Efficacy of Commercially Available Entomopathogenic Agents against the Polyphagous Shot Hole Borer in South Africa.

The invasive ambrosia beetle, Euwallacea fornicatus, was first reported in South Africa in 2018. The beetle has now spread to eight provinces of the country and has had a devastating impact on both native and non-native tree species. This is especially true for trees located in urban and peri-urban environments. Recent predictions are that the South African E. fornicatus invasion will cost an estimated ZAR 275 billion (approx. USD 16 billion) if it continues to spread uncontrollably, justifying an urgent need for its effective management in the country. One option is biological control, which is preferred over the use of chemicals due to its lower environmental impact. We tested two broad-spectrum fungal entomopathogenic agents, Eco-Bb® and Bio-Insek, which are commercially available in South Africa, for efficacy against E. fornicatus. Initial laboratory assays yielded promising results. However, beetle infestation trials using treated pieces of woody castor bean stems showed little effect on beetle survival and reproduction.

Ecological disequilibrium drives insect pest and pathogen accumulation in non-native trees.

Non-native trees have become dominant components of many landscapes, including urban ecosystems, commercial forestry plantations, fruit orchards, and as invasives in natural ecosystems. Often, these trees have been separated from their natural enemies (i.e. insects and pathogens) leading to ecological disequilibrium, that is, the immediate breakdown of historically co-evolved interactions once introduced into novel environments. Long-established, non-native tree plantations provide useful experiments to explore the dimensions of such ecological disequilibria. We quantify the status quo of non-native insect pests and pathogens catching up with their tree hosts (planted Acacia, Eucalyptus and Pinus species) in South Africa, and examine which native South African enemy species utilise these trees as hosts. Interestingly, pines, with no confamilial relatives in South Africa and the longest residence time (almost two centuries), have acquired only one highly polyphagous native pathogen. This is in contrast to acacias and eucalypts, both with many native and confamilial relatives in South Africa that have acquired more native pathogens. These patterns support the known role of phylogenetic relatedness of non-native and native floras in influencing the likelihood of pathogen shifts between them. This relationship, however, does not seem to hold for native insects. Native insects appear far more likely to expand their feeding habits onto non-native tree hosts than are native pathogens, although they are generally less damaging. The ecological disequilibrium conditions of non-native trees are deeply rooted in the eco-evolutionary experience of the host plant, co-evolved natural enemies, and native organisms from the introduced range. We should expect considerable spatial and temporal variation in ecological disequilibrium conditions among non-native taxa, which can be significantly influenced by biosecurity and management practices.

Evolutionary dynamics of tree invasions: complementing the unified framework for biological invasions.

Evolutionary processes greatly impact the outcomes of biological invasions. An extensive body of research suggests that invasive populations often undergo phenotypic and ecological divergence from their native sources. Evolution also operates at different and distinct stages during the invasion process. Thus, it is important to incorporate evolutionary change into frameworks of biological invasions because it allows us to conceptualize how these processes may facilitate or hinder invasion success. Here, we review such processes, with an emphasis on tree invasions, and place them in the context of the unified framework for biological invasions. The processes and mechanisms described are pre-introduction evolutionary history, sampling effect, founder effect, genotype-by-environment interactions, admixture, hybridization, polyploidization, rapid evolution, epigenetics, and second-genomes. For the last, we propose that co-evolved symbionts, both beneficial and harmful, which are closely physiologically associated with invasive species, contain critical genetic traits that affect the evolutionary dynamics of biological invasions. By understanding the mechanisms underlying invasion success, researchers will be better equipped to predict, understand, and manage biological invasions.

Ganoderma species, including new taxa associated with root rot of the iconic Jacaranda mimosifolia in Pretoria, South Africa.

Jacaranda mimosifolia trees have been progressively dying due to Ganoderma root and butt rot disease in Pretoria (the "City of Jacarandas") for many years. Ganoderma austroafricanum was described from these trees previously but this was based on a single collection. This study treats a substantially expanded collection of isolates of Ganoderma made from all dying trees where basidiomes were present in a Pretoria suburb. DNA sequences were obtained from the ITS and LSU region for the isolates and compared against sequences on GenBank. Phylogenetic analyses were used to compare sequences with those for other Ganoderma species. Based on sequence comparisons and morphological characters, two new Ganoderma species were discovered and these are described here as G. enigmaticum and G. destructans spp. nov. Interestingly, the previously described G. austroafricanum was not found, G. enigmaticum was found on only one Ceratonia siliqua tree and G. destructans was found on all other trees sampled. The latter species appears to be the primary cause of root rot of J. mimosifolia in the area sampled.

Identifying and naming plant-pathogenic fungi: past, present, and future.

Scientific names are crucial in communicating knowledge about fungi. In plant pathology, they link information regarding the biology, host range, distribution, and potential risk. Our understanding of fungal biodiversity and fungal systematics has undergone an exponential leap, incorporating genomics, web-based systems, and DNA data for rapid identification to link species to metadata. The impact of our ability to recognize hitherto unknown organisms on plant pathology and trade is enormous and continues to grow. Major challenges for phytomycology are intertwined with the Genera of Fungi project, which adds DNA barcodes to known biodiversity and corrects the application of old, established names via epi- or neotypification. Implementing the one fungus-one name system and linking names to validated type specimens, cultures, and reference sequences will provide the foundation on which the future of plant pathology and the communication of names of plant pathogens will rest.

Knoxdaviesia proteae is not the only Knoxdaviesia-symbiont of Protea repens.

Two polyphyletic genera of ophiostomatoid fungi are symbionts of Proteaceae in southern Africa. One of these, Knoxdaviesia, includes two closely related species, K. proteae and K. capensis, that have overlapping geographical distributions, but are not known to share Protea host species. Knoxdaviesia capensis appears to be a generalist that occupies numerous hosts, but has never been found in P. repens, the only known host of K. proteae. In this study, extensive collections were made from P. repens and isolates were identified using DNA sequence comparisons. This led to the surprising discovery of K. capensis from P. repens for the first time. The fungus was encountered at a low frequency, suggesting that P. repens is not its preferred host, which may explain why it has not previously been found on this plant. The basis for the specialisation of K. proteae and K. capensis on different Protea species remains unknown.

Reconsidering species boundaries in the Ceratocystis paradoxa complex, including a new species from oil palm and cacao in Cameroon.

The Ceratocystis paradoxa complex accommodates a group of fungal pathogens that have become specialized to infect mostly monocotyledonous plants. Four species currently are recognized in this group, including C. paradoxa, which has a widespread distribution and broad host range. In this study, multigene phylogenetic analyses involving sequences of the ITS, ß-tubulin and TEF-1α gene loci, in combination with phenotypic and mating studies, were used to characterize purported C. paradoxa isolates from Cameroon and to compare them with isolates from elsewhere, including protologs and type specimens of known species. We show that the C. paradoxa complex comprises substantially greater species diversity than previously recognized. One new species in this group is described from Cameroon as Ceratocystis cerberus, while C. paradoxa sensu stricto (s. str.) and four other species are redefined. Lectotypes are designated for C. ethacetica and Endoconidium fragrans (synonym of C. ethacetica), while epitypes are designated for C. paradoxa s. str., C. ethacetica and C. musarum. A neotype is designated for Catenularia echinata (synonym of C. ethacetica) and two species, previously treated in Thielaviopsis, are transferred to Ceratocystis.

DNA extraction techniques for DNA barcoding of minute gall-inhabiting wasps.

DNA extraction from minute hymenopterans and their larvae is difficult and challenging because of their small size indicating a low amount of starting material. Hence, 11 DNA extraction methods were compared to determine their efficacy in isolating DNA. Success of each method was scored on a 2% agarose gel after PCR of the cox 1 mitochondrial locus. A silica-membrane-based approach was the most successful, followed by a method using a combination of incubation buffers and a method using magnetic beads. The method using buffers was the most cost- and time effective. Using this method, larvae from Eucalyptus seed capsule galls could be assigned a role (parasitoid, gall former or inquiline) in the gall-inhabiting complex.

Characterization of a novel dsRNA element in the pine endophytic fungus Diplodia scrobiculata.

Diplodia scrobiculata and Diplodia pinea are endophytic fungi associated with dieback and cankers of mainly Pinus spp. in many parts of the world. These two fungi are closely related and have, in the past, been considered to represent two morphological forms (A and B morphotypes) of D. pinea. dsRNA elements are known to occur in both D. scrobiculata and D. pinea. Two dsRNA elements from D. pinea, SsRV1 and SsRV2, have been characterized previously. The aim of this study was to characterize a third dsRNA element that is most commonly associated with D. scrobiculata and to determine its phylogenetic relationship to other mycoviruses. The 5018-bp genome of this element was sequenced, and it is referred to as D. scrobiculata RNA virus 1, or DsRV1. It has two open reading frames (ORFs), one of which codes for a putative polypeptide with a high degree of similarity to proteins of the vacuolar protein-sorting (VPS) machinery, and the other for an RNA-dependent RNA polymerase (RdRp). Phylogenetic comparisons based on amino acid sequence alignments of the RdRp revealed that DsRV1 is closely related to a dsRNA element isolated from Phlebiopsis gigantea (PgV2), and they grouped separately from virus families in which mycoviruses have previously been described. Although D. pinea and D. scrobiculata are closely related, DsRV1 does not share high sequence identity with SsRV1 or SsRV2, and they probably have different recent evolutionary origins.

Causes and consequences of variability in peptide mating pheromones of ascomycete fungi.

The reproductive genes of fungi, like those of many other organisms, are thought to diversify rapidly. This phenomenon could be associated with the formation of reproductive barriers and speciation. Ascomycetes produce two classes of mating type-specific peptide pheromones. These are required for recognition between the mating types of heterothallic species. Little is known regarding the diversity or the extent of species specificity in pheromone peptides among these fungi. We compared the putative protein-coding DNA sequences of the 2 pheromone classes from 70 species of Ascomycetes. The data set included previously described pheromones and putative pheromones identified from genomic sequences. In addition, pheromone genes from 12 Fusarium species in the Gibberella fujikuroi complex were amplified and sequenced. Pheromones were largely conserved among species in this complex and, therefore, cannot alone account for the reproductive barriers observed between these species. In contrast, pheromone peptides were highly diverse among many other Ascomycetes, with evidence for both positive diversifying selection and relaxed selective constraint. Repeats of the α-factor-like pheromone, which occur in tandem arrays of variable copy number, were found to be conserved through purifying selection and not concerted evolution. This implies that sequence specificity may be important for pheromone reception and that interspecific differences may indeed be associated with functional divergence. Our findings also suggest that frequent duplication and loss causes the tandem repeats to experience "birth-and-death" evolution, which could in fact facilitate interspecific divergence of pheromone peptide sequences.

Paleogene radiation of a plant pathogenic mushroom.

BACKGROUND: The global movement and speciation of fungal plant pathogens is important, especially because of the economic losses they cause and the ease with which they are able to spread across large areas. Understanding the biogeography and origin of these plant pathogens can provide insights regarding their dispersal and current day distribution. We tested the hypothesis of a Gondwanan origin of the plant pathogenic mushroom genus Armillaria and the currently accepted premise that vicariance accounts for the extant distribution of the species. METHODS: The phylogeny of a selection of Armillaria species was reconstructed based on Maximum Parsimony (MP), Maximum Likelihood (ML) and Bayesian Inference (BI). A timeline was then placed on the divergence of lineages using a Bayesian relaxed molecular clock approach. RESULTS: Phylogenetic analyses of sequenced data for three combined nuclear regions provided strong support for three major geographically defined clades: Holarctic, South American-Australasian and African. Molecular dating placed the initial radiation of the genus at 54 million years ago within the Early Paleogene, postdating the tectonic break-up of Gondwana. CONCLUSIONS: The distribution of extant Armillaria species is the result of ancient long-distance dispersal rather than vicariance due to continental drift. As these finding are contrary to most prior vicariance hypotheses for fungi, our results highlight the important role of long-distance dispersal in the radiation of fungal pathogens from the Southern Hemisphere.

The Ascomycota tree of life: a phylum-wide phylogeny clarifies the origin and evolution of fundamental reproductive and ecological traits.

We present a 6-gene, 420-species maximum-likelihood phylogeny of Ascomycota, the largest phylum of Fungi. This analysis is the most taxonomically complete to date with species sampled from all 15 currently circumscribed classes. A number of superclass-level nodes that have previously evaded resolution and were unnamed in classifications of the Fungi are resolved for the first time. Based on the 6-gene phylogeny we conducted a phylogenetic informativeness analysis of all 6 genes and a series of ancestral character state reconstructions that focused on morphology of sporocarps, ascus dehiscence, and evolution of nutritional modes and ecologies. A gene-by-gene assessment of phylogenetic informativeness yielded higher levels of informativeness for protein genes (RPB1, RPB2, and TEF1) as compared with the ribosomal genes, which have been the standard bearer in fungal systematics. Our reconstruction of sporocarp characters is consistent with 2 origins for multicellular sexual reproductive structures in Ascomycota, once in the common ancestor of Pezizomycotina and once in the common ancestor of Neolectomycetes. This first report of dual origins of ascomycete sporocarps highlights the complicated nature of assessing homology of morphological traits across Fungi. Furthermore, ancestral reconstruction supports an open sporocarp with an exposed hymenium (apothecium) as the primitive morphology for Pezizomycotina with multiple derivations of the partially (perithecia) or completely enclosed (cleistothecia) sporocarps. Ascus dehiscence is most informative at the class level within Pezizomycotina with most superclass nodes reconstructed equivocally. Character-state reconstructions support a terrestrial, saprobic ecology as ancestral. In contrast to previous studies, these analyses support multiple origins of lichenization events with the loss of lichenization as less frequent and limited to terminal, closely related species.

Polyphyly and two emerging lineages in the rust genera Puccinia and Uromyces.

The phylogenetic validity of Puccinia and Uromyces, Pucciniaceae, and closely related genera was evaluated using nucLSU rDNA sequences. Using a wide range of rust species with different life cycles and with different host specificities, Puccinia and Uromyces were shown to be highly polyphyletic and to also include representatives of the genera Aecidium, Cumminsiella, Dietelia, Endophyllum, Miyagia, and Uredo. Furthermore, the structure of the phylogenetic data did not reflect previous sub-generic delimitations based on teliospore pedicel structure, but rather suggests that at least two major lineages have evolved within Puccinia/Uromyces: Rusts with telial states on Poaceae were exclusively found in one of these groupings and those with telial states on Cyperaceae resided in the other lineage. This might suggest that the two lineages evolved in close association with these host groups in different biomes.

Two new species of Fusarium section Liseola associated with mango malformation.

Mango malformation is an economically important disease of Mangifera indica globally. A recent DNA-based study indicated that two distinct, phylogenetic lineages previously identified as Fusarium subglutinans are associated with this disease in South Africa. The objective of this study was to characterize Fusarium isolates associated with mango malformation, including the two different F. subglutinans groups, based on morphological characteristics. For this purpose we examined Fusarium strains isolated from diseased mango inflorescences from diverse geographical origins. We also used sexual compatibility tests to determine whether sexual reproduction among the strains was possible. The morphological characters considered were shape of the conidia, presence of mono- and/or polyphialides, origin of the conidiophores from the substrate, presence of chlamydospores and the presence of sterile coiled hyphae. Three unique Fusarium spp. were identified. In this paper, we provide formal descriptions for the two new taxa in the section Liseola that we have named F. mangiferae and F. sterilihyphosum. Fusarium mangiferae is conspecific with strains that were previously identified as F. subglutinans and reported to be the causal agent of malformation in mango growing areas throughout the world. Fusarium sterilihyphosum, on the other hand, has been isolated only from malformed mango tissue in South Africa.