PATHOGENS ASSOCIATED WITH DISEASES. OF PROTEA, LEUCOSPERMUM AND LEUCADENDRON SPP. Lizeth Swart Thesis presented in partial fulfillment of the requirements for the degree of Master of Science in Agriculture at the University of Stellenbosch Supervisor: Prof. P. W. Crous Decem ber 1999 Stellenbosch University https://scholar.sun.ac.za DECLARATION 1, the undersigned, hereby declare that the work contained in this thesis is my own original work and has not previously in its entirety or in part been submitted at any university for a degree. SIGNATURE: DATE: Stellenbosch University https://scholar.sun.ac.za PATHOGENS ASSOCIATED WITH DISEASES OF PROTEA, LEUCOSPERMUM ANDLEUCADENDRONSPP. SUMMARY The manuscript consists of six chapters that represent research on different diseases and records of new diseases of the Proteaceae world-wide. The fungal descriptions presented in this thesis are not effectively published, and will thus be formally published elsewhere in scientific journals. Chapter one is a review that gives a detailed description of the major fungal pathogens of the genera Protea, Leucospermum and Leucadendron, as reported up to 1996. The pathogens are grouped according to the diseases they cause on roots, leaves, stems and flowers, as well as the canker causing fungi. In chapter two, several new fungi occurring on leaves of Pro tea, Leucospermum, Telopea and Brabejum collected from South Africa, Australia or New Zealand are described. The following fungi are described: Cladophialophora proteae, Coniolhyrium nitidae, Coniothyrium proteae, Coniolhyrium leucospermi,Harknessia leucospermi, Septoria prolearum and Mycosphaerella telopeae spp. nov. Furthermore, two Phylloslicla spp., telopeae and owaniana are also redecribed. The taxonomy of the Eisinoe spp. associated with scab disease of Proteaceae in Australia, California, South Africa and Zimbabwe is elucidated in chapter three. General morphology, symptomatology and phylogenetic analysis based on random amplified polymorphic DNA (RAPD) profiles and DNA sequence of the S.8S rDNA gene and its flanking ITS 1 and ITS2 regions were used. The study provides the first evidence that several distinct Eisinoe spp. are associated with scab disease of Proteaceae. The isolates from Leucospermum, Protea and Banksia represent three distinct species. The isolates from Protea in Zimbabwe represent an additional species. The isolates from Leucadendron, Leucospermum and Serruria in South Africa and Australia, and the isolates from Leucospermum in California and Zimbabwe are representative of the same species. Stellenbosch University https://scholar.sun.ac.za In chapter four, fungal endophytes occurring in leaves and stems of a species of Protea, Leucospermum and Leucadendron were investigated in three localities in the Western Cape province. The aim of the study was to determine if Botryosphaeria proteae was an endophyte of Proteaceae, and is so, how the role of water stress would influence canker development. B. proteae was routinely isolated as an endophyte but was not regarded as a dominant taxon. Inoculation studies were done on non-stressed plants, as well as plants with a leaf water potential of -1.0 MPa (moderately stressed) and -2.0 MPa (severely stressed). From the results of the study it was concluded that Botryosphaeria proteae is primarily an endophyte and can cause leaf necrosis of Protea, and is not a serious stem canker pathogen. A new disease of cultivated Pro/ea in southern Africa, Fusarium wilt, is described in chapter five. The disease is caused by Fusarium oxysporum. It occurs on various Pro/ea cultivars in the North-Western province and in Zimbabwe. Disease symptoms first become visible as necrotic leaves on infected plants. Subsequently, a dark lesion develops from the roots along the stem, usually visible only on one side of the stem. The vascular tissue is discoloured, leading to branch die-back and plant death. Glasshouse trials were conducted to prove Koch's postulates on six Pro/ea cultivars. Forty-five rooted plants of each of six cultivars were inoculated with isolates of F. oxysporum derived from the same cultivar. Disease symptoms similar to those occurring in the field, developed 6 weeks after inoculation. This is the first record of Fusarium wilt on Pro/ea plants. In chapter six, Pestalotiopsis leaf spot disease of Proteaceae in Zimbabwe is described. Pes/a/optiopsis Steyart causes necrotic leaf spots in Leucospermum R. Br. and Pro/ea L. species in Zimbabwe. Inoculation studies conducted to prove pathogenicity, confirmed the Pes/a/otiopsis sp. as the causal agent of the disease. A description of the fungus is given, and it is compared to other Pestaiotiopsis spp. associated with Proteaceae. In conclusion, the present study has shown that several umque species of fungal pathogens are associated with the Proteaceae. Several of these have proven to be new to Stellenbosch University https://scholar.sun.ac.za science, and are described in this thesis. It is clear, however, that the taxonomy of some of these pathogens, their host range and distribution needs to be further investigated. Furthermore, much more research needs to be done on the biology, epidemiology and control of the diseases of the Proteaceae. Stellenbosch University https://scholar.sun.ac.za PATHOGENS ASSOCIATED WITH DISEASES OF PROTEA, LEUCOSPERMUM . AND LEUCADENDRON SPP. OPSOMMING Die tesis bestaan uit ses hoofstukke wat handel oor navorsing op verskillende siektes en nuwe rekords van siektes van die Proteaceae wereldwyd. Die swambeskrywings wat in hierdie tesis verskyn is nie effektief gepubliseer nie, en sal dus elders in wetenskaplike joernaie gepubliseer word. Hoofstuk een is 'n oorsig wat 'n in diepte beskrywing gee van die belangrikste swampatogene van die genera Protea, Leucospermum en Leucadendron, soos aangeteken tot 1996. Die patogene word gegroepeer volgens die siektes wat hulle op die wortels, blare, stamme en blomme veroorsaak, asook die kanker veroorsakende swamme. In hoofstuk twee word verskeie nuwe swamme wat op blare van Protea, Leucospermum, Te/opea en Brabejum voorkom, en versamel is in Suid-Afrika, Australie of New Zealand, beskryf. Die volgende swamme word beskryf: C/adophia/ophora proteae, Coniothyrium nitidae, Coniothyrium proteae, Coniothyrium /eucospermi, Harknessia /eucospermi, Septoria protearum en Mycosphaere//a telopeae spp. nov. Verder word twee Phy/Josticta spp., te/opeae en owaniana herbeskryf. Die taksonomie van die E/sinoe species wat geassosieer word met skurfbassiekte van Proteaceae in AustraIie, Kalifornie, Suid-Afrika en· Zimbabwe, word in hoofstuk drie vasgestel. AJgemene morfologie, simptomatologie en filogenetiese analise, gebaseer op RAPD-analise en DNA volgorde bepaling van die S.8S rDNA geen en sy ITSI en ITS2 areas is gebruik. Die studie verskaf die eerste bewys dat verskeie species van E/sinoe geassosieer word met skurfbassiekte van Proteaceae. Die isolate van Leucospermum, Protea en Banksia verteenwoordig drie verskiIlende species. Die isolate van Protea in Zimbabwe verteenwoordig 'n verdere species. Die isolate van Leucadendron, Leucospermum en Serruria in Suid-Afrika en AustraIie, en die isolate van Leucospermum in Kalifornie en Zimbabwe verteenwoordig dieselfde species. Stellenbosch University https://scholar.sun.ac.za In hoofstuk vier is die swam endofiete wat in die blare en stamme van 'n species van· Protea, Leucospermum en Leucadendroll in drie lokaliteite in die Wes-Kaap voorkom, ondersoek. Die doel van die studie was om vas te stel of Botryosphaeria proteae 'n endofiet van die Proteaceae is, en indien so, vas te stel hoe waterstres kanker­ ontwikkeling sal belnvloed. B. proteae is gereeld as endofiet gelsoleer, maar is nie beskou as 'n dominante taxon nie. Inokulasie studies is op nie gestreste plante, asook plante met 'n blaarwaterpotensiaal van -1.0 MPa (gematig gestres) en -2.0Mpa (uiters gestres), uitgevoer. Uit die resultate van die studie kan die gevolgtrekking gemaak word dat Botryosphaeria proteae hoofsaaklik 'n endofiet van Protea is en blaamekrose kan veroorsaak, en nie 'n belangrike stamkankerpatogeen is nie. 'n Nuwe siekte van verboude Protea in suidelike Afrika, Fusarium verwelk, word in hoofstuk vyf beskryf Die siekte word deur Fusarium oxysporum veroorsaak' Dit kom op verskeie Protea kultivars in die Noord-westelike provinsie en in Zimbabwe voor. Siekte-simptome word sigbaar as nekrotiese blare op die geYnfekteerde plante. Kort daarna ontwikkel 'n donker letsel vanaf die wortels opwaarts langs die stam, gewoonlik sigbaar slegs aan een kant van die stam. Die vaskulete weefsel is verkleur en lei tot takterugsterwing en plantafsterwing. Glashuisproewe is uitgevoer ten einde Koch se postulate op ses Protea kultivars te bewys. Vyf-en -veertig gewortelde plante van elk van ses kultivars is gelnokuleer met isolate van F. oxysporum van dieselfde kultivar. Siektesimptome soortegelyk aan die in die veld, het ses weke na inokulasie ontwikkel. Dit is die eerste rekord van Fusarium verwelk op Protea plante. In hoofstuk ses word Pestaiotiopsis blaarvlek op Proteaceae in Zimbabwe beskryf Pesta/otiopsis Steyart veroorsaak nekrotiese blaarvlekke in Leucospermum R. Br. en Protea L. species in Zimbabwe. Inokulasiestudies, uitgevoer ten einde patogenisiteit te bewys, bevestig Pesta/otiopsis as die veroorsakende agent van die siekte. 'n Beskrywing van die swam word gegee, en dit word vergelyk met ander Pesta/otiopsis spp. wat al met die Proteaceae geassosieer is. Stellenbosch University https://scholar.sun.ac.za Ten slotte, die studie het getoon