Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Very little is known about the diversity of fungi associated with Afromontane forests of the Cape Floristic Region (CFR) of South Africa. The ophiostomatoid fungi include many species, some known as pathogens in the CFR, while others are well-known saprophytes important in wood degradation. This study focused on the biodiversity and ecology of tree-associated ophiostomatoid fungi (Ophiostomatales) in the CFR. In addition to this, mites and subcortical beetles associated with the CFR trees were collected, regardless of whether they were associated with ophiostomatoid fungi or not. A relatively high diversity of ophiostomatoid fungi were collected from native trees, ten of which were newly described here. Three further fungal species, two of which are probably new to science, were also collected from exotic Pinus species growing in these forests. Four Ophiostomatales species (including three newly described species) were associated with subcortical beetles on Rapanea melanophloeos and Olea capensis ssp. macrocarpa. These were Sporothrix pallida, Sporothrix aemuluphilus, Raffaelea scabbardiae and Raffaelea rapaneae, associated with the beetles Lanurgus sp. 1, Ctonoxylon sp. 1, Xyleborinus aemuluphilus and a Platypodinae species. This represents a first study to explore the associations between subcortical beetles and ophiostomatoid fungi on native trees in the CFR. In addition to fungi associated with subcortical beetles, several members of the Ophiostomatales associated with wounds on Rapanea melanophloes trees were also collected. These included Ophiostoma stenoceras, Sporothrix reniformis, S. rapaneae, S. lunateae and S. noisomeae. All but O. stenoceras were new to science, and were formally described here. All of these wound-associated species from R. melanophloeos belong to the Sporothrix schenckii – O. stenoceras complex, except for S. noisomeae that was provisionally placed in the S. lignivora complex. Besides fungal taxa collected from wounds on Rapanea melanophloeos, other fungi were also collected from wounds on other host trees species. Three more previously undescribed ophiostomatoid fungal species were collected from this niche. They included Sporothix capensis collected from O. capensis ssp. macrocarpa, Graphilbum roseus collected from many different, unrelated host trees and Graphium ilexiense (Microascales), isolated from wounds on Ilex mitis. The latter represented the first isolation of an ophiostomatoid fungus from this host tree species. Two possibly new fungal species (Sporothrix sp. 1, Ceratocystiopsis sp. 1) and Ophiostoma ips, associated with three bark beetles (Orthotomicus erosus, Hylurgus ligniperda and Hylastes angustatus), were collected from Pinus. Several fungal species were collected from both native trees and non-native trees. These included Sporothrix fusiforme from Brabejum stellatifolium and Acacia mearnsii, O. quercus and O. pluriannulatum-like fungus from several native trees and from A. mearnsii. This suggests a possibility for host shifting of some of these fungi between native and non-native hosts or even between different native hosts. Eight non-ophiostomatoid fungi associated subcortical beetles taxa were found also to infest native trees in the Afromontane forests and in total more than 4500 beetle individuals were collected. Some species of ophiostomatoid fungi collected in this study were found to be associated with other arthropods such as mites. Four phoretic mites species associated with ophiostomatoid fungi (Dendrolaelaps quadrisetus, Histiogaster sp. 3, Elattoma sp. 1 & 2) were collected. In addition, sixteen species of tree wound-associated mites were collected from 12 native trees. Of these, nine were associated with several ophiostomatoid fungi (Graphilbum roseus, O. pluriannulatum-like, O. quercus) that were isolated from several different host trees. This suggests that they may aid in the transport of these fungi from one host species to another. The possible consequences of transfers of Ophiostomatales species between hosts were tested using pathogenicity tests, which highlighted that some fungi are pathogenic on several different trees. Transfers seemed most likely in fungal species isolated from wounds, especially those associated with mites, because the mites may aid in the vectoring of these. When phoretic mites were tested for their specificity to their vector beetles, they proved to be highly specific. Although some of the fungi associated with these mites and their sub-cortical beetles were also pathogenic, it is less likely for these fungi to be transferred to other host tree species due to the high specificity of their arthropod associates. This study represents one of a few studies that focused on ophiostomatoid fungi, subcortical beetles and mites associated with trees in the Afromontane forests of South Africa. Although we collected a high diversity of Ophiostomatales members, many more still await discovery. It is recommended that future studies focus on the complex inter-organismal interactions in many of the systems uncovered in this study. / AFRIKAANSE OPSOMMING: Baie min is bekend oor die diversiteit van fungi wat met die Afromontane woude van die Kaapse Floristiese Streek (KFS) van Suid Afrika geassosieer is. Die ophiostomatoïde fungi sluit baie spesies in, sommiges bekend as patogene in die KFS, terwyl ander bekende en belangrike saprofiete in houtdegradasie is. Hierdie studie het op die biodiversiteit en ekologie van die boom-geassosieerde ophiostomatoïde fungi (Ophiostomatales) in die KFS gefokus. Daarbenewens is myte en subkortikale kewers wat met die KFS bome geassosieer word ook versamel, ongeag of hulle geassosieerd was met ophiostomatoïde fungi of nie. „n Relatief hoë diversiteit van ophiostomatoïde fungi is van inheemse bome versamel, tien waarvan hier nuut beskryf is. Drie verdere fungi spesies, twee waarvan ook waarskynlik nuut is tot die wetenskap, is ook vanaf Pinus spesies versamel wat in hierdie woude gegroei het. Vier Ophiostomatales spesies (insluitend drie nuut beskryfde spesies) wat met subkortikale kewers op Rapanea melanophloeos en Olea capensis L. ssp. macrocarpa geassosieer is, is ook versamel. Hulle was Sporothrix pallida, Sporothrix aemuluphilus, Raffaelea scabbardiae en Raffaelea rapaneae, geassosieer met die kewers Lanurgus sp. 1, Ctonoxylon sp. 1, Xyleborinus aemuluphilus en „n Platypodinae spesie. Hierdie verteenwoordig die eerste studie wat die assosiasies tussen subkortikale kewers en ophiostomatoïde fungi op inheemse bome in die KFS ondersoek. Addisioneel tot fungi geassosieer met die subkortikale kewers, is verskeie lede van die Ophiostomatales vanaf wonde op Rapanea melanophloes bome versamel. Hulle sluit in Ophiostoma stenoceras, Sporothrix reniformis, S. rapaneae, S. lunateae en S. noisomeae. Almal behalwe O. stenoceras was nuut tot die wetenskap, en is hier formeel beskryf. Al hierdie wond-geassosieerde spesies vanaf R. melanophloeos behoort aan die Sporothrix schenckii – O. stenoceras kompleks, behalwe vir S. noisomeae wat voorlopig in die S. lignivora kompleks geplaas is. Benewens fungi taxa wat van die wonde op Rapanea melanophloes versamel is, is ander fungi ook vanaf die wonde op ander gasheer boom spesies versamel. Drie verdere ophiostomatoïde fungus spesies is in hierdie nis versamel. Hulle sluit in Sporothix capensis wat vanaf O. capensis ssp. macrocarpa versamel is, Graphilbum roseus wat vanaf baie verskillende, onverwante gasheer bome versamel is en Graphium ilexiense (Microascales), wat vanaf wonde op Ilex mitis versamel is. Laasgenoemde verteenwoordig die eerste isolasie van „n ophiostomatoïde fungus vanaf hierdie gasheer boom spesie. Twee moontlik nuwe fungus spesies (Sporothrix sp. 1, Ceratocystiopsis sp. 1) en Ophiostoma ips, geassosieer met drie baskewers (Orthotomicus erosus, Hylurgus ligniperda en Hylastes angustatus) is vanaf Pinus versamel. Verskeie fungi spesies is van beide inheemse en nie-inheemse bome versamel. Hulle het Sporothrix fusiforme vanaf Brabejum stellatifolium en Acacia mearnsii, O. quercus en O. pluriannulatum-like fungus vanaf verskeie inheemse bome en vanaf A. mearnsii ingesluit. Dit suggereer die moontlikheid van gasheer-skuiwing van sommige van hierdie fungi tussen inheemse en uitheemse gashere of selfs tussen verskillende inheemse gashere. Agt nie- ophiostomatoïde geassosieerde subkortikale kewers was ook versamel en in totaal is meer as 4500 kewer indiwidue versamel. Sommige ophiostomatoïde fungus spesies wat in hierdie studie versamel is, was met ander geleedpotiges soos myte geassosieer. Vier foretiese myt spesies wat met ophiostomatoïde fungi geassosieer is (Dendrolaelaps quadrisetus, Histiogaster sp. 3, Elattoma sp. 1 & 2), is versamel. Nege addisioneële myt spesies was met verskeie ophiostomatoïde spesies vanaf verskeie boomspesies geassosieer (Graphilbum roseus, O. pluriannulatum-like, O. quercus). Dit suggereer dat myte die vervoer van hierdie fungi van een gasheer spesie na die ander mag bewerkstellig. Die moontlike gevolge van die oordrag van Ophiostomatales spesies tussen gashere is getoets deur patogeniteitstoetse. Dit het beklemtoon dat sommige fungi patogenies is op verskeie onverwante boomspesies. Oordraag van spesies is mees waarskynlik in fungi spesies wat vanaf wonde geisoleer is, veral dié wat met myte geassosieer is, want die myte mag hierdie fungi help vervoer. Toe foretiese myte getoets is vir hulle spesifisiteit tot hulle vektore, is hulle hoogs spesifiek bevind. Alhoewel sommige fungi wat met hierdie myte en hulle geassosieerde kewers geassosieer word wel patogenies is, is dit minder waarskylik dat hulle na ander gasheer bome sal verskuif as gevolg van die hoë spesifisiteit van hulle geleedpotige assosiate. Hierdie studie verteenwoordig een van net enkele studies gefokus op ophiostomatoïde fungi, subkortikale kewers en myte wat met bome van die Afromontane woude van Suid-Afrika geassosieer is. Alhoewel ons „n hoë diversiteit van Ophiotomatale lede versamel het, wag baie meer fungi spesies waarskynlik nog op ontdekking. Daar word voorgestel dat toekomstige studies fokus op die komplekse inter-organismiese interaksies in baie van die sisteme wat in hierdie studie blootgelê is.

This work presents a survey of blue-stain fungi found in Northwestern Ontario, characterization of a homing endonuclease gene within Grosmannia piceiperda and finally an examination of the introns and homing endonuclease genes found in the large ribosomal subunit gene in species of Ceratocystis; using molecular techniques and phylogenetic analysis, we studied the molecular evolution of these mobile genetic elements. The blue-stain fungi of Northwestern Ontario were identified based on phylogenic analysis of rDNA internal transcribed spacer region sequences. This data was supplemented with morphological characteristics of the fungal cultures. The second project was an examination of a LAGLIDADG homing endonuclease and its IC2 group I intron. This intron is uniquely positioned within the group I intron-encoded rps3 gene of the large subunit ribosomal RNA gene. The final chapter is an investigation of the large subunit ribosomal RNA gene in species of Ceratocystis. The 3’ segment of this gene contains several novel introns and homing endonuclease genes. There is also much diversity between strains despite their close relation on the rDNA internal transcribed spacer region phylogenetic tree. Further, our data also suggest that the single motif LAGLIDADG homing endonuclease of the rDNA mL1923 intron is likely to be an ancestor to other homing endonucleases in the area. The results of these studies demonstrate the role that these elements play in the genetic diversity observed in the blue-stain fungi.

This work presents a survey of blue-stain fungi found in Northwestern Ontario, characterization of a homing endonuclease gene within Grosmannia piceiperda and finally an examination of the introns and homing endonuclease genes found in the large ribosomal subunit gene in species of Ceratocystis; using molecular techniques and phylogenetic analysis, we studied the molecular evolution of these mobile genetic elements. The blue-stain fungi of Northwestern Ontario were identified based on phylogenic analysis of rDNA internal transcribed spacer region sequences. This data was supplemented with morphological characteristics of the fungal cultures. The second project was an examination of a LAGLIDADG homing endonuclease and its IC2 group I intron. This intron is uniquely positioned within the group I intron-encoded rps3 gene of the large subunit ribosomal RNA gene. The final chapter is an investigation of the large subunit ribosomal RNA gene in species of Ceratocystis. The 3’ segment of this gene contains several novel introns and homing endonuclease genes. There is also much diversity between strains despite their close relation on the rDNA internal transcribed spacer region phylogenetic tree. Further, our data also suggest that the single motif LAGLIDADG homing endonuclease of the rDNA mL1923 intron is likely to be an ancestor to other homing endonucleases in the area. The results of these studies demonstrate the role that these elements play in the genetic diversity observed in the blue-stain fungi.