 1994), vascular wilt of Eucalyptus caused by Ceratocystis jimbriata Ell. & HaIst. (Roux et al. 2000), pink disease caused by Erythricium salmonicolor (Berk. & Broome) Burds. (Sharma, Mohanan & Florence 1984, Roux et at. 2001, Alemu, Roux & Wingfield 2002) and Leaf blotch caused by Mycosphaerella spp. (Park & Keane 1982, Crous 1998) are examples of diseases in commercial Eucalyptus plantations. Recently, a serious stem canker disease caused by Coniothyrium zuluense Wingfield, Crous & Coutinho has also been described causing losses to Eucalyptus trees in various countries (Wingfield, Crous & Coutinho 1996, Roux, Wingfield & Cibrian 2002, Van Zyl et at. 2002). 104 Stem canker caused by C. zuluense was reported for the first time in 1989 from an E. grandis clone in South Africa (Wingfield et al. 1996). Trees affected by Coniothyrium stem canker develop small, discrete, necrotic lesions on the young, green bark (Wingfield et al. 1996, Roux et al. 2002, Van Zyl et al. 2002). The canker disease has been found on several E. grandis clones, on hybrids of E. grandis with E. urophylla S. T. Blake and on E. camaldulensis, which is generally believed to be a relatively disease tolerant species (Wingfield et al. 1996). Initially, the pathogen was believed to be native to South Africa. It has, however, recently been described from Eucalyptus spp. in Thailand (Van Zyl et al. 2002) and Mexico (Roux et al. 2002). During a disease survey of plantation forestry species in Western and South Western Ethiopia, several pathogens were identified (Alemu, Roux & Wingfield 2003). Symptoms of stem canker similar to those of Coniothyrium canker were observed on E. camaldulensis trees at a number of these localities (Alemu et al. 2003). Coniothyrium spp. are difficult to identify and morphological characteristics are generally considered insufficient to identify C. zuluense with certainty. This study was, therefore, conducted to confirm the identity of the causal agent of the canker disease of E. camaldulensis. An additional objective was to determine the phylogenetic relationship between the fungus occurring in Ethiopia and isolates from other parts of the world. To achieve this DNA sequences of the ITS regions of the rRNA operon and ptubulin gene were used. MATERIALS AND METHODS Sample collection and isolation Samples were collected from infected E. camaldulensis trees planted in Southern and South Western Ethiopia (Figure 1). Disease symptoms were used to select infected trees for sampling. Samples were collected from symptomatic plant parts including twigs, branches and stems of infected trees. Collections were made from plantations, community woodlots, and from E. camaldulensis trees planted around farmlands and homesteads. Segments of plant parts with disease symptoms were incubated in moist chambers at room temperature to induce sporulation. Masses of spores emerging from pycnidia were transferred to petri plates containing malt extract agar (MEA, 20 g Biolab Malt Extract; 15 g Biolab Agar), spread on 105 the agar surface with sterilised water and incubated at 25°C for two weeks. Stock cultures of all isolates were maintained on 2% MEA slants at 5 0c. Coniothyrium cultures collected from Ethiopia are maintained in the culture collection (CMW) of the Forestry and Agricultural Biotechnology Institute (F ABI), University of Pretoria, South Africa. DNA extraction Total genomic DNA was extracted from isolates (Table 1) grown in liquid MY medium (2% Malt Extract, 0.3% Yeast Extract Agar) for two weeks at 25°C. Mycelium was harvested by centrifugation (8000 rpm, 30 min), freeze dried and ground to a fine powder in liquid nitrogen using a pestle and mortar. A modified version of the method of Raeder and Broda (1985) was used to extract DNA from the mycelium. Approximately 0.5 g of powdered mycelium was placed in 1.5 III Eppindorf (Epps.) tubes and 1000 III extraction buffer (100 mM TrisHC1, pH 8; 50 mM EDTA; 500 mM N aCl; 5 g CTAB) was added to each tube. These suspensions were then incubated in a 60°C water bath for 2 hours, and frequently mixed by inverting the tubes. Phenol (500 Ill) was added and the solution was mixed using a vortex mixer. Thereafter, 300111 chloroform was added and mixed. The cell debris were removed by centrifugation at 12500 g, for 60 min at 4°C. The upper aqueous layer of this mixture was transferred to new tubes, whereafter a further phenol:chlorophorm extraction was carned out by adding 200 III phenol and 200 III chloroform. This mixture was centrifuged at 12500 g for 5 min at 4 °c and the upper aqueous layer transferred to a fresh tube. To remove the excess phenol it was washed with 400 III chloroform and centrifuged at 12500 g for 5 min at 4 0c. This step was repeated until a clear interface was obtained. Next, 0.1 volume of 3M NaAc (PH 5.5) and two volumes of absolute ethanol were added and the mixture was centrifuged for 30 min at 4 °c to precipitate the nucleic acid. The liquid phase was discarded and the precipitated nucleic acid was washed with 70% ethanol and centrifuged for 5 min at 4 °c to obtain a DNA pellet. The DNA pellets were vacuum dried to remove excess ethanol and resuspended in 50 III water. RNase A (1mg/ml) (Roche Diagnostics, South Africa) was added to the DNA solution to remove the contaminating RNA and incubated at 37°C in a water bath over night. The presence of DNA in the samples was detected by using agarose gel electrophoresis in a 1% agarose gel, stained with ethidium bromide and visualised under UV light. 106 PCR amplification The internal transcribed spacer (ITS) regions of the ribosomal RNA operon and the 5.8S gene were amplified using the polymerase chain reaction (PCR). PCR was conducted using primers ITS 1 (5' TCC GTA GGT GAA CCT GCG G '3) and ITS 4 (5' TCC TCC GCT TAT TGA TAT GC '3) to amplify the ITS 1, ITS 2 and 5.8S genes of the ribosomal RNA operon (White et al. 1990). The PCR reaction mixture contained DNA polymerase (Taq, 2.5 U /IlI, Roche Diagnostics, South Africa), 2.5 mM dNTP's, PCR Buffer (10 mM TrisHCl, 1.5 mM MgCh, 50 mM KCl). 2.5 mM MgCh, 0.15 mM of each primer, approximately 1 III of DNA and 37 III water to make up a final volume of 50 Ill. Denaturation was performed at 96°C for 1 min. This was followed by 35 cycles of primer annealing at 55 °c for 30 s, chain elongation was undertaken at 72 °c for 1 min and denaturation was conducted at 92 °c for 1 min. Final chain elongation was carried out at 72 °c for 5 min. The ~-tublin gene was partially amplified using the forward primer Bt2a (5' GGT AAC CAA ATC GGT GCT GCT TTC 3') and the reverse primer Bt2b (5' ACC CTC AGT GTA G TG ACC CIT GGC 3') (Glass & Donaldson 1995). The PCR reaction mix included DNA polymerase (Taq , 2.5U/ lll ), 0.2 mM dNTP's, PCR buffer (10 mM TrisHCl, 1.5 mM MgCh, 50 mM KCl), 2.5 mM MgCh, 0.15 mM of each primer, 1 III of DNA and 37 III sterilised water to make up a final volume of 50 Ill. Amplification was conducted using the following PCR reaction conditions: an initial denaturation at 94°C for 1 min, which was followed by 40 cycles at 94 °c for 1 min, primer annealing at 51°C for 30 s, chain elongation at 72 °c for 1 min and an additional chain elongation step at 72°C for another 1 min. All PCR products were detected using agarose gel electrophoresis on 1% agarose gels stained with ethidium bromide under UV illumination. DNA sequencing The PCR products of both the ITS regions and the ~-tublin gene were purified using the High Pure PCR Product Purification Kit (QUIAGEN, GmbH, Hilden, Germany). The PCR products were sequenced in both directions using the Big Dye Cycle Sequencing kit with Amplitaq® DNA Polymerase FS (PerkinElmer, Warrington, UK), according to the manufacturer's protocol, on an ABI PRISM™ 3100 DNA Autosequencer (PerkinElmer). Primers ITS 1 and ITS 4 were used for sequencing the ITS regions and for the ~-tublin gene, 107 Bt2a and Bt2b were used. The sequences for with Ethiopian were compared DNA sequences obtained from GenBank [National US Information National Institute (http:/www.ncbi.nlm.nih.gov/BLAST]. Once the determined a of the Health identity additional Mycosphaerella spp. were included for Biotechnology Bethesda the fungus was of Coniothyrium and ITS and ~-tublin and analyzed. Sequence analysis The and 1998). ~-tublin sequences were manually were manually PAUP 4.0 (Swofford, treated as missing were analysed using parsimony with trees generated by searches, Reconstruction (TBR) branch swapping. addition and Confidence intervals were determined usmg DNA BOOTSTRAP analysis (Bootstrap confidence intervals on DNA parsimony) (1000 replicates) 1993). Mycosphaerella molleriana (Thumb.) Lindau. (Cooke) were used as an outgroup homogeneity test was sets combined the combinability of the and set M. Partition ~-tubiln Cunningham 1996). et Pathogenicity test An inoculation study was conducted on an ISmonthold (approximately 1 cm diameter) m to inoculation, 1). Cultures were grown on to remove the bark with the test were wounds were covered contamination and desiccation. trees were house at a temperature 0c. 14) Prior to conditions for one Ethiopia were randomly Coniothyrium grandis clone for the study two weeks before inoculation. A 9 mm cork borer was the cambium. Mycelial with the of equal size, overgrown the wood. parafilm (Pechiney Plastic Packaging, Chicago) to prevent was inoculated on 10 trees and an additional ten MEA to serve as controls. 108 After development inoculated trees. A was examined by measuring the lesion ANOVA was conducted 1995) to statistically on for Windows (Statsoft. <-",~'v.u development with isolates and control. RESULTS Sample collection and Isolation Symptoms Coniothyrium stem were observed localities in South and South and between Wolkite Ethiopia. Sodo (Figure 1). lima, and E. camaldulensis trees planted were seriously camaldulensis trees liren plantation near stem canker. About 50% of E. by and trees growing at Stem malformation and extensive discoloration were evident on most stems (Figure developed on young necrotic areas were between Woliso and lima woodlots as well as around localities had symptoms of camaldulensis bark 2a, 2d). When developed on the stems, observed in the wood trees. Initially, small discrete n+~,t>1i and twigs with the lesions coalesced, 2b). lesions on infected trees pockets were 2c). After one day in moisture chambers, pycnidial structures, producing slimy spore masses were found in the sunken necrotic collected from infected trees. A Coniothyrium isolated from these lesions and this South olive in colour. was morphologically similar to In culture, colonies of most was slowly colonies were had similar growth and colour in culture. peR amplification and analysis ofsequence data Amplification of study yielded a fragment tubulin gene regions and gene for the Coniothyrium isolates about 500 base pairs (bp) in in this Amplification of the partial P- of approximately 400 bp. 109 The ITS regions and 5.8S gene were sequenced and after alignment yielded a total of 551 characters of which 495 characters were constant, 40 variable characters were parsimony uninformative and 16 characters were parsimony informative. A total of 485 characters were obtained when the ~-tublin gene was aligned. Of these, 397 characters were constant, 67 were parsimony uninformative and 21 characters were parsimony informative. Comparison of the ITS and 5.8S gene sequences to sequences available in the NCBI data base revealed that the sequences of the samples from Ethiopia are most similar to that of C. zuluense (98%) followed by Mycosphaerella vespa (Carnegie & Keane) and M molleriana (96% homology) and M nubilosa (94% homology). Analysis of the ITS sequence data, using sequences obtained from Genbank and the data set from Van Zyl et al. (2002) produced 1 tree. The tree had a CI = 0.976 and RI = 0.944 (Figure 3), and showed that the Coniothyrium isolates from Ethiopia and Uganda grouped together in the larger C. zuluense clade (83% bootstrap). Two distinct subclades, were however, apparent. Isolates from South Africa, Thailand and Mexico grouped in one clade (97% bootstrap) and isolates from Ethiopia and Uganda grouped in another (80% bootstrap). C. zuluense isolates grouped more closely with M molleriana and M nubilosa, than with other species of Coniothyrium, including C. ovatum Swart and C. fuckelii Sacco A partition homogeneity test showed that the ITS and ~-tublin sequences could be combined (P value = 1). The combined sequences had a total of 956 characters of which 796 characters were constant, 116 variable characters were parsimony uninformative and 44 characters were parsimony informative. Analysis of the combined data sets generated 1 tree (Figure 4). The tree generated from the combined data set had a consistency index (CI) of o. 969 and retention index (RI) of 0.942. Ethiopian and Uganda isolates grouped together with C. zuluense (100% bootstrap). Two subclades were, however, produced within C. zuluense (Figure 4). Isolates from South Africa, Thailand and Mexico grouped together in clade I with a 96% bootstrap support. This clade represents C. zuluense. Clade II contained the Coniothyrium isolates from Ethiopia and Uganda with a 100% confidence limit. The Coniothyrium isolates grouped separately from any of the Mycosphaerella isolates. 110 Pathogenicity test Small lesions developed on E. grandis trees inoculated with Ethiopian Coniothyrium isolates (Figure 2f). Lesion lengths differed statistically from those of the control (P<O.OOO 1) (Rsquare = 0.48). No variation was observed in lesion development between the C. zuluense isolates used in the inoculation study (Table 3, Figure 5). DISCUSSION Coniothyrium stem canker, caused by C. zuluense is considered to be one of the most important new threats to plantation grown Eucalyptus species. Until recently, this disease was known only from South Africa (Wingfield et al. 1996), Thailand (Van Zyl et at. 2002) and Mexico (Roux et al. 2002). Although observations based on symptoms and morphology of the fungus have led to suggestions that the disease is present in Ethiopia (Alemu et al. 2003), this study provides the first clear evidence for its occurrence in the country and expands the geographic distribution of this important disease. This is particularly important, as it is virtually impossible to identify C. zuluense with certainty without DNA based comparisons. Symptoms of Coniothyrium stem canker were first observed on E. camaldulensis in Ethiopia during a survey of plantation forestry diseases in 2000 and 2001 (Alemu et al. 2003). The disease is restricted to specific areas in Western Ethiopia, and is causing large-scale damage to trees in plantations, woodlots and around homesteads. It has not been found on other species of Eucalyptus in Ethiopia. This is probably due to the fact that they are planted in cooler areas, which would not be conducive to the development of C. zuluense. In South Africa Coniothyrium stem canker is only a problem in wanner sub-tropical areas (Wingfield et al. 1996) while the only other reports of this disease is from tropical areas such as Thailand (Van Zyl et al. 2002) and Mexico (Roux et al. 2002). Comparison of ITS and the 5.8S gene sequences showed that Ethiopian isolates were most similar to those of C. zuluense. The next closest relatives were Mycosphaerella spp., including M. vespa, M. molleriana and M. nubilosa. This is particularly interesting as other Coniothyrium spp. such as C. ova tum and C. jitckelii were more distantly related to C. zuluense than the group of Mycosphaerella spp. noted above. Van Zyl et al. (2002) provided 111 the first DNA sequence data for C. zuluense and used C. ovatum and C. fuckelii as outgroup taxa. Our study, however, strongly suggests that C. zuluense is more closely related to Mycosphaerella spp., than to other Coniothyrium spp. for which sequence data are available. It was for this reason that we choose Mycosphaerella spp. as outgroup taxa. Our data provide preliminary evidence to suggest that C. zuluense is an anamorph of Mycosphaerella. This is particularly interesting, as many Mycosphaerella species are pathogens of Eucalyptus leaves and stems. Results of our combined sequence data set separated the C. zuluense isolates into two distinct groups. One of these groups mainly constituted authentic C. zuluense isolates from South Africa, Thailand and Mexico. The Ethiopian isolates and one isolate from Uganda were identical and resided in a separate clade. These data might suggest that C. zuluense represents a species complex, and this deserves further scrutiny. Pathogenicity tests showed that Ethiopian Coniothyrium isolates are pathogenic to E. grandis. Only very small lesions were produced, but they differed significantly from the controls. Wingfield et at. (1996) reported similar results for South African isolates in artificial inoculations. During an extensive survey of Eucalyptus diseases in Western and Southern Ethiopia (Alemu et al. 2003), Coniothyrium stem canker was not observed on E. grandis, or any other species than E. camaldulensis. The pathogenicity of C. zuluense under field conditions and on E. camaldulensis, however, needs to be investigated further. E. camaldulensis is one of the most widely planted Eucalyptus spp. in Ethiopia. This species appears to be highly susceptible to Coniothyrium stem canker. The disease is wide spread in E. camaldulensis growing areas between Wolkite and Sodo as well as between Woliso and Jima. Near Jima, the disease was found on most E. camaldulensis trees in the Jiren plantation, east of Jima, whereas E. camaldulensis planted on the other side of the town showed no signs of infection. 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CMW11220, CMW7399 CMW11221,CMW7459 CMW11225 CMW 11226 CMW11227 CMW11228 CMW11230 CMW11231 CMW5232 CMW5234 CMW3032 CMW8575 a CMW numbers refer to South Africa. ______~_sion ti~: ITS Btubilin South Africa Coniothyrium zuluense E. grandis L.M. van Zyl AF376823 AY244383 South Africa " AF376816 AY244384 Ethiopia Alemu Gezahgne & 1. Roux A Y244415 AY244390 Ethiopia " AY244413 AY244391 Ethiopia " A Y244414 AY244392 Uganda Eucalyptus spp. 1. Roux AY244416 AY244389 Mexico M. J. Wingfield & 1. Roux AF385610 A Y244385 Mexico " AF38561I AY244386 Thailand M. J. Wingfield & van Zyl AF376828 A Y244387 Thailand " AF376825 AY244388 South Africa Mycosphaerella nubilosa E. bicostata P.W. Crous AY244393 Chile M molleriana E. globulus R. Ahumada AY244394 the culture collection numbers of the Tree Pathology Cooperative Programme (TPCP), FABI, University of Pretoria, Origin Species Host Collector 115 Table 2. Results of inoculation of an E. grandis clone with Ethiopian Coniothyrium isolates Isolates Mean Lesion 95% Confidence Limits Length (mm) CMW11223 17.2 CMW11234 17.9 a 16.3519.45 CMW11233 16.6 a 15.0518.15 CMWl1238 16.7 a 15.1518.25 CMW11238 17.9 a 16.3519.45 CMW11225 16.8 a 15.2518.35 CMWl1235 18.8 a 17.2520.35 Control 11.0 b 9.4512.55 a 15.65 18.75 Each mean lesion length is the average of 10 measurements. RSquare =0.48. Mean values with the same letters did not differ significantly at P = 0.05. 116 '" \ SOdan i \ LakeTana Addis Ababa SOMALIA o Wolisso • Jima ..... Wolkite CJ So do SOMALIA Figure 1. Map of Ethiopia showing the plantation areas where surveys were conducted. 11 7 Figure 7. Aligned sequences of the ITS and ~-Tubln genes of isolates used i n this study. Gaps , (.)= homologous nu c leoti des (N)= Unknown bases. 20 10 CMWl1220 South Africa CMWl1221 South Africa CMfJll11230 Mexico CM(oJl 1231 Mexico CMW5232 Thailand CMW5235 Thailand CMWl1228 _Uga nda CMWl1226_Ethiopia CMW l1 227_Ethiopia CMWl1225_E t hi opia Mycosphaerella_ffiolleriana Mycosphaerella nu bil osa 40 50 60 70 80 TCCGTAGGTG GAACCTGCGG AGGGATCATT ACTGAGTGAG GGCGCAAGCC CGACCTCC A ACCCCATGTT TTCCAACCAT .................... . ........ .......... . . .. .. . . .. . . . .. . . .. .. .. . . .. .. .. .................. .. .. .. .. .. . .. .. . .............. . . ... T. . .. .. . . .. .. . . .. .. .................. .. . . .. . . . .. . .. . . .. .. . . .. . . .. .. ................ . ................ .. .... . . .... ...... · .. T. NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN N......... · .. T. .. .................. .. .............. . . .... ................ . .. .. .. . . .. . . . NNNNNNNNNN NNN ....... .. . . .. . . . . .. . ...... . . . ...... . .. .. . . . .. . .. .. . .. . .. . . . .. . . .. . . .. . . .. .. .. .. . .. . . .. . .. .. . .. .. . .. . . . .. . . .. . .. . . .. . . . .. .. 90 CMWl1220 South Africa CMWl1221 South Africa CMfJll11230 Mexico Cl"M1123l Mexico CMW5232 Thailand CMW5235 Thailand CMW11228 Uganda CMWl1226_Et hi opia CMWl1227 _Et hiopia CMWl 1 225_Ethiopia Mycosphaerella_ffiolleriana Mycosphaere ll a_nubilosa 30 () = .. .. .. . .. . . . .. .. . .................... . . .. . G .... . .............. .. ........ .... ...... .. ... G... . ................ . ...... .... ........ ..... G.... ................ .. .... .... .......... ... . . G .... ................ .. . ........ . . .... .. . .. .. .. .. .. . .. .. ................ .. ...... . .. . .. .. .... GC .... . . . . . . . . T. C .. . ..... .. .. . .. .. . .. .. .. . . . . .. . . .. .. .. . . .. . . .. . . . . .. .. .. .. .. . . .................. .. .. . .. .. .. .. .. . .. · .C ... . ... 100 110 120 . . .. .. .............. . .. .. . .. . .. .. . .. . .... . C ... ... .. . C ... ..... . C ... ...... C ... .. .. . .. . .. . .. .. .. 130 . 140 150 . C . A ..... C · C ... .. . C 160 GTTGCCTCGG GGGCGACCCG GCCATCGCGC CGGGGCCCCC GGTGGACCCC TCCAACTCTG CATCTTTGCG TCTGAGTCAC .............. ... . . . . .. . . C ..... · .... . .... · ......... · .. . . .... . ... . C ... .. · .. . ...... · ....... TC · ......... ... . C .... . · ......... · ....... . . · .. ... .... ... . C ..... · ......... · ......... · ......... .. . GC ..... · ......... · . ... ... . . · ......... .. . CC ..... · ...... . T . .. .. .. .. . .. .. . . . .. . .. .. .. . .. .. · · · · . C ....... .C ....... .C ....... .C ....... · . .... .. " .CA.A ..... · . · ...... · . · ...... · . · .. . ... · . · ...... · . · ...... ..... C. . .... C . . .... C . ..... C . ..... C . · . ... GGCT GGATC.GTGC GTG .... A.T 122 70 CMWl12 South Africa 12 1 South Africa 230 co 1231 Mexico 32 land la molleriana la nubilosa o 1 00 210 20 2 24 AAAATAAAAT CAATCAAAAC TTTCAACAAC GGATCTCTTG GTTCTGGCAT CGATGAAGAA CGCAGCGAAA TGCGATAAGT •.••• C • . . .C .. CC .. . 28 310 250 260 90 300 AATGTGAATT GCAGAATTCA GTGAATCATC GAATCTTTGA ACGCACATTG CGCCCTCTGG TATTCCGGAG GGCATGCCTG 23 123 CMW5232 Thailand .•. C •••.•• ... C .•.... 123 CMWl l22 0 CMW l1221 CMW 11 230 CMWll 23l Sout h Africa South Africa Mexico Mexico CMv~523 Thailand CMW5235 Thailand CMW11228_Uganda CMWl l2 26_Ethi opia CMW ll227_Ethi opia CMW ll2 25_Ethi opia Mycosphaerella_ffiolleriana Mycosphaerella nubilosa CMWll220 South Africa CMWll22l South Africa CMlAll 1230 Mexic o CMW 11 23 l Mexico CMW 52 32 Thail and CMW5235 Thailand CMW 11 228 _Uga nda CMWll 226_Ethi opi a CMW l1227 Ethlopla CMWl1 225_Ethi opia Myc os phaerella_ffio ll eria na Mycosphaerella_nubilosa 370 350 360 380 390 330 340 400 TTCGAGCGTC ATTACACCAC TCCAGCCTCG CTGGGTATTG GGCGCCGCGG CCTCCGCGCG CCTT AATGT CTCCGGCCGA · ......... · ......... · ......... · ......... · ......... · ......... · ...... .. . · ......... · . . . . . . . . ? · ......... · .... . .... · ........ . · ......... · ......... · ......... · ......... · ......... ... G...... · ......... · .... ..... · ......... . .. CG. · ......... · .. T ...... ....... C .. · .T . . ..... · ......... · ......... . . . C- 450 460 47 0 480 420 330 440 41 0 GCCGACCGTC TCCAAGCGTT GTGGCACAAC TGTTTCGCTT TCGGG ACCG GTCCGGCGAC GCGCCGTTAA ACCCTTTCAC · ......... · ......... · ......... · ......... · ........ . · ......... · ......... · ......... · .T ....... · ......... · ......... · . T ....... · .......... · ...... . .. · . T ....... · ......... · ......... · .T ...... i. · ......... . . . . . . . . . . · ......... · ........... · .. . ...... · . TC ...... · ...... T .. · ......... · ......... · ......... · . . .. .. . .. · .... ... .. · .......... · .... · .... · .... · .... · .. .. · ... · ... · ... · ... · . .. ......... G A .... G.... · .. T. ... T. . .. T. · .. T. ... T .... G. .. . T. 124 CMWl 1220 South Afr i ca CMW l1 22 1- South Af r i ca CMW 11 230 Mex i co CMW 11 23 1 Mexic o CMW5232 Thai l and CMW523 5 Tha il and CMW 11 228 Ug an da CMW l1 226 Et hlop la 01\'1111227 Eth i op i a CMW 11 225 Et hi op i a Myc osphaere l la_m o ll er i ana Mycosp ha ere ll a nub il osa CMW l1 220 South Afr i c a CMW l 12 21 South Af r ic a CMW 11 230 Me xi co CMW 112 3 1 Mexico CMW5 2 32 Thai l and CMW5235 Th a il and CMW11 228 _ Ugand a CMW l1 22 6_Eth i opia CMW l1 227 Eth l op i a CMWl1225 Eth l op l a Myco sp haere ll a _ moll eriana Mycosphae re lla nubilo sa 52 0 530 5 10 4 90 50 0 540 550 560 CAAAGGTTGA CCTCGGATCA GGTAGGGATA CC CG CTGAAC TTAAGCATAT CAAT TAAAGC GGAGGAT GGT AACCAAA - · . . ... .. .. · ...... ... · . . .. .... . · .. ...... . · ... . ..... · . . ... . . . . · ....... .. · .. .. ..... · ... . ..... · .. . .. .. .. · ... ... ... · . . . .. .. .. · . .. .. .... · .. .. ..... · .. .. .... . · ... ..... . · ........ . · . . ..... .. · .. . .... .. .???????? ? ?????? · . T. A. GCG . AG . A. · ..... . ... · .... . . . .. · . .... .... · ... .. .. .. · . ... . ... . · ......... · . - . ... . .. . . ... .. . . . . ... .. .. A- · .. ... . ... · . ... . . .. . · . . ... . . . . · .. . .. . ... · ....... . . . .. .... .. · . .. . ... . . · ........ . ?????????? ?????? ? ??? ??????? ? ?? ?????? ... . . . . . . . A · ....... . . · ......... · ..... . . . . ????? ? ???? ?????? ? ??? ?????????? ????????? ..... .. AA- 61 0 620 630 57 0 580 590 600 64 0 TCGGTGCTGC TTT CT GGCAG AACATC TCCG GCGAG CACGG CCTCGACG GC TCCGGCG TGT AGGTC TAGCA GGAGTGGGAT · ......... · .. . .. . ... · .. .. ..... · . . ... . . . . ... .... .. T · ... .. .... · .... ... .. · ...... .. . · . . . . . . ? . · ......... · .......... ... . ..... G · ..... .. .. · ........ . · ........ . · ..... . ... · .. .. .. ... · . . ... . . .. .. .. . .. . . G · .. ... . ... · . . . . ... .. · ........ . · . . . . .... . · .. .... .. . · ..... ... . · . ....... . · . ... . ... . · .. . ..... . · .... . .. . . ..... . . . . T · . ..... . . . · .. . .... ... · . .. ..... . . .. . . . T ... . . . . . . . . . . · .. ... . ... · . ... . .. .. .. . ... T ... · ......... · ... .. .... · . . .. . . . . . .. . ... T ... · ... . ... . . . . . . . . . . . . · ...... .. .. · ..... T . .. · ....... .. · . . .. ..... · . ..... T .. · ... . ..... · ....... . . . C . . ... ... ... . A . . T .. · ... . . . . . T · .... . . ... · ..... . ... · .. . .... .. · .. ..... . . . . . ... GA .. ... A . . A .. G ... . .. GA .. . .. A .. A . . G ..... . GA . . .. . A . . A .. G . ..... GA . . .. . A .. A.. G · . T ....... . T . • G .. ..• AT GC ... C.G G . . . . . . . . . GA . . G .. A.. ACGC GAAAGA 125 CMWl1 220 South Afr i ca CMW l1 221 So ut h Af r i ca CMW1l 230 Mex i c o CMW1l 2 3 l Mexico CMW5232 Tha il and CMW5235 Tha il a n d CMlfH1 2 2 8_Uganda CMWl 1226_E th i op i a CMW l1 22 7_Eth i op i a CMW l1 225_Et h i op i a Mycosphaere lla_ffio ll er i ana Myco s p haere ll a n u b il osa CMW l1 220 So ut h Af r i ca CMWl 1 22 1 South Af r i ca CMW1l 2 30 Mex i co CMW1l231 Mex i co CMW5232 Tha il a n d CMW5235 Tha i la n d CMW1l228 _ Uganda CMW l1 22 6_ Eth i op i a CMW l1 227 _Et h i op i a CMWl1 225 _ Eth io p i a Myco s phaere ll a _ ffi oll e ri ana Mycosp h aere ll a nub il osa 650 6 60 67 0 68 0 690 700 71 0 72 0 CGAAGGAGAA GAGGATAC TG ACGCGAGGCA GG TACAATGG CACGTCTGAC CTCCAGC TCG AGCGCATGAA CG TGTAC TT C . , .. T . . . . G. .. . G. · ... T. · . G. A ... G. · . G.A . . . G. · .G. A ... G. · . G.A ... G. T. G... . ... . C . . . C . .. . ..... . . A . . GCC T. AG . . . CGC .. C .... · TAT. GT ... eo .. .. . .. .. ... ... . ..... T ... .. . . .. . . ... ... . . . . . ..... .. . . .. .. .. . . . .. C . 770 78 0 790 730 740 750 7 60 800 AACGAGGTAT GGCCTGAG GC AGCAACTATC TCCAATCCA CACAC --- --TAACGCGA TACGCAGGCA TCCGGCAACA · . .. .. ... . · . .. . T . . .. · ... . ... . . C . T ... · . ......... · .. .. T .... .. . . . . . . . . C . T . · . ... . . . . . · . .. . T .. . . · .. .. .... . . C . T. . · . .. . T . ... · .. . .... . . · .. .. T .... · .... . .. . . · .... T .... · . . ..... . . · .... T ... . · ... . ...... . . .. . T . .. . · ....... . . · .. . .... .. .... . C .... .. . C. T .... ....... . GC .A .ACCGC T . TT TC CA .. .. · ...... . .. · . .... . . . .. . . ... ... .. · .. .. .. ... .. . . . . . . . . . G. T .. C-. C. T .. . - . . . C . T .. . - ... C . T .. . - ... C . T . .. - .. . CC T.T.- .A . . . ACA- - - CC AGG . T . - TGG . . GTGAGGAT - - . .. T . . . . -- . .. T .... - - . .. T .. . . -- . .. T .... AC.G .. CGC . AC . G .. T .. C C. C. C. C. A .. AT .. . .. .. T . A .. A ..... G 126 810 South Africa South Africa o 20 870 50 880 AGTATGTCCC GCGTGCCGTC CTCGTCGACT TGGAGCCGGG CACCATGGAC GCTGTCCGCG CTGGTCCGTT CGGTCAGCTC .. G •••••• 0 .Co .C . . . . . . . 0 . .T. .G . . . . . . . o. 0 •• 000 0 0" . · - . ,. '". ~. " .•... C .• • C .•.•• C •• . Co .C .• . ...... T ......... 0" mol a nubil ••••••••••••••••••••••••••••• T . . . . . . . . . . . C. o " . . . . . A... o 940 00. 0" 58 ACAACTTCGT CTTCGGTCAG TCGGGTGCTG GCAACAACTG GGCCAAGGGT CACTACACT GAGGGTA CMW52 235 Thailand 1 .... A. . . . . . . . . . . . . . . . . T . . . . . . . . . . T . . . . . . . . . . . . . . . . T . . . . 10 CMWI 0 South Africa CMWl1221 Africa 0 •••••. C .•• 8 ... ~, .00 ••• ~ · C .. •..•. . C ••. 0 • • • • • • • • ~ ~ . .. 0 ..... C.•.......... . T . . . . . . . . . . • • . C ... . .... NNNNN NNNNNNN .A . . . . . . . · ... AC . . . . . . . . . . 127 ABSTRACT of Eucalyptus spp. plantation as fuel and to the E. and E. camafdulensis, commonly planted. more than 30% a are the most spp. in 2000 and 1, Botryosphaeria stem canker was observed m most plantations. Characteristic symptoms included dieback, coppice kino exudation. The aim and stem characterised this study was to identify the species responsible stem Ethiopia. morphology, as involving ribosomal DNA and regions of the elongation identify isolates. Pathogenicity field conditions. Results showed canker of country. The results I(){) gene, were used to Ialpha were conducted in the greenhouse and under B. par>'a is responsible spp. in Ethiopia. This is the first report Botryosphaeria stem the fungus from the greenhouse and field inoculation Ethiopian isolates are highly virulent. Careful are needed to reduce as impact of this U1"'''''''''' showed that the selection in Ethiopia. 129 INTRODUCTION Ethiopia, the planting of Eucalyptus globulus LabiI. the late 1890's (Pohjonen & spp., ex Maid and citriodora Hook are major proportion 100 000 1990 (Pohjonen poles Sm., Hill commonly planted today. they the plantation resource, which covered an Pukkala 1990, Persson 1995). The wood from these as a source IS 1990, Persson camaldulensis Dehnh., E. constitute U""".HJAh> commenced with the introduction of construction timber and the production of posts. Fungi in the genus Botryosphaeria are associated with fungi are known as saprophytes, opportunistic hosts. On Eucalyptus spp., and (Davison Tippett on a wide range Tay 1 Barnard et al. 1987, Smith, Kemp & Wingfield 1 Smith, Wingfield Petrini 1996). Damage due to Botryosphaeria spp. is more pronounced when plants are stress water 1980, 1990). Recently, it symptomless endophytes Wingfield 1987, 1989, at. et as Eucalyptus spp. For example B. dothidea (Moug.) Ces. & Corda) reported as an endophyte in Maid.) Maid. in England (Fisher, Petrini & Sutton 1993) and in gran dis, E. camaldulensis, et & been recognised that Botryosphaeria spp. also Not. nitens (Deane and nitens and 1996). When trees or tree parts are smithii T. in South Africa (Smith by become active and can cause taxonomy Botryosphaeria spp. is complicated and has considerable debate (Sivanesan 1984, Butin 1993, because many the Furthermore, the subject Rehner 1998). This is are almost impossible to distinguish from each other in structures are morphologically similar to describe new Botryosphaeria spp. on the for collections from different hosts and this 130 has caused substantial confusion. Names such as B. doth idea , B. ribis Gressenb. & Dugg., (anamorph = Fuscicoccum ribis Gressenb. & Dugg.) and B. berengeriana De Not. have been used interchangeably (Slippers et al. 2003). In recent years, molecular techniques, particularly DNA sequencing have been used to clarify questions pertaining to Botryosphaeria taxonomy (Jacobs & Rehner 1998, Zhou & Stanosz 2001, Slippers et al. 2003). These data are showing that names used in past descriptions of diseases, must be treated with some circumspection. Botryosphaeria ribis has been found associated with Eucalyptus spp. in different countries. In Florida, B. ribis has been associated with seed capsule abortion and twig dieback of E. camaldulensis, where it subsequently resulted in the abandonment of commercial seed production (Webb 1983). Infection by B. ribis has also been found associated with basal cankers and coppice failure of E. grandis in Florida (Barnard et al. 1987). In Australia, B. ribis is associated with twig, branch and stem cankers of E. marginata Donn. ex Sm. (Davison & Tay 1983). This fungus was also responsible for the death of E. radiata Sieb. ex DC. in species selection trials in Western Australia (Shearer et al. 1987). In Africa, Botryosphaeria dieback and canker, caused by B. doth idea, B. rhodina (Cooke) Von Arx (anamorph = Lasiodiplodia theobromae (Pat.) Griffon & Maubl.) and B. eucalyptorum Crous, H. Smith et M. J. Wingf. (anamorph = Fusicoccum eucalyptorum Crous, H. Smith et M. J. Wingf.) has been reported from several countries including South Africa, Republic of Congo and Uganda (Smith et al. 1994, 2001, Roux et al. 2000, 2001). In South Africa, widespread twig dieback and stem cankers were observed on E. grandis, E. nitens and E. smithii, clones of E. grandis, hybrids of E. grandis with E. camaldulensis as well as with E. urophylla S. T. Blake. B. dothidea and B. eucalyptorum were identified from symptomatic trees (Smith et al. 1994, 2001) and are considered to be the most common cause of Eucalyptus disease in South Africa. In the Republic of Congo, B. rhodina was found associated with root disease on E. grandis (Roux et al. 2000). Similarly, B. rhodina was associated with stem cankers on Eucalyptus spp. in the Republic of Congo and Uganda (Roux et al. 2000, 2001). 131 A recent plantations of Ethiopia In symptoms, typical canker the of spp. associated with stem study is to identify species in of this, morphological characterization, Ethiopia. To DNA on dieback are spp. (Alemu, Roux & Wingfield 2003). The shown analysis and were used. MATERIALS AND METHODS Symptoms, sample collection andfungal isolation In 2000 2001, were conducted in plantations of Eucalyptus spp. in U''''",<>'0v and South Western Munessa 1). Eucalyptus spp. Wondo were of symptomatic plant on were incubated in moist days to enhance development of fruiting structures. chambers Biolab Malt 2S°C. and 1 Biolab from symptomatic tissue was also Isolations were twigs collected Botryosphaeria of water and incubated at directly onto MEA fruiting structures occurring on Eucalyptus forest All isolates in the culture Institute (F ABI), collection (CMW) of the University of IJr"tAr'I were then South Africa. were inoculated on (2% Biolab Agar) H"",U"_oJ dishes placed on the weeks at induce sporulation. Conidial masses from fruiting structures were to on MEA in a sterile drop of water. Germinating single conidia were isolated 1 16 hI. 132 Jlorphological characterisation were initially based on culture morphology on were a Zeiss light for each Widths and lengths and and width in lactophenol and ten conidia were measured length:width ratios were calculated each isolate ''fU.UHU.''V on mean 1). extraction genomic DNA was extracted from (" fable were Mycelium used BOl1yosphaeria to rprlrp,opn different morphological DNA extraction was scraped directly from MEA plates covered with mycelium a sterile scalpel and placed into 1.5 III Eppendorf tubes. DNA extraction method of and (1985), modifications, was used to extract DNA. Extraction buffer, 200 III (1 8; 500 mM NaCl; 5 g CTAB) was added to mycelium Eppendorf was 800 III buffer was added water bath for one hour. Thereafter, 500 III phenol and mixed. The 300 III chloroform was out by adding 200 chloroform. at 12000 g mixture was was into chloroform was added to remove ,.",rnrnlPrI at 4 upper this, excess phenol. When a III interface was for 30 min at 4 °c to precipitate the nucleic nucleic acids were ethanol (70%) and centrifuged for 5 min at 4 and the DNA pellets vacuum pellet was 5 phenol and 200 III tubes. ethanol was discarded and ~Ll ~tl NaAc (pH 5.5) and two volumes of absolute ethanol were mixture addition of 60 into new tubes and a further phenol:chlorophorm extraction was added in a debris was precipitated by centrifugation (12000 g, min) at 4°C. The upper aqueous layer was obtained, 0.1 volume and the with a into small broken, a 60 mM 50 The ethanol was to remove excess ethanoL water. A (1 was added to 133 peR amplification internal transcribed spacer regions and gene of ribosomal RNA operon Botryosphaeria isolates used 1-00 of were amplified Reaction (PCR). this amplifY the OCO 0 '3) and 4 OC '3) (White et at. 1990) were For the OAAOTICOA 003') Iex gene, forward primer EF 1 reverse (5'TAC (Carbone, Anderson The PCR was used AAO OAA CCC Kohn 1999). mixture contained DNA polymerase (Taq, 2.5U/I.t1, Roche), 0.2 mM DNTP's, 1 with 1 mM MgCh supplied by the mM MgCb and 0.75 mM of each primer and approximately 1111 of DNA. Ill) was added to a total reaction volume water (37 50 Ill. Denaturation was performed at for 1 min. This was followed by 35 cycles of primer annealing at 96 at s, for 1 min. Final chain for 1 min and denaturation at 92 elongation was carried out at for °c for 5 min. Restriction Fragment Length Polymorphisms (RFLP) The ITS amplicons Botryosphaeria isolates to CYo I DNA template, 0.5 III enzyme and 2.5 III a water bath were gel stained with ethidium bromide were vVIHI"'U ,",,,""I11a. with those published of the buffer. The mixture was digested at 6 molecular marker was used to the RFLP reaction mix contained 20 III (Jacobs 2002, Slippers et 37°C this study were on a visualised under UV light. A standard 100 bp the Jacobs (2002) and banding et (2002). 134 DNA sequencing PCR products were purified usmg the High Pure PCR Product Purification Kit (QUIAGEN, GmbH, Hilden, Germany) and sequenced in both directions. The Big Dye Cycle Sequencing kit with Amplitaq® DNA Polymerase, FS (PerkinElmer, Warrington, UK), was used following the manufacturer' s protocols, on an ABI PRISM™ 377 DNA Autosequencer (PerkinElmer). Primers ITS 1 and ITS 4 were used for sequencing the ITS rDNA regions, and primers EF l728F and EF 1986R were used to sequence the EF Iex gene. Sequence analysis The possible identity of the Ethiopian isolates was considered by comparing their ITS sequences with those in the GenBank database [National Centre for Biotechnology Information (NCBI) US National (http:/www.ncbi.nlm.nih.govIBLAST)]. Institute of Health Bethesda The Ethiopian Botryosphaeria sequences were aligned against Botryosphaeria sequences obtained from GenBank and from Slippers et al. (2003). Alignment of both ITS and EF Iex gene sequences was done manually using PAUP version 4.0b (Swofford 1998). Gaps were treated as missing data. The sequences were analysed using parsimony, with trees generated by heuristic searches with simple addition and Tree Bisection Reconstruction (TBR) branch swapping. In the phylogenetic analysis Guignardia philoprina (Ellis) Viala & Ravaz was used as outgroup. Confidence intervals were determined using DNA BOOTSTRAP analysis (Bootstrap confidence intervals on DNA parsimony) (1000 replicates) (Felsenstein 1993). A partition homogeneity test (Farris et al. 1995, Huelsenbeck, Bull & Cunningham 1996) was used to consider whether the data sets for the ITS and EF Iex sequences could be combined. Pathogenicity tests Greenhouse inoculation studies were conducted on an l8monthold E. grandis clone (ZG 14). The trees were maintained in a greenhouse for one week to allow them to 135 to greenhouse Ethiopia were Isolates from Botryosphaeria greenhouse inoculation trial (Table 3). for inoculation tests were grown on with a Qlame:rer 9 mm was used to remove the bark inoculation. Mycelial plugs mycelial for ten equal facing the xylem. A cork borer expose the wood for wound with were placed into inoculation, were covered with (Pechiney Plastic Packaging, Chicago) to prevent contamination desiccation of also IV,"" ... l«,,",U inoculum. isolate was inoculated on ten trees. Ten trees were MEA to serve as a """ITT" with weeks, development was evaluated measunng lesion 1'vUF;U10 on inoculated trees. These measurements were subjected to statistical analysis (Oneway ANOV A) whether Statistica for Windows lesions associated with the various from For isolates differed other. field trials, three (CMWl1059, CMWII065 CMWl1073) were trial Inc. 1995) to determine results of the inoculation of lesion sizes (Table 3). Isolates were these are representative inoculated onto two-year-old coppice stems citriodora trees in a plantation at Wondo trees was into 20 trees were with sterile MEA as a control. A 9 mm cork borer was used to remove the same protocol used in the greenhouse trial was covered with after 8 LU,"",JL>." A determine statistically, the was using Dunnett's t wounds were Lesion to variance bark and (P<O.OOO1) was was evaluated out to in lesion development. Comparison of means method available in for Windows Inc. 1995). 136 RESULTS Symptoms, sample collection and fungal isolation Symptoms of Botryosphaeria canker were commonly found in Eucalyptus plantations at Munessa Shashemene, Wondo Genet, lima and Menagesha. Disease symptoms were found on different Eucalyptus species including E. globulus, E. sa ligna, E. grandis and E. citriodora. Symptoms of Botryosphaeria stem canker were observed on both coppice stems and first generation stands and on trees of all ages (Figure 2ad). Bark cracking, production of copious amounts of kino (Figure 2a, 2d), stem discoloration and malformation, tip die-back and death (Figure 2b), as well as the occurrence of kino pockets in the xylem were the most common symptoms observed. When the bark was removed from symptomatic trees, well-developed kino pockets (Figure 2d) were visible in the cambium and xylem. Of all Eucalyptus spp., E. citriodora plantations at Wondo Genet and limaiBelete were the most severely damaged by this stem canker disease. Large basal cankers (Figure 2a), as well as two to three layers of black kino rings (Figure 2c) were commonly found on E. citriodora trees, indicating different seasons of infection. Isolates of Botryosphaeria associated with these stem cankers were easily collected from all samples. Morphological characterisation The Ethiopian Botryosphaeria isolates grown on MEA showed some variation in colony growth and morphology. Some of the isolates had fluffy light brown aerial mycelium, whereas others had flat colony growth with little aerial mycelium (Figure 3d, 3e). Considerable variation was observed between the conidial lengths of Botryosphaeria isolates obtained from Ethiopia (Table 1). The lengths of the individual conidia for all isolates ranged from 12.5 /lm to 27.5 /lm and the average conidial length for different isolates ranged from 15.25 /lm to 24.25 /lm. The widths of the conidia showed limited variation and ranged from 5 /lm to 7.5 ).lm. The conidia were grouped into three categories, namely (a) Those with long, narrow conidia, (b) those with long, wide conidia and (c) those with short conidia (Figure 3a-c). No teleomorph structures were observed for isolates examined in this study. 137 peR amplification Amplification of the ITS regIOns and 5.8S gene yielded a PCR product with a fragment length of approximately 500 base pairs (bp). For the EF 1(){ gene, fragments of approximately 300 base pairs were obtained. Restriction Fragment Length Polymorphisms (RFLP) All of the Botryosphaeria isolates from Ethiopia produced the same banding pattern (Figure 4) when the ITS PCR products were cut with Cfo 1. This suggested that these isolates might represent the same species, even though they displayed substantial morphological variation. Comparison of the RFLP pattern for the Ethiopian isolates with banding patterns described for Botryosphaeria spp. (Jacobs 2002, Slippers et ai. 2002) showed that the Ethiopian isolates had a banding pattern similar to that of B. parva (Figure 4). DNA sequencing and analysis When compared with sequences in GenBank, the ITS sequences of the Ethiopian Botryosphaeria isolates, most closely matched those of B. ribis. Aligrunent of these sequences with sequences of B. ribis and with representative sequences of other Botryosphaeria spp. (Slippers et ai. 2003) yielded a total of 518 characters. Analysis of the data set for the EF 1(){ sequences produced a total of 343 characters. The partition homogeneity test revealed that the ITS and EF 1(){ data sets could be combined. The phylogenetic tree generated for the combined sequences of the ITS and EF 1(){ produced five clades (Figure 5). Based on this tree, the Ethiopian Botryosphaeria isolates resided within the B. parva group. Other clades were similar to those defined by Slippers et ai. (2003) and included B. ribis (Clade II), B. eucaiyptorum (Clade III), B. iutea (clade IV) and B. dothidea (clade V). All clades were supported with bootstrap values of 100%. This phylogenetic tree was generated 13 8 based on a total of 855 characters, where 254 variable characters were parsimony uninformative and 194 characters were parsimony informative. The phylogenetic tree generated from the combined sequences had a CI value of 0.928 and RI value 0.905. Pathogenicity tests All Ethiopian Botryosphaeria isolates used in the greenhouse inoculation trial produced lesions on the E. grandis clone (Figure 6a, b). The mean lesion lengths produced ranged from 24.9 mm and 91.8 mm (Table 3). Isolate CMWll073, produced the largest lesions while the smallest average lesions were associated with isolate CMWl1065. No lesions developed on seedlings inoculated with the sterile MEA. Statistical analysis showed that the lesions produced by the majority of isolates were significantly different from the control (P<O.OOOI) (Table 3). An Rsquare value of 0.47 was recorded for the data obtained in the greenhouse trial. Isolates CMWII073, CMW10095, CMWII066, CMW11064, CMWI1063, CMW11069, CMWI1059, CMW10094, CMW11067 and CMW11068 produced lesions that were significantly different from the control. The average lesion lengths associated with isolates CMW11071, CMWl1070 and CMWl1065 (Table 3, Figure 7) were not statistically different from the controls. The three isolates used in the field inoculation trial produced lesions ranging In average length between 63 mm and 255.1 mm. The largest lesion was recorded for isolate CMWI1073 (average = 255.1mm) and the smallest lesion (average = 63.35) was that associated with CMWI1065 (Table 4). Some trees inoculated as controls also developed lesions. However, the controls were statistically different (P=O.OOOI) to those where Botryosphaeria isolates were used for inoculation (Table 4, Figure 6ce). An Rsquare value of 0.71 was recorded for the data obtained from the field study. The results of the field inoculation trial also showed that the lesions associated with isolates CMW 11073 and CMW 11059 were statistically different to those of the control. CMWI1065 produced lesions that did not vary significantly from the control (Table 4, Figure 8). The field and greenhouse trials, therefore, gave similar results. 139 DISCUSSION Results of this study and a prior survey in 2000/2001 have shown that Botryosphaeria canker is the most common disease of Eucalyptus in Ethiopia. This disease affects all the major Eucalyptus spp. including E. globulus, E. gra ndis, E. saligna and E. citriodora (Alemu et al. 2003). The results of the current study have, furthermore, shown that B. parva is the major cause of Botryosphaeria canker in Ethiopian Eucalyptus plantations. This is the first report of this fungus from Ethiopia. Ethiopian Botryosphaeria isolates used in this study showed some variation in colony growth, as well as in conidial length and shape. Based on culture morphology two groups could be distinguished. When the morphology of the conidia was considered, three morphological groups emerged. The morphological variation detected in this study, was however, not consistent with the results of the DNAbased comparison, which showed that the Ethiopian Botryosphaeria isolates represent a single species. Results of this study support the view (Jacobs & Rehner 1998, Smith & Stanosz 2001 , Slippers et al. 2003) that morphological characteristics are insufficient to identify many Botryosphaeria spp. with confidence. They also provide additional evidence to suggest that names used for Botryosphaeria spp. in the past, are questionable. Analysis of the banding patterns of the RFLP of ITS rDNA peR product has been successfully used to distinguish between Botryosphaeria spp. obtained from different hosts (Jacobs 2002, Slippers et al. 2002). In this study the RFLP analysis showed that all Ethiopian isolates might represent a single species. It was, however, not useful in determining a species name for the fungus because B. ribis and B. parva have the same banding pattern (Slippers et al. 2002). Ethiopian Botryosphaeria isolates had identical ITS sequences, which were sufficient only to determine that the isolates represented either B. ribis or B. parva. Inability to distinguish between these two species based on ITS sequences has been reported previously by Smith & Stanosz (2001) and Zhou & Stanosz (2001). However, the combination of the ITS rDNA and EF l()( sequence data was useful to separate B. 140 ribis and B. parva and showed that Ethiopian isolates belong to B. parva. These combined sequences were also used by Slippers et al. (2003) who showed that B. ribis and B. parva represent two distinct species. It is interesting that only one species of Botryosphaeria is associated with dieback in Ethiopia, while three species, B. parva, B. dothidea and B. eucalyptornm are associated with dieback on this host in South Africa (Smith et al. 1994, Smith et al. 2001, Slippers et al. 2003). Botryosphaeria parva was first recorded in 1985 as a new species from Kiwifruit in New Zealand (Pennycook & Samuels 1985). There has, however, been considerable controversy surrounding its taxonomic status. It has, for example, been suggested that B. parva represents a synonym of B. ribis (Smith & Stanosz 2001, Zhou & Stanosz 2001). More recent studies have, however, shown that B. ribis and B. parva are distinct (Zhou, Smith & Stanosz 2001, Slippers et al. 2003). Botryosphaeria parva was previously most frequently found associated with fruit trees (Pennycook & Samuels 1985) and little information is available on the importance of this species in Eucalyptus plantations. Recently, Slipperset al. (2003) showed that B. parva is dominant in plantations of Eucalyptus spp. in South Africa. The results of the current study also showed that this fungus is important in Eucalyptus plantations of Ethiopia. Greenhouse and field inoculation trials revealed that most Botryosphaeria isolates obtained from Eucalyptus in Ethiopia are pathogenic to E. grandis (clone ZG 14) and to E. citriodora. The B. parva isolates used in this study showed variation in pathogenicity both in the greenhouse and field study. Development of lesions on some trees inoculated as controls might have been due to contamination at the time of inoculation, wound stress or entophytic infections. The variations in virulence of the three isolates were concordant between greenhouse and field inoculation studies. These findings are similar to those of Jacobs (2000) who showed that B. parva is pathogenic to Mango, but isolates varied substantially in pathogenicity. Botryosphaeria spp. have long been recognised as stress related opportunistic pathogens (Schoeneweiss 1981, Pusey 1989). A contemporary view is that they are latent pathogens that commonly occur in leaf and branch tissues of healthy woody 141 plants, and cause disease when trees are stressed (Fisher et al. 1993, Smith et al. 1996). In this respect, they are very similar to fungi such as Sphaeropsis sapinea (Fr.:Fr.) Dyko & Sutton (Syn=Diplodia pinea (Desm.) Kickx), which biologically and phylogenetically is a typical species of Botryosphaeria (Smith et al. 1996). The latter fungus is commonly found in healthy pine tissue but causes serious damage under conditions of stress such as after hail damage (Zwolinski, Swart & Wingfield 1990). Hence, B. parva must be considered an important pathogen in Ethiopia, where it almost certainly resides in healthy trees, but causes dieback and death of trees under stress conditions. Plantations in Ethiopia are commonly developed on marginal sites where moisture stress is a limiting factor for tree growth. This could favour disease development associated with B. parva. Moreover, the association of Botryosphaeria canker with Eucalyptus coppice stands is of great concern, because regenerating Eucalyptus species by coppicing is widely practiced in Ethiopia, particularly by small scale tree growers. 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(2001) Differentiation of Botryosphaeria species and related anamorphic fungi using Inter Simple or Short Sequence Repeat (ISSR) fingerprinting. Mycological Research 105: 919926. Zwolinski, 1. B., Swart, W. 1. & Wingfield, M. 1. (1990) Intensity of dieback induced by Sphaeropsis sapinea in relation to site conditions. European Journal of Forest Pathology 20: 167174. 145 Table 1. Conidial sizes of Botryosphaeria isolates from Eucalyptus in Ethiopia Isolate CMWlO088 CMWI0089 CMWI0092 CMWI0093 CMW10094 CMWI009S CMWI0096 CMWI10S9 CMWII060 CMW11061 CMWII062 CMWII063 CMWII064 CMWII066 CMWll068 CMWII069 Origin Wando Genet Wando Genet Menagesha Wando Genet Wando Genet Wando Genet Wando Genet JimaJBelete JimaJBelete JimaJBelete JimaJ Belete JimaJBelete JimaJBelete JimaJBelete Munessa Menagesha Host Eucalyptus sp. Eucalyptus sp. E. globulus E. saligna E. saligna E. grandis Eucalyptus sp. E. citriodora E. citriodora E. citriodora E. citriodora E. citriodora E. citriodora E. citriodora E. globulus E. globulus Range and A verage Length (!lm) (15) 18.3 (22.S) (20) 24.3 (27.S) (IS) 17.3 (20) (12.5) 15(17.S) (17.5) 19 (20) (IS) 1S.3 (17.5) (1S) 16.3 (17.25) (1S) 17.S (20) (17.S) 18.3 (20) (IS) 17.8 i22.S) (17.S) 19.5 (22.5) (15) 16.S (17.5) (17.S) 21.8 (25) (17.5) 20.8 (2S) (15) 18.S (22.S) (IS) 16.8 (20) Range and Average Width (!lm) (S.25) S (S.5) (7) 7.S (7.75) (5) S.8 (7.S) (4.75) 5 (5.S) (5) S (5.25) (5) S (5.S) (S) S (S.2S) (5) S (5.S) (4.7S) S (S.S) (S) S.3 (7.S) (7) 7.5 (7.S) (4.7S S (S.S) (5) S (S.S) (5) S.3 (7 .S) (S) S (S .5) (S) S.8 (7.S) (S.2S) S (S .5) CMWII070 Menagesha E. globu Ius i17.S) 17.S (20) CMWII071 Menagesha E. globulus (17.5) 18.5(20) (5) 6.S (7.5) CMW110n Menagesha E. globulus (17 .S) 19.8 (22.S) (S) S (S.5) Each mean values and ranges are based on measurements from 10 conidia. CMW numbers are culture collection numbers of the Forestry and Agricultural Bioteclmology Institute, University of Pretoria. 146 Length: Width (ratio) 3.6S 3.23 3.17 3 3.8 3.0S 3.25 3.5 3.17 3.38 2.6 3.3 4.3S 3.95 3.7 2.91 3.S5 2.8S 3.9S Table 2. Isolates used in the DNA sequence analysis. Culture No. CMW7780 CMW8000 CMWIOl25 CMWI0126 CMW992/3 CMW9076 CMW7772 CMW7773 CMW9071 CMW994 CMW9077 CMWI0122 CMWII060 CMWII062 CMWII064 CMWlO089 CMWI0095 CMWI0094 CMW7063 Identity B. dothidea B. dothidea B. eucalyptorum B. eucalyptorum F. luteum B.lutea B. ribis B. ribis B. parva B. parva B. parva B. parva Botlyosphaeria sp. " Host Fraxinus excelsior Prunus sp. E. grandis E. grandis Actinidia deliciosa Malus X domestica Ribis sp. Ribis sp. Ribis sp. Malus sylvestris Actinidia deliciosa E. grandis E. citriodora " " " E. globulus " E. grandis " E. saligna " Guignardia philoprina Taxus baccata Origin Switzerland Switzerland S. Africa S. Africa New Zealand New Zealand New York New York Australia New Zealand New Zealand S. Africa Ethiopia Collector B. Slippers B. Slippers H. Smith H. Smith G. 1. Smuels S. R .Pennycook B. Slippers/ G. Hudler B. SliQpers/ G. Hudler M. 1. Wingfield G. 1 Samuels S. R. Pennycook H. Smith Alemu Gezahgne & 1. Roux " " " " " " " Netherlands " " " H. A. van der Aa Accession No. EF I-a ITS AY236947 AY236896 AY236949 AY236898 AF283686 AY236891 AF283687 AY236892 AF027745 AY236894 AY236946 AY236893 AY236935 AY236877 AY236936 AY236878 AY236938 AY236880 AY243395 AY236883 AY236939 AY236884 AY236882 AF283681 AY210474 AY210480 AY210475 AY210481 AY210476 AY210482 AY210477 AY210483 AYS20478 AY210485 AY210479 AY210484 AY236979 AY236905 I I I I i I Isolates from Ethiopia were sequenced in this study. All other sequences are from the study of Slippers et al. (2003) . CMW numbers are culture collection numbers ofthe Forestry and Agricultural Biotechnology Institute, University of Pretoria. 147 Table 3. Mean lesion lengths and confidence limits for greenhouse inoculations using Botryosphaeria isolates from Ethiopia. Isolate CMW11059 CMW11063 CMW11064 CMW11065 CMW11066 CMW11067 CMW11068 CMW11069 CMW11070 CMW11071 CMW11073 CMW10095 CMW10094 Control Mean Lesion length (mm) 54.2OCd 66.0 aoc 71.7 ao 24.9 de 72.8 ao 48.1 ocd 43.5 OCd 60.8 abc 34.9 cde 39.6 cde 91.8 a 83.23b 49.5 OCd 10.g e 95% Confidence limits 38.3570.05 49.2972.71 55.8587.55 9.0540.75 56.9588.65 32.2563.95 27.655935 44.9576.65 19.0550.75 23.7555.45 75.95107.65 67.3599.05 33.6565.35 4.9526.75 CMW numbers are culture collection numbers of the Forestry and Agricultural Biotechnology Institute, University of Pretoria. Means are averages of 10 measurements. Means with the same letter are not significantly different from each other at P<0.05 significance level. 148 Table 4. Mean lesion lengths and confidence limits for trees inoculated with Botryosphaeria sp. on E. citriodora in the field. Isolates Mean Lesion 95% Confidence limits length (mm) a CMW11059 197.95255.65 226.8 b 34.5092.20 CMW11065 63.35 255.1a CMW11073 226.25283.95 0.5058.20 Control 29.35 b CMW numbers are culture collection numbers of the Forestry and Agricultural Biotechnology Institute, University of Pretoria. Means are averages of 20 measurements. Means with the same letter are not significantly different from each other at P<0.05 significance level. 149 \ Sudan LakeTana Addis Ababa Menagesha SOMALIA DO o Wolisso • Jima o Munessa Shashemenne = Wondo Genete SOMALIA Figure 1. Map of Ethiopia showing the plantation areas where samples were collected. 150 Figure 9. Aligned sequence s of the ITS and Elongation Fac t or ( Ialpha ) genes o f Botryosphaeria isolates. () = Gaps , ( .) = homo l ogous nucleotides (N) = Unknown b ases B_parva_CMW9071 B PARVA CMW9078 B PARVA CMW9077 B DOTHI DEA CMW7780 B DOTH I DEA CMW8000 CMWI0094ETHIOPIA CMWI 008 9ETHIOP IA CMWII064ETHIOP I A CMWll060ETH I OPIA CMWII062ETHIOPIA CMWI0095 ETH I OP IA B_ eucalyptoruffi_CMWI0125 B_ eucalyptoruffi_CMWI0126 B l ute a CMW90 7 6 B lutea CMW992 B RI BIS - CMW 7772 3 RIBI S CMW7773 G PHILOPRINA CMW7063 10 20 30 GAAGTTCGAG AAGGTAAGAA AGTTTTTC .... G. - --. . - . .......... .... G. - - -- .. T .... -. '" .G.---- --- - - ... C. C . CA .. . . -. . ... G. ---- ----- ... C . C . CA . ... - . ., .. G.---- .. T. - .. T . ... . G.---- .. T. .. , .G . - - -- .. T .. .. - . . , .. G. ---- .. T. · ... G----- .. T .. . . -. · ... G----.... ...... ...... .... . .................. - . AT .... -. .................... . .. .. . . .. .. .. . . - .AT .... -. .. .. . . .. .. .. . .. .. .................... G. - T .. .. -. . .. . .. . . . . .. . .................... G. -T . ... -. - . . T .... - . . , .. G. ---- .. T . . . . -. · ... G. ---... . G.- - -C . ------ 40 70 50 60 CTT CCGCTG CACGCGC T GGGTGCCAGG TGCTGGGT-- TG.G .. -- .. TG.G. - .. .............. - .. .............. -G ....... . -G ........ - ................ - ................ ----------------- . .. . . TTCC. ..... TTCC. . C ... . . GA. .C ..... GA . . ............ · .- . . . · . - . .. --- . ... A- --- .. .. A-· ..... TG .. .. ...... GC . . .... TG .. .... . .. . GC . .. . .. .. .. . - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - --- - -- 158 B_parva CMW 907 1 B PARVA CMW9078 B PARVA CMW9077 B DOTHIDEA CMW7780 B DOTHIDEA CMW8000 CMW10094ETH IO PI A CMW1008 9 ETHI OPIA CMW11064ETHIOPIA CMW 110 60 ETHIOPIA CMW11062ETHI OP IA 0 11iH00 95ETHIOPI A B_e uca 1 yp tor um_CMW1 0 1 25 B_e uc a1ypt orum_ CMW 101 26 B lutea CMW9076 B- lutea CMW99 2 B RIBIS CMW 7772 B RIBI S CMW7773 G PHILOPRINA CMW 70 63 100 90 110 120 130 140 80 TCCCGCAC TCAATTTGCC TTAT CGCT TCGGTGAGG G GCATTTT GGTGGTGGGG ---------- · ......... · ..... . ... -- ... T . . G. CGG....... -- ... T . . G. CG ........ ---------· ....... · ....... T. ..... A .. CT ........ . . . . . A .. CT . . ...... . ... A. ---------- · ....... . .. A... C. . .. A... C . -------------------------- - ------------------------------------- TGGGTG CTGG TG GGTG CTGG · ... . ... · . . ....... · ....... · ......... · ....... · ......... · ....... · ......... · ....... · . ........ . G...... .G .. . ... GT ........ GT ........ ---------- -------- · . . ....... · ......... ........ . . · .A. · . A ....... · ....... T. ..... . . . T. · ......... · ......... · ......... · ......... · . C .... A .. C.CA.A .. ... C.. C .. C .CGC A.CT CACA- 159 8_parva_CMW9071 B PARVA CMW9078 B PARVA CMW90 77 8 DOTHIDEA CMW7780 B DOTHIDEA CMW80 00 CMW I0 094 ETHI OP IA CMWI0089ETH I OPIA CMW II 064ETHI OP IA CMW II 0 60ETH IOP I A CMW II 062E THIOPIA CMWI 00 95 ETHIOPIA B_eucalyptorum_CMWI0125 B_eucalyptorum_CMWI0126 B lut ea CMW907 6 S l utea CMW992 8 RIBIS CMW7772 B RIBIS CMW 777 3 G PHILOPRINA CMW 706 3 1 90 170 15 0 160 180 200 210 TTGG CCCGC GCTAAG CCTC GTTTGGGCT CG GCAAAATG TCCGCATC TGGTTTTTTT GCGACCGGCG C ........ C ........ . .. . . . . . .............. · .............. · ..... T .. T . . . . . . . . . C · ......... · ..... T .. T . ........ C · ................ . . . . .. . ........ . .. .. . .. . . . . .. A ...... ... A...... · T. · T. ? . . . - ................ · ..... T. - .............. - ................ ................ .C ....... · .... T ... G . C ... .... · .... T ... G . C ..... .. . .. . . . . . .. .. . . C ....... . . . . . . . . . . ................ . .................. . . . . . . . . . .................. CC .... ATT. TG.GCC ... ? .... T ... T .... T ... T .... T ... T .... T ... T .. C ..... .. C ... . . .. ACCC .. C . · · . · · . . . ...... C ... . T. . ........ C .... T. ......... C ......... C . ........ . ........ . .................. . . . .. . . . ................ ~ .TCA ..... .AA T ....... · T. G.. CT TT 160 B_parva _ CMW 9071 B PARVA CMW9078 B PARVA CMW9077 B DOTHIDEA CMW7780 B DOTHIDEA CMW 8000 CMW I 009 4ETHIOPIA CMWI00 89E THIOPI A CMW11064ETHIOPIA CMW 11 0 6 OET HI OPI A CMW11062E THI OPIA CMWI00 95 ETHI OPIA B_eucalyptoruffi_CMWlO125 B_eucalyptoruffi_CMWlO126 B lutea CMW90 7 6 B l ut ea CMW992 B RIBIS CMW7772 B RIBIS CMW77 73 G PHILOPRINA CMW706 3 220 230 2 40 25 0 260 270 280 TGCGACCGA AGCGCGCC CC TC GCCAGA CACG CCAC GCATGTGC GACCA · ........ · .. .................... ------ ------ .. . . . .............. ------ ------ . . . · .. ...... ... · .. C ... AA.A .. ... . A ... . CGCTTC .. . ... . TCACGT TC.T C .A. · ........ · .. C... AA.A .. ... . A ... . .......... · .. · ................ .......... · .. .. ............. · ............... · ....... · .. ......... · .. · ............ .............. · ......... · .. · ....... . . · ...... · .. · . ..... · .. .AT. .... · . . ...... ........ · .. .AT. -- . ... · .......... · ......... · .. C... .A .. · ............. · ....... · .. C ... .A .. · ........... · ...... · .. .... .A .. · ........... · ....... · .. .... .A .. · .......... . . . TA.TG GG . C CA.AA. CGCTT C .. . ... . TCACGT TC.TC.A . ------ ------ ------ ------ ------ ------ ------ ------ ------ ------ ------ TG .. TG .. CTCG .. . CT CG .. . ------ . ... AC . . . . AC ------ ------ ------ ------ . CGG . .CGG. .ATCGG .ATCG G- · . C ...... GTTCTCG.TA .... C.CA AGG.- 161 B_parva _ CMW907 1 B PARVA CMW9 0 78 B PARVA CMW9077 B DOT HI DEA CMW7780 B DOTHI DEA CMW8000 CMW1 0094 ETHI OPIA CMW 1 0089 ETH I OPI A CMW 11 064 ETHI OPIA CMWll 0 60E THI OPI A CMW11 06 2 ETHIOP I A CMW 1 OO95ET HIOP I A B_ e uc alyp t oruffi_CMW10 1 25 B_ eucalypt oruffi_CMW1 01 26 B l utea CMW9076 B l utea CMW99 2 B- RI BIS CMW7772 B RI BI S CMW7 77 3 G PH I LOPR I NA CMW7 063 3 10 320 290 300 33 0 340 350 GACGCTAAC AGC CA TCCC AGG AAGCCACCGA GTT GATTCGA GC TCCGGC T CGACT CTCCC · ... . .... · ........ T.T .... .. CA .. G C . A . AAC ... T .T ...... CA .. G C. A.AAC ... · ..... . .. · . .. .. ... G . . . C · ........ · . ... .... · . .... . .. . · ......... · .. ..... · . . . . ..... · . .. . ..... · . .. .. .. · ... . ... .. · ...... . . G . . . . . . . . C ... TC CT . · .. . .. . . . . · . .. .... . G . .. . .... c .. . TC CT . ... . . NN NNN NN NNNN NNNN NNNNNNNNNN NN NN . · ..... . .. . · ..... . .. . · . .. .. . . · .. .. .. . .. · ... . .... . · ....... . ...... . T G C . .. · . .. .... .. ... .. C .. . . · .. .. ... . ..... . . T GC . .. · . ... . .. .. ..... C . ; . . . . . . . . GA . . G . . T. . . . . . . . . . GA .. G . . T. . · .. .. .... . · ... . . . ... · .... . .. · ... . .... G .... · ... . ..... · .. .. ... .. · . ... ... · . ....... G.. . . . G .. . . GCGC TGA . AG .. . . . AA .T AGG .AT .. . A . C .. G. C .. T ... GG . A. 162 B_parva_CMW9071 B PARVA CMW9078 B PARVA CMW9077 B DOTHIDEA CMW7 78 0 B DOTHIDEA CMW8000 CMW 100 94 ETHIOPI A CMW10089ETHIOPIA CMW1 1064ETHI OP IA CMWll 0 60 ETH I OP I A CMWll0 62 ETHI OPI A CfViW1 0 0 95ETH I OP IA B_euca1yptoruffi_CMW10125 B_eucalyptoruffi_CMW10126 B lutea CMW9076 B lutea CMW 99 2 B RIBIS CMW7772 B RIBIS CMW7773 G PH ILOPR INA CMW7063 370 400 410 420 360 380 390 ACCCAATGTG TACCTACCT CTGTTGCTTT GGCGGGCCGC GGTCCTCCGC ACCGG CGC CCTTCGGG · .. . T. · ......... · ... · .... · ......... · ......... · ......... · .... · .. · . · .. CA .. CT.TGT GTA.C ..... · ......... · " ........ . . . . . . . . . . GG .... C .C. · .TCC .C. CA .. CT.TGT GTA.C ..... · ......... · ......... . . . . . . . . . . GG .... C.C. .. TC C . C . · ... T. · ......... · ... - · .... · ......... · ......... · ......... · ... T ..... · ... - · .... · ......... · .. . ...... · ......... · ... T. · ... T ..... · ... · .... · ......... · ......... · .. . · .... · .... T .... . , .. T ..... · ... · .... · ......... · ... T ..... · ... - · .... · ......... .... C ..... · ... · .... · . .. ...... .... C ..... · ... · .... · ......... · ......... · ... · .... · ......... · ......... · ... · .... · ..... .. .. · ......... · ......... · ... TT ... T ., . AA ..... T . . . . . . . . . · .... C .. . · ......... · ......... · ........ . · ......... · ......... · ......... · ......... · ......... · ....... . . . . . . . . . . . . · ......... · ......... · ......... · ......... · .... · .. · . · .. · .... · .. · . - · .. · .... · .. · . · .. · .... · .. · . · .. ..... . T . ..... . T. .... AC.C. .... AC. C . · .... · .. · .... · .. . .. G. AA G .. AAC .. G · . T. · . T . . .G ... .G ... · . ... G. · . ... G. ..C.. - 163 B parva _ CMW90 71 B PARVA CMW9078 B PARVA CMW9077 B DOTHIDEA CMW7780 B DOTHIDEA CMW8 000 CMWI009 4ETHI OP I A CMWI0089ETHIOPIA CMWII064ETHIOPIA CMW II0 60ET HI OPIA CMWll062ETH I OP I A CMW I0 095E THI OP I A B_eucalyptorum_CMWl0125 B_eucalyptorum_CMWl0126 B l utea CMW9076 B lutea CMW 99 2 B RI BIS CMW7772 B RIBIS CMW7773 G PHILOPRINA CMW7063 430 440 450 470 480 490 460 GGGCTGGCCA GC GCC CGCCA GAGGACCAT AAAACTCCAG TCAGTGAAC TTCGCAGTCT GAAAAACAAG ......... C .••.. . ..• C . ... C ..... •... C ..•.. · .. T . . . . . . . . . . . . . . · .. T . . . . . . . . . . . . . . · ................. . . . . . . .. C C C C . . . . . A ... G A. - . . . . . .. . . . . . . . . -T . . . . . A ... G A .- . . . . . . . . . . . . . . . -T . . . . . A ... G •... . A .•• G . . . . . . . . . . . . . . A ... G . . . . . . .. . . . . . . A ... G . . . . . . . . . . • G .•...•. . • G .... . . . .... T . . . .. G. . . . . . . . . . . . .C ....... ATA .T.TTA .. .TC . . . . . . GT..T.T 164 B_ parva CMW90 71 B PARVA CMW9 078 B PARVA CMW9077 B DOTH IDEA CMW7780 B DOTHID EA CMW8000 CMW 1 0094 ET HIOPIA CMW 1 0089 ET HI OP IA CMW 110 64 ET HI OPIA CMW 11 060 ET HIOP I A CrvlW 11 0 62 ETHI OPIA CMW1 0095 ET HIOP I A B_eucalyp t orum_CMW 10 1 25 B_ euca lyp torum_ CMW 1 0 126 B l utea CMW9076 B l u t ea CMW992 B RI BIS CMW77 72 B RI BI S CMW777 3 G PHI LOPRI NA CMW70 63 510 52 0 550 500 530 540 5 60 TTAATAAAC T AAAACTTT CA ACAACGGATC TCT TGGTTCT GGCAT CGATG AAGAACGCAG CGAAAT GC GA .... . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... A.. . .. GT . 165 B_parva_CMW9071 B PARVA CMW9078 B PARVA CMW9077 B DOTHIDEA CMW7780 B DOTHIDEA CMW8000 CMW10094ETHIOPIA CMW10089ETHIOPIA CMWll064 ETHIOPIA CMWll060ETHIOPIA CMW110 62ETH I OPIA CMW10095ETHIOPIA B_eucalyptoruffi_CMW10125 B_ eucalyptoruffi_ CMW10126 B lutea CMW9076 B lutea CMW992 B RIBIS CMW7772 B RIBIS CMW7773 G PHILOPRINA CMW7063 570 590 580 600 610 620 630 TAAGTAATGT GAATT GCAGA ATTCAGTGAA TCATCGAATC TTTGAACGCA CATTGCGCCC CTTGGTATTC · . ..... .. . · ......... · .... ..... · ......... · . ...... .. · ..... .. .. · ......... · . .... .... · ......... · ......... · ...... ... · ......... T. T. · ......... · ......... · ......... · ......... · ......... · ......... .C. 166 640 B_parva CMW907 8 B PARVA 650 660 0 690 GCGTCATTTC AACCCTCAAG CTCTGCTTGG TATTGGGCCC CGTCCTCCAC B B DEA CMW7780 CMW80 0094ETHIOPIA 0089ETHIOPIA CMliIJll ETHIOPIA • • 9 • ~ ~ ~ . . . . . . TT • . . . . . . . . A . . . . . . . T- T CMWI06~THOPA 1 62ETHIOPIA 95ETHIOPIA CMWIOI eucalyptorum CMWIOI B lutea CMitJ907 B lutea CMitJ992 B RIBI CMliJ7 2 CMvH B S TT. TT . . . . . . . -. T. . . . . . . . T. T. o •• 0 •••• 0000. 3 G PHILOPRINA CMW7 . ..... -. T- .. T . . . . . . . ~ . ~ . . .. . ~ o 0 ••••• o •••••• 0 •••• 0 • •• 0 • ... AC . . . 67 B_ par va_CMW907 1 B PARVA CMW9078 B PARVA CMW9 0 77 B DOTHIDEA CMW 778 0 B DOTHIDEA CMW8000 CMW 1 0094ET HIOPIA CMW 10 089E THIOP IA CMWllO 6 4 ET HIO PI A CMW 11 060ET HIOP IA CMW1 1062 ETHI OPIA CMW1 00 9SETH I OP I A B_eucalypt or um_ CMW 1 0 12 5 B_e uca lypt orum_CMW1 0 1 26 B l u tea CMW9076 B lu tea CMW99 2 B RIB I S CMW7772 B RI BIS CMW7773 G PH ILOPR INA CMW7063 710 73 0 74 0 750 760 770 72 0 GGACGC G CC TCAAAGAC CTCG GCGGTG GC GT CT TGCC TCAAGCGTAG TAGAAAAC ACC TCGC TT T · .... . · .. T . · CGG .. GC . · CGG .. GC . · ......... · . . ....... · ... .... .. · . . .... . .. · . . .... . .. · . ...... . .. · .. . . ..... · . . ... · . .... · . . ... · ... . . · ..... · ..... ... T. · .. T. · .. T. ... T. · .. T. · . . T. e •• •• •• • •• .TG. ACGC . · . ... . . .. . · ...... . .. · . ........ .TG. ACGC . · . ...... .. · ...... . . . · . ........ . TG. ACGC . . , . . G ..... · . .. . . .... · .... . .... .TG. ACG C . ... . G ..... · .... . ..... · .... . .... . ... T. · ..... · . .... · . . T. · . GT .. . .. . T ... AT. AGT . ... . .. C .. . .. G . . T · ......... · . ... . .... · .... . .... · ......... . .. . . CAT .. . .... CAT .. · · · · . T ... .... C . T ....... C .... . T. .... . T. .... A. . .... A . . · . ..... . ... . .. T . . TT . 168 B_parva_CMW9071 B PARVA CMW9078 B PARVA CMW90 77 B DOTHIDEA CMW7780 B DOTHIDEA CMW80 00 CMW I00 94 ETHI OPIA CMWI0089ETHIOPIA CMIiH 10 64 ETHIOPI A CMW II 060 ETHI OP I A CMIjH 10 62E THIOPI A CMvVlO0 95ETHI OPIA B_eucalyptorum_CMWI0125 B_eucalyptorum_CMWlOI26 B l utea CMW90 76 B l utea CMW992 B RIBIS CMW77 72 B RIBIS CMW 777 3 G PHILOPRIN A CMW70 63 79 0 780 800 8 10 820 830 833 GGAGCGCACG GCGT CGCCCG CC GGACGAAC CTTTGAATT ATTTCTCAAG GTT GAC CTC G GAT · . .. ...... · ... ..... . ....... . G . ....... . G . · ....... .. o •••••• • •• o ••••••••• · .... . .... · ......... ... CT ... C . - · ........ . ... CT ... C . · .. . ...... · .... . ... . · ......... · ......... · ......... .... . ... T. · .. . .... T. · ... . ..... · .......... · ........ . · ......... · ......... · ......... · ......... · ..... . ... · ......... ... . TC.GG. CGAG.. T .. T G.CA .-- ... . CCC --.-- . .. . . T. T. 169 ABSTRACT patula is In recent disease In surveys a dark grey fungus resembling Diplodia sp. was isolated from cones of patula in a cosmopolitan distribution on Pinus spp. and Diplodia pinea is an important pathogen on exotic Pinus spp. in Hemisphere. tropics and Southern pinea is a stress related opportunistic pathogen that can branch and shoot dieback, root disease and blue stain including two (A and C), and associated with Pinus spp. of the sapwood. m pinea, scrobiculata are study was to dark obtained from the cones of P. patula in Ethiopia. These were compared based on morphology and based techniques. Morphological comparisons isolated pine cones a pinea. DNA sequences for the identification of the parva or pinea isolates is tests lesions but as as ptubulin gene regions confinned the pinea and showed that the is closet to of that with the Fusicoccum anamorph rib is. Analysis Ethiopia represent SSR A morphotype of showed that fungus, which in countries where pines are grown as exotics. Pathogenicity that isolates D.pinea both Fusicoccum and pinea were to cause were most pathogenic. 171 INTRODUCTION In Un'JUiU. P. patula Schiede & LJvIJU'v and radiata D. have been been introduced into the country more than 100 for afforestation, patula is been abandoned of due to Dothistroma cover 30 000 UV,"',",,",, radiata has blight. Plantations of Pinus of which the major proportion (Anonymous 1994). Information available on P. patula plantations is very limited. In a recent disease survey conducted Ethiopia, a resembling (Fr.:Fr.) Dykco & pinea (Desm.) Kick cones of P. patula (Alemu, Roux & Wingfield 2003). Sutton) was isolated a worldwide distribution and an extensive host Diplodia among conifers (Birch 1 Pinus spp., & Toes 1 pathogen (Marks 1985). Gibson 1979) damage, predispose trees to caused by stems as an endophyte et it Minko 1 & associated with environmental conditions without necessarily mainly Eldridge 1961, Gibson 1979). This fungus is known are planted is known as an cones Sphaeropsis '_",.~ pinea. exists in pine the plant 1996), disease symptoms. mechanical of commences when the trees are under stress (Swart et al. 1985, Smith et al. 1996, Stanosz et al. 1997), as that caused by hail et al. 1978, et al. 1985, et al. 1985). & Wingfield 1991). include shoot blight, (Gilmour 1964, Marks & Minko 1969, Swart et al. I root (Wingfield Eldridge 1961). .:>"",""UIUil'. KnoxDavies 1980) and South Africa, root rot (Wingfield (Swart et al. 1985). in South Minko 1969, is associated with various disease symptoms (Puntinhalingam Diplodia vU}'ll"''''' drought or frost (Marks example, dieback, stem Stanosz et al. 1997), Costa 1955, pinea was KnoxDavies 1980) and associated with hail damage reason, P. radiata is not (Swart, Wingfield & n.HV"J.JU. VluUC.... U 1987, areas 1). 172 In past, four morphotypes (A, pinea (Wang C and I) have been et al. 1985, Palmer, Swart & Wingfield 1987, De Wet et at. 2002, Hausner et 1999). Wingfield & Wingfield (2001) showed that I morphotype anamorph of Botlyosphaeria obtusa (Schw.) study, in S. Wet et ai. (2003) showed ,HU/LW",U. the a recent and C morphotypes, once are closely related to each other and treated them in S. sapinea represent a new B morphotype recently as sp. scrobiculata 1. de Wet, B. Slippers & M. 1. Wingfield (De Wet et al. 2003). All of the broadly treated as D. Pinus pathogenic to a wide are known to (Wang et al. 1985, Palmer et al. 1987, Wet et al. 2002). The C morphotype D. pinea is more pathogenic than the scrobiculata (De Wet et al. 2002). The A morphotype is morphotype or to <if. occur on a wide range conifers worldwide (Morelet & Chandelier 1993, Smith et Stanosz 1995, Hausner et ai. 1999, De known the North ai. 1985, Smith & scrobiculata is best 2000). is only mildly (Wang et Smith 1999, De Wet et ai. 2000, 1995, Stanosz, Swart et ai. 2001). The C morphotype has been only Indonesia Wet et al. 2002). diseases affecting P. patuia in Ethiopia. Very little information is available In a recent survey, Alemu et al. (2002), identified a fungus similar to Armillaria as a cause of m Alemu et ai. (2003), and South Western patula in Western plantation reported presence of D. pinea in cones of P. patuia in Ethiopia, but these authors did not identify morphotype the Diplodia This was DNA based techniques. 31m study was to identify both on morphological characteristics and Pathogenicity of isolates was also considered in 173 MATERIALS AND METHODS Fungal isolations and morphological characterisation During a disease survey conducted in 2000, cones were randomly collected from the floor in a P. patula plantation at Munessa Shashemene and isolations were from them. pith cones were opened mm were cones. with (100%), washed with (20 g Biolab Malt Extract, 15 g Biolab Agar) and sterile water, plated onto incubated at were taken from mycelium, typical of Isolates and transferred to MEA in culture collection (CMW) pinea were in the Pure the and Agricultural Biotechnology Institute, University of Pretoria. (WA) (15 g were transferred to water lL H 2 0) with pine needles laid on the surface and incubated of fruiting structures. structures. three weeks to promote Single conidial was were by spreading spore masses on a drop of sterile water and incubating plates for 24 hr at germinating spores were picked and widths of the a were selected randomly from VVA . . . . . " " , were After hr, single to MEA and incubated at 25 Conidia were mounted in lactophenol and ""'.LAH.U'......... microscope. Ten 0c. Axioskop light isolate and the lengths and (Table 1). DNA extraction Total DNA was different (Table Mycelium for DNA extraction was obtained by of the agar into 1.5 with a sterile Eppendorf tubes. The powder in liquid nitrogen a and placing mycelial mats was and mortar. to a The method of Raeder Broda (1985) was used to extract DNA from the mycelium. 174 The DNA pellets were vacuum dried to remove excess "'~C<HV and resuspended III sterilised water. RNase A (1 to the DNA solution to remove water bath over night. 50 was contaminating RNA and incubated at concentration of comparison with a standard on a 1% fi<>r,,,"p DNA the III a was detected by gel, stained with ethidium bromide and visualised under UV light peR amplification internal transcribed spacer (ITS) 5.8S of the ribosomal RNA operon and the were amplified using conducted using chain reaction (PCR). ITS 1 GCT GTA GGT CCT GCG G '3) and ITS 4 '3) (White et al. 1990). TGA TAT contained DNA polymerase (Taq, 2.5U/1l1, (l0 mM TrisHCl, primer, 1 III of DNA and 1.5 mM at denaturation at 1 50 mM KCl), 0.15 mM of volume Ill. 1 min. This was followed at 72 for 30 s, chain °c PCR reaction mM dNTP III sterilised water to make up a Denaturation was performed at 96°C was chain was carried out at for 5 min. ~-tublin was (5' GGT CAA ATC AGT GT A G 3') and the reverse primer Bt2b ACC 3 ') & Donaldson 1995). PCR reaction mix included DNA polymerase (Taq, 2.5U/lll), 2.5 mM dNTP's, lOx Buffer, supplied by the manufacturer, 0.15 mM each primer, 1 III of DNA and 37 III water. PCR reaction conditions involved an 1 min, which was followed by 40 30 s, chain elongation at °c at 94 mM for 1 min, primer at 94 at 51 1 min and an additional chain elongation at an additional minute. 175 Morphotype determination sets of unlabelled Simple (SSR) et were used in PCR reactions to determine the morphotype The sets of the used included CAT 3'), 5' GAT CAG TCG CAT TGA AAT GCC 5' GAC TTG TCT CCT ACC 3') and reverse primer 3') as well as TB41 5' 10 (forward 5' CAG primer 5' CAG (forward primer the D. pinea isolates. (forward primer TB23 5' GAC AGA 3' and reverse primer ACG 2001) 3' and reverse CGA et al. 2001). CGG ...,....,,"v,•.., and conditions used were similar to those described by al. (200 All PCR were bromide and on 1% et with ethidium under UV illumination. DNA sequencing PCR products were purified using the (QUIAGEN, GmbH, Germany). The Pure Product Purification products were the kit with Amplitaq® DNA Polymerase, FS (PerkinElmer, Warrington, UK), according to the manufacturer's protocol, on an 1 and PRISM™ 3100 DNA Autosequencer (PerkinElmer). 4 were used genes were whereas the l3tubulin primers determination and Bt2b. For morphotype mentioned above were used. Sequence analysis identity isolated was determined by the rDNA Ethiopian [~ationl Centre Biotechnology Information (NCB I), Health Bethesda (http:/www.ncbi.nlm.gov/BLAST)]. l3tubulin gene sequences National Institute of rDNA and the isolates were combined and aligned manually using 76 PAUP 4.0b (Swofford 1998) against the sequence data set of D. pinea and Botryoshpaeria spp. obtained from De Wet et al. (2000) and Slippers et al. (2003) (Table 2). Gaps were inserted manually and were treated as missing data. The sequences were analysed using parsimony, with trees generated by heuristic searches with simple addition and Tree Bisection Reconstruction (TBR) branch swapping. Confidence intervals were determined using DNA BOOTSTRAP analysis (Bootstrap confidence intervals on DNA parsimony) (1000 replicates) (Felsenstein 1993). Guignardia philoprina (Ellis) Viala & Ravaz as well as Lasiodiplodia theobromae (Pat.) Griff. & Maubl. were used as outgroup taxa in the phylogenetic analysis . SSR sequences of the Ethiopian isolates were aligned against each other and with representative sequences of the three morphotypes of D. pinea obtained from De Wet et al. (2003). The sequences were analysed using parsimony, with trees generated by heuristic searches with simple addition and Tree Bisection Reconstruction (TBR) branch swapping. A phylogenetic tree showing the relationships of the isolates was obtained using the mid point rooting option. Pathogenicity trials Greenhouse inoculation trials usmg isolates CMW10717, 11240, 11246, 11250, 11252 and 11253 were conducted to evaluate the pathogenicity of the fungi in question. The greenhouse pathogenicity tests were conducted on 2yearold P. taeda seedlings. Prior to inoculation, the trees were kept in the greenhouse for ten days to allow them to acclimatise to the environment. Six isolates were selected to represent the two conidial forms emerging from morphological and DNA sequence results. Isolates used in the inoculation trials were grown on MEA for ten days. A 9 mm cork borer was used to wound the trees and expose the cambium. Mycelial plugs of equal size were placed in the wounds with mycelium facing the exposed cambium. Each isolate was inoculated onto 20 trees. Plugs of sterile MEA, were also inoculated onto 20 trees, to serve as controls. The inoculated wounds were covered with Parafilm (Pechiney Plastic Packageing, Chicago, USA) to prevent desiccation. 177 After six were measured to evaluate inoculated plants. Oneway analysis development on variance (ANOV A) using Statistica Windows (StatSoft. Inc. 1995) was carried out to evaluate statistical differences ""'T""""'''' treatments. Mean variation was in Statsitica for Dunnett's T test available (StatSoft. Inc. 1995). RESULTS Fungal isolations and morphological characterisation coloured were obtained from cones collected patula plantations at Munessa Shashemene. Cultures showed growth on 01H""-"lr'''' cultures also had a mycelial growth of the Petri dish, No isolated fungi. In total, was detected 'UfP'nnr isolates, to black mycelial whole mycelial growth from a different cone were retained study. Of the isolates inoculated onto water the pine only 15 produced fruiting structures on and conidial morphology was determined for these. Evaluation of conidial morphology that two different (Figure 1a) to conidia similar to were present. One group had of pinea (Figure 1b). other of the conidia in the latter group were smooth and the of resembled and from 16 Jlm to 17 Jlm. The length ranged from 17 Jlm to 19 Jlm and to the conidia that Fusicoccum spp. average widths ranged from 11 conidia that Jlm (Table 1). Their widths (Table 1). No sexual structures were found. Four conidia had Jlm to 5.7 Jlm the 15 had the isolates had to Fusicoccum peR amplification A fragment rDNA of DNA sequence comparisons of approximately 500 base pairs (bp) was obtained when the pinea and the Fusicoccum were with 1 and 178 ITS4. Partial amplification of the ~-tublin gene of the D. pinea and Fusicoccum isolates with primers Bt2a and Bt2b produced a fragment size of approximately 400 bp. The ITS sequence data of the D. pinea isolates were compared with sequences in Genbank. Sequences of Diplodia isolates showed that they were closely related to D. pinea. Similarly, when sequences of the Fusicoccum isolates were compared, they showed a high degree of homology to sequences of Botryosphaeria ribis Grossenb. & Dugg. (anamorph =Fusicoccum ribis Grossenb. & Dugg.) and B. parva (Pennycook & Samuels). The ITS rDNA and ~-tublin sequences of the isolates used in this study were combined and aligned against each other and against sequence data obtained from De Wet et al. (2002) and Slippers et at. (2003). After alignment a total of 1029 characters was obtained (Figure 6). Of these 486 characters were constant whereas 278 characters were variable and parsimony uninformative, while 265 characters were parsimony informative. Phylogenetic analysis using parsimony produced 9 trees. The topologies of these trees were the same with only minor variation in arrangements within the groups. The phylogenetic tree (CI=0.886, RI=0.886) (Figure 2) showed that sequences of the Ethiopian isolates grouped together with D. pinea isolates with a 100% bootstrap value and the Ethiopian Fusicoccum isolates resided in a clade containing B. parva and B. ribis with 97% bootstrap value. The Ethiopian Fusicoccum isolates, however, formed their own subgroup of which the exact identity is unclear (Figure 2). This clade was supported by a bootstrap value of 100%. Morphotype determination To determine the morphotype of the D. pinea isolates a further sequence analysis was conducted using three SSR markers. Alignment of the combined SSR sequences of the D. pinea isolates with sequences obtained from De Wet et al. (2003) produced a total of 1051 base pairs (Figure 7). Sequence analysis using mid point rooting produced a single tree. This phylogenetic tree showed that the D. pinea isolates from Ethiopia group together with the 'A' morphotype of D. pinea (Figure 3). 179 Pathogenicity trials All m P. taeda the 0 ........''UL,U.A'' two inoculation studies produced (Figure The mean lesion lengths produced inoculation trial was in a ""',,VH,U on twoyearold these m of 30 mm to 56.8mm (Table 3) and in the mm and inoculation trial to mm (Table Both fungi developed (P>O.OOOl) compared to the Analysis of were controls (Table 5). variance showed statistically significant differences, in pathogenicity for and were more pinea than DISCUSSION Pinus patuLa is the only Pinus sp. currently planted in Ethiopia and is of importance to the country. During a was isolated D. the conducted P. patula plantations cones (Alemu et study showed that two fungal 2003). are associated with P. patula cones in Ethiopia. Based on morphological characteristics and and D. identified as a IS morphotype of Ethiopia. of a Botryosphaeria sp. from Pinus spp. in Our results, the analysis, they were preliminary, that in contrast to other sp. is more commonly with patula cones than D. pinea. of Use of sequence comparisons confirmed of the and in determining the morpho type of ~-tublin sequence, however, could not assist pinea in Ethiopia. Further sequence analysis the patula from plantations at Munessa on morphology. obtained Ethiopia, morphotype. De Wet et al. (2003) used SSR sequences to determine cones to relationships 180 of the morphotypes of Diplodia. The A morphotype has been frequently found associated with seed and seed chaff (Anderson, Belcher & Miller 1984), indicating the endophytic nature of the fungus. The A morphotype is also the most widely distributed morphotype of D. pinea (Wang et al., 1985, De wet et al. 2000). Its presence in Ethiopia is, therefore, not surprising. Identification of the Fusicoccum isolates to species level, was not possible using only conidial morphology. Evaluation of the ITS and ~-tublin sequences, however, showed that the Fusicoccum sp. associated with P. patula in Ethiopia is closely related to B. ribis and B. parva. Several Fusicoccum spp. are known anamorphs of Botryosphaeria spp. (Sutton 1980, Pennycook & Samuels 1985). Botryosphaeria spp. are also known as opportunistic wound and stress related pathogens and as symptomless endophytes on several hosts (Smith et al. 1996). It has been shown that Botryosphaeria spp. cause dieback and cankers on a wide range of woody plants including Eucalyptus spp. (Smith, Kemp & Wingfield 1994). In Hawaii, B. dothidea has been found associated with wilting and dying P. taeda and P. elliottii Engelm. (Hodges 1983). The importance of the Fusicoccum sp. in pine plantations of Ethiopia, however, needs further investigation. The results of the greenhouse inoculation studies showed that both D. pinea and the Fusicoccum sp. are pathogenic to P. taeda. The D. pinea isolates, however, produced larger lesions than those of the Fusicoccum sp. The A morpho type of D. pinea has been shown to be highly pathogenic to several Pinus spp. (Wang et al. 1985, Palmer et al. 1987). In Swaziland symptoms similar to those of D. pinea were observed on P. elliottii Englem and P. taeda (Wingfield & KnoxDavies 1980) suggesting that P. taeda is susceptible to D. pinea infection. This was confirmed in our study where P. taeda was used as a substitute for P. patula, due to the lack of available trees. The occurrence of D . pinea in Ethiopian P. patula plantations could have a serious impact on the management, utilisation and future development of P. patula. It has been shown that D. pinea was introduced into several countries, apparently with seeds (Burgess et at. 2001, Smith et al. 1996). It is therefore, essential to manage future introductions of pine seed into Ethiopia to minimize the risks of introduction of the 181 other morphotypes of D. pinea. For example, the C morphotype of this fungus is considerably more pathogenic than isolates of the A morphotype (De Wet et al. 2002). Multiple introductions increase the clonal diversity and risk of disease from this pathogen. Species site matching and selection for disease resistance have to be considered to minimise severe damage from D. pinea. The importance of the Fusicoccum sp. in P. patula plantation also needs further investigation. REFERENCES Alemu Gezahgne, Coetzee, M. P. A., Roux, J., & Wingfield, M. J. (2002) Armillaria root rot in Ethiopia. South African Journal ofScience 98: IV. (Abstract). Alemu Gezahgne, Roux, J. & Wingfield, M. J. (2003) Diseases of Exotic plantation Eucalyptus and Pinus species in Ethiopia. South African Journal of Science 99: 2933. Anderson, R. L., Belcher, E. & Miller, T. (1984) Occurrence of seed fungi inside slash pine seeds produced in seed orchards in the United States. Seed Science and Technology 12: 795799. Anonymous (1994) Ethiopian Forestry Action Plan Final report, vol. I, pp 14 and Vol. II, pp 34. Addis Ababa, Ethiopia. Bega, R. V., Smith, R. S., Martinez, A. P. & Davis, C. 1. (1978) Severe damage to Pinus radiata and P. pinaster by Diplodia pinea and Lophodermium spp. on Molokai and Lanai in Hawaii. Plant Disease Reporter 62: 329331. Birch, T. T. C. (1937) Diplodia pinea in New Zealand. Review ofApplied Mycology 16: 148. Burgess, T., Wingfield, B. D. & Wingfield, M. J. (2001) Comparison of genotypic diversity in native and introduced populations of Sphaeropsis sapinea isolated from Pinus radiata. Mycological Research 105: 13311339. Currie, D. & Toes, E. (1978) Stem volume loss due to severe Diplodia infection in a young Pinus radiata stand. New Zealand Journal ofForestry 23: 143148. Da Costa (1955) Effects of blue stain on the strength of Pinus radiata. CSIRO Forest Products Newsletter 209, Austra1ia. De Wet, J., Wingfield, M. J., Coutinho, T. A. & Wingfield B. D. (2000) Characterisation of Sphaeropsis sapinea isolates from South Africa, Mexico, and Indonesia. Plant Disease 84: 151156. 182 Wet, 1., Wingfield, M. 1., Coutinho, the 'c' & Wingfield B. D. (2002) Sph a erops is morphotype of the Management 161: 1811 0., Wingfield, B. Wet, (2003) Multiple & , " , u " , •. v.. M. J. markers reflect and relationships between morphotypes of Sphaeropsis sapinea and of Diplodia. Mycological Research (Submitted). new Eldridge, a 1. (1961) Significance Diplodia pinea in plantations. Review of Applied Mycology 41: J. (1993) DNABOOTBootstrap 3.1. Seattle, University as exoUcs in widely southern hemisphere. Part II. Gilmour, J. W. Interval on Washington DC. I. A. S. 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Mycota);:on 24: 445448. 183 Punithalingam, Waterson, 1. M. (1970) Diplodia pinea. CMI description Commonwealth Mycological plant pathogenic fungi and Bacteria and Association of Applied Biologist, Kew, (1 Rapid preparation of England. from filamentous lo/1icrobiology 1: 1 W" Wingfield, B. D. Coutinho, Wingfield M. 1. (2003) Combined multiple characters and phenotypic differentiating previously identified as Botryosphaeria dothidea. Mycologia (In press). Smith, 1. Kemp, Wingfield M. 1. (1994) Eucalyptus m South Pathology by Bot;yosphaeria doth idea. 10311034. & Stanosz, Smith, D. and dieback of R. (1995) Confirmation north sapinea two distinct populations United states Phytopathology 85: 699704. Smith, H., A. (l M. 1., Crous, P. W. & Coutinho, Sphaeropsis doth idea endophytic in Pinus spp. and Eucalyptus spp. m G. South African Journal ofBotany Smith, R., Guthmiller, M. 86-88. 1. & Persistence (1 of Sphaeropsis sapinea on or in asymptomatic stems of red pine nursery seedlings. l\1ycologia 89: Stanosz, R., Swart, W. 1. Smith, D. R. (1999) RAPD marker and characterization of Sphaeropsis sapinea from diverse coniferous hosts and locations. il!fycological Research 103: 1 Sutton, B. (1980) 202. Commonwealth Mycological Swart, W. 1., KnoxDavies, P. 1. & Wingfield, M. 1. (1985) Sphaeropsis sapinea, with special ,.",T,""""'''''' to occurrence on spp. in South Africa. South Journal 35: 18. Swart, W. 1. & Wingfield, M. 1. (1991) Biology and control of Sphaeropsis sapinea in Pinus South W.l., 75: KnoxDavies, P. 1. (1987) with Sphaeropsis sapinea infection of pine trees in South Phytophylactica 19: M. 1. Plant 10. 184 Swofford, D. L. (1998) PAUP*. Phylogenetic Analysis Using Parsimony. (* and other methods). Version 4.0. Beta version. Sinauer Associates. Sunderland, Massachusetts. Wang, CG., Blanchette, R. A. , Jackson, W. A. & Palmer, M. A . (1985) Difference in conidial morphology among isolates of Sphaeropsis sapinea. Plant Disease 69: 83884l. White, T. 1., Bruns, T., Lee, S. & Taylor, 1. (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In peR protocols: a guide to methods and applications. (M. A. Innis, D. H. Gelfand, J. J. Snisky & J . W. White eds.). San Diego: Academic Press. Wingfield, M. J. & KnoxDavies, P. S. (1980) Association of Diplodia pinea with a root disease of pines in South Africa. Plant Disease 64: 221223. 185 Table 1. Average conidial lengths of fungal isolates obtained from Pinus patula cones. Isolate No. Species A verage Conidial Length CMWl1249 CMWl1250 CMWl1252 CMW10717 CMWl1240 CMWl1241 CMWl1242 CMWl1243 CMWl1244 CMWl1245 CMWl1246 CMW11247 CMW11248 CMW11251 CMWl1253 Diplodia pinea " " " Fusicoccum sp. " " " " " " " " " " (~m) Average Conidial Width (~m) 34.6 15.6 33.6 16.5 33.2 16.1 34.3 15.8 17.4 5.5 18.8 5.7 17.1 5.5 17.5 5.3 18.2 5.7 17.2 5.2 16.8 5.7 17.2 5.5 18.5 5.2 18.9 5.5 18.3 5.7 CMW numbers are those of the culture collection of the Forestry and Agricultural Biotechnology Institute (FAB!), University of Pretoria. Values of conidial length and width are averages of 10 measurements. 186 Tables 2. Isolates used in the DNA sequence analyses Culture Nr. ...... ...-. -.. " ~ . CMWl 89 CMWl90 CMW4876 CMW11250" CMW11246" CMWI07l7" CMW489I CMW7780 CMW8000 CMWlOl25 CMWIOl26 CMW992/3 CMW9076 CMW7772 CMW7773 CMW9071 CMW994 CMW9077 CMW907 l CMWlOl22 CMW11246" CMWI0717" CMW7060 CMW7774 CMW7775 CMWlOl30 CMW9074 CMW7063 Identity Diplodia pinea Lasiodiplodia theobromae B. dothidea B. dothidea B. eucalyptoru111 B. eucalyptorul11 F. luteum B. lutea B. rib is B. ribis B. parva B. parva B. pm-va B. parva B. pm-va B. parva B.pm-va B. stevensii B.obtusa B. obtusa B. rhodina B. rhodina Guignardiaphyiloprina Morhotype B A C A A A Host Pinus resinosa P. banksiana P .patula P. patufa P. patufa P. patula Fraxinus excelsior Prunus sp. E. grandis E. grandis Actinidia deliciosa Malus X domestica Ribis sp . Ribis sp. Ribis sp. Malus sylvestris Actinidia deliciosa Ribis sp. E. grandis Pinus patula Pinus patula Fraxinus ecelsior Ripes spp. Ripes spp. Vitex donniana Pinus sp. Taxus baccata Origin United States United States Indonesia Ethiopia Ethiopia Ethiopia South Africa Switzerland Switzerland S. Africa S. Africa New Zealand New Zealand New York New York Australia New Zealand New Zealand Australia S. Africa Ethiopia Ethiopia Netherlands New York, USA New York, USA Uganda Mexico Netherlands Collector M . A. Palmer M. A. Palmer M . 1. Wingfield Alemu Gezahgne & Jolanda Roux Alemu Gezahgne & Jolanda Roux Alemu Gezahgne & Jolanda Roux W. A. Smith B. Slippers B. Slippers H. Smith H. Smith GJ. Smuels S.R .Pennycook B. Slippers/ G. Hudler B. Slippers/ G. Hudler MJ. Wingfield G.J Samuels S.R. Pennycook M.J. Wingfield H. Smith Alemu Gezahgne & Jolanda Roux Alemu Gezahgne & Jolanda Roux H. A. van der Aa B. Slippers/G. Hudler B. Slippers/G. Hudler J. Roux T. Burgess H.A. van der Aa a/ Sequences of the isolates from Ethiopia were obtained in this study. All other sequences are those from the studies of Slippers et at. (2003) and De Wet et at. 2000, 2003. 187 Table 3. Lesion lengths and confidence limits for trees inoculated with D. pinea and Fusicoccum sp. obtained from Pinus patula cones in Ethiopia. Isolates CMW11250 CMW10717 CMWl1252 CMWl1246 CMWl1240 CMWl1253 CONTROL Species Diplodia pinea " " Fusicoccum sp. " " Trial 1 Tria12 Mean Lesion 95% Confidence Mean Lesion 95% Confidence Length (rom)! limits! Length (mmi limits 56.80 a 48.81964.781 38A5 bc 30.87346.026 54.30 a 46.31962.281 51.3 b 43.73258.876 48 .65 ab 40.66956.631 95.93 a 87.185104681 38.85 b 30.86946.831 52.30 b 44.72359.876 37.90 b 29.91845.881 37 AO bc 29.82344.976 30.00 bc 22.01937.981 29 .75 c 22.17337.326 14.35 c 6.36922.331 11.95 d 4.37319.526 Each value is the average of 20 measurements. Means followed by the same letters are not significantly different from each other at P=<0.05 significance level. 188 2 6. s s 0 The ITS rONA and ~-tublin s (.)= Homologous nucleotides, (N)= Unknown bases. o D. ( ) = g 1 ribis 2 B ribi _parva CMW994 CMWI 25 B eucalyptoruffi_CMW101 lutea 6 B 780 000 01 B stevens 11 6 Et 0 253 Et NNNNNACCAA GCTTTCTGGT TTGTTGCCAA NNNNN ••... NNNNN •. " . ........ . NNNNNNNNNN NNNNNNNNNN . . . . . • . . . • NNNNN . . . . . NNNNNNNNNN NNNNN •... NNNNN •..•• NNNNN •.... NNNNN ..•.. NNNNN . . . . . NNNNN ...•• NNNNN .. ... NNNNN .••.• NNNNN ..••. o 6 CTCCCGCGCC CCCGCTGA ....... .. . .. T .•... • .•. T ..••• • ••••• G ••• •.••.• G ••. . . . . . . . . A. . . . . . . . . A. • .•..••. T . . • • T • • . • · •.....• T . • • . . T •••.• .•• C .••••. · .G • • . . . . . . . • c ..... . · .G • . . . . . . . . . . . . • . . .. • CC . . . . . NNNNN . • • . . • . • . . • . . • . CMW189 (B) 90 (A) CHW4 6( ) 07 ETHIOPIA ETHIOPIA LasioBt G a 20 coccum sp. f CMW70 TGGTA . . . . . . A . . . . . . . . TGGTA. . . .. . . . . . . . . . . TGGTA .... . TGGTA .... . TGGTA ..... TGGTA ..... NNNNN ••.•• NNNNN ...•• · .G . . . . . . . · . . . . . . T. • G • • . • • . • . • • C ...••. . •• C ••.•. ... . · .G . . • . . . . . . ... ..... ~ ~ ~ • .G • • . . . . . . . . C • . . . . • · ., . . . . . T. .. ...... . .. c ..... . 95 80 B ribi s CMW7772 B ribis CMW77 7 3 B_parva_CMW9 94 B_parva_CMW9071 B_parv a_CMW9077 B_euca1yptorum_CMW10125 B_ euca1yptorum_CMW10126 B 1u t ea CMW9076 B 1u tea CMW 9 92 B dothidea CMW77 80 B d o thidea CMW8000 B rhcdina CMW101 30 B rhcdina CMW9074 B obtusa CMW7774 B obtus a CMW 7774 B s tev ens ii - CMW 70 60 CMW1124 6_Et hi o p i a CMW1 1253 Ethlopia CMW189 (B) CMW190 (A) CMW 4786 (C) CMW10717 ETHIOPIA CMW11252 ETHIOPIA CMW l1 250 ETHIOPIA Lasi oB t2 G p hi1 opr ina_CMW 7063 90 100 llO 1 20 130 140 CGCGAATCGA CAC CACAGGC AGACCATTT C CGGCGAG CAC GGCCTG GACG GCT CTGGCG T GTGAGTCTGC · ... .. .... · ......... · ......... · ......... · ......... · ......... · ......... · ......... · ......... · ......... · ......... · . ... G... · . . .. G... .. . C ...... .. . C ...... . C.C ...... · ......... · ......... .. . C . ..... ... C...... ... C ...... .. . C ... G.. .. . C ...... ... C . . . G .. · . G .. G... ---------- · . . ....... · ......... · ......... · ......... ..... T .... · ......... · .. .. ..... · ......... .... G..... · ......... .... G.. . .. · ......... · ......... ....... C.. · ......... ....... c .. T ......... · ......... T ......... · . .... . ... T .... .. ... · .. . . ..... T .. T .. A... · ......... T .. T .. A... · .......... T . ........ · ......... T .... . .... · ......... · ......... · ......... · ......... · ......... · ......... · ......... · ......... · ....... . . · ......... · ......... · ......... · ......... · ......... .... . T .... . ...... C.. · . ........ · .... T .... ....... C.. · ......... · ......... ... . T .. C.. T ......... · ......... . ... T . . C .. T ......... · .. - . . . . . . ....... C.. T ....... .. · ......... · ......... T .... .. ... · ......... · ...... . .. T . ........ · ......... . ... T .. C .. T ......... · ......... . ... T .. C.. T ... . ..... · . . . . . . . . ... . T .. C .. T ......... · ......... ... . T .. C.. T .... . .... · ......... · ... T .. C .. T ......... · .. ....... ... . T .. C .. T ......... · .... T .... . ..... . C .. · ......... ------...... . C .. T ......... . ....... T . ... . C .. T .. . ....... T . ... . C .. T .. · ......... .... C..... · ......... . ... C ..... · .. . ...... · ..... ... · ......... . ...... A.. · ........ . · ... . ..... " · ....... T. · . . ... ... . · ......... · ......... · ......... · ..... . ... . ....... T. ... . . C ... A .... C ..... . ... C ..... · ... C .. T .. .... C ..... ... . C ..... . ... C ..... . ... C .. T .. .. AA ... T .. · .A. · .A . · . A. · .A. · . A. · .A. · .A. · . A. · .A. · .A ... G. · .A ... G. · . A ... T. · .A . .. T . · .A ... G. · . A. · .A. · . A ... T . · . A ... T. · . A ... T . · . A ... T . · .A . .. T . · . A ... T . · .A ... G. C .ACA . 196 1 50 8 ribis CMW7772 8 :rib i s CMW7773 8_parva_CMW994 8_parva_CMW 907 1 8 _ parva_ CMW9077 8_e uca 1 yptorum_CMW 1 0 1 25 8_euca1 yptorum_CMW10126 8 lutea CMW9076 8 l utea CMW992 8 d o th i dea CMW7780 8 doth i de a CMW8000 8 rhodina CMW 1 0130 8 rh od ina CMW9074 8 o btu sa CMW777 4 8 o bt usa CMW7774 8 st evensii CMW7060 CMWl1 2 46_Ethiopia CMW l1 253_Et hi o pia CMW 1 89 (8) CMW 1 90 (A) CMtJll4786 C) CMW10717 ETHIOP I A CMWl1 2 52 ETHI OP IA CMWl 1 250 ETHIOPIA Las i o 8t2 G p hilopr i na _ CMW7063 160 l70 1 80 200 190 2 10 GCCGT TT C CCGCGC GAA T GG CAATGGCTGA CCCGTAGCA GCTA CAATGGCACC . T. . T. . TT .TT .A . . AT .A .. . TC A ... TGG GA ... CA . . A. AT. A ... TC A ... TG G GA ... CA . . CT CC G ..... . . . . . CA . .C. . CTCC G . .. .. . . . . . CA . .C. . . . . C. · T .. C . TT. G. . . . . . TC . · T .. C . TT. G. . . . . . TC . . C. .C. · T .. C . TT. G. . . . . . TG . .T TC . . TTC .T .. C.TT . G. . . . . . . . . . . TC . . . ... C . .T .. C .TT. G. . . . . . . . . . . T C . . . .. . C . · T .. C. TT. G. ... . ... . .. TC. . . ... C. .T .. C.T T. G. . . . . . . . . .. T C. . . . . . C . .T .. C .TT. G .. .. .. .. . .. TC. . . . . . C . . T .. C .TT. G. . . . . . . . . . . T C . . . . . . C . . CT . . . . . . . CGCGC AT .. . . . . . . C . .C. .C. .C. .C.A. .C.A . .C . .C. AA .T. AA.T . . . T. ..T. .TTG. . TTG . .T .TCG . T .ACC . T. ACC. .TT.. .TTG. .TTG . . TTG . .TTG. .TTG . ..TGT. .T .T .AGCAGC. . AGCAGC . .A .T . AG C. .T. 197 220 B ribis CMW 7772 B ribis CMW7773 B_parva_CMW994 B_parva_CMW9071 B parva_CMW9077 B_eucalypto ruffi_CMWl O1 25 B_eucalyptoruffi_CMWlO126 B l utea CMW 9076 B l utea CMW9 92 B dothidea CMW7780 B dot hi dea CMW8000 B rhodina CMWI0130 B rhodina CMW907 4 B obtusa CMW7774 B obtusa CMW777 4 B stevensii CMW7060 CMWl1246_Ethi op i a CMWl1253_Ethiopia CMW189 (B) CMvH 90 (A) CMW4786 (C) CMWI0717 ETHIOPIA CMWl1252 ETHI OPIA CMWl1250 ETHIOPIA LasioBt2 G_philoprina CMW7063 230 240 250 260 270 280 TCCGACCTGC AGC TCGAGCG CATGAACGTC TACTTCAACG AGGTACTCTC TCACTAATT GCACAAACAC ...... . . C . ........ C. ........ C. . ....... C. · . G..... T. · .G ..... T. · . G..... C . · .G ..... C . . . . . ... . C. . .. .... . C . ....... . C. o ••••••••• o ••••••••• o ••••••• o ••••••••• • • · .......... · .......... . A .. G..... . A . . G ..... ... . G ..... ... . G .. . .. . A .. G..... · ......... · ......... · ......... · ......... · ......... · ......... · ......... · ........ . · ... . ..... · ..... .... · ......... · ......... · ......... · .. ....... · .. .. ... . . · ... . ..... · .......... · ......... · . . ....... · .T .. . .... · . T ....... · ......... · ..... . ... · .. ... .. .. · ....... .. · ......... · ......... · ......... . . . . . . . . . N · ......... · ......... · ......... · ..... .. .. · .... . .... ........ C . ... . G ..... · ... .... .. · ......... ........ C . ... . G ..... · ... . ..... · ......... ....... . C . ....... . C . ....... . C . ........ C . · .G ..... C. .... . G .. C. ... . G ..... ... . G ..... ... . G . . ... ... . G ..... . A .. G..... o •• 0 •••••• • •••••• 00" • .. 0 0 • .... 0 0 • 000 • •••• ••• .0 0 •• 0 0 • 000000 ••• 0 0 0 .. 0 0 0 ••• 0 0 0 .. 0 • 0 •• 0 •• 0 •• • 0 0 0 • • • • • • • • • • 0 0 • • • •• 0 0 0 o • 0 •••• • • • • • • • • • •• •• 0 0 · ......... · ......... · ......... · ......... · ......... · ......... · ......... · ......... · .. ....... · ..... ... . · ......... ......... C . . ....... C . .... .... C . ........ C . ......... · ......... · . C. ..... · . C. .. .. . · ..... G.. · ..... G.. · TG ...... . C . .C. · ..... G.. · ..... G.. · - ..... G.. · ..... G.. · ..... G.. · ..... G.. · . C. ..... · .... . G · . C . GACCGA 0 0 ... 0 0 • • •• 0 ... • •• 0.0 • • ••••• • 0 0 • 0 •••• 0 0 • 0 0 • • • • • 0 0 0 •• A. A. .. , .G. ., .. G. AG. AG. AG. AG. AG . AG. AG . AG . AG. AG . AG . AG. AG . AG . .. TTC.CATA 198 290 B ribis CMW7772 B ribis CMW7773 B_parva_CMW994 B_par v a_CMW9071 B_parva_CMW9077 B_eucalyptoruffi_ CMW10125 B_eucalyptoruffi_CMW10126 B lutea CMW9076 B lutea CMW992 B dothidea CMW7780 B dothidea CMW8000 B rhodina CMW10130 B rhodina CMW9074 B ob tusa CMW7 77 4 B obtu sa CMW7774 B stevensii CMW7060 CMWl1246 Ethiopia CMWl1253_Ethiopia CMW189 (A) CMW190 (B) CMW4786 (C) CMW10717 ETHIOPIA CMWl1252 ETHIOPIA CMWl1250 ETHI OP IA LasioBt2 G philoprina CMW706 3 300 310 320 330 340 350 GTAAAGTATG GCAATCT TCT GAACG CGCAGCAGG CGTC C AACAACAAG T ACGTT CC TCG A. A. · ... .. .. .. · .... .. ... · ......... · ......... · ...... G. · ......... · .. . .. . .. . . . .. ... G. · ......... · ....... " . . . . . . . . . . · ......... · ......... · ... .. .... · .. . ..... . · . .... .. .. · ..... . .. . · ......... · ......... · . ........ · ......... · ..... . ... . C ........ · . ....... . · .. ....... · ......... · . . ...... . · ......... · ......... · .. . . .. . .. · ......... · ......... · ......... · ......... . A ....... . ..... T . . A . . ...... · .... T . .T . · ......... · ... .. .. . . · . T . .. . G. · . . ....... · ......... · -. T .. . . G. · ......... · ......... · . T . . .. G. · . .. ...... · ......... .-. T . ... G. · ......... · . . ....... · . T .... G. · ......... · .. .... ... · -. T .... G. T.CTG .. GAT TTTCATC.TC TG .. CGAGA TTTGG.T.TA G. C .TCCGGC · · · · · · .... T . .... T. .... T. .... T. .... T. .... T. . T. 199 360 B ribis CMW7772 B ribis CMW 7773 B_parva_CMW994 B_pa rva_CMW9071 B_parva_CMW9077 B_eucalyptoruffi_CMW10125 B_euca l yptorurrI_ CMW10 12 6 B lu tea CMW9076 B lut ea CMW 992 B dothidea CMW7780 B do t h i dea CMW8000 B rhod i na CMW 1 0130 B rhodina CMW9074 B obtusa CMW7774 B ob t usa CMW7774 B s t evens ii CMW70 60 CMW l12 46_Ethi opia CMWl 1 253_E thiopia CMW189 (A) (B) CMW190 CMW4786 (C) CMW107 1 7 ETHIOPIA CMWl1252 ETHI OP IA CMWl1250 ETHIOPIA LasioBt2 G ph il oprina_CMW7063 370 380 390 400 410 420 TGCCGTCCTC GTCGACC TCG AGCCCGGCAC CATGGATG CC GTCCGCGCCG GCCCC TTCG G CCAGC TCTT C · ......... · . T. · ......... · . T. · ......... · . .... .. . . · .... . .... G......... · . ........ . ....... .. . ..... T. · ..... . ... · ......... · . ... .. . .. G......... · . ... .. ... . . . . . . . . . . ...... T. · · · · · .. T. . . T. .. T ..... . · . T. .. T . .. ... · . T. .. T ...... · . T ....... · . ... " T .. · . . . ... ... · ......... · ......... · .. T . .. . . . · .. T ...... · .. T ...... · . . T ...... · .. T ...... · .. T ...... · .. T. C .. T ...... · ......... . .... T. · ... .... T. · . T. · . T. · .T. · .T ....... .. T. · . T. · ......... · ......... · ....... . .. ....... NNN NNNNNNNNNN · .... T .. T. · ...... T .. · ... . ..... · .. . . T . . T. .A .. T. 200 430 8 ribis CMW7772 8 ribis CMW777 3 8_parva_CMW994 8_parva_CMW9071 8_parva_CMW9077 8_eucalyptoruffi_CMW10125 8_eucalyptoruffi_CMWlO126 B l u tea CMW9076 8 lutea CMW992 B dothidea CMW7780 8 dothidea CMW8000 8 rhodina CMWI0130 8 rhodina CMW9074 8 obtusa CMW7 774 B obtu s a CMW7774 8 stevensii CMW7060 CMWl1246_Ethiopia CMWl1253_Ethiopia (A) CMW189 (8) CMW190 CMW4786 (C) CMWI0717 ETHIOPIA CMW11252 ETHIOPIA CMWl1250 ETHIOPIA Lasi o8t2 G_philoprina CMW7063 440 450 460 470 480 490 CGCCCTGACA ACTTCGTCTT CGGTCAGTCT GGCGCCGGTA ACAACTGGGA AGGATCATTA CCGAGTTGAT · ......... · ......... ..... C.... ..... C.... .... . C .... ..... C .... ..... C.... ..... C.... ..... C.... . . .. . C .... · . T .. C.... · .T .. C.... · . T .. C.... · . T .. C.... .. T . . C .... · .T .. C.... NNNNNNNNNN · .T .. C.... · .T .. C .... .... . C .... ..... C.... ....... NNN NNNNNNNNNN NNNNNNN. .. . C ...... · ... T ..... .. . C ...... · ... T ..... . .. C ...... .. . . T . .... . .. C...... · ......... . ........ C · ......... ........ . C · ......... . .. C ...... · ......... . .. C...... . . . . . . . T .. ... C ...... · ...... T .. . .. C...... ...... . T .. .. . C ...... ... . T . . . . . · ...... T .. . . . . . . . T .. · ...... T .. NNNNNNNNNN ...... . T .. · ...... T .. · ......... ...... . T .. .. . C ...... . .. C ...... .. . C ...... NNNNNNNNNN . .. C ...... .. . C ...... .. . C ...... ........ . C · · · · · · · · · · · . T ....... . T ....... . T. .T. . T. . T. . T. . T. . T ....... . T ....... . T ....... · .T ..... NN · .T ..... NN .. T ..... NN NNNNNNNNNN · .T ..... NN · .T ..... NN · .T ..... NN · . T .. T .. C . ... . T ..... · ... T ..... · ......... . ........ C · ......... . ........ C · ......... · .N ....... · ...... C · ......... · ......... · ...... C NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN · · · · · · · ......... · ......... · ...... C NNNNN ..... NNNNN ..... NNNNN ..... NNNNN ..... NNNNN ..... NNNNN ..... NNNNN . ...... C ...... C ...... C ...... C ...... C ...... C 201 500 8 ribis CMW77 72 B ribis CMW7773 8_parva_CMW994 8_p arva_CMW9 071 8 parva_CMW9077 8_eucalyptorufn_CMWlO125 8 eucalyptorufn_CMWlO126 8 lute a CMW907 6 B lutea CMW 992 8 dothidea CMW7780 8 dothidea CMW8000 8 rhodina CMWI0130 8 rhodina CMW9 074 8 obtusa CMW7774 8 obtusa CMW7774 B steve nsii CMW7060 CMWl1 246_Ethiopia CMWl1253_ Ethiopia CMW189 (8) CMW 1 90 (A) CMW 47 86 (C) CMW I 071 7 ETHIOPIA CMWl1252 ETHIOPIA CMWl1250 ETHIOPIA Lasio8t2 G_philoprina CMW7063 510 530 520 540 550 560 TCGAGCTCCG GCTCGAC TCT CCC ACCCAATG TGTACCTACC TCTGTT GCTTTGGCGG · ......... · ......... · .. ... .... · ......... · ....... . . · ......... · ......... · ......... .. . G ...... .. . G ...... .. . G ... T .. · ......... · ......... · ......... · ......... · .. C.... . . .. C ...... · .. C... . . ... C ...... · ......... · ......... · ......... • .•.• N •..• · ......... · ... T. · ... T. .... C. .... C . · ... TT. · ... TT. · ... TT .. · ... TT .. · ... TT .. · ... TT .. · ... TT .. · ......... · ......... · ......... · .... .. ... · ......... · ......... · ......... · ......... · ......... · ......... · ......... .. . G ... T .. · .. G... T .. · ...... T .. .. . G ... T .. · .. G... T .. · .. G... T . . .. . G ... T .. · .. G... T .. ... G... T .. . T .. ....... . A. ........ A. ....... . A. ........ A . ........ A. ....... . A. ....... . A. ....... . A. ....... . A. A . ... C.G .T . .ACA .. T .... TC .. .... TC .. .... TC .. .... TC .. .... TC .. .... TC .. CGAC .. TC .. C .. CCAA · · · · · · · ... TT .. ... TT .. ... TT .. ... TT .. ... TT .. ... TT .. ... TT .. · .... .... · · · · · · · · · · · · · .A .. G. .A .. G. .A .. A. . A .. A. .A .. A . .A .. A.... .A .. A.... .A .. A. '... .A .. A.. .. .A .. A.... .A .. A.... .A .. G.... .A .. A... . TACC. TACC . TCTG.TGC. TCTG.TGC. TCTG.TGC . TCTG. TG .. TCTG.TGC. TCTG . TG .. TCTG. TGC .. TA ... TGT.G C. C. . G.CG .C CT . CG .CG . 202 570 B ribis CMW7772 B ribis CMW7773 B_parva_CMW 994 B_parva_CMW9071 B_ parva_CMW9077 B euca1yptorum_CMW10125 B_ eucalyptorum_CMW10126 B lutea CMW9076 B lutea CMW992 B dothidea CMW77 8 0 8 dothidea CMW8000 8 rhodina CMWI0130 8 rhodina CMW9074 8 obtusa CMW7774 8 obtusa CMW7774 8 stevensii CMW7060 CMWl1246_Ethiopia CMWl1253_Ethiopia CMW189 (8) CMW190 (A) CMW4786(C) CMWI0717 ETH IOP IA CMW 1 1252 ETHIOPIA CMWl1250 ETHIOPIA Lasi o Bt2 G_philoprina _ CMW7063 580 590 600 610 620 630 GCCGCGG TC C T CCGC AC CG G CGCCC TTCGGGG GGGCTGGCCA GCGCC CGCCAGAGGA · ......... · ..... . . . . · ...... ... .... .. ... · ......... · . . . .. . .. . · .. . ...... · .. . ...... · . ........ · .. ....... .. · ...... . .. · .. ....... · ... . ..... · ......... · .... T ... T · ... . . . . . · ......... · .... T ... T · ... . . . . . o . .. T . .. T . .. T C .. T C .. T C .. T C . . T C .. T C .. T T .. . A.T · TG .... G. .T G . .. . G. .TG .... G. · TG .... G. · TG .... G. · TG . ... G. .G .... C.A . G .. C.G -- --- - ----- . ... . .. · .. T. . .... . . · . . T. · ......... · . AC . C . . G. · ... . .. . .. .. . C. .. . C. · ......... · .AC.C .. G. ...... . GG . ....... GG . . . .NG .. .GG · TG .... G . · TG .... G. .. G.... GT ....... A . · ... .... . .. · ......... · . ....... . · ......... .. . C.C .. .. .. . C . C .. .. . . .. AG ..... .. AG .. .. . . . AG ..... · .. C .... T ... C . ... T .. AG . ... T .. AG ... . T .. AG .. .. T .. AG .... T .. AG .... T .. AG .... T .. G. .. CGC . T • ••• • •• •• . CCC . . CCC . . ..... ..... A. . .. . . . .. ... A. -- - - - - - - -- -- - - - - - - - - - - - - . .... C ... CC C CC .. · .... C ... CC CCC .. · - ... AAAJl.A . C CCC .. C · .... G... . C. · .... G... C . ... C . . . CCC . CCA .GC .... C ... CCC.CC . GC . . .. C ... CCC . CC . GC .... C ... CCC.CC.GC .. . . C .. . CCC . CC .GC .... C .. . CCC . CC . GC · · · · · . ... , . TTT. ..... TTT. ..... TTT. ..... TTT. . .... TTT. ..... TTT. - ---------- CGTGT.CCCC .. ATCA . G.G C ...... .. .. TAG.A. -- --- ---------- · . .. GCTTT. · .. . GCTTT . · T .. GCT. T . 203 640 B rib is CMW7772 B r i bis CMW77 7 3 B_parva_ CMW99 4 B_parva_CMW9071 B_par v a _ CMW9077 B eucalypt o rum_CMW10125 B_eucalyptorum_CMW10126 B lutea CMW9076 B lutea CMW992 B dothidea CMW 7 780 B doth i dea CMW8 0 00 B rh o dina CMW10 1 30 B rhodina CMW90 7 4 B obtusa CMW777 4 B o btusa CMW7774 B stevens ii CMW 7 060 CMWl1246 Ethlopla CMWl1253_Ethiopia CMW189 (B) CMW1 90 (A) CMW4 786 (C) CMW10 71 7 ETHIOPIA CMWl1252 ETH I OP I A CMWl1250 ETHIOPIA LasioBt2 G_ phil o prina_CMW7063 650 670 660 6 80 690 700 CCATAAAAC TCCAGTCAGT GAACTTCGC AGTCTGAAAA ACAAGTTAA TAAACTAAAA CTTTCAACA · .. C ..... ... C ..... · .. C .. ... · .. C ..... · ... C .. . . · ... C ... . · . T. C ... . · . T. C ... . · . T.C ... . · . T. C ... . · . T. C ... . .. T. C .. T. · . T . C ... . · . T . C . . . . .. T. C ... . · . T.C ... . · . T. C ... . · . T. C ... . A. T .. .. . · .... . .... A... G.. · ......... A... G.. . . . . . . . . . . A . .. G .... . . . . . . . . . . A.. . G .... · ......... A... GA . .. · ......... A ... GA. .. . . . . . . . . . . A.. . GCA . A · . . . . ..... A... GCA . A · . . . . . . . .. A ... G . . . A · . . . . . . . . . A... G ... A · .... . .... A... G ... A ...... . G . . ... ... G. . . . .. . .... . .......... . ... T. . ... T. C ...... T. C .... . . T. C ...... T . C ...... T. C. · ........ . A... G.CGA C...... T .. .. T ... .... .. . G . · ...... .. . A... G. CGA C .... . . T. · . . . . . . . .. A... G . CGA · . . . . . . . .. A ... G . CGA ... ....... A ... G.CGA · .... ... .. A... G.CGA . . . . . . . . . . A..GCAG · . TT .. TTTA TTTTGGAAT C ...... T . C ...... T . C .. . ... T. C .. .... T . C ...... T. CT ..... GT. GTTTTTAC .. AT .. A ..... A . 204 710 B ribis CMW7772 B ribis CMW7773 B_parva_CMW994 B_parva_CMW9071 B_parva_CMW9077 B_euca1yptorum_CMW10125 B eucalyptorum_CMW10126 B l utea CMW9076 B 1 utea CMW992 B dothidea CMW7780 B dothidea CMW8000 B rhodina CMW10130 B rhodina CMW9074 B obtusa CMW7774 B obtusa CMW7774 B stevensii CMW7060 CMWl1246_Ethiopia CMWl1253_Ethiopia CMW189 (B) CMW190 (A) CMW4 78 6 (C) CMW10717 ETHIOPIA CMWl1252 ETHIOPIA CMWl1250 ETHIOPIA LasioBt2 G_philoprina CMW7063 720 730 740 750 760 770 ACGGATCTCT TGGTTCTGGC ATCGATGAAG AACGCAGCGA AATGCGATAA GTAATGTGAA TTGCAGAATT 205 78 0 B ribis CMW7772 B ribis CMW7773 B_p arva _CMW994 B_parva_CMW9071 B_ parva_ CM\rI7 9077 B_ eu c alypt o ruffi_CMWI0125 B_ eucalypto ruffi_CMWI 0 1 26 B l u tea CMW9076 B l utea CMW9 9 2 B dot hi dea CMW7780 B doth idea CMW8000 B rhod in a CMWI 0 1 30 B rhodina CMW9074 B obtusa CMW7774 B obtusa CMW7774 B s t evens ii CMW7060 CMW l124 6 Ethlopia CMWl 1 253 Ethiopla CMW189 (B) CMW 1 9 0 (A) CMW4 78 6 (C) CMWI0717 ETHIOPIA CMW 11 252 ETHIOPIA CMW11 250 ETH I OPI A LasioBt2 G_ph il oprina_CMW7063 790 800 810 820 830 840 CAGTGAATCA TCGAATCTTT GAACGCACAT TGCGCCCCTT GGTATTCCGA GGGG CATGC CTGTTCGAGC A........ . A ........ . · . . .. .. T .. · • . • . . . T .. ••.••.•.. G . ..... . •. G . . ...•. . C .•. C ...... G .•.• .. .• C • . . C .. . .... G · . C ...... . . .. . C ...•• .• . • C . . . . . . . . . . . - •.. • G . . . . . · .... . . GCC A.... .. T. . C .. G. . . .. · · · · · · · .C . . . . . . . C ...... . C .. . . . . .C . . .. . . . C ...... .C . . . . . . .•.....• . ... CT ... . 206 850 B ri bis CMW 7772 B ribis CMW7773 B_ pa r va_CMW994 B_parva CMW9071 B_parva_CMW9077 B_eucalyptorum_CMW10125 B_euca l yptorum_CMW1 01 26 B l ut e a CMW9076 B l utea CMW992 B- dothidea CMW7780 B dothidea CMW8 0 00 B rhod ina CMW101 30 B rhodina CMW907 4 B obtusa CMW7774 B obt us a CMW77 74 B stevens ii CMW7060 CMW l1 2 46 Ethlopia CMW l125 3_Ethi op i a CMW1 89 (B ) CMW1 90 (A) CMW4786 (C) CMW 10717 ETHI OPIA CMWl 1 252 ETH I OPIA CMWl1250 ETHIOPIA LasioBt2 G_ phi l op ri na CMW70 63 86 0 87 0 880 890 900 910 GTCATTTCAA CC CT CAAGCT CT GCTT GGTATTGGGC TC CGTCCTCC A CGGAC GCGC CTTAAA · ......... · ....... . . · ....... . . · ......... · ........ . · ......... · ......... ..... . A ... ...... A... ..... . A ... ..... . A ... .. . .. . A ... . .. .. . A ... ..... . A . .. · ........ . · ......... · ......... · ......... · ......... · ......... · ......... · . ........ · ..... . .. . · ......... · ......... · .. . .. . ... · ...... . .. · . ........ · . .. ...... · ......... · ......... · ......... · ......... · ......... · ......... · ......... · ......... · ......... · .... . .... · .... . .... · .. ....... · .... . .... · ... . . . ... T . ........ · ......... T ........ . · . ..... . .. · ......... · ......... · .. ..... . .. · ..... . . . . · ........ . .......... · ......... · ... . ...... · . .. . ..... · .A ....... · ...... ... · . A ....... · ......... · . .. . .. . .. · ......... · ......... · ......... · ......... · ......... · ......... · . . ....... · ......... · ......... · ... ...... · ......... · ......... · ......... · . ....... . · ... . ... . . · ......... · ......... · ......... · .. . ...... · ......... · ......... · .A ....... ...... A... .. ... . A ... ...... A... . .. .. . A ... ..... . A ... ..... . A .. . · ......... ..... . A ... · ... ... . .. · ... .. .... · ..... T .. C . CTAGG .. G. C. C ......... C......... C ........ C ........ .. .. .. . .. . .. ...... A....... T A....... T A....... . A A........ A G........ G . . ...... GA . .. .. . . C. C. G........ G . ..... . . G........ G . ....... G........ G........ A........ A · .. G..... G AT .. G.. AAA . . .. .... .. ...... C. . ......... ...... C. .. TGT .... . . .... C. .. TGT .... ..... . C . .. TGT .... .. .. . . CG. .. TGT .... .. ... . CG . . . TG ... G . . ..... C . .. TG .. . G . ..... . C . . CTG .... . . ..... C. . CTG ..... .. ... . C . T CTG . T CTG . TCT G . .... . . .... C. T CTG . T CTG . T CTG. T CTG . T CTG . TCTG . . CTG . GCCCG .. AGG . AC GGCCGGC 207 920 B ribis CMW7772 B r i bis CMW7773 B_parva_CMW994 B_parva_CMW9071 B_parva_CMW9077 B_euealypto r um_CMW10 125 B_ e u ealyptoruffi_ CMW10 126 B l utea CMW9 0 76 B lutea CMW99 2 B dot hid ea CMW7780 B dot hi dea CMW8000 B rh od i n a CMW10 1 30 B rhodina CMW 9074 B o b tusa CMW7774 B obtusa CMW 7 774 B st evensii CMW70 6 0 CMW l1246 Ethiopla CMW l125 3_Eth i op i a CMW 189 (B) CMW190 (A) Cl'1W 4 786 (e) CMW 10 7 17 ETHI OPIA CMWl 12 52 ETHIOPIA CMWl 1 250 ETHIOPIA Las i o Bt 2 G philopr ina_CMW7063 930 940 950 97 0 960 980 GACC TCGGCG GTGGCGTCT TGCC TCAAG CGTAGTAGAA AACACC TC GC TTT GGAGC GCACGGCGTC · . . . . . . . . · ......... · ......... · .. . ...... . T ..... . . · ......... . .. T. · ......... · ......... · ......... · ......... . T ....... · .......... . .. T. ~ · ......... · ......... · ......... · ......... · ......... · ......... · ......... · ......... · . .. .... . . ..... T .. TC A .. . C . . . . . · ......... · ......... · ......... · . . ...... . · ......... · ......... · ...... . .. · .... . .... · ......... · ......... · ......... · ......... · ......... · . ..... . . . - C .. · .... T .. TC .. ... T .. TC . . .. . T . . TC ... . . T .. TC · .... GTCT . . : ... GTCT. · .... T .. TC · ... . T .. TC · .... T . . TC · .... T .. TC .... . T .. TC · .... T .. TC · .... T .. TC . AAATC TA.TGGCGG A ... C..... A .. . C . . . . . A .. . C . . . . . A .. . C . . . . . GC . ... . .. GC . ...... A . . . C . . . .. A .. . C . . . . . A... C... . . A ... C..... A .. . C . . . . . A... C.. . .. A ... C.. ... AC .. G.. GT. · ......... · ......... · ......... ... . A ..... · ........ . · ... . ..... · ......... · . . ....... · ......... · ...... . .. · ..... . ... • •••• '4' 4 ••• ...... • ••• GCCTCCTCTG C .TA .. T ... C. TA .. T ... TA...... TA ... .... TA ...... TA .. . ... TA...... . . A...... ..A. TA . ..... TA ...... TA...... T A... : .. TA .... . . TA...... TA ...... CGAAGTAG. G .. . G. . .. G. · ......... .GTT. · .. .. ..... . GTT. · ......... .GTT. · ........ . .GT T. · ......... .GTT . ... . C . . . . . .. , .C . . . . . · .. .... ... . .... . . C . · ......... . ....... .. · . . ....... · ... . ..... · ........ . .GTT. . GTT . . GT T. .GTT. . GTT . . GTT. . GTT. ATA.. CCGCA T. GGA. A. CG • .4 • ...... ' . . . . . . . 4 .... 4 208 990 8 ribis CMW7772 8 ribis CMW7773 8_par va_CMW994 8_parva_CMW9071 8 pa r va CMW9077 8 euca1yptoruffi_CMW10125 8 eucalypto r uffi_CMW10126 8 lutea CMW9076 8 lutea CMW992 8 - dot hidea CMW77 8 0 8 d oth i dea CMW8000 8 rh od ina CMW10130 8 rh od in a CMW9074 8 obt usa CMW77 74 8 obt us a CMW7774 8 stevensii CMW7060 CMWl1246_Ethiopia CMWl1253_Ethiopia (8) CMW189 CMW190 (A) CMW47 8 6 (C) CMW10717 ETHIOPIA CMWl1 2 52 ETHIOPIA CMWl1250 ETHI OPIA Las io 8 t2 G_philoprina CMW7063 1000 1010 1020 1029 GCCCGCCGGA CGAACC TTT GAATTATTT CTCAAGGTTG ACC TCGGAT ..... .... ...... .... . .. . .. . . .. .. .. .. . . .. .. .. .. . . .. .. ....... . C . ........ C . ........ C . ........ C. . ... .. . . C . ....... . C . .. . .... . C . .. . .. . . .. .. . . .. .. .. .. .. .. .. .. .. . .. . .. . . . . . . . .. .. .. . .. .. .. .. .. . .. .. . .. .. . .. . . .. . .. . . .. .. . . .. . .. ... ... . . C . ........ C. . . .. . .. .. . . . . . ..... . . C. . .. .. .. .. . .. .. . . ........ C . .. . . .. .. .. .. . .. . ..... . .. C. . . . .. .. .. .. .. .. . ... . . ... C. . .. . .. . .. . . . . ........ C . A.GA ... CCT GC CGTTAAAC .......... ........ . ...... . ............ · · · · · · · . . C. .. C . .. C . .. C. . . C. .. C . .. C . . . . . . . . · .. C . .... · .. C. .... · .. C. .... · .. C . .... · .. C. .... · .. C. ... . · .. C . .... CCCCA .C .. T ... .. . . NNN ...... . NNN ..... .. NNN ....... NNN . ..... . NNN ....... NNN ...... . NNN ................ NNNNNNNNN NNNNNNNNN NNNNNNNNN NNNNNNNNN NNNNNNNNN NNNNNNNNN NNNNNNNNN ..... A. 209 Figure 7. Alignment of combined SSR sequences of D. pinea sequenced with SS7, SS9 an d SSlO markers. ()= gaps, (.)= Homologous nucleotides, (N)= Unknown bases. 10 CMW190A CMW11250 CMW11252 CMW10717 CMlrJ4 78 6 CMW1898 NNNNNNNNN 40 50 60 70 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN · ... - .. . .. T ......... 90 ... . C ... CA ....... T .. 100 110 . G .. G..... A......... A... C . . T. 120 130 140 CAAAAGCAGT GAAGTCGTAA GGCCCGGACC CTA-GAAGAG GCGCTTTCCT CTCCACGGAG TAACCACCGG · ......... NNNN ...... GG ... A.... 150 CMW190A CMW11250 CMW11252 CMW10717 CMW4786 CMW1898 30 GACAAGACAT CTAGGCCCTG CCGGTCCCG- TCCCCGTCTC CAGGCTCACA TGGAAACAAA -CTGT ACAGG 80 CMW190A CMW11250 CMW11252 CMW10717 CMW4786 CMW1898 20 · ......... · ......... · ......... · ......... · .... T .... · ......... 160 l70 . .-GA ... . . 180 . ...... G.. T ......... C. 190 200 210 CTCGGCTACG CTAGAAAGCA AATTCCCCGA TCTTAGTGGC ATTTTTTCTT TTGCATCATT CCCGGGCCTC ......... C · ......... · ......... G.... G.... G.. A...... ... C . .. . C ...... .. . . ... . .. .. . ... .... . ....... A . 210 220 CMW190A CMW11250 CMW11252 CMW10717 CMW4786 CMW189B 250 · ......... · ......... · ...... · ......... · ......... · ...... · ......... · ......... · ...... · ......... · ......... T .... · ...... ........ A. 260 27 0 28 0 · G. . . . . . .. TTTTG. T ... 300 310 · .. " .. TCT 320 · ........ . · ......... · ......... · .... .. ... · ......... · ......... · ..... .. .. · ..... .. .. . . . . . . . . . . · ......... 330 340 ---------------------- .. TCC. . TCCTCCTC 350 TCTCTTTCT CAACACGAGG CT CACCAATC ACGATGACGA CGACGACGCC GCTGAGAATG AGCGGAAAAT C ......... 360 CMW190A CMW11250 CMW11252 CMW10717 CMW4786 CMW189B 240 TTTGGAAATT GCTTTTTTTT GATT T TGATTTT CTTCTTTTCC TCCTCCTCCT CC - 290 CMW 1 90A CMW11250 CMvJl1252 CMW10717 CMW4786 CMliJl898 230 · ......... . . . . . . . .. 370 TATCCGAGAA TCATTCCAC · ......... ......... C · ......... ......... C · .. ...... . ......... C · ......... ....... .. C · ......... ... C ... G.C . . · ......... · ......... 380 TTCACCGNNN ....... GAT .. .. ... GAT ....... GAT ....... GAT ....... GAN 390 NNNNNNNNNN GGGCCCCTCG GGGCCCCTCG GGGCCCCTCG GGGCCCCNNN NNNNNNNNNN 400 NNNNNNNNNN TCTCGACCCG TCTCGACCCG TCTCGACCCG NNNNNNNNNN NNNNNNNNNN · ..... T ... 410 NNNNNNNNCA ACTGATCANN ACTGATCANN ACTGATCANN NNNNNNNNNN NNNNNNNNNN . ....... . C 420 GCGGTTTCAT NN .... C ... NN .... C ... NN .... C ... N. . . . . . . . . N. . . . . . . . . 211 CMW 1 90A CMvH12 5 0 CMW1125 2 CMW10717 CMvv 4 786 CMW1898 CMW190A CMlr\111250 CMW 11 252 CMW10717 CMW47 8 6 CMIII)1898 440 TGAAATGCCA · A ... A ... C GA ... A . . . C · A .. . A ... C TCTTCAG TAT . T ... CAG .. . T ... CAG .. . T ... CAG . . CTTGGATATC TTTTTTTTTT TTTTTGATGA GTGCGCGCGC ACACTGCGTT T ..... A .. T T . . ... A .. T T .... . A .. T · .. .... .. . .G ........ · ......... 500 510 520 460 · . . ... 480 ----- .... C . . . . . . . . . . 530 540 550 490 . . . . C . T. 560 GAGTGAGGAC GGTG TG CTGG TGGCGG---T GATGTATGTG TGTTGTTGGT GG TG---T GG GTAGTGTGTG . .. . . . .... . ... TGG . · ......... .. . A ...... · .. T .. TGG. 570 CMW190A CMW11 2 50 CMW11252 CMW10717 CMW4786 CMW1898 450 470 430 580 590 600 610 GATGGAGTGG ATGGAGGAAG GGGTCCGGGA GTGTTGGTTG TTGTATCTGC · ... . .... . · ......... · .A . ... ... .. . .. ...... . . CN- ... .. · .. ..... .. · ......... · . A ....... · ........ . .. CN- . . .. . · .. .. .. .. . · ..... . ... · .A ....... · ......... CGACTG .... 620 -------- 630 TCTTCGGGCG AGAGAGAGTC . AGNGAN . . AGNGAN. . AGNGAN. --- .. T. 212 650 660 670 680 CAAGGAAGAA GGAAG TG G · ......... .AA.NNNNNN · . . ....... .AA.NNNNNN · ......... .AA.NNNNNN · . . . . . . . .. ANNNNNNNNN ... A...... AG. NNN NNN N GAATCGGTAG NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN GAGACAAGTC NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN GCCAACCCTA .......... .......... .......... .......... .G ........ 730 740 750 640 CMW190A CMW1l250 CMW1l252 CMW10717 CM W4 786 CMW189B 710 CMW190A CMW1l250 CMW1l252 CMW1 07l 7 CMW4786 CMW 1 89B 700 ATGCTTCCAT AGAAACCAAT · ........ . . ..... G... · ......... . . . . . . . . . . · ......... . . . . . . . . . . .......... C......... .......... C......... 760 770 TGACGGCGGA AAGACAAAGG AGCTTACACC GCAGCACCAT TCCCTCCCAC AATCCCTGGT CACAAGACAC A. C ......... C......... 780 CMW190A CMW1l 2 50 CMW1l252 CMW10717 CMW4786 CMW189B 720 690 · ......... . . . . . . . . . . · .. T ...... 790 . ........ . . . . . . . . . . . ........ A .......... 800 810 . ........ C .A ...... G. . ........ C 820 830 840 ATACAGACAC ACACACACAC ACACACACAC ACCCAACACA CACATACAAC CTCTCCAACT CACCACCACG · ...... T .. · ......... · ......... .......... . ........ T .... G. 213 850 CMW190A CMW1l250 CMW1l252 CMWI0717 CMW4786 CMW1898 870 880 890 900 910 GCGCCTTCAA CGCCCCGATC TGTTCCCTCG GACCACCCAG CAGCAGCATG AACTCCCGCG CACCGTCACT · .... .. ... · ......... · ......... · ......... ...... . T .. · ......... . . . . . . . . T . · ......... · .... ..... · ......... · ......... · ...... T .. · .. ....... . .... G. · ......... · ......... · .. .... ... · ......... · ... ... T .. ......... ..... G. · · ......... T ......... 920 CMliVl90A CMW1l250 CMW1l252 CMWI0717 CMW4786 CMW1898 860 930 AACCTCCCTT CCTTCATCGA · ......... ... ... .. A. · ... " .. ... .A ..... NNN ........... . A .. G.. NNN · ......... · ......... · ......... · ........ . · ......... .G. 940 950 960 970 980 CTCCTGGCGC T ... G.. G .T NNNNNNNNNN NNNNNNNNNN TTCCACCGCC .......... NNNNNNNNNN NNNNNNNNNN GCCGAAGTGG .. . A . G.G .. NNNNNNNNNN NNNNNNNNNN CAGAACCCTC · .A ....... NNNNNNNNNN NNNNNNNNNN CAGACCGCAA ..T .... G.G NNNNNNNNNN NNNNNNNNNN · ........ . · ......... · ......... · ......... · .......... · .... .. ... · ......... . . . . . . . . . . · ......... · .NNNNNNNN NNNNNNNNNN NNNNNNNNNN 989 CMW190A CMW1l250 CMW1l252 CMWI0717 CMW4786 CMW1898 TCGCGGTTG · ... NNNNN NNNNNNNNN NNNNNNNNN ... . C.C .. NNNNNNNNN 214 ABSTRACT Several damaging leaf pathogens are known from Eucalyptus spp., worldwide. Of these, Mycosphaerella spp. are among the most important. Characteristic symptoms of Mycosphaerella leaf blotch disease (MLD) include leaf spot, premature defoliation, stunting, shoot and twig dieback as well as twig and stem cankers. Recent disease surveys conducted in Ethiopian Eucalyptus plantations have revealed disease symptoms similar to those caused by Mycosphaerella spp. These symptoms were restricted to E. globulus trees growing in several localities in South, South Western and Western Ethiopia. The aim of this study was to identify the fungi associated with this disease. This was achieved by examining the germination patterns of the ascospores and by sequencing the ITS region of the rRNA operon, for representative isolates. Several different ascospore germination patterns were observed, suggesting that more than one Mycosphaerella sp. is responsible for MLD on E. globulus, in Ethiopia. Analysis of sequence data showed that three Mycosphaerella spp., M marksii, M. nubilosa and M. grandis were present. This is the first report of these three species from Ethiopia and it is also the first report of M. grandis from a country other than Australia. M. grandis and M. nubilosa were the most common species associated with leaf blotch in Ethiopia. Given the fact that these fungi are well- recognised pathogens of Eucalyptus, we assume that they are the most important cause of MLD on E. globulus in Ethiopia. 216 INTRODUCTION Plantations of exotic tree species are widely utilised in the tropics and subtropics for the production of solid timber products and pulp. Pinus, Eucalyptus, Cupressus and Australian Acacia spp. are among the most widely planted exotic species in these situations. Plantations of Eucalyptus spp. alone cover approximately 10 million ha of land worldwide (Eldridge et al. 1997). In Ethiopia, planting of exotic species commenced with the introduction of Eucalyptus globulus Labill. about 110 years ago (Persson 1995). Thereafter, several Eucalyptus spp. including E. camaldulensis Dhen., E. saligna Sm., E. grandis Hill ex Maid and E. citriodora Hook were introduced. It has been estimated that plantations of Eucalyptus spp. constitute about one third of the total plantation area in the country (Anonymous 1994). The wood from plantations of Eucalyptus species is commonly used for construction purposes, for fuel, poles and posts and is an important resource for subsistence farmers. Plantations of Eucalyptus spp., though displaying tremendous promise in areas where they have been planted as exotics, are threatened by various pathogens (Wingfield 1990, Persson 1995). Several foliage diseases have been recorded on Eucalyptus spp., both in their areas of origin and also in several areas where they have been introduced as plantation species. These include, for example, foliage diseases caused by Pseudocercospora eucalyptontm Crous, Wingfield, Marasas & Sutton (Crous et al. 1989), Phaeoseptoria eucalypti Hansf. emend. Walker (Chipompha 1987), diseases caused by Cylindrocladium spp. (Sharma & Mohanan 1982, Crous, Phillips & Wingfield 1991, Schoch et al. 1999) and leaf blotch caused by several Mycosphaerella spp. (Park & Keane 1982a, Crous 1998). Mycosphaerella spp. are important leaf pathogens of Eucalyptus spp. and they are distributed worldwide (Corlett 1991, Crous 1998). They include both saprobes and aggressive pathogens (Von Arx 1983). ThirtyTwo Mycosphaerella spp. have been described associated with diseases of Eucalyptus spp. (Crous 1998, Carnegie 2000, Milgate et al. 2001, Hunter et al. 2003). Of these, 12 have been described associated with Eucalyptus spp. in different African countries (Crous 1998, Hunter et al. 2003). For example, in South Africa nine Mycosphaerella species have been reported associated with different Eucalyptus species (Crous 1998, Hunter et al. 2003) and 217 thirteen species have been recorded from Australia (Carnegie 2000, Milgate et al. 2001). Similarly five Mycosphaerella spp. have been identified from E. globulus and E. nitens (Deane & Maid.) Maid. plantations in Chile (Ahumada 2002). The most important symptoms of Mycosphaerella leaf disease (MLD) include leaf spot, defoliation, stunting, stem canker, twig and shoot dieback (Beresford 1978, Dick & Gadgi1 1983, Lundquist & Purnell 1987, Crous 1998). MLD reduces the photosynthetic capacity of the plant, causes shoot dieback, resulting in multistemmed trees and reduces growth and yield of trees (Park & Keane 1982b, Dick 1982, Carnegie 2000). Lundquist & Purnell (1987) showed that MLD causes a reduction in growth of E. nitens trees in South Africa. Similarly, a positive correlation between severity of M nubilosa infections and growth of E. grandis was observed in Australia (Carnegie et al. 1994). It has also been shown that the provenances of some Eucalyptus spp. such as E. globulus, E. nitens and E. regnans F. Muel!. vary in resistance to jVfycosphaerella infection (Dick & Gadgil 1983, Purnell & Lundquist 1986, Carnegie et al. 1994). In South Africa, for example, it is recommended that the New South Wales provenances of E. nitens are planted, as they are considerably more tolerant to infection than those from areas such as Victoria (Purnell & Lundquist 1986, Wingfield & Roux 2000). Several different Mycosphaerella spp. can infect individual Eucalyptus trees. Similarly, more than one MycosphaereUa sp. can be found on a single leaf and even on the same lesion (Crous & Wingfield 1996). Milgate et al. (2001), for example, showed that M. grandis Carnegie & Keane was found associated with older lesions of M tasmaniensis Crous & M.J. Wingfi., M nubilosa (Cooke) Hansf. and M. cryptica (Cooke) Hansf. It has also been shown that lesions of M. marksii Carnegie & Keane coalesce with those of M. cryptica and M molleriana (Thurn.) Lindau. Park & Keane (1984) also indicated the association of M parva R. F. Park & Keane, a saprophytic species, with M nubilosa (Park & Keane 1982b, Crous et al. 1993, Carnegie & Keane 1994). In this manner, multiple infections of trees can take place, compounding the impact of MLB on susceptible trees. Such, multiple infections often result in defoliation (Park & Keane 1982b) and they also complicate identification of the causal agents. 218 The occurrence of Mycosphaerella spp. on Eucalyptus leaves can vary with the age of the leaves. Some Mycosphaerella spp. infect both juvenile as well as mature leaves and others even infect twigs and branches (Park 1988, Crous 1998). Some Mycosphaerella spp., including M nubilosa, M molleriana and M juvenis Crous & M. 1. Wingf. are commonly associated with severe defoliation of juvenile leaves (Crous & Wingfield 1996, Carnegie & Keane 1998), whereas M. cryptica and M suberosa Crous, F. A. Ferreira, Alfenas & M. 1. Wingf. are found mainly on mature leaves (Park & Keane 1982a, Crous et al. 1993, Carnegie et al. 1994). Succession of infections by different Mycosphaerella spp. thus results in susceptible trees being affected at all stages of their rotation. In cases where only juvenile leaves are attacked, for example, M. nubilosa on E. nitens in South Africa, trees can outgrow the problem as they change to their mature leaf stage, normally during their second year of growth (Lundquist & Purnell 1987). In Ethiopia, symptoms of MLD have been reported from several plantations of E. globulus. It has been observed that the disease causes severe damage on juvenile E. globulus leaves, in most areas where this tree species is planted (Alemu, Roux & Wingfield 2003). The Mycosphaerella spp. involved in causing the disease have, however, not been identified. This study was, therefore, conducted to identify the fungi associated with MLD on E. globulus in Ethiopia. To accomplish this, a suite of identification techniques, including examination of ascospore germination patterns, cultural characteristics as well as sequencing of the Internal Transcribed Spacer (ITS) regions of the ribosomal RNA operon, were used. MATERIALS AND METHODS Sample collection and isolations In a prevIOUS survey conducted in Eucalyptus plantations in Ethiopia, symptoms similar to those of MLD were observed in most E. globulus plantations investigated (Alemu et al. 2003). The samples used in the current study were thus collected from E. globulus plantations in South, South Western and Western Ethiopia (Table 1, 219 Figure 1). At each locality where trees showed leaf blotch symptoms, five to ten symptomatic leaves per tree were colleted from three to ten trees, depending on the size of the stand of trees. The method described by Crous (1998), was used to isolate the Mycosphaerella spp. Two to four leaves were selected from each sample and four leaf discs containing lesions were excised from each leaf. These discs were then immersed in water for two hours to moisten the pseudothecia, facilitating spore release. The discs were then attached to the insides of Petri dish lids with the pseudothecia facing downwards over malt extract agar (MEA) (2% Biolab malt extract, 1.5% Biolab agar). The Petri dishes were kept in the dark at room temperature for 24 hours. After 24 hr, plates were examined for the germination of ascospores. Single germinating spores were picked up and transferred to 2% MEA plates and incubated at 25°C in the dark. Cultures resulting from germinated ascospores were incubated at 25°C under continuous light. Isolates obtained in this study are maintained in the culture collection (CMW) of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa. Morphological characterisation Growth of the fungi on MEA, germination patterns and anamorph associations were used to differentiate the Mycosphaerella spp. associated with MLD in Ethiopia. Colony colour was determined using Rayner's (1970) colour charts. Germinating ascospores for each sample were mounted in lactophenol on microscope slides and the germination patterns noted. Ascospore germination patterns were studied using a light microscope (Zeiss Axioskope) and compared with those described for Mycosphaerella spp. on Eucalyptus (Crous 1998). To identify the anamorph states of the Mycosphaerella spp., isolates were grown on water agar (1.5% Biolab agar) containing sterilised carnation leaves at 25°C under near ultra violate light (NUV) 250 nm. 220 DNA extraction Isolates for DNA extraction were selected based on differences detected in culture morphology and ascospore germination patterns. Mycelium used for DNA extraction was scraped directly from the surface of cultures on agar plates. Mycelium was placed in Eppendorf tubes and freeze dried under vacuum. A modified version of the DNA extraction method described by Raeder and Broda (1985) was used to isolate DNA. A repeated Phenol:chlorophorm purification step was conducted to remove cell debris. Sodium Acetate (3M NaAc, pH 5.5) (0.1 v/v) and two volumes of absolute ethanol were added to the clean aqueous phase to precipitate the nucleic acids. The precipitated DNA was washed with 70% ethanol. The DNA pellets obtained were vacuum dried to remove the remaining ethanol and the pellets were resuspended in 50 III sterile water. The contaminating RNA was removed by digesting the isolated DNA with RNase A (Roche, South Africa) in a 37°C water bath overnight. The DNA in each sample was visualised under ultraviolate light after electrophoresis on a 1% agarose gel containing ethidium bromide. peR amplification Specific DNA fragments from isolates included in this study were amplified using the Polymerase Chain Reaction (PCR). The Internal Transcribed Spacer (ITS) region and 5.8S genes of the ribosomal RNA operon were amplified using Primers ITS 1 (5'TCC GTA GGT GAA CCT GCG G -3') (White et al. 1990) and LR 1 (5'- GGT TGG TTT CTT TTC CT-3') (Vilgalys & Hester 1990). The PCR mix consisted of 1 ilL DNA, 0.25 mM dNTP's, PCR Buffer (10 mM Tris-HCI, 1.5 mM MgCb, 50 mM KCI, pH 8.3) (Roche, South Africa), 0.2 mM of each primer, 2.5 U Taq DNA polymerase (Roche Diagnostic, South Africa) and 37 ~l sterilised water. The PCR reactions consisted of an initial denaturation step at a temperature of 96°C for 2 min, followed by 40 cycles of template denaturation at 94 °c for 30 s, primer annealing for 30 s at 55°C and chain elongation for 2 min at 75 °C. This was followed by a final elongation at 75°C for 7 min. PCR amplicons were electrophoresed on a 1% agarose gel, stained with ethidium bromide and viewed under UV illumination. Sizes of he PCR fragments were estimated using a 100 bp molecular weight marker (XIV) 221 (Roche). Prior to sequencing the PCR products were cleaned with the High Pure PCR product purification kit (Roche, South Africa). DNA sequencing and phylogenetic analysis The PCR products obtained were used as templates for DNA sequencing using an ABI Prism, Big Dye Terminator Cycle sequencing reaction kit (Perkin Elmer Biosystems, USA) according to the manufacturers protocol. Primers ITS 1 and LR 1 were used to sequence both strands of the amplicons. Sequencing reactions were analysed using an ABI PRISMTM 3100 automated DNA sequencer (Perkin Elmer, Norwalk, Con). DNA sequences of the Ethiopian isolates used in this study were compared with sequences deposited in GenBank [National Centre for Bioteclmology Information (NCBI), US National http:/www.ncbi .nlm.nih.gov/BLAST] Institute for of preliminary Health Bethesda, identification. Sequence Navigator (Version 1.0.1) was used to align sequences and gaps were inserted manually and treated as missing data. Sequences were aligned against those of Mycosphaerella spp. from and extensive inhouse database emerging from previous studies (Hunter et at. 2003, Crous et al. 200 I) and those obtained from GenBank (Table 1). Phylogenetic analysis of the aligned sequences was conducted using PAUP (Phylogenetic Analysis Using Parsimony) version 4.0b (Swofford 1998). The sequences were analysed using parsimony, with trees generated by heuristic searches, simple addition and Tree Bisection Reconstruction (TBR) branch swapping. Bootstrap values for the branching points were calculated using 1000 replicates (Felsenstein 1993). In the phylogenetic analysis, Ramulispora anguoides (Nirenberg) Crous was used as the outgroup taxon. RESULTS Sample collection and isolation Symptoms of MLB were found on E. globulus at several localities, including Wondo Genet, Hossana, Endibir, Bedele, Menagesha, Holeta and Addis Alem (Table 1). 222 Disease symptoms, including shoot dieback and leaf blotch (Figure 2a2d) were common. In some cases, nearly 100% of the leaves on a tree and nearly 100% of the leaf surfaces of these trees were affected (Figure 2c). Lesions varied in size from small to large spots spreading over the whole leaf surface. Some lesions coalesced to form larger lesions (Figure 2a, 2b). The lesions were light brown in colour and had raised brown margins. On some leaves, lesions were confined to the margins of the leaves. Other samples had leaf spots that extended through the leaf laminas, with lesions visible on both leaf surfaces with a light brown colour and a faint red margin (Figure 2). Ascospores germinated within 24 hours. Mycosphaerella spp. were successfully isolated from samples collected from 16 trees. Ascospores from a number of samples failed to germinate, while some isolates died shortly after germination. Representative isolates were, however, obtained from all areas sampled. Morphological characterisation When the growth of the fungi on MEA was considered, three culture morphologies were found (Figure 3a, b, c). Four Mycosphaerella isolates (CMW10186, CMW10189, CMWI0376, CMW10187) obtained from E. globulus leaves collected from Addis Alem, Endibir and Hossana (Table 1) had similar colony morphology and constituted one group designated as Group I (Figure 3a). The colony colour of this group is olivaceous black 27""'m. Group II isolates (CMWll148, CMWl1149, CMW10377, CMWll150), obtained from Hossana, Endibir, Holeta, and Bedele (Table 1) showed a dark olivaceous grey colour, 23'''''i (Figure 3b). The third group included only one isolate (CMW1 0 190) obtained near Hossana. This isolate had a pale olivaceous grey colour (23"'''f) (Figure 3c). Examination of the ascospore germination of JvJycosphaerella isolates obtained from Ethiopia showed three different germination patterns. These germination patterns could be directly correlated to the morphological groups defined based on culture morphology (Figure 4). Isolates belonging to morphotype I (CMW1 0 186, CMW10189, CMW10376 and CMW10187) had an ascospore germination pattern 223 closely resembling a Type F pattern (Figure 4a). This pattern is characteristics of M. juvenis. The four isolates in morphotype Group II (CMWl1148, CMW11149, CMWI0377, CMWl1150) had Type C (Crous 1998) germination patterns (Figure 4b). This type of germination is characteristic of M. heimii, M. gregaria Carnegie & Keane, Ai molleriana and M. nubilosa (Crous 1998). The isolate obtained from Hossana (CMW10190) had a Type B germination pattern (Figure 4c) which is associated with M. gracilis Crous & Alfenas and M. marksii (Crous 1998). No anamorph structures were found for any of the Ethiopian Mycosphaerella isolates. DNA sequencing and phylogenetic analysis Amplification of the ITS reglOn of the rRNA operon produced a similar sized fragment of approximately 600 bp for all Mycosphaerella isolates obtained from Ethiopia. A BLAST search using sequences of Ethiopian Mycosphaerella isolates showed that these isolates were closely related to three different Mycosphaerella speCIes. When the sequence data were incorporated into a larger data base of sequences from previous studies including those in GenBank and analysed, 12 trees were generated. These trees had the same topology. The number of characters in the analysed data set was 705 bp's, of which 267 characters were constant, 131 variable characters were parsimony uninformative and 307 characters were parsimony informative. The phylogenetic tree generated using a heuristic search had CI and RI values of 0.698 and 0.861 respectively. In all parsimonious phylogenetic trees (Figure 5) one of the Ethiopian Mycosphaerella isolates (CMW10190), grouped with M. marksii with 100% bootstrap support. Four of the isolates (CMWI0186, CMW10189, CMW10376 and CMWI0187) grouped with M grandis (100% bootstrap support) and the remaining four isolates, (CMW11148, CMW11149, CMWI0377 and CMW11150) resided in the M. nubilosa clade (100% bootstrap support). DISCUSSION Mycosphaerella leaf blotch was the most common foliage disease observed on E. globulus in Ethiopia, during surveys in 2000 and 2001 (Alemu et al. 2003). Results 224 of the present study provide the first identification of this group of fungi on Eucalyptus in Ethiopia. Three Mycosphaerella spp., namely M. grandis, M. nubilosa and M. marksii were thus identified and this study represents the first report of these Mycosphaerella spp. on Eucalyptus spp. from Ethiopia. This study also represents the first report of M grandis from a country other than Australia. Ascospore germination patterns present a useful method to differentiate between Mycosphaerella spp. (Park & Keane 1982a). Crous (1998) described 14 types of ascospore germination patterns for Mycosphaerella spp. Examination of the ascospore germination patterns revealed that three different species of lvfycosphaerella were linked to MLD in Ethiopia. The occurrence of 3 different species was supported by DNA sequence data confirming the value that germination patterns have when identifying Mycosphaerella spp. Mycosphaerella marksii was found only from a single leaf sample collected from E. globulus near Hossana. Previous studies have shown that M marksii occurs on several Eucalyptus spp., including E. globulus, E. grandis, E. nitens and E. saligna (Carnegie et al. 1994). This fungus was first described in Australia and it is now known to occur in South Africa, Indonesia, Portugal and Uruguay (Carnegie et al. 1994, Crous & Wingfield 1996, Carnegie & Keane 1997, Crous 1998). This fungus is common in Australia and South Africa, but has not been reported to cause significant damage (Carnegie 2000, Hunter et at. 2003). Because the fungus was collected only from a single leaf, it is probably not an important component of the MLD problem in Ethiopia. Mycosphaerella grandis was found on samples collected from Addis Alem, Endibir and Hossana. This fungus was first described from Australia on E. grandis, E. globulus and E. nitens (Carnegie & Keane 1994, Carnegie 2000). According to Carnegie & Keane (1994), this pathogen is a common cause of necrotic lesions at the margins of leaves. This type of symptom was common in Ethiopia, suggesting that M. grandis is one of the more important components of MLD in the country. This is the first report of this species outside of Australia, and given its occurrence m Ethiopia, it might be expected to be found in neighbouring countries in the future. 225 Mycosphaerella nubilosa was found in several areas including Endibir, Holeta, Hossana, and Bedele. This species mostly affects juvenile leaves of E. globulus (Park & Keane 1982a, Purnell & Lundquist 1986, Carnegie et al. 1994). This is also one of the most common and destructive foliage pathogens of Eucalyptus in Australia, New Zealand and South Africa (Park & Keane 1982a, Dick & Gadgil 1983, Purnell and Lundquist 1986, Hunter et al. 2003). M nubilosa and M. molleriana were once regarded as the same fungus (Crous, Wingfield & Park 1990), but it has been shown that they represent distinct species (Crous & Wingfield 1997, Crous 1998, Crous et al. 1999). The presence of M nubilosa in Ethiopia explains the serious defoliation of E. globulus in this country. This fungus should be placed on the list of more important constraints to E. globulus propagation in the future. This study has shown that MLD is common, wherever E. globulus is grown in Ethiopia. Previous studies have shown that infection by Mycosphaerella spp. not only causes premature defoliation and retarded growth, but can also lead to the abandonment of planting certain Eucalyptus spp. (Lundquist & Purnell 1987). In South Africa, for example, the planting of E. globulus was terminated in the 1940's as a result of MLB (Lundquist & Purnell 1987). As E. globulus is a widely planted species in Ethiopia, the discovery of M nubilosa is of concern, especially given the fact that plantations of this species are likely to be expanded in the future. Selection of species and provenances with tolerance to MLB might help to minimise loss of yield caused by Mycosphaerella species in the future. REFERENCES Ahumada, R (2002) Diseases in commercial Eucalyptus plantations in Chile, with special reference to Mycosphaerella and Botryosphaeria species. M. Sc. Thesis. The Faculty of Natural and Agricultural Science. University of Pretoria, South Africa. Alemu Gezahgne, Roux, J. & Wingfield, M. J. (2003) Diseases of exotic plantation Eucalyptus and Pinus species in Ethiopia. South Aji-ican Journal of Science 99:2933. 226 Anonymous (1994) Ethiopian Forestry Action Program. Final Report, Vol. I, pp14 and vol. II, pp34. Addis Ababa Ethiopia. Beresford, R. M. (1978) Mycosphaerella nubilosa (Cooke) Hans! on Eucalyptus delegatensis R. T Baker: Further studies of epidemiology in the northern island ofNew Zealand., Ph.D Thesis. University of Auckland, New Zealand. Carnegie, A. 1. & Keane, P. 1. (1994) Further Mycosphaerella species associated with leaf diseases of Eucalyptus. Mycological Research 98: 413418. Carnegie, A. J., Keane, P. 1., Ades, P. K. & Smith, I. W. (1994) Variation in susceptibility of Eucalyptus globulus provenances to Mycosphaerella leaf disease. Canadian Journal ofForestry Research 24: 17511757. Carnegie, A. 1. & Keane, P. 1. (1997) A revised Mycosphaerella gregaria nom. nov. for j\1. aggregata on Eucalyptus. Mycological Research 101:843-844. Carnegie, A. 1. & Keane, P. J. (1998) Mycosphaerella vespa sp. nov. from diseased Eucalyptus leaves in Australia. Mycological Research 102:1274-1276. Carnegie, A. 1. (2000) A study of the species of Mycosphaerella on Eucalyptus in Australia and the impact of the Mycosphaerella leaf diseases on Eucalyptus globulus Labill. Ph. D Thesis. The University of Melbourne, Australia. Chipompha, N. W. S. (1987) Phaeoseptoria eucalypti: A new pathogen of Eucalyptus in Malawi. South African Journal ofScience 142: 1012. Corlett, M. (1991) An armotated list of the published names of Mycosphaerella and Sphaerella. Mycologia Memoir 18: 1328. Crous, P. W., Wingfield, M. 1., Marasas, W. F. o. & Sutton, B. C. (1989) Pseudocercospora eucalyptorum sp. nov. on Eucalyptus leaves. Mycological Research 93: 394398. Crous, P. W., Wingfield, M. 1. & Park, R. F. (1990) Mycosphaerella nubilosa, a synonym of M molleriana. Mycological Research 95: 628632. Crous, P.W., Phillips, A. J. L. & Wingfield, M. 1. (1991) The genera Cylindrocladium and Cylindrocladiella in South Africa, with special reference to forestry nurseries. South African Forestry Journal 157: 6885. Crous, P. 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(2001) ITS rONA phylogeny of selected Mycosphaerella species and their anamorphs occurring on Myrtaceae. Mycological Research 105: 425431. Dick, M. (1982) Leaf inhabiting fungi of Eucalyptus in New Zealand. Journal of Forest Science 12: 525537. Dick, M. & Gadgil, P. D. (1983) Eucalyptus leaf spots. Forest Pathology in New Zealand 1: 17. Eldridge, K., Davidson, 1., Harwood, C. & Van Wyk, G. (1997) Eucalyptus domestication and breeding. Oxford Science Publications. Fe1sentein, J. (1993) DNABOOTBootstrap Confidence Interval on DNA parsimony 3.1. Seattle, University of Washington DC. Hunter, G. C, Crous, P. W., Roux, 1., Coutinho, T. A., Wingfield, B. D . & Wingfield M. 1. (2003) Identification of Mycosphaerella spp. associated with leaf disease of Eucalyptus nitens in South Africa. Australian Journal of Pathology (Submitted). Lundquist, 1. E. & Purnell, R. C. (1987) Effects of Mycosphaerella leaf spot on growth of Eucalyptus nitens. Plant Disease 71: 10251029. Milgate, A. W., Yuan, Z. 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C. & Lundquist, 1. E. (1986) Provenance variation of Eucalyptus nitens on the Eastern Transvaal Highveld in South Africa. South African Forestry lournal138: 23-31. Raeder, U. & Broda, P. (1985) Rapid preparation of DNA from filamentous fungi. Applied Microbiology 1: 1720. Rayner, R. W. (1970) A mycological colour chart. Commonwealth Mycological Institute, Kew. Schoch, C. L., Crous, P. W., Wingfield, B. D. & Wingfield, M. 1. (1999) The Cylindrocladium candelabntm species complex includes distinct mating populations. Mycologia 91: 286298. Sharma, J. K. & Mohanan, C. (1982) Cylindrocladium spp. associated with various diseases of Eucalyptus in Kerala. European lournal ofForest Pathology 12: 129136 Swofford, D. L. (1998) PAUP*. Phylogenetic Analysis Using Parsimony (*and other methods). Version 4.0. Beta version. Sinauer Associates. Sunderland, Massachusetts. Vilgalys, R. & Hester, M. (1990) Rapid genetic identification and mappmg of enzymatically amplified ribosomal DNA from several Cryptococcus species. Journal ofBacteriology 172: 42384246. Von Arx, J. A. (1983) Mycosphaerella and its anamorphs. Proceedings of the Koninklijke Nederlandse Akademie van Wetenschappen, Series C 86: 1554. White, T. J., Bruns, T., Lee, S. & Taylor, 1. (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR 229 protocols: a guide to methods and applications. San Diego, Academic Press. Wingfield, M. 1. (1990) Pathogens in exotic plantation forestry. International Forestry Review 1: 163168. Wingfield, M. 1. & Roux, J. (2000) Plant disease of South African forest plantations. In South African Forestry Handbook (D. L. Owen ed):pp 241252. The South African Forestry Institute. Pretoria, South Africa. 230 Table 1. Mycosphaerella isolates used in this study. Isolates Species Host Origin Collector Accession No. CMW 10 190a M marksii Eucalyptus globulus Ethiopia Alemu Gezahgne & J. Roux A Y244404 a CMWI 0 186 M grandis Ethiopia Alemu Gezahgne & J. Roux A Y244405 M. grandis Ethiopia Alemu Gezahgne & J. Roux A Y244406 CMWI0187 a a CMWI 03 77 M. nubilosa Ethiopia Alemu Gezahgne & J. Roux A Y244408 Mgrandis Ethiopia Alemu Gezahgne & J. Roux A Y244412 CMW 10 189 a CMWI 03 76 a M. grandis Ethiopia Alemu Gezahgne & J. Roux A Y244407 CMW11148 a M. nubilosa Ethiopia Alemu Gezahgne & J. Roux A Y244409 CMW 11149 a M nubilosa Ethiopia Alemu Gezahgne & J. Roux A Y244411 CMW 11150a M. nubilosa Ethiopia Alemu Gezahgne & J. Roux A Y24441 0 CMW9090 M marksii E. grandis South Africa M. J. Wingfield AF468870 M marksii " M . J. Wingfield AF468871 CMW9091 " M . J. Wingfield AF468872 M marksii CMW9092 CMW3358 M. parkii E. grandis Australia A. J. Carnegie AF309590 CMW4945 M. africana E. viminalis South Africa P. W. Crous AF309602 CMW4942 M heimii Eucalyptus spp. Madagascar P. W. Crous AF309606 CMW5705 M. heimii Eucalyptus spp. Brazil P. W. Crous AF452508 CMW5224 M jlexuosa E. globulus Colombia M. J. Wingfield AF309603 CMW4937 M. juvenis E. grandis South Africa M. J. Wingfield AF309604 " M . J. Wingfield AF309605 M juvenis CMW4036 CMW3282 M. nubilosa E. globulus Australia A. J. Carnegie AF309618 CMW4940 Portugal S. McCare AF309620 M. molleriana E. globulus California (USA) M. J. Wingfield AF309619 M molleriana E. globulus CMW2734 aIsolates collected from E. globulus in Ethiopia and sequenced in this study. All isolates are maintained in the culture collection (CMW) of the Forestry and Agricultural Biotechnology Institute (F ABI), University of Pretoria. All other sequences are obtained from Crous et al., (2001) and Hunter et al. (2003). 231 \ t S~dan \ LakeTana Menagesha Bedele Jima =• = DO 0 Addis Ababa SOMALIA Wolisso Hossana o Munessa Shashemenne Wondo Genete = SOMALIA Turkana Figure 1. Map of Ethiopia showing the plantation areas where samples were collected. 232 Aligned ITS sequence o f (N)= unknown bases Figure 6. Mycosphaerella spe c ies. 10 CMWI 0 190Ethi op ia Mycosphaerella_marksiiCMW9090 Mycosphaerella_marksiiCMW9091 Mycosph aerella_rnarksi i CMW9 092 Mycosphaerella_parkii353 Mycosphaerella_africana794 Mycosphaerella_flexuosall09 AY045516M.grandis CMW I01 86Ethiop ia CMWI0187Ethiopia CMWI0189Ethiopia Mycosphaerella_juvenisl004 Mycosphaerella_juvenisl005 Mycosphaerella_heimii760 Mycosphaerella_heimiiCMW5705 Mycosphaerella_nubilosa937 CMWll148Ethiopia CMW ll149Ethi opia CMW ll15 0E thiopia CMWI0377Et hi opia CMWI0376Ethiopia Mycosphaerella_molleriana784 Myc ospha erella_mo lleriana 1214 Ramulispora angui oides 20 30 () =Gaps I 40 TCCGTAGGTG AACCTGCGG AGGGATCATT ACC GAG C . . • . . • . G .. · ........ . . G...... . ........ . A · ........ · ........ · .T ...... · .T ...... • • G..•.... · . T ...... · ........ · ........ ........ N......... G.AAC .... . · ........ · ........ · .... . ... · . T ...... · .T ...... ......... G.A ...... . · .T . . . . . . - ( • ) 50 GGAGGGCCC T ..... TTT . T ..... TTT. T ..... TTT. T ..... TTTC T ...... TC T ..... . TC T ....... TC T ....... TC T ....... TC T ....... TC T ...... TC T ...... T C T ...... TA. T ...... TA. T ...... G T .C .... G = Homologous nucleotides 60 70 CGGCCCG ACCTC CG ... ... . . ........ . A.C .. ... . A .. ..... . CG . ..... . CG .... T .. . CG .... T .. . CG .... T .. . CG .... T . .. CG. ..... . CG. ..... . . G. . T ... . CG ..T ... . G.A ...... . C.A ...... . . . . ...... T NNNNNNNNNN NNNNC .... . · . T . . . . . . T. C .... G C . A . . . . . . . ....... . CT NNNNNNNNNN N.. ..... . · .T ...... T.C .... G C.A ...... . NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNG C.A ...... . ......... · . T ...... · .T ...... .A . . . . . . . . . ATA ... CAA T ....... T. T ...... G T ...... G T ... C.T. AG C ... G.T .. . CAA ...... . CAA ...... . .C. C .... G GAGCA .TC . 237 90 80 CMW1 0 19 0 Ethi op ia Mycosphaere ll a _mark s ii CMW9090 Mycosphaerella_marksiiCMW9091 Mycosphaerel1a_marksiiCMW9092 Mycosphaerel1a_p ark ii35 3 Mycosphaerel1a africana794 Mycosphaere lla_fle x uosa 11 09 AY045516M.grandis CMW 101 86 Ethi op i a CMW 1 0 187 Ethi op i a CMW10189Ethiopia Mycosphaere lla_ju ven is1 004 My cosph ae re lla_juven i s1005 Mycosphaerel1a helmii760 My c ospha e re lla_heimiiCMW5 7 05 Mycosphaerella_nubilosa937 CMWl l1 48 Ethi opi a CMvH 114 9Ethiopi a CMW ll15 0 BEthi op ia CMW10377Ethiopia CMW 1 0376 Ethiop ia Mycospha er e lla_m o l1erian a784 Mycosphaerel1a_molleriana1214 Ramulispora ang ui o i des 10 0 1 20 110 13 0 1 40 CAA CCCT TT GT GAA TCA- . . . . . . . . . . ................. . .. . . . .. . . .. . . . . . .. .. . . .. . . .. . . .. . . .. . . . .. . .. .. . .. .. . .. . .. . . . .. .. .. .. . . . .. .. . . . . . . .. . .. .. .. .. . . . . .... . C .... . . .. .. . . . .. . . . . . .. . .. .. . . . . . .. .. .. . .. .. .. .. . . .. .. . .. .. .. . .. CT .... .. C ..... .. A CC ... . .. A C ..... .. A C ..... . . A C ..... .A ...... C. A ...... C.A TGGGGG.C .C . . .. . . .. .. . . . . .. . .. . .................. . . . .. ... C...... ... C ...... ... C ...... ... C ...... ... C ...... ., . C ...... ... . A ..... ... . A ..... ... CA ..... . ... A..... ... . A. ... . . . . A. ... . . . . .. .. .. .. . .. . . . .. . .. . .. . .. . ... C... . .. ... C ...... ... C ...... ... C...... ... C ...... ... C...... ... C ...... ... C ...... .. TCCTCGGA . . . . . . .. . . . . .. . . .. . . .. .. . ... A. ... ... . A. ... .... A. ... ... . A. ... .. , .A. ... ... . A ..... . . . . A. ... ... . A. ... GGGT T. AGAG .. ................. . ................ . ................ .. . .... ........ . . .. .. . . . . . . . . . .. .. .. · ........ A ......... A . ........ A · ........ A TTCC . TTC C . TT CC ..... TT CC . . ... - . . . . . . . .. .. . ... . . .. . ..... C ... C ............... ..... C . .. .... TT CG ACCT. . . .............. .... .. . . .. . . .. . .. . .. .. .............. . . . .. .. .......... .. . .. .................. .. .. .. .. .. . . . . . .................. .. ...... . .......... . .......... . ...... . .. . .. .. . .. . . .. .................... . . .. .. . .. .. .. .. .. · ..... .. . T .................... .. . .. . . .. . .. . . . ........ A ................ .... .. .. . . . . .. . . .. AC .. CGAGCC .. . . . .. .. . . .. .................. .. TTT C... .. ................. TTTC ... .. .................. TTTC ... . ................ . TTTC .. . . .................. TTT C... . ................. TT ..... .. .................. .TTC .. . . .................. . TT C.. . . .............. TCTCGGA .. . GCTCGG TT CA .. , .T CT.C · ... TCT. C ..... C . ..... C . ..... C .. CC ..... C . . CC · .... .. CC ..... C .. CC ..... C .. CC ..... C.G . · ... CAA. CC · .. . CAA . CC GACCT CC 238 15 0 CMWI0190Ethiopia Mycosphaerella_marksiiCMW9090 My cosph ae re lla_marksi i CMW5296 Mycosphaerel l a _mark s iiCMW5 29 9 My c ospha e re ll a _p ark ii3 53 Mycosphaere ll a_af ri cana 7 94 Mycosphaerella_flexuosall09 AY045516M.grandis CMW I01 86 Ethiop i a CMWI 018 7E th i op i a CMWI 0189 Ethio p i a Mycosphaerella_juvenisl004 Mycosph a erel la ] uvenisl005 Myc o sphaere lla_he im ii76 0 Mycosphaerella_heimiiCMW5705 Mycosphaerel1a nu bil osa 9 37 CMW1 1148Et h i op i a CMW1114 9E thiop ia CMW 111 50Ethiopi a CMWI 037 7 Ethio pi a CMW I 0376 Eth iopia Mycosphaerella_molleriana784 Mycosphaerella_molleriana1214 Ramulispora angu i oides AACCT A. C.T .... A.C .. .. . . A.C. T .... A. . T .... · . . T .... · . T .... · c. T .... · C. T .... · C . T .... · c . T .... · . C . T .... · .C . T .. .. A. . T . ... A. . T .... A C .... AC .... A C . . .. AC .... A C .... 16 0 l70 180 1 90 2 00 210 GTT GCTTCG GGGGCGAC CC T GCC GTTCGC GGCGA · . ........ . . . . . . . . . . · . ... ..... · ......... · ......... · ... C . · ........ . . . . . . . . . . . · ......... · ......... · ......... · ... c. · ....... GT T ......... · . ... ..... · ......... · ..... .. .. · ... C . · .... .. ... T ..... . ... ...... . . C . T ......... · ......... ...... C ... · ... .... CT T ..... C ... · . ...... CT T ..... C... · ....... CT T ..... . .. · ... .... CT T ..... C ... ....... . C. T ..... C ... ....... . C. T ... .. C ... · . .... . . . . T ......... · . .. .. .... T ..... . ... · ......... . . .... C ... · ......... . ..... C ... · ......... ..... . C ... · ......... ...... C ... · ... .. ... . ..... . C ... · . . . . . . . . · .... . .. CT T ... . . C ... AC .... · ......... .. .. . . C ... AC .... ......... . ..... C... ACC.T.GAAT AAAAAACCTT T ... .... T . · · .. ..... . . · .. .. ..... · ... ...... · . ........ · .. . ...... · ......... · ... . ..... · .. . ...... · ......... " . ATC. ..C ....... · . G . ... CTC · . G . ... TT C · . G .... TT C · .G .... TTC · . G . .. . TT C · ..... T ... · . G .... CTC · ..... T .. . · .G .... CTC · . ... ..... · .... . .... · ... . . .. .. · ......... · ......... · ......... · ......... · ......... · ......... · ......... · ......... · ......... · . G.... CCC · . G .... CCC · .G .... CCC · . G .... CCC · . G .... CCC · .G ...... T · . G . ..... . · . G ..... . . . CA .. A.. C . T.GC ... AGC · .. T .. . G .. T ... G.T . G.GC G .G.GC G .G. GCG . G.GC G TG. G.G TG. G.G .G . C. TG .G . G. C .T C . G . G. C. .G.C. .G. C . . G. C . .G. C . TC .. .. G . G . C . G.C GG . C. TC. G . A. . ..C. TC. TC. TC. TC . CC . CC. C .GT .... G C .GT . ... G CC . CC . CC . CC. CC . C. T C . C ... CC . C... CC . CT.T TGAG . 239 220 CMWI 0 1 90 ETHI OP IA Mycos ph ae r e lla_mar ksi i CMW9090 Mycosp h a e re ll a _ma rks ii CMW909 1 Mycosph a ere ll a _m ar ksii CMW 9092 Myc osp h aere ll a _ park ii 353 Myco sp haere ll a _ af ri cana 7 94 Mycosph a ere ll a f l ex uo sa ll 09 AY0 4 55 1 6M .g rand i s CMW I 0 1 86 Ethi o pi a CMW I 0 187 Et hiop i a CMW I 0 18 9Eth i opia Mycosph ae r e ll a ]uven isl O04 Myco s p h aere ll a ]u v en i s l O05 Mycosph a ere ll a h e imii 760 Mycos p haer ell a _h e im i i CMW 5705 My cosphaere l l a_nub il o s a93 7 CMWll1 48Et hi op i a CMWll14 9Ethi op i a CMW ll1 50 Ethi op i a CMW I 0377 Et hi o p i a CMWI 0376E THI OPI A Mycos ph ae r e ll a_ mo ll er i ana784 Mycospha er e ll a _ mo ll e ri ana 1 214 Ramu li spora angu i oides 2 30 2 40 250 260 270 28 0 GGC GCCCC CGGGGGAAA TCAAAC A CTGC GT CA AT TTGT GTC GGAG TA CTT ........ .. . . G . .. .. · . . G ..... · .. G .. . .. · .. G ..... · .. G .. . .. · .. G .... . · .. G . .. , . · . . G .. .. . C ......... C . . .... . .. · .. G .... T · . . G .. . . T · .. G ... . T · .. G .. . . T · .. G .... T · .. G . . . . . · . . G ... . . · .. G . . ... . T GC .TG. · .. A.. . T · .. A . . TC .. . C ... C . . . T ... CC. ... T ... CC. .. . T ... CC . ... T . .. CC . . . . C .. . CC . .. . C ... CC . . . . A .. CC. .. . A .. CC. · . CA. A. CCC · . CA ... CCC · . CA .. . CC C · . CA . .. CCC · . CA . .. CCC . .. T ... CC . .. . T . . . CCC .. . T ... CCC . A. A .. . CCA AC. T . ... A ... ..... . ACC ... ... A......... A.. ... .. . . A....... . . A.... . .... A.. C .. . .. A.. C . . ... ATT. .. . . ATT. .... CTC . .. . CTC. . .. CTC. ... CTC . .. . CTC. ... ATC . .. A . TC . ... . TC . . .. C... ... C ... . . A . . . . .. .. . A .. T .... A.. T .. .. A . . T . . .. A . . T ... . A .. T . .. . A .. T . ... A . . T . ... A.. . . . .. . A ... . .... . A . . . GG ... GA . . G . .. .. A.. G... .. A . . G.. . .. A . . G.. . .. A . . C .T CTGCATC T ..... A . . T . . . .. A.. TCT .. TT . TT ... . C ... . . . .. C . .. ..... C ... .... AC ... .... AC . .. .... AC ... .. . . AC ... .G ... C .. . .G . .. . . . . .. . C ... . ... C ... . G . .. C .. .G . .. C ... . G . . . C ... . G... C ... . G . .. C .. . .. TGACG T . C... C ... .C . .. C . .. . G . . A . .. . . . . .. . A . .. TT .. A. . . . . . ... . . . .. AAAG TA T .. . .. TGA T T ..... AA .T T . .. .. AA . T T . .. . . AA .T T .. . .. AA .T T . . .. . AA. T T ..... AA.T . ..... AA .. ..... . AA . . . ..... A.TA . .. . .. A.TA ..... . A. TA . . . ... A.TA . ... .. A.TA CTGAG TAAAT T . .. . . . CA T .... .. CA T ...... CTA AT ... . . GAA AT . . .. GAA AT . .. . GAA AT. ... GAA AT. .. . GAA AT . . ... GAA AT .... . GAA . .. AG . . .. AG . C. AA . C. AA . C . AA. C. AA. C. AA. AT. . . .. GA C . AA . C . AA . TA . AATAG . 240 290 CMW I019 0Eth io p i a Mycosphaerella_marksi iC MW9090 Myco sp hae rella_ma rk si iCMW 909 l Mycosphaer e lla_marksiiCMW909 2 Mycosphaerella_parki i3 53 Mycos ph ae r e lla_afri cana 794 Mycosphaer e lla flexuosall09 AY0455 l 6M .grandis CMW1 0 18 6Ethi o pia CMW 101 87Ethiopia CMW 10 89 Eth i o p ia Mycosphaerella_juvenislO04 Mycos ph ae r e lla Juveni s lO 05 Mycospha er e ll a he im i l7 60 Mycos phaerella_he i mi iCMW5705 My cos pha ere lla_nubil osa937 CMW ll14 8Ethiopia CMW ll149Et h iop ia CMWll15 0 Et h i o pia CMW10377Ethiop ia CMW1 037 6E th i opa JVlycosphaer ella _mo ll er i a na 7 8 4 [Vjy cos p haere lla_mo ll er iana12l4 Ram uli spo r a anguioides 300 31 0 320 330 34 0 3 50 GTTAA TAAA CAAA AC TTT CAACAAC GGATCTCTTG GTTCTGGCAT CGATGAAGAA · .... .... · .AA. .... TCAA. TCAA. TCAA . TCAA. TCAA . TCAAA TCAAA T . AAA .... T .AAA .... CCAA . CCAA. CCAA. CCAA. CCAA. ATCAA · . AA. · . AA . · .. C. T. A. .T. · T .... · T .... .T .... · T .. .. .T. .T. A . .... A ..... .T . .. .T ... .T ... .T .. . . T ... · T .... .T ... T .T ... T ---- ---- · ........ . · ......... · ....... . . · ......... . . . · .. T. .. T. .. T. .. T. · ......... · . ........ . ......... · . ........ · ....... . . . . . . . . . . . . ., .. CA . .... CA. .TTAAAAC .TTAAAAC .TTAAAAC .TTAAAAC .TTAAAAC · ...... . .. . .. T. CAATCAAAAC CAA TCAAAAC ...... .. · .AA ... TTT CA. CAA . GGA TC ... T.GT . . TGGC .TCG . 241 3 60 CMWI0190Ethiopia Mycosphaere11a_marksiiCMW9090 Mycosphaere11a_marksiiCMW9091 t/iycosphaere11a_mark s ii CMW9092 Mycosphaerel1a_parkii353 Mycosphaere11a_africana794 ~yco s p ha er1 a f1e xuo sall 09 AY045516M.grandi s CM WI 0 186 Ethi op i a CMW10187Ethiopia CMW10189Ethiopia Mycosp haere1 1a juven i s l 004 Mycos p hae re11 a_juven i s l0 05 My cosphaer e 1la heimil760 Mycosphaere11a helmii CMW5 705 Mycosphaere11a_nubi1osa937 CMWll148Ethiopia CMW ll1 4 9Ethi op ia CMWll150Ethiopia CMWI 0377 Ethi op ia CMWI0 3 76Ethi op ia Mycosphaere11a_mo11eriana784 Mycosphaere11a_mo11eriana1214 Ramu 1i spora anguioides 370 380 390 400 410 420 CGCAG CGAAA TGCGATAAGT AATGTGAATT GCAGAATT CA GTGAATCATC GAATCTTT GA ACGCACATTG •.• C . . • • . . ......... C T.A .. AACG C A .... A.T.C G.. AA.T.A . . TGA . T. G. . . AATTCAG.G A .TCA . CGA . T.TTTG.AC. 242 430 CMW1 0 1 90 Et h iopia tJiyco sphaerell a _m ar ksiiCtJiW90 90 Mycosphaerella marksliCMW9091 Mycosphaerella_marksiiCMW9092 Mycosphaerella_parkii353 My cosph a e rell a _ a fri cana79 4 Mycosphaerella fle xuo s all09 AY045516M.grandis CMW 101 86Ethi op ia CMW10187Ethiopia CMW 1 0189 Ethi opia My cospha erella_j uv eni s l O0 4 Myc ospha er ella_juvenisl O05 Mycosphaerella_heimii760 Mycosphaerella_heimiiCMW5705 Mycosph aer e lla_nubil osa93 7 CMWll14 8 Ethi op i a CMWll14 9Ethiopia CMWl l1 50Eth i op ia CMW10377Ethiopia CMW 1 0376 Ethiopia Mycosphaerella_molleriana784 Mycosphaer ella_mo lleriana1 21 4 Ramu lis pora anguioides 440 45 0 46 0 47 0 480 490 CGCCCCGTGG TATTC CGCGG GGCATGCC TG TTCGAGCGTC ATTTCACCA CT CGAG T C TGACT CGGTA · ........ . . . . . . . . . . . · .T. · .. ....... . . . . . . . . . . · ......... ....... G.. · .. . ...... ...... . A .. · ......... ...... . A .. · ......... ...... T ... ...... TC .. ..... . TC .. · ..... T ... ..... . TC .. · ..... T .. . ..... . T ... · .... TC . .. · .... TC ... .... . TC ... · .... TC ... · .... TC ... · .. .. TC ... ..... TC ... · ..... TC .. · .... TC .. . · .... TC . .. .A.ATT.C.C ...... . A .. ...... . A .. ...... . G . . ....... G.. ...... . G .. ...... . G .. ...... . GA. ...... . GA . ...... . GA. ...... . GA . ....... GA. ...... . A .. ...... . GA. ...... . GA . CC. CTG.TAT · ......... · .......... · . ........ · ... . ...... · ....... . . ... A.... C . · ......... · ......... . .. A.... C. · ......... · ......... . .. A.... C. · ......... · ......... ... A.... C . · ......... · ......... . .. A.... C . · ......... · .......... " .A .... C . · ......... · ..... . .. . . ., . A .. .. C . · ... . ..... · ......... .. . A .... C . · ......... · ..... ... .. · ......... · ......... ... A.... C . . . . A .... C . ... C .... C . ... C .... C . ... C .... C . . .. C .... C . . .. C .... C. . .. A.... C . . .. A.... . . . .. CG.C . CT " " . .. A... . .. ... CG . C . CT " " " " " TC.GG. GGGC A .GCC TGT .. GAGCG TCAT T A.AACCAC .. · .... . ... . · . ........ · ......... · ......... · ......... · .......... · . . . . . . . . · ......... · .... . .... · ......... · ...... . .. · ......... · ......... · ......... · . ........ · . ... . " . ... " .. " · . " . ... " ~ · ....... .. · ......... · .... .. ... · ......... · ......... · .. . ...... · ..... .. .. . AG .. T. · . G .. T . .. G.. T . · .G .. T. .. G.. T. .. G.. T. ..G .. T. · . G .. T . ..G .. T. .. G.. T . CCG.. T . CCG .. G.. . C CCG .. G... C CCG .. G... C CCG .. G... C .. G.. T. CG .. G . CG .. G. AAG ... TCG 243 500 CMW 1 0 1 90Ethiopia Mycosphaerella_marksiiCMW99090 Mycosphaerel l a_ma r ksiiCMW9091 Mycosphaerella_marksiiCMW9092 Mycosphaerel l a_parkii353 Mycosphaerella_africana7 94 Mycosphaerella_flexuosa1109 AY045516M . grandis CMW10186Ethiopia CMW10187Ethiopia CMW10189Ethiopia Mycosphaerella Juven i s 1004 Mycos phaere1la_juvenis1005 Mycosphaerella he lm li760 Mycosphaerella_heimiiCMW5705 Mycosphaerella_nubilosa937 CMWll148Ethiopia CMWl l149Ethiopia CMWll150Ethiopia CMW10377Ethiopia CMW10376Ethiopia Mycosphaerella_molleriana784 Mycosphaerella _m olleriana1214 Ramulispora anguioides TTGGGCGCCG · ...... T .. ....... T .. · ...... T .. .. . . . . . T .. · .... .. T .. .... .. . AG. · ......... · . ...... .. · ......... · . . .. . .. . . . ...... AG. ....... AG . .. . . .. . T .. · ...... T .. · . ....... . · ......... · ......... · ......... · ......... · ......... · ...... . .. · ......... C .T .. TATT. 510 520 CG TTT CG ACGCGCGCC · . ... . . ... · . T. · ....... . . · . T. · ......... .. T. · .GCT .. C .. . C ........ · .. · .GT .. C .. · .GCT .. C .. GC .. . C ... · .. · .G.T.GC .. · .. · . G .T. GC .. · . G. T .GC .. · .. · .. · .G.T.GC .. · . GCT ... · ... . . . · . GCT ... · ... . .. ·.. . . GCT .C .. · .GCT.C .. · ..... C · .GCC .. C .. · ... G C . · .GCC .. C .. · .. . GC . · .GCC . . C .. · ... GC. · .GCC . . C .. · ... GC. · .GCC . . C .. · ... GC. · .G.T.GC .. · ..... C · .GC. ... TC .... T.GC. ... TC .... G.G.TCGCG. TTT .. 530 540 550 560 TTA AAGTTTCCG GCTG GACCG TCCGTCTCCG ....... . C. .... C..... . ..... G.A . C . · ......... ... . C ..... ..... AG.A . . T ...... TA ........ C. .... C .. ... ........ . A .. .. ... TA . ....... C . . ....... C . ........ C . ....... . C . GCCCGCC . C . GCC CGCC.C . · ... .... . . · . ........ . ....... C . ....... . C . . .... .. . C. . . ..... . C . . ... ... . C. ..... ... C . · .. CGCC. CG ... CGCC . CG · .CGGCC ... . C ..... ... . C ..... ... . C ..... ... . C ..... ... . C .. ... ... . C .. .. . · .. . C. T ... · ... C . T ... · T .. C ..... .T .. C..... · T .. C .... . · T .. C ..... · T .. C..... ... . C ..... .... C..... ... . C ..... . C.AAAC · .... AG .. A . .... AG .. A · .... AG .. A · .... AG . . A ....... . G. . .. . ... . G. . . ... AG.T . .... . AG .T. · . C . . AG ... · .C .. AG .. . · .C .. AG ... · . C .. AG ... · . C .. AG ... · .... AG .. A · . C .. AG ... · . C .. AG ... T.A.T.G.G. A. T ..... TA A.T ... .. TA A . T ..... TA A. T ..... TA AT ...... TA AT . .. ... TA . . .. .... TA . . . ..... TA A....... TC A .. ..... TC A ....... TC A . .. . . .. TC A ....... TC A . T ..... TA A........ A A .. . G .... A .G.C.G .. G. 244 5 70 CMW10 1 90Eth i op ia Mycosphaerella_marksii CMW9090 Mycosphae r e ll a_mar ks ii CMW 9091 Mycospha er e l1 a_marks ii CMW9092 My cosph a e rell a_pa rkii 353 Myc o sphaere ll a a f r i cana794 Mycos phaerella flexuosa1109 AY04 5 5 1 6M.grand i s CMW10186Ethiopia CMW 1 0187Ethiop i a CMW 10 189 Eth i op ia Mycosph aerella Juvenis1004 Mycosphae r e ll a Ju ven i s1005 Mycosphaerel l a _h ei mi i760 Myco s phaere ll a_he i miiCMW570 5 Mycosphaerel la_nub il osa937 CMW ll1 48Ethiopia CMW l l1 49 Eth i op i a CMWll150Ethiopia CMW1 0377E t h i op ia CMW 10 376ETHIOPA Mycosphaere ll a_mo ll e ri ana78 4 Mycosp h aere lla_mo lle r i ana12 1 4 Ramulispora anguioides 58 0 590 600 610 62 0 630 AGCGTTGTG G CATCT GTC TC GC T A GGGAGTC GCGGAGGGC G TT GGCC · ........ . · ...... . .. .. . . . . . . . . . · .... .. ... · ... . ..... .. . ...... A · .. ..... .. · ... ...... · . .. ... ... · ..... . ... ..... ... . A ........ . A · ......... · .......... · . .. ...... · ..... . .. . · . .T . .T T TT TTT TTT TTT .T .. G .. · T .. G .. ------- · ........ . - - - - - - -----· . ........ ----· .... .. ... ---· . .. ...... -----· .... . .... -- - --· ......... · .. . ... . .. TTT · ......... .. CAAC.G TT · ......... · . CAAC.GT T CT .TAC . C.T AG .AATA CT · .... . .... · ....... . . · .......... · ... . ..... · . ........ · . ........ ACA . GT TC .. ATA .. TT ... AAA .. TTG GA AATCAT .... AATCAT .... AATCAT .... AA TCAT . . .. ... . . TT GGA ..... TTGGA AACTAT .... AACTAT .... · . CTACTGTT · . CTACTGTT · . CTACTGT T · . CTACTG TT · . CTACTGTT AATCAT .... T. CG ... CTT T . CG . .. CTT CC .... . TCG ...... . C. .. .... . C . ...... . C . · .... . ... . · . ..... .. . . .... C... . · . C . ...... . G .. -.C .. . TGT -. C .. . TGT -. C.. . TGT -. C . . . TGT -. C .. . TGT -.C .. . TGC - .C .. . TGC TC ... T.C T C ... T. C · . GCT. G.. · . GCT. G.. · . GCT . G .. · . GCT. G.. · . GCT . G .. -.C .. . TGT · .. GGG .. · .. GGG . . . GAT .... · . . . C . .. · .. TT. · . .. C . T. .C .. . C . · . A . . . . A .. C . · .A .... A . . C . · . A .... A . . C . · .A .... A . . C. ... · . CCTC . · . CCT C . · .. T . . C .. · .. C . · .. T . . C .. · .. C. · . TCT .... . . CGC . · . TCT .... .GCGC. · . TCT .... . GCGC . · . TCT .... . GCGC . · . TCT .... .GCGC . · .A .... A .. C. C- . . . . · . TCT . . .. . GCGC . C- . . .. · . TCT . ... . GCGC . .. . G A.T CC .. TA . GG TTTA. AA .... T . ...... AT .. . . . AT. ..... AT . . . ... AT. ..... AT. ...... A . .. ... . A. · . AG . . . . · . AG . . . . C .. G.G AC .. C.. G.GAC .. C . . G. GAC .. C .. G.GAC .. C.. G. GAC .. .AG. . .. 245 6 40 CMW101901Ethiopia Mycosphaerella_marksiiCMW9090 My cosphaere lla_ma r ks iiCMW9091 Mycosphaerella_marksiiCMW9092 My cosphaere lla_pa r kii35 3 Mycosphaerella_africana794 Mycosphaerella_flexuosall09 AY0455 16M.grandis CMW101 86Eth i op ia CMW10187Ethiopia CMW10189Ethiopia Mycosphaerella Juvenlsl004 Mycosphaerella_juvenisl005 Mycosphaerella_heimii760 Mycosphaerella_heimiiCMW5705 Mycosphaerella_nubilosa937 CMW ll14 8E thiopia CMWll149Ethiopia CMWll 150Ethiopia CMW10377Ethiopia CMW1037 6E thiopa Mycosphaerella_molleriana784 Mycosphaerella_molleriana1214 Ramulispora anguioides 650 660 6 70 680 690 697 GT TAAAC A CCCCATCAA AGGTTGACCT CGGAT CAGGT AGGGATA · .•. T . . . . . .... TC .... TTTC ... TT. -------· .TTATTAC. ------· .TTATT.C. -------· .TTATT.C. -------· .TTATT. C . -------· .TTATT.C. -------..... CC ... TTTTAT .. C. -------..... CC ... TTTTAT .. C. -------.... TC .... TTT ..... C. ------· ... TC. . .. TTT ..... C. ------..... CC ... TTT ..... C. . . ------A. ..... CC ... TTT ..... C. . . ------A. ..... CC ... TTT ..... C. . . ------A. ..... CC ... TTT ..... C. . . ------A. ..... CC ... TTT ..... C. . . ------A. · ... -C. . .. .. TTATT. C. -------..... CC ... TT ... T .. C. . -------A. ..... CC ... TT ... T .. C. . -------A. TTGCCAAC-- - .. C .CCCAA TTTTTT.AC- -------- - 246 SUMMARY In Ethiopia, the planting of exotic species commenced with the introduction of Eucalyptus globulus approximately 110 years ago. Today several different Eucalyptus, Pinus, Cupressus and Australian Acacia species are planted to provide wood for fuel/energy and raw material for furniture and construction. In many areas, people are dependent solely on wood to provide for their basic fuel and construction needs. Despite this, little attention has been given to improve the silvicultural and management practices of plantations in Ethiopia. In particular, disease surveillance and management has never received due attention. The aim of the studies that make up this thesis have been to address the issue of diseases of plantation trees in Ethiopia. Studies have thus focused on the prevalence, identity and importance of major diseases of especially Eucalyptus and Pinus spp. As a background to this thesis, available information on diseases of exotic tree species in Africa has been reviewed and this is presented in the first chapter. In the review, diseases of the major exotic plantation species including Eucalyptus, Pinus, Cupressus and Acacia species have been considered. A section was also devoted to highlight tree diseases reported from Ethiopia. The review shows clearly that there is a great lack of information on diseases of exotic plantation species in most African countries, with the exception of South Africa. This suggests the need for more pathology studies in African plantations. The review also highlights the importance of diseases in plantation forests. In Ethiopia, little information is available on tree diseases in plantation forests. To partially address this problem, disease surveys were conducted in 2000 and 2001 in Eucalyptus and Pinus plantations in South and South Western Ethiopia. The results of this survey showed that a number of pathogens, known from other countries, including Armillaria root rot, stem canker and foliage diseases are found in plantations of Ethiopia. The major diseases discovered during the survey are discussed in Chapter two of this thesis and an indication is given of their impact and distribution. During the disease survey, Armillaria root rot was found to be associated with both exotic and native tree species. Morphological and molecular identification techniques revealed that the Armillaria sp. collected in this study is A. jitscipes. This is discussed in chapter three, where I also provide preliminary data regarding the host range and distribution of Armillaria root rot in Ethiopia. Prior to this study it was suggested that A. mellea is responsible for Armillaria root rot of 247 hard woods in Ethiopia. The current study, however, showed that at least two Armillaria spp., A. mellea and A. Jusicpes are causing Annillaria root rot in the country. Of significance is the fact that A. jilscipes was isolated from two indigenous tree species, A. abyssinica and J excelsa. Chapter four of this thesis deals with the identity of the fungus causing stem canker on Eucalyptus camaldulensis. Disease symptoms identical to those caused by Coniothyrium zuluense were commonly found on E. camaldulensis in restricted areas in Western Ethiopia. The causative agent was detennined based on DNA sequence analysis of the ITS 1, ITS 2 and 5.8S gene region and 0- tubulin genes. According to the phylogenetic tree generated for these sequence data, the Ethiopian Coniothyrium isolates seem to be closely related to C. zuluense, however, the Ethiopian isolates fonned a separate group. This may suggest that C. zuluense represents a species complex, but this needs further investigation. Coniothyrium canker is considered to be one of the most serious diseases of Eucalyptus spp. especially to the sawn timber and construction industry as it weakens and flaws the timber. It occurrence in Ethiopia is, therefore, of great importance. Disease symptoms similar to those of Botryosphaeria canker on Eucalyptus were commonly observed in all the areas where surveys were conducted. Botryosphaeria spp. are known as opportunistic stress related and endophytic pathogens on a wide range of woody plants, worldwide. In Ethiopia, symptoms similar to those associated with Botryosphaeria infection elsewhere, were found in almost all plantations surveyed. The disease was found on several Eucalyptus spp. including E. globulus, E. saligna, E. grandis and E. citriodora. Both morphological and molecular identification techniques were used to detennine the identity of the fungus and the results are presented in chapter five. It was shown that B. parva is responsible for Botryosphaeria stem canker of Eucalyptus spp. in Ethiopia and the pathogenicity of Ethiopian isolates was also tested. This pathogen can have a serious effect on Eucalyptus in Ethiopia, as growing conditions in the country are often harsh and many people rely on coppicing to reproduce their stands. All these factors are conducive to stress and thus to Botryosphaeria infection. Diplodia pinea is a fungus that commonly resides in the cones of Pinus spp. and it tends to move from these sites to infect stems, when trees are under stress. Therefore, isolations were made from Pinus patula cones to detennine whether D. pinea was present in these structures in Ethiopia. Chapter 6 of the thesis provides results of this study. It was expected that D. pinea would be the most common inhabitant of the cones. Contrary to this, a Fusicoccum sp. was found more 248 frequently than D. pinea. The results presented in this chapter show clearly that the A morphotype of D. pinea is found in cones of P. patula in Ethiopia. The Fusicoccum sp. found associated with P. patula cones is most closely related to B. parva. Results of greenhouse inoculation studies showed that both these fungi are pathogenic to Pinus tadea, with D. pinea being the more pathogenic. Serious leaf spot and shoot dieback symptoms were observed on leaves of E. globulus at several localities. The leaf blotch symptoms closely resemble those caused by Mycosphaerella spp. Even though 30 different Mycospha erella spp. are known to be associated with Eucalyptus species worldwide, the cause of Mycosphaerella leaf blotch on E. globulus in Ethiopia is not known. Morphological and DNA based comparisons were used to determine the identity of the species found in Ethiopia and the results are provided in chapter seven. I was thus able to show that three Mycosphaerella spp. namely, M marksii, M. grandis and M nubi/osa are involved in causing Mycosphaerella leaf disease of E. globulus in Ethiopia. This is the first report of these species from Ethiopia and the first report of M grandis from a country other than Australia. The results presented in the various chapters making up this thesis provide the first detailed studies on diseases of plantation trees in Ethiopia. Most tree diseases discussed in the thesis are first reports for the country. The thesis provides information on the identity of the pathogens and their significance in plantation development in Ethiopia. It also highlights the need for adequate management and silvicultural practices, as well as the need for selecting disease tolerant provenances and/or individuals. The information presented in the thesis also expands the host range and geographic distribution of all the pathogens included in the study, giving the study international significance. 249

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Diseases of exotic plantation forestry trees in Ethiopia

Diseases of exotic plantation forestry trees in Ethiopia

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