Botryosphaeriaceae on Syzygium cordatum across a latitudinal gradient in South Africa.

The Botryosphaeriaceae is a family of endophytic fungi, many of which are latent pathogens of woody plants. Although extensively sampled in some parts of the world, little is known regarding their occurrence across different environmental conditions. This study considered the presence of the Botryosphaeriaceae on Syzygium cordatum trees across a latitudinal gradient. We examined the relative importance of different environmental factors on the presence of the Botryosphaeriaceae across this latitudinal gradient. Specifically, Botryosphaeriaceae community composition and species richness were analysed. The optimal growth temperature of the most common Botryosphaeriaceae isolates and its relation to isolate origin was also tested in culture. We identified 14 Botryosphaeriaceae species including seven each of Lasiodiplodia and Neofusicoccum species. The maximum historical temperature emerged as the environmental factor that best predicted the presence of Botryosphaeriaceae species in S. cordatum trees, specifically influencing Botryosphaeriaceae community composition. For all the Botryosphaeriaceae species studied in vitro, temperature strongly influenced mycelial growth and they all had an optimal growth temperature of 25 °C. Contrary to our hypothesis, the optimal growth temperature was not related to isolate origin. These results contribute to understanding the presence of the Botryosphaeriaceae in trees and our ability to detect these latent pathogens.

A new species in the Mycosphaerellaceae from Cecidomyiidae leaf galls on Avicennia marina in South Africa.

During studies to investigate the health of mangrove trees in South Africa, high numbers of Avicennia marina were found with leaf galls caused by unidentified adults and larvae of midges (Cecidomyiidae). Fungal fruiting structures were commonly observed on the abaxial areas of the galls. To determine the identity of the fungi associated with the gall midges, phylogenetic analyses using multigene sequence data were used. The nuclear large subunit (LSU), internal transcribed spacer (ITS), and a portion of the actin gene region (ACT), were amplified and analyzed. The results revealed that the fungal fruiting structures represent a new taxon in the Mycosphaerellaceae described here as Zasmidium mangrovei sp. nov. This is the first report of a species in the Mycosphaerellaceae associated with cecidomyiid leaf galls on A. marina.

Phytophthora Species Associated with Roots of Native and Non-native Trees in Natural and Managed Forests.

Roots act as a biological filter that exclusively allows only a portion of the soil-associated microbial diversity to infect the plant. This microbial diversity includes organisms both beneficial and detrimental to plants. Phytophthora species are among the most important groups of detrimental microbes that cause various soil-borne plant diseases. We used a metabarcoding approach with Phytophthora-specific primers to compare the diversity and richness of Phytophthora species associated with roots of native and non-native trees, using different types of soil inocula collected from native and managed forests. Specifically, we analysed (1) roots of two non-native tree species (Eucalyptus grandis and Acacia mearnsii) and native trees, (2) roots of two non-native tree species from an in vivo plant baiting trial, (3) roots collected from the field versus those from the baiting trial, and (4) roots and soil samples collected from the field. The origin of the soil and the interaction between root and soil significantly influenced Phytophthora species richness. Moreover, species richness and community composition were significantly different between the field root samples and field soil samples with a higher number of Phytophthora species in the soil than in the roots. The results also revealed a substantial and previously undetected diversity of Phytophthora species from South Africa.

Poroid Hymenochaetaceae associated with trees showing wood-rot symptoms in the Garden Route National Park of South Africa.

Poroid Hymenochaetaceae associated with wood rots of trees in three timber-harvesting compartments of the Garden Route National Park (GRNP), South Africa, were investigated using multilocus phylogenetic analyses and morphology of the basidiomes. Results revealed the presence of 10 species belonging to five genera. Six of the species are known, but four are described as new. The known species include Fomitiporia capensis, Fuscoporia gilva, Sanghuangporus microcystideus, Tropicoporus tropicalis, Inonotus rickii, and Inonotus setuloso-croceus. The new species are described as Fomitiporia tsitsikamensis, Fulvifomes elaeodendri, Fuscoporia pulviniformis, and Phellinus guttiformis.

Quantification of Outcrossing Events in Haploid Fungi Using Microsatellite Markers.

Species in genera of the fungal family Ceratocystidaceae are known to have different mating strategies, including heterothallism and homothallism. Of these, species of Ceratocystis, typified by the pathogen Ceratocystis fimbriata all undergo unidirectional mating-type switching. This implies that the pathogens possess the ability to self, but also to undergo sexual outcrossing between isolates of different mating types. In this study, we extended the recently developed microsatellite-based technique to determine the extent to which outcrossing occurs in ascospore masses of haploid fungi to two field collections of Ceratocystis albifundus. In this way, the role of reproductive strategies in shaping population structure and diversity could be better understood. Results showed that a high frequency of outcrossing occurs in isolates of the pathogen from both non-native and native areas. This explains the high level of genetic diversity previously observed in this population despite the fact that this pathogen has the ability to self.

Cryphonectriaceae associated with rust-infected Syzygium jambos in Hawaii.

Syzygium jambos (Myrtales, Myrtaceae) trees in Hawaii are severely affected by a rust disease caused by Austropuccinia psidii (Pucciniales, Sphaerophragmiaceae), but they are commonly co-infected with species of Cryphonectriaceae (Diaporthales). In this study, S. jambos and other trees in the Myrtales were examined on three Hawaiian Islands for the presence of Cryphonectriaceae. Bark samples with fruiting bodies were collected from infected trees and fungi were isolated directly from these structures. Pure cultures were produced and the fungi were identified using DNA sequence data for the internal transcribed spacer (ITS) region, part of the ß-tubulin (BT1) gene and the transcription elongation factor-1α (TEF1) gene. Five species in three genera of Cryphonectriaceae were identified from Myrtaceae tree samples. These included Chrysoporthe deuterocubensis, Microthia havanensis and three previously-unknown taxa described here as Celoporthe hauoliensis sp. nov., Cel. hawaiiensis sp. nov. and Cel. paradisiaca sp. nov. Representative isolates of Cel. hauoliensis, Cel. hawaiiensis, Cel. paradisiaca, Chr. deuterocubensis and Mic. havanensis were used in artificial inoculation studies to consider their pathogenicity on S. jambos. Celoporthe hawaiiensis, Cel. paradisiaca and Chr. deuterocubensis produced lesions on young S. jambos trees in inoculation trials, suggesting that, together with A. psidii, they may contribute to the death of trees. Microsatellite markers were subsequently used to consider the diversity of Chr. deuterocubensis on the Islands and thus to gain insights into its possible origin in Hawaii. Isolates of this important Myrtaceae and particularly Eucalyptus pathogen were found to be clonal. This provides evidence that Chr. deuterocubensis was introduced to the Hawaiian Islands as a single introduction, from a currently unknown source.

Taxonomy and species diversity of Ganoderma species in the Garden Route National Park of South Africa inferred from morphology and multilocus phylogenies.

Ganoderma is a cosmopolitan genus that encompasses species with cultural, economic, and pathogenic importance. Despite the importance of this genus, knowledge pertaining to the species diversity of Ganoderma in South Africa is limited. This study aimed at elucidating the identity and phylogenetic placements of Ganoderma samples obtained during a survey of wood-rotting fungi in the Garden Route National Park (GRNP) of South Africa, supplemented with isolates obtained from other localities across the country. Identification was achieved by means of multilocus phylogenetic inference combined with morphological evaluation. In total, eight distinct species of Ganoderma were recovered from different hosts and localities across the country. Of these, Ganoderma cf. cupreum and Ganoderma cf. resinaceum represent possible new records for South Africa. Two novel species are described, namely, G. eickeri. and G. knysnamense. Ganoderma eickeri, sp. nov., is characterized by a triquetrous and broadly attached basidiome, a sulcate or zonate yellowish brown to brown pilear surface, and ovoid to ellipsoid basidiospores. Ganoderma knysnamense is distinguished by an applanate to ungulate, sometimes convex, dimidiate to broadly attached basidiome, a chocolate-brown pilear surface covered with a hard woody-like crust and ellipsoid, broadly ellipsoid to ovoid basidiospores. The discovery of two new Ganoderma species in this study raises the known Ganoderma species in South Africa to 13.

Nine novel species of Huntiella from southern China with three distinct mating strategies and variable levels of pathogenicity.

The ascomycete genus Huntiella (Microascales) has a cosmopolitan distribution and occurs on a wide range of woody plants. Little is known regarding the identity, diversity, origin, or impact of these fungi in China. Recently, isolates of Huntiella spp. were collected from stumps of freshly felled trees or wounds on plantation-grown Eucalyptus in Guangdong, Guangxi, Fujian, and Hainan provinces of southern China. Additional isolates were obtained from stumps of Acacia confusa near Eucalyptus plantations in Hainan Province. The aim of this study was to identify these Huntiella species and to test their pathogenicity on Eucalyptus seedlings. Morphology and multigene phylogenies of the nuclear rDNA internal transcribed spacer (ITS1-5.8S-ITS2 = ITS) region and partial ß-tubulin (BT1) and translation elongation factor 1α (TEF1α) genes revealed nine previously unknown Huntiella species, eight from Eucalyptus and one from A. confusa. The mating types of these species were determined, showing that seven are heterothallic, one is homothallic, and one is unisexual (MAT1-2-1 gene). Pathogenicity tests showed that the nine Huntiella species can produce lesions on Eucalyptus seedlings, larger than wounds caused by controls on these plants. This study provides a basic understanding of the distribution, diversity, and pathogenicity of Huntiella species in southern China.

Non-Mendelian segregation influences the infection biology and genetic structure of the African tree pathogen Ceratocystis albifundus.

The African fungal tree pathogen, Ceratocystis albifundus, undergoes uni-directional mating type switching, giving rise to either self-fertile or self-sterile progeny. Self-sterile isolates lack the MAT1-2-1 gene and have reduced fitness such as slower growth and reduced pathogenicity, relative to self-fertile isolates. While it has been hypothesized that there is a 1:1 ratio of self-fertile to self-sterile ascospore progeny in relatives of C. albifundus, some studies have reported a significant bias in this ratio. This could be due to the fact that either fewer self-sterile ascospores are produced or that self-sterile ascospores have low viability. We quantified the percentage of self-sterile and self-fertile ascospores from ascospore masses in C. albifundus using real-time PCR. Primers were designed to distinguish between spores that contained the MAT1-2-1 gene and those where this gene had been deleted. A significant bias towards the self-fertile mating type was observed in all single ascospore masses taken from sexual structures produced in haploid-selfed cultures. The same result was observed from a disease outbreak situation in an intensively managed field of cultivated native trees, and this was coupled with very low population diversity in the pathogen. This was in contrast to the results obtained from ascospore masses taken from the crosses performed under laboratory conditions or ascomata on native trees in a non-disease situation, where either self-fertile or self-sterile ascospores were dominant. The results suggest that reproductive strategies play a significant role in the infection biology and genetic structure of C. albifundus populations.

Calonectria species isolated from Eucalyptus plantations and nurseries in South China.

Diseases caused by species of Calonectria (Ca.) represent a serious threat to the growth and sustainability of Eucalyptus plantations in China. Symptoms caused by these fungi mainly include leaf blight on trees in plantations and rotting of stems and leaves in nurseries. Extensive surveys have recently been conducted where Calonectria species were collected in Eucalyptus plantations and nurseries in the FuJian, GuangDong, GuangXi, and YunNan Provinces of South China. Additional isolates were baited from soil samples in the Hong Kong Region. The aim of this study was to identify the 115 Calonectria isolates obtained using comparisons of DNA sequence data for the ß-tubulin (tub2), calmodulin (cmdA), histone H3 (his3) and partial translation elongation factor-1α (tef1) gene regions as well as their morphological features. Seven known species were identified, including Calonectria arbusta, Ca. asiatica, Ca. chinensis, Ca. eucalypti, Ca. hongkongensis, Ca. mossambicensis and Ca. pentaseptata. In addition, six novel taxa were collected and are described here as Ca. aciculata, Ca. honghensis, Ca. lantauensis, Ca. pseudoturangicola, Ca. pseudoyunnanensis, and Ca. yunnanensis spp. nov. Overall, the results reflect a high diversity of Calonectria species in China.

Overlap of latent pathogens in the Botryosphaeriaceae on a native and agricultural host.

Some species of the Botryosphaeriaceae are capable of infecting a broad range of host plants. We studied the species diversity of Botryosphaeriaceae associated with marula (Sclerocarya birrea subsp. caffra, Anacardiaceae) trees in South Africa over two seasons, as well as species common to both S. birrea and adjacent mango (Mangifera indica, Anacardiaceae) trees in a subset of sites. Gene flow amongst populations of Botryosphaeriaceae shared on these tree species was tested using microsatellite markers. Twelve species were identified from S. birrea and eleven species were found on M. indica trees. From isolations done in 2006, the dominant species on S. birrea was Neofusicoccum vitifusiforme, while N. parvum was the dominant species isolated from M. indica. Neofusicoccum parvum was dominant in isolations from both hosts in 2012. Isolates of Botryosphaeria fabicerciana, Lasiodiplodia mahajangana, L. pseudotheobromae, L. theobromae, N. mediterraneum, and N. umdonicola were also collected from both hosts. Population genetic analyses on isolates of N. parvum suggested that three populations were present, each comprising isolates from both hosts. There was significant gene flow between N. parvum populations on these hosts. This ability to infect multiple hosts and to migrate amongst them facilitates the establishment and spread of species and genotypes of the Botryosphaeriaceae, such as N. parvum, in new areas.

Endophytic Botryosphaeriaceae, including five new species, associated with mangrove trees in South Africa.

Little is known regarding the fungi, especially fungal pathogens, associated with mangroves in Africa. This includes fungi in the Botryosphaeriaceae that comprise numerous opportunistic, stress-associated pathogens often associated with trees affected by environmental and anthropogenically generated stresses, such as those affecting mangroves. We investigated the occurrence of endophytic Botryosphaeriaceae along the entire distribution of mangroves in South Africa. Asymptomatic branches were collected from ten localities and six mangrove species. Isolates resembling species of Botryosphaeriaceae were identified based on multi-gene sequence data of the internal transcribed spacer regions (ITS), including the 5.8S nrRNA, the beta-tubulin (tub2), partial translation elongation factor 1-alpha (tef1-α), and DNA-directed RNA polymerase II second largest subunit (rpb2) gene regions. Inoculation trials were conducted on healthy branches of Avicennia marina and Bruguiera gymnorrhiza to evaluate the potential pathogenicity of the collected species. Fourteen species in the Botryosphaeriaceae belonging to four genera, Botryosphaeria, Diplodia, Lasiodiplodia, and Neofusicoccum were collected, including five new species. Neofusicoccum was the most prevalent genus followed by Lasiodiplodia, with species of Diplodia and Botryosphaeria being the least frequent. The inoculation studies revealed that one of the new species, Lasiodiplodia avicenniae is highly pathogenic to A. marina and could pose a threat to the health of these trees.

Phylogenetic species recognition and hybridisation in Lasiodiplodia: A case study on species from baobabs.

Lasiodiplodia species (Botryosphaeriaceae, Ascomycota) infect a wide range of typically woody plants on which they are associated with many different disease symptoms. In this study, we determined the identity of Lasiodiplodia isolates obtained from baobab (Adansonia species) trees in Africa and reviewed the molecular markers used to describe Lasiodiplodia species. Publicly available and newly produced sequence data for some of the type strains of Lasiodiplodia species showed incongruence amongst phylogenies of five nuclear loci. We conclude that several of the previously described Lasiodiplodia species are hybrids of other species. Isolates from baobab trees in Africa included nine species of Lasiodiplodia and two hybrid species. Inoculation trials with the most common Lasiodiplodia species collected from these trees produced significant lesions on young baobab trees. There was also variation in aggressiveness amongst isolates from the same species. The apparently widespread tendency of Lasiodiplodia species to hybridise demands that phylogenies from multiple loci (more than two and preferably four or more) are compared for congruence prior to new species being described. This will avoid hybrids being incorrectly described as new taxa, as has clearly occurred in the past.

Ecology and population structure of a tree wound-infecting fungus in a native South African forest environment.

Ceratocystis tsitsikammensis was first isolated from bark harvesting wounds on two indigenous tree species in the Afromontane forests of the Western Cape Province of South Africa. Inoculation studies indicated that it is a potential pathogen of native Rapanea melanophloeos trees. In this study, we investigated the distribution, ecology and biology of C. tsitsikammensis in the Garden Route National Park of South Africa. Isolates were obtained from wounds on R. melanophloeos, three non-native hosts as well as from nitidulid and staphylinid beetles visiting wounds on these trees. The genetic diversity and population biology of the fungus was examined using microsatellite markers. Its mating strategy was also determined by amplifying its mating type genes and the fungus was shown to be homothallic. Despite the homothallic nature of the fungus, high levels of random mating and absence of genetic structure was found in the investigated population, suggesting a strong effect of gene flow, probably linked to insect dispersal. The gene diversity of C. tsitsikammensis was similar to that of a related fungus, Ceratocystis albifundus, that is known to be native in Africa. This, together with the fact that C. tsitiskamensis is not known elsewhere, within or outside South Africa, suggests that it is native and endemic to the Cape Afromontane region.

Ophiostomatoid fungi associated with mangroves in South Africa, including Ophiostoma palustre sp. nov.

Mangrove trees are continuously under stress due to environmental and/or anthropogenic pressures, which expose them to attack by pathogens, compromising their survival. Ophiostomatoid fungi cause sap stain and diseases of a wide spectrum of tree species globally. These fungi infect trees through natural, insect, animal and/or human made wounds. During routine surveys of mangrove trees in South Africa, wounds on branches and stems of Avicennia marina were regularly monitored for the presence of ophiostomatoid fungi at ten study sites in the country. The stems of four mangrove species, A. marina, Bruguiera gymnorrhiza, Rhizophora mucronata and Barringtonia racemosa were also wounded and evaluated for the appearance of these fungi. Ophiostomatoid fungi were obtained from the mangrove associate B. racemosa, but not from any of the true mangroves. Analyses of DNA sequence data for the internal transcribed spacer, ß-tubulin, calmodulin and translation elongation factor gene regions revealed that the fungi isolated from the wounds on B. racemosa belong to three species in the Ophiostomataceae, including a new taxon described here as Ophiostoma palustre sp. nov. These results suggest that the mangrove associate B. racemosa is more prone to colonization by ophiostomatoid fungi than the true mangroves.

Endophytic Cryphonectriaceae on native Myrtales: Possible origin of Chrysoporthe canker on plantation-grown Eucalyptus.

Chrysoporthe austroafricana (Cryphonectriaceae) is a damaging canker pathogen on Eucalyptus species in Southern Africa. Recent studies have shown that the fungus occurs on native Syzygium species and that it has apparently undergone a host range expansion from these native trees to infect non-native Eucalyptus. The aim of this study was to consider whether Chr. austroafricana and other Cryphonectriaceae might exist as endophytes in native Myrtaceae, providing a source of inoculum to infect non-native Myrtales. Healthy branches were collected from Myrtaceae in Mozambique, incubated in florist foam, allowed to dry gradually and monitored for the appearance of fruiting bodies resembling species in the Cryphonectriaceae. Isolates were identified based on DNA sequence data. Two species in the Cryphonectriaceae were obtained, representing the first evidence that species in the Cryphonectriaceae occur as endophytes on native Myrtales, thus providing a source of inoculum to infect non-native and susceptible trees. This has important implications regarding the movement of planting stock used by ornamental tree and forestry enterprises.

The genetic landscape of Ceratocystis albifundus populations in South Africa reveals a recent fungal introduction event.

Geographical range expansion or host shifts is amongst the various evolutionary forces that underlie numerous emerging diseases caused by fungal pathogens. In this regard, Ceratocystis albifundus, the causal agent of a serious wilt disease of Acacia mearnsii trees in Africa, was recently identified killing cultivated Protea cynaroides in the Western Cape (WC) Province of South Africa. Protea cynaroides is an important native plant in the area and a key component of the Cape Floristic Region. The appearance of this new disease outbreak, together with isolates of C. albifundus from natural ecosystems as well as plantations of nonnative trees, provided an opportunity to consider questions relating to the possible origin and movement of the pathogen in South Africa. Ten microsatellite markers were used to determine the genetic diversity, population structure, and possible gene flow in a collection of 193 C. albifundus isolates. All populations, other than those from the WC, showed high levels of genetic diversity. An intermediate level of gene flow was found amongst populations of the pathogen. The results suggest that a limited number of individuals have recently been introduced into the WC, resulting in a novel disease problem in the area.

Novel ophiostomatalean fungi from galleries of Cyrtogenius africus (Scolytinae) infesting dying Euphorbia ingens.

Euphorbia ingens trees have been dying in large numbers in the Limpopo Province of South Africa for approximately 15 years. The ambrosia beetle Cyrtogenius africus is often found infesting diseased and dying trees. The aim of this study was to identify the ophiostomatoid fungi occurring in the galleries of C. africus. Logs infested with this beetle were collected from the KwaZulu-Natal, Limpopo, Mpumalanga, and North West Provinces of South Africa. Fungi belonging to the Ophiostomatales were identified based on morphology and comparison of sequence data for the ß-tubulin, ITS1-5.8S-ITS2 and LSU gene regions. A novel species of Ophiostoma and a novel genus in the Ophiostomatales were identified. Inoculation studies with these fungi produced lesions in the branches of healthy E. ingens trees.

Three genetic groups of the Eucalyptus stem canker pathogen Teratosphaeria zuluensis introduced into Africa from an unknown source.

The Eucalyptus stem canker pathogen Teratosphaeria zuluensis was discovered in South Africa in 1988 and it has subsequently been found in several other African countries as well as globally. In this study, the population structure, genetic diversity and evolutionary history of T. z uluensis were analysed using microsatellite markers to gain an enhanced understanding of its movement in Africa. Isolates were collected from several sites in Malawi, Mozambique, Uganda and Zambia. Data obtained were compared with those previously published for a South African population. The data obtained from 334 isolates, amplified across eight microsatellite loci, were used for assignment, differentiation and genetic diversity tests. STRUCTURE analyses, θ st and genetic distances revealed the existence of two clusters, one dominated by isolates from South Africa and the other by isolates from the Zambezi basin including Malawi, Mozambique and Zambia. High levels of admixture were found within and among populations, dominated by the Mulanje population in Malawi. Moderate to low genetic diversity of the populations supports the previously held view that the pathogen was introduced into Africa. The clonal nature of the Ugandan population suggests a very recent introduction, most likely from southern Africa.