A new species of Raffaelea from beetle-infested Leucaena leucocephala

Species of Raffaelea (Ophiostomatales: Ascomycota) are obligate symbionts of ambrosia beetles, some of which pose a substantial threat to forest trees. Leucaena leucocephala is a small mimosoid tree species that is considered as an invasive weed in most of its introduced range globally. During a field expedition on the French island of Réunion, dying L. leucocephala trees were observed. Samples were taken from these trees and isolations made from symptomatic wood tissues that included beetle tunnels, but in the absence of the beetles themselves. Multiple isolates of a fungus resembling a Raffaelea species were obtained from the discoloured wood associated with the beetle tunnels. To determine their identity, microscopic examination was performed and DNA sequences for three gene regions (ITS, LSU, TUB) were obtained. Phylogenetic analyses based on these gene regions revealed that the isolates represent a new species of Raffaelea, described here as R. borbonica sp. nov. A pathogenicity test was conducted with the fungus, which was shown to cause lesions on the inoculated seedlings, but with a low level of aggressiveness.https://fuse-journal.orgpm2021BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant Patholog

Ophiostomatoid fungi associated with fungus farming insects in South Africa

Symbiosis is the term used to describe the different forms of communal life that can exist between two unlike organisms. Primarily this involves interaction in one of three forms: mutualism where both partners benefit from their association; antagonism where one or both partners are harmed by their association; and commensalism where one partner derives benefit but the other is neither harmed nor profited from their association. Many different forms of symbioses have been described between insects and microbes and these include short, simple interactions as well as obligate associations. Aside from humans, sophisticated agricultural farming practices have only been found for three insect groups, colloquially known as fungus-farmers. These three groups – the attine ants, the macrotermitinae termites, and the ambrosia beetles - each independently evolved an obligate mutualism with fungal partners that they actively cultivate and maintain within their nests and utilize as a primary source of nutrition. The primary focus of this PhD thesis was that of the association that exists between bark and ambrosia beetles and their respective fungal partners and the genomic signatures of these associations. Additionally, the unexpected and interesting discovery of Ophiostomatoid fungi from fungus-growing macrotermitinae termites was also investigated. Very little research on ambrosia beetles and their fungal partners has been conducted in South Africa. However, the accidental introduction of the devastating Polyphagous Shot Hole Borer into the country has sparked renewed interest into this topic. The primary focus of the research included in this thesis has consequently been on ambrosia beetles in South Africa. This is also the topic of the literature review (Chapter 1) at the start of the thesis. However, given the many commonalities regarding their associations with fungi, we have also included a study that led to the discovery of Ophiostomatoid fungi associated with fungus-farming termites. The background literature relating to this association is provided in the introduction to that study and it is also a topic that has been thoroughly treated in a number of recent reviews. In Chapters 2-4 I explore the diversity of Ophiostomatoid fungi associated with fungusfarming insects in South Africa. In Chapter 2, I report four species of Ophiostomatalean species associated with commonly found ambrosia beetles in the country. One of these species is reported from South Africa for the first time and two are described as novel species. In Chapter 3, I report for the first time the presence of the granulate ambrosia beetle and its Microascalean associate, Ambrosiella roeperi, in South Africa and highlight the potential threat that this beetle and its fungal symbiont poses to South Africa’s agricultural industry. In Chapter 4, I investigate three species of Ophiostomatalean fungi discovered on the abandoned Termitomyces fungus combs of fungus-farming termites. Using both culture- based and genomics techniques, I describe these newly discovered Ophiostomatalean fungi and investigate their distinct lifestyle. In Chapter 5, I delve more deeply into the relationship shared between Sordariomycete fungi known to be associated with arthropods. In this chapter, I attempt to elucidate how the relationship with their arthropod partners has influenced their genomic evolution. Using whole genome sequences and comparative genomics methods I investigate the traits shared amongst the Ascomycete ambrosia fungi, as well as investigate how these fungi differ genetically from their close relatives. Finally, as a supplementary chapter to this thesis, I provide a comprehensive record of bark and ambrosia beetle species present in South Africa. This is justified by the fact that other studies included in this thesis involved extensive trapping of bark and ambrosia beetles, many of which had not previously been recorded in the country. This record increases the currently recorded number of these insects from South Africa from 163 to 260. Additionally, 22 species are reported from the county for the first time. It was decided to place this chapter as supplementary material and not within the main body of the thesis because it did directly capture the common theme of Ophiostomatoid fungi associated with fungus-farming insects.Thesis (PhD (Microbiology))--University of Pretoria, 2021.Microbiology and Plant PathologyPhD (Microbiology)Unrestricte

Taxonomy and biology of the plant pathogenic fungus Thielaviopsis basicola

The Ascomycete species Thielaviopsis basicola is a well-known pathogen of multiple important crop and ornamental plant species. This pathogen has been known for more than 150 years and has been extensively studied during this time, but important questions surrounding its taxonomy and mating behaviour remained to be answered. The first aim of this project was to resolve the taxonomic placement of the species using a multi-gene phylogenetics approach. Our research to address this aim revealed that the species represented a lineage distinct from all other genera in the Ceratocystidaceae, that we proceeded to describe as the new genus Berkeleyomyces. Our phylogenetic analyses also separated the collection of T. basicola isolates into two well-supported lineages within the genus, that we recognized as two distinct species. The first of these represented T. basicola, which was provided with a new combination, namely B. basicola. The second was described as a new species, named B. rouxiae. From the range of genera in which T. basicola was treated over the years, the name Milowia was technically available to accommodate the two species. However, we submitted a formal proposal to reject the name of the type species of this genus, M. nivea, and all names for which it serves as basionym, due to the absence of a type specimen and its dubious identity based on varying descriptions and illustrations by the same author across different publications. The second aim of our study was to determine the sexuality of these species, as some researches had suggested that T. basicola might exist exclusively asexually. To address this aim, we sequenced and assembled the whole genome sequence of the reference specimen of Berkeleyomyces basicola, designated when we described the genus. Using information from this genome we were able to determine the mating strategy of both species in the genus. Our results showed that both B. basicola and B. rouxiae contain all the typical genes required for heterothallic mating, but despite our efforts to obtain the sexual state in laboratory crosses, the sexual state of both species remains unknown. As a whole, the research conducted as part of this thesis has contributed to the global knowledge of these important pathogens by clarifying and settling the confusing taxonomy of the species, and by elucidating the mating strategy of these fungi. This will no doubt assist in improved diagnosis of the disease and a better understanding of the risks posed by introduced populations of these pathogens.Dissertation (MSc)--University of Pretoria, 2017.National Research Foundation MSc Scarce Skills Bursary SFH150628121614Genomics Research Institute University of PretoriaMicrobiology and Plant PathologyMScUnrestricte

Ophiostomatalean fungi associated with wood boring beetles in South Africa including two new species

Ambrosia beetles are small wood inhabiting members of the Curculionidae that have evolved obligate symbioses with fungi. The fungal symbionts concentrate nutrients from within infested trees into a usable form for their beetle partners, which then utilize the fungi as their primary source of nutrition. Ambrosia beetle species associate with one or more primary symbiotic fungal species, but they also vector auxiliary symbionts, which may provide the beetle with developmental or ecological advantages. In this study we isolated and identified ophiostomatalean fungi associated with ambrosia beetles occurring in a native forest area in South Africa. Using a modified Bambara beetle trap, living ambrosia beetle specimens were collected and their fungal symbionts isolated. Four beetle species, three Scolytinae and one Bostrichidae, were collected. Five species of ophiostomatalean fungi were isolated from the beetles and were identified using both morphological characters and DNA sequence data. One of these species, Raffaelea sulphurea, was recorded from South Africa for the first time and two novel species were described as Ceratocystiopsis lunata sp. nov. and Raffaelea promiscua sp. nov.The University of Pretoria, members of the Tree Protection Co-operative Programme (TPCP), the DSI-NRF Centre of Excellence in Plant Health Biotechnology (CPHB) and the National Research Foundation (NRF) of South Africa.http://link.springer.com/journal/104822022-03-06hj2022BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant Patholog

Black root rot : a long known but little understood disease

Black root rot caused by the pathogen Thielaviopsis basicola has been known since the mid 1800s. The disease is important on many agricultural and ornamental plant species and has been found in at least 31 countries. Since its description, the pathogen has had a complex taxonomic history that has resulted in a confused literature. A recent revision of the Ceratocystidaceae following the advent of DNA sequencing technology has made it possible to resolve this confusion. Importantly, it has also shown that there are two pathogens in the Ceratocystidaceae that cause black root rot. They reside in the newly established genus Berkeleyomyces and are now known as B. basicola and B. rouxiae. This review considers the taxonomic history of the black root rot pathogens, and their global distribution. Prospects relating to the serious diseases that they cause and the likely impact that the era of genomics will have on our understanding of the pathogens are also highlighted.Table S1. Hosts reported to be susceptible to black root rot infection.Table S2. Variation in host susceptibility to black root rot infection by the fungus formally known as Thielaviopsis basicola.The University of Pretoria, the members of Tree Protection Co‐operative Programme (TPCP), the DST‐NRF Centre of Excellence in Tree Health Biotechnology (CTHB) and the National Research Foundation.https://onlinelibrary.wiley.com/journal/136530592020-06-01hj2019BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant Patholog

The polyphagous shot hole borer (PSHB) and its fungal symbiont Fusarium euwallaceae: a new invasion in South Africa

The polyphagous shot hole borer (PSHB), an ambrosia beetle (Coleoptera: Curculeonidae: Scolytinae) native to Asia, together with its fungal symbiont Fusarium euwallaceae, has emerged as an important invasive pest killing avocado and other trees in Israel and the United States. The PSHB is one of three cryptic species in the Euwallacea fornicatus species complex, the taxonomy of which remains to be resolved. The surge in the global spread of invasive forest pests such as the PSHB has led to the development of programmes utilising sentinel tree plantings to record new host-pest interactions. During routine surveys of tree health in botanical gardens of South Africa undertaken as part of a sentinel project, an ambrosia beetle/fungal associate was detected damaging Platanus x acerifolia (London Plane) in the KwaZulu-Natal National Botanical Gardens, Pietermaritzburg. Identification of the beetle by sequencing part of the mitochondrial cytochrome oxidase c subunit 1 (COI) gene confirmed its identity as PSHB, and specifically one of the invasive haplotypes of the beetle. The associated fungus F. euwallaceae was identified based on phylogenetic analysis of elongation factor (EF 1-α) sequences. Koch’s postulates have confirmed the pathogenicity of fungal isolates to P. x acerifolia. This is the first report of PSHB and its fungal symbiont causing Fusarium dieback in South Africa. This report also represents the first verified case of a damaging invasive forest pest detected in a sentinel planting project, highlighting the importance of such studies. Given the potential impact these species present to urban trees, native biodiversity and agriculture, both the PSHB and its fungal symbiont should be included in invasive species regulations in South Africa.The South African National Department of Environment Affairs, through the South African National Biodiversity Institute’s Invasive Species Programme.http://link.springer.com/journal/133132019-03-02hj2018Forestry and Agricultural Biotechnology Institute (FABI)Microbiology and Plant Patholog

Phylogenetic and phylogenomic analyses reveal two new genera and three new species of ophiostomatalean fungi from termite fungus combs

The Ophiostomatales (Ascomycota) accommodates more than 300 species characterized by similar morphological adaptations to arthropod dispersal. Most species in this order are wood-inhabiting fungi associated with bark or ambrosia beetles. However, a smaller group of species occur in other niches such as in soil and Protea infructescences. Recent surveys of Termitomyces fungus gardens (fungus combs) of fungus-growing termites led to the discovery of characteristic ophiostomatalean-like fruiting structures. In this study, these ophiostomatalean-like fungi were identified using morphological characteristics, conventional molecular markers, and whole genome sequencing. In addition, the influence of the extracts derived from various parts of Termitomyces combs on the growth of these fungi in culture was considered. Based on phylogenomic analyses, two new genera (Intubia and Chrysosphaeria) were introduced to accommodate these ophiostomatalean species. Phylogenetic analyses revealed that the isolates resided in three well-supported lineages, and these were described as three new species (Intubia macrotermitinarum, I. oerlemansii, and Chrysosphaeria jan-nelii). Culture-based studies showed that these species do not depend on the Termitomyces comb material for growth.The National Research foundation of South Africa (NRF), The University of Pretoria, the Tree Protection Co-operative Programme (TPCP), and Department of Science and Innovation (DSI)-NRF Centre of Excellence in Plant-Health Biotechnology (CPHB), and the DSI-NRF South African Research Chairs Initiative Chair for Fungal Genomics.http://www.tandfonline.com/loi/umyc202022-09-03hj2022BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant Patholog

The polyphagous shot hole borer beetle : current status of a perfect invader in South Africa

The polyphagous shot hole borer (PSHB) beetle is a recent invader in South Africa. Together with its fungal symbiont, Fusarium euwallaceae, it can rapidly kill highly susceptible host plants. Its impact is most profound in urban areas, but it has also been found infesting important forestry, agricultural crop and native species. Since its first detection in 2012, PSHB has spread to all but one province in the country. The beetle–fungus complex has several biological traits that enhance its anthropogenically mediated dispersal, establishment and survival in novel environments – factors that have likely facilitated its rapid spread across the country. We review the history of the PSHB invasion in South Africa, its taxonomic status and the reasons for its rapid spread. We highlight its potential impact and challenges for its management. Finally, we provide an updated distribution map and list of confirmed host plants in South Africa. Of the 130 plant species identified as hosts, 48 of these (19 indigenous and 29 introduced) are reproductive hosts able to maintain breeding PSHB populations. These reproductive hosts may succumb to beetle infestations and act as ‘pest-amplifiers’. The economic impact on urban forests, plantation forestry and agricultural crops may be severe, but the ecological impact of PSHB invasion in native ecosystems should not be underestimated. SIGNIFICANCE : • We provide an updated host list and distribution map for South Africa of the globally significant tree pest, the polyphagous shot hole borer (PSHB, Euwallacea fornicatus). The South African PSHB invasion represents the largest outbreak of this pest in its global invaded range. PSHB was confirmed to infest 130 plant species in urban, agricultural, and native ecosystems in South Africa, including 44 previously unreported hosts. Impact in South Africa is in its infancy but will likely be substantial to local economies and ecosystems. Mitigation has proven difficult, but numerous research projects have been initiated throughout the country.South African Department of Forestry, Fisheries and the Environment, University of Pretoriahttp://www.sajs.co.zahj2021BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant Patholog