GNPS-Guided Discovery of Madurastatin Siderophores from the Termite-Associated Actinomadura sp. RB99.

In this study, we analyzed if Actinomadura sp. RB99 produces siderophores that that could be responsible for the antimicrobial activity observed in co-cultivation studies. Dereplication of high-resolution tandem mass spectrometry (HRMS/MS) and global natural product social molecular networking platform (GNPS) analysis of fungus-bacterium co-cultures resulted in the identification of five madurastatin derivatives (A1, A2, E1, F, and G1), of which were four new derivatives. Chemical structures were unambiguously confirmed by HR-ESI-MS, 1D and 2D NMR experiments, as well as MS/MS data and their absolute structures were elucidated based on Marfey's analysis, DP4+ probability calculation and total synthesis. Structure analysis revealed that madurastatin E1 (2) contained a rare 4-imidazolidinone cyclic moiety and madurastatin A1 (5) was characterized as a Ga3+ -complex. The function of madurastatins as siderophores was evaluated using the fungal pathogen Cryptococcus neoformans as model organism. Based on homology models, we identified the putative NRPS-based gene cluster region of the siderophores in Actinomadura sp. RB99.

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.

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.

Phylogenomic Analysis of a 55.1-kb 19-Gene Dataset Resolves a Monophyletic Fusarium that Includes the Fusarium solani Species Complex.

Scientific communication is facilitated by a data-driven, scientifically sound taxonomy that considers the end-user's needs and established successful practice. In 2013, the Fusarium community voiced near unanimous support for a concept of Fusarium that represented a clade comprising all agriculturally and clinically important Fusarium species, including the F. solani species complex (FSSC). Subsequently, this concept was challenged in 2015 by one research group who proposed dividing the genus Fusarium into seven genera, including the FSSC described as members of the genus Neocosmospora, with subsequent justification in 2018 based on claims that the 2013 concept of Fusarium is polyphyletic. Here, we test this claim and provide a phylogeny based on exonic nucleotide sequences of 19 orthologous protein-coding genes that strongly support the monophyly of Fusarium including the FSSC. We reassert the practical and scientific argument in support of a genus Fusarium that includes the FSSC and several other basal lineages, consistent with the longstanding use of this name among plant pathologists, medical mycologists, quarantine officials, regulatory agencies, students, and researchers with a stake in its taxonomy. In recognition of this monophyly, 40 species described as genus Neocosmospora were recombined in genus Fusarium, and nine others were renamed Fusarium. Here the global Fusarium community voices strong support for the inclusion of the FSSC in Fusarium, as it remains the best scientific, nomenclatural, and practical taxonomic option available.

Targeted Discovery of Tetrapeptides and Cyclic Polyketide-Peptide Hybrids from a Fungal Antagonist of Farming Termites.

Herein, we report the targeted isolation and characterization of four linear nonribosomally synthesized tetrapeptides (pseudoxylaramide A-D) and two cyclic nonribosomal peptide synthetase-polyketide synthase-derived natural products (xylacremolide A and B) from the termite-associated stowaway fungus Pseudoxylaria sp. X187. The fungal strain was prioritized for further metabolic analysis based on its taxonomical position and morphological and bioassay data. Metabolic data were dereplicated based on high-resolution tandem mass spectrometry data and global molecular networking analysis. The structure of all six new natural products was elucidated based on a combination of 1D and 2D NMR analysis, Marfey's analysis and X-ray crystallography.

Actinomadura rubteroloni sp. nov. and Actinomadura macrotermitis sp. nov., isolated from the gut of the fungus growing-termite Macrotermes natalensis.

The taxonomic positions of two novel aerobic, Gram-positive actinobacteria, designated strains RB29T and RB68T, were determined using a polyphasic approach. Based on 16S rRNA gene sequence analysis, the closest phylogenetic neighbours of RB29T were identified as Actinomadura rayongensis DSM 102126T (99.2 % similarity) and Actinomadura atramentaria DSM 43919T (98.7 %), and for strain RB68T was Actinomadura hibisca DSM 44148T (98.3 %). Digital DNA-DNA hybridization (dDDH) between RB29T and its closest phylogenetic neighbours, A. rayongensis DSM 102126T and A. atramentaria DSM 43919T, resulted in similarity values of 53.2 % (50.6-55.9 %) and 26.4 % (24.1-28.9 %), respectively. Additionally, the average nucleotide identity (ANI) was 93.2 % (94.0 %) for A. rayongensis DSM 102126T and 82.3 % (78.9 %) for A. atramentaria DSM 43919T. dDDH analysis between strain RB68T and A. hibisca DSM 44148T gave a similarity value of 24.5 % (22.2-27.0 %). Both strains, RB29T and RB68T, revealed morphological characteristics and chemotaxonomic features typical for the genus Actinomadura, such as the presence of meso-diaminopimelic acid in the cell wall, galactose and glucose as major sugar components within whole-cell hydrolysates and the absence of mycolic acids. The major phospholipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and phosphatidylinositol mannoside. Predominant menaquinones were MK-9(H6) and MK-9(H8) for RB29T and MK-9(H4) and MK-9(H6) for RB68T. The main fatty acids were identified as 10-methyloctadecanoic acid (10-methyl C18:0), 14-methylpentadecanoic acid (iso-C16:0), hexadecanoic acid (C16:0) and cis-9-octadecanoic acid (C18 : 1 ω9c). Here, we propose two novel species of the genus Actinomadura: Actinomadura rubteroloni sp. nov. with the type strain RB29T (=CCUG 72668T=NRRL B-65537T) and Actinomadura macrotermitis sp. nov. with the type strain RB68T (=CCUG 72669T=NRRL B-65538T).

Nocardia macrotermitis sp. nov. and Nocardia aurantia sp. nov., isolated from the gut of the fungus-growing termite Macrotermes natalensis.

The taxonomic positions of two novel aerobic, Gram-stain-positive Actinobacteria, designated RB20T and RB56T, were determined using a polyphasic approach. Both were isolated from the fungus-farming termite Macrotermes natalensis. Results of 16S rRNA gene sequence analysis revealed that both strains are members of the genus Nocardia with the closest phylogenetic neighbours Nocardia miyunensis JCM12860T (98.9 %) and Nocardia nova DSM44481T (98.5 %) for RB20T and Nocardia takedensis DSM 44801T (98.3 %), Nocardia pseudobrasiliensis DSM 44290T (98.3 %) and Nocardia rayongensis JCM 19832T (98.2 %) for RB56T. Digital DNA-DNA hybridization (DDH) between RB20T and N. miyunensis JCM12860T and N. nova DSM 44481T resulted in similarity values of 33.9 and 22.0 %, respectively. DDH between RB56T and N. takedensis DSM44801T and N. pseudobrasiliensis DSM44290T showed similarity values of 20.7 and 22.3 %, respectively. In addition, wet-lab DDH between RB56T and N. rayongensis JCM19832T resulted in 10.2 % (14.5 %) similarity. Both strains showed morphological and chemotaxonomic features typical for the genus Nocardia, such as the presence of meso-diaminopimelic acid (A2pm) within the cell wall, arabinose and galactose as major sugar components within whole cell-wall hydrolysates, the presence of mycolic acids and major phospholipids (diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol), and the predominant menaquinone MK-8 (H4, ω-cyclo). The main fatty acids for both strains were hexadecanoic acid (C16 : 0), 10-methyloctadecanoic acid (10-methyl C18 : 0) and cis-9-octadecenoic acid (C18 : 1 ω9c). We propose two novel species within the genus Nocardia: Nocardia macrotermitis sp. nov. with the type strain RB20T (=VKM Ac-2841T=NRRL B65541T) and Nocardia aurantia sp. nov. with the type strain RB56T (=VKM Ac-2842T=NRRL B65542T).

Streptomyces smaragdinus sp. nov., isolated from the gut of the fungus growing-termite Macrotermes natalensis.

The taxonomic position of a novel aerobic, Gram-positive actinobacteria, designated strain RB5T, was determined using a polyphasic approach. The strain, isolated from the gut of the fungus-farming termite Macrotermes natalensis, showed morphological, physiological and chemotaxonomic properties typical of the genus Streptomyces. Based on 16S rRNA gene sequence analysis, the closest phylogenetic neighbour of RB5T was Streptomyces polyrhachis DSM 42102T (98.87 %). DNA-DNA hybridization experiments between strain RB5T and S. polyrhachis DSM 42102T resulted in a value of 27.4 % (26.8 %). The cell wall of strain RB5T contained ll-diaminopimelic acid as the diagnostic amino acid. Mycolic acids and diagnostic sugars in whole-cell hydrolysates were not detected. The strain produced the following major phospholipids: diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol-mannoside and phosphatidylserine. The menaquinone profile showed hexa- and octahydrogenated menaquinones containing nine isoprene units [MK-9(H6) and MK-9(H8)]. The strain exhibited a fatty acid profile containing the following major fatty acids: 12-methyltridecanoic acid (iso-C14 : 0) 12-methyltetradecanoic acid (anteiso-C15 : 0), 13-methyltetradecanoic acid (iso-C15 : 0) and 14-methylpentadecanoic acid (iso-C16 : 0). Here, we propose a novel species of the genus Streptomyces - Streptomyces smaragdinus with the type strain RB5T (=VKM Ac-2839T=NRRL B65539T).

Polyhalogenation of Isoflavonoids by the Termite-Associated Actinomadura sp. RB99.

Based on high-resolution tandem mass spectrometry (HR-MS2) and global natural products social molecular networking (GNPS), we found that plant-derived daidzein and genistein derivatives are polyhalogenated by termite-associated Actinomadura species RB99. MS-guided purification from extracts of bacteria grown under optimized conditions led to the isolation of eight polychlorinated isoflavones, including six unreported derivatives, and seven novel polybrominated derivatives, two of which showed antimicrobial activity.

Euwallacea perbrevis (Coleoptera: Curculionidae: Scolytinae), a confirmed pest on Acacia crassicarpa in Riau, Indonesia, and a new fungal symbiont; Fusarium rekanum sp. nov.

Non-native Acacia plantations in Indonesia were first reported to be infested by a native ambrosia beetle species, identified as Euwallacea fornicatus in 1993. Recently the level of infestation in these plantations by ambrosia beetles has steadily increased. The recent redefinition of the taxonomic parameters of the Euwallacea fornicatus species complex has resulted in the identity of the ambrosia beetle species in these plantations becoming unclear. This is also true for their obligate fungal associates. Therefore, the aim of this study was to identify the ambrosia beetle species, as well as its corresponding fungal associate/s, infesting Acacia crassicarpa plantations in Riau, Indonesia. Morphological identification and phylogenetic analysis of the mitochondrial cytochrome oxidase c subunit I (COI) gene, revealed that the beetles are E. perbrevis, previously a synonym of E. fornicatus and commonly referred to as the Tea Shot Hole Borer A (TSHBa). Multi-locus phylogenetic analyses of the fungal associate of E. perbrevis revealed a Fusarium sp. that is among members of the Ambrosia Fusarium Clade (AFC), but that is genetically distinct from other previously identified Fusarium symbionts of Euwallacea species. This novel fungal species is described here as Fusarium rekanum sp. nov.

Unexpected placement of the MAT1-1-2 gene in the MAT1-2 idiomorph of Thielaviopsis.

Sexual reproduction in the Ascomycota is controlled by genes encoded at the mating-type or MAT1 locus. The two allelic versions of this locus in heterothallic species, referred to as idiomorphs, are defined by the MAT1-1-1 (for the MAT1-1 idiomorph) and MAT1-2-1 (for the MAT1-2 idiomorph) genes. Both idiomorphs can contain additional genes, although the contents of each is typically specific to and conserved within particular Pezizomycotina lineages. Using full genome sequences, complemented with conventional PCR and Sanger sequencing, we compared the mating-type idiomorphs in heterothallic species of Thielaviopsis (Ceratocystidaceae). The analyses showed that the MAT1-1 idiomorph of T. punctulata, T. paradoxa, T. euricoi, T. ethacetica and T. musarum harboured only the expected MAT1-1-1 gene. In contrast, the MAT1-2 idiomorph of T. punctulata, T. paradoxa and T. euricoi encoded the MAT1-2-1, MAT1-2-7 and MAT1-1-2 genes. Of these, MAT1-2-1 and MAT1-2-7 are genes previously reported in this idiomorph, while MAT1-1-2 is known only in the MAT1-1 idiomorph. Phylogenetic analysis showed that the Thielaviopsis MAT1-1-2 groups with the known homologues of this gene in other Microascales, thus confirming its annotation. Previous work suggests that MAT1-1-2 is involved in fruiting body development, a role that would be unaffected by its idiomorphic position. This notion is supported by our findings for the MAT1 locus structure in Thielaviopsis species. This also serves as the first example of a MAT1-1-specific gene restricted to only the MAT1-2 idiomorph.

Biodiversity and ecology of flower-associated actinomycetes in different flowering stages of Protea repens.

Actinomycete bacteria have previously been reported from reproductive structures (infructescences) of Protea (sugarbush/suikerbos) species, a niche dominated by fungi in the genera Knoxdaviesia and Sporothrix. It is probable that these taxa have symbiotic interactions, but a lack of knowledge regarding their diversity and general ecology precludes their study. We determined the diversity of actinomycetes within Protea repens inflorescence buds, open inflorescences, young and mature infructescences, and leaf litter surrounding these trees. Since the P. repens habitat is fire-prone, we also considered the potential of these bacteria to recolonise infructescences after fire. Actinomycetes were largely absent from flower buds and inflorescences but were consistently present in young and mature infructescences. Two Streptomyces spp. were the most consistent taxa recovered, one of which was also routinely isolated from leaf litter. Lower colonisation rates were evident in samples from a recently burnt site. One of the most consistent taxa isolated from older trees in the unburnt site was absent from this site. Our findings show that P. repens has a distinct community of actinomycetes dominated by a few species. These communities change over time and infructescence developmental stage, season and the age of the host population. Mature infructescences appear to be important sources of inoculum for some of the actinomycetes, seemingly disrupted by fire. Increased fire frequency limiting maturation of P. repens infructescences could thus impact future actinomycete colonisation in the landscape. Streptomyces spp. are likely to share this niche with the ophiostomatoid fungi, which merits further study regarding their interactions and mode of transfer.

Natalenamides A⁻C, Cyclic Tripeptides from the Termite-Associated Actinomadura sp. RB99.

In recent years, investigations into the biochemistry of insect-associated bacteria have increased. When combined with analytical dereplication processes, these studies provide a powerful strategy to identify structurally and/or biologically novel compounds. Non-ribosomally synthesized cyclic peptides have a broad bioactivity spectrum with high medicinal potential. Here, we report the discovery of three new cyclic tripeptides: natalenamides A⁻C (compounds 1⁻3). These compounds were identified from the culture broth of the fungus-growing termite-associated Actinomadura sp. RB99 using a liquid chromatography (LC)/ultraviolet (UV)/mass spectrometry (MS)-based dereplication method. Chemical structures of the new compounds (1⁻3) were established by analysis of comprehensive spectroscopic methods, including one-dimensional (¹H and 13C) and two-dimensional (¹H-¹H-COSY, HSQC, HMBC) nuclear magnetic resonance spectroscopy (NMR), together with high-resolution electrospray ionization mass spectrometry (HR-ESIMS) data. The absolute configurations of the new compounds were elucidated using Marfey's analysis. Through several bioactivity tests for the tripeptides, we found that compound 3 exhibited significant inhibitory effects on 3-isobutyl-1-methylxanthine (IBMX)-induced melanin production. The effect of compound 3 was similar to that of kojic acid, a compound extensively used as a cosmetic material with a skin-whitening effect.

Isolation, Biosynthesis and Chemical Modifications of Rubterolones A-F: Rare Tropolone Alkaloids from Actinomadura sp. 5-2.

The discovery of six new, highly substituted tropolone alkaloids, rubterolones A-F, from Actinomadura sp. 5-2, isolated from the gut of the fungus-growing termite Macrotermes natalensis is reported. Rubterolones were identified by using fungus-bacteria challenge assays and a HRMS-based dereplication strategy, and characterised by NMR and HRMS analyses and by X-ray crystallography. Feeding experiments and subsequent chemical derivatisation led to a first library of rubterolone derivatives (A-L). Genome sequencing and comparative analyses revealed their putative biosynthetic pathway, which was supported by feeding experiments. This study highlights how gut microbes can present a prolific source of secondary metabolites.

Two new Leptographium spp. reveal an emerging complex of hardwood-infecting species in the Ophiostomatales.

Species of Leptographium are generally characterized by mononematous conidiophores and are commonly associated with bark beetles and weevils. These species are responsible for sapstain and in some cases serious diseases on a range of primarily coniferous trees. In comparison with coniferous trees, the occurrence of Leptographium species on hardwood trees has been poorly studied in Europe. During a survey of ophiostomatoid fungi on various tree species in Norway and Poland, three unusual species, which fit the broader morphological description of Leptographium spp., were found in association with Scolytus ratzeburgi, Dryocoetes alni and Trypodendron domesticum on a variety of hardwoods, and from wounds on Tilia cordata. Phylogenetic analyses of sequence data for three gene regions (ITS2-LSU, ß-tubulin, and TEF1-α) showed that these Leptographium species are phylogenetically closely related to each other and form a well-supported lineage that included Grosmannia grandifoliae and Leptographium pruni. The first species could be distinguished from the other Leptographium species based on conidiophores arising from spiral hyphae, chlamydospore-like structures and a hyalorhinocladiella-like synanamorph in culture. The second species differs from the previous one by having distinctly shorter conidiophores and smaller conidia. This species also produces a well-developed sporothrix-like synanamorph with denticulate conidiogenous cells. Based on these unusual morphological characteristics and distinct DNA sequences, these fungi were recognised as new taxa for which the names Leptographium trypodendri sp. nov. and L. betulae sp. nov. are provided. The third group of isolates belonged to Grosmannia grandifoliae, representing the first report of this species outside of the USA. The newly defined G. grandifoliae complex is the first species complex in Leptographium s.l. consisting of only hardwood-infecting species.

Complementary symbiont contributions to plant decomposition in a fungus-farming termite.

Termites normally rely on gut symbionts to decompose organic matter but the Macrotermitinae domesticated Termitomyces fungi to produce their own food. This transition was accompanied by a shift in the composition of the gut microbiota, but the complementary roles of these bacteria in the symbiosis have remained enigmatic. We obtained high-quality annotated draft genomes of the termite Macrotermes natalensis, its Termitomyces symbiont, and gut metagenomes from workers, soldiers, and a queen. We show that members from 111 of the 128 known glycoside hydrolase families are represented in the symbiosis, that Termitomyces has the genomic capacity to handle complex carbohydrates, and that worker gut microbes primarily contribute enzymes for final digestion of oligosaccharides. This apparent division of labor is consistent with the Macrotermes gut microbes being most important during the second passage of comb material through the termite gut, after a first gut passage where the crude plant substrate is inoculated with Termitomyces asexual spores so that initial fungal growth and polysaccharide decomposition can proceed with high efficiency. Complex conversion of biomass in termite mounds thus appears to be mainly accomplished by complementary cooperation between a domesticated fungal monoculture and a specialized bacterial community. In sharp contrast, the gut microbiota of the queen had highly reduced plant decomposition potential, suggesting that mature reproductives digest fungal material provided by workers rather than plant substrate.

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.

The Ophiostoma clavatum species complex: a newly defined group in the Ophiostomatales including three novel taxa.

Two species of blue-stain fungi with similar morphologies, Ophiostoma brunneo-ciliatum and Ophiostoma clavatum, are associates of bark beetles infesting Pinus spp. in Europe. This has raised questions whether they represent distinct taxa. Absence of herbarium specimens and contaminated or mistakenly identified cultures of O. brunneo-ciliatum and O. clavatum have accentuated the uncertainty regarding their correct identification. The aim of this study was to reconsider the identity of European isolates reported as O. brunneo-ciliatum and O. clavatum by applying DNA-based identification methods, and to provide appropriate type specimens for them. Phylogenetic analyses of the ITS, ßT, TEF-1α and CAL gene sequences revealed that the investigated isolates represent a complex of seven cryptic species. The study confirmed that ITS data is insufficient to delineate species in some Ophiostoma species clusters. Lectotypes and epitypes were designated for O. clavatum and O. brunneo-ciliatum, and three new species, Ophiostoma brunneolum, Ophiostoma macroclavatum and Ophiostoma pseudocatenulatum, are described in the newly defined O. clavatum-complex. The other two species included in the complex are Ophiostoma ainoae and Ophiostoma tapionis. The results suggest co-evolution of these fungi in association with specific bark beetles. The results also confirm the identity of the fungus associated with the pine bark beetle Ips acuminatus as O. clavatum, while O. brunneo-ciliatum appears to be mainly associated with another pine bark beetle, Ips sexdentatus.

Wounds on Rapanea melanophloeos provide habitat for a large diversity of Ophiostomatales including four new species.

Rapanea melanophloeos, an important canopy tree in Afromontane forests, is commonly utilised for medicinal bark harvesting. Wounds created from these activities provide entrance for many fungi, including arthropod-associated members of the Ophiostomatales and Microascales (ophiostomatoid fungi). In this study we assessed the diversity of wound-associated Ophiostomatales on storm-damaged R. melanophloeos trees in the Afromontane forests of South Africa. Five species were identified based on micro-morphological and molecular phylogenetic analyses. These included Ophiostoma stenoceras and four newly described taxa Sporothrix itsvo sp. nov., S. rapaneae sp. nov., S. uta sp. nov. and O. noisomeae sp. nov. Four of these are members of the S. schenckii-O. stenoceras complex (O. stenoceras, S. itsvo sp. nov., S. rapaneae sp. nov., S. uta sp. nov.) while O. noisomeae groups basal in the Ophiostomatales alongside the S. lignivora complex and Graphilbum. In addition to other taxa known from this host, the present study shows that there is a rich, yet still poorly explored, diversity of Ophiostomatales associated with R. melanophloeos in Afromontane forests. More taxa are likely to be discovered with increased research effort. These must be assessed in terms of pathogenicity towards this ecologically and economically important tree.

Taxonomy and phylogeny of the Leptographium procerum complex, including Leptographium sinense sp. nov. and Leptographium longiconidiophorum sp. nov.

Leptographium procerum (Ophiostomatales, Ascomycota) is a well-known fungal associate of pine root-infesting bark beetles and weevils, occurring in several countries of the world. The fungus is not a primary pathogen but has been associated with white pine root decline in the USA and with serious damage caused by the introduced red turpentine beetle (RTB) Dendroctonus valens in China. Several species closely related to L. procerum have been described during the past decade. The aim of this study was to reevaluate species boundaries in the L. procerum complex using multigene phylogenetic analyses and morphological comparisons. Phylogenetic analyses of seven gene regions (ITS2-LSU, actin, ß-tubulin, calmodulin, translation elongation factor 1-α, and the mating type genes MAT1-1-3 and MAT1-2-1) distinguished between nine species in the complex. These included L. procerum, L. bhutanense, L. gracile, L. profanum, L. pini-densiflorae, L. sibiricum, L. sinoprocerum, as well as two new species described here as Leptographium sinense sp. nov. from Hylobitelus xiaoi on Pinus elliottii in China, and Leptographium longiconidiophorum sp. nov. from Pinus densiflora in Japan. Leptographium latens is reduced to synonymy with L. gracile, and an epitype is designated for L. procerum, because a living culture associated with the holotype of L. procerum did not exist. Amplification patterns of the mating type genes suggest that all known species in the L. procerum complex are heterothallic, although sexual states have not been observed for any of the species. The results also suggest that Eastern Asia is most probably the centre of species diversity for the L. procerum complex.