Molecular phylogenetic analyses reveal three new Ceratocystis species and provide evidence for geographic differentiation of the genus in Africa

The emergence of wattle wilt disease on non-native Acacia mearnsii trees in Africa, caused by the indigenous fungus Ceratocystis albifundus, has highlighted a need to better understand the diversity, ecology and distribution of Ceratocystis species in natural African environments. In this study we applied phylogenetic inference to identify and characterize isolates of Ceratocystis collected in a natural savanna ecosystem in South Africa. Three new species were recognized and are described as C. cryptoformis sp. nov. in the C. moniliformis complex, as well as C. thulamelensis sp. nov. and C. zambeziensis sp. nov., both residing in the C. fimbriata complex. Incorporating the new species into global phylogenies of Ceratocystis provided insights into the patterns of evolution and biogeography of this group of fungi. Notably, the African continent was identified as an important centre of diversification of Ceratocystis spp., from which several lineages of these fungi were shown to have radiated.     

A new wilt and die-back disease of Acacia mangium associated with Ceratocystis manginecans and C. acaciivora sp. nov. in Indonesia

Species of Ceratocystis are well-known wound related pathogens of many tree species, including commercially planted Acacia spp. Recently, several Ceratocystis isolates were collected from wilting A. mangium in plantations in Indonesia. The aim of this study was to identify these Ceratocystis isolates and to investigate their ability to cause disease on two plantation-grown Acacia spp. using greenhouse and field inoculation experiments. For identification, morphological characteristics and comparisons of DNA sequence data for the ITS, β-tubulin and TEF 1-α gene regions, was used. Ceratocystis isolates were identified as C. manginecans, a serious pathogen of mango trees in Oman and Pakistan and a previously undescribed species, described here as C. acaciivora sp. nov. Both fungi produced significant lesions in inoculation experiments on A. mangium and A. crassicarpa, however, C. acaciivora was most pathogenic suggesting that this fungus is the primary cause of the death of trees under natural conditions.     

Evolutionary dynamics of introns and their open reading frames in the U7 region of the mitochondrial rnl gene in species of Ceratocystis

The mtDNA rnl-U7 region has been examined for the presence of introns in selected species of the genus Ceratocystis. Comparative sequence analysis identified group I and group II introns encoding single and double motif LAGLIDADG open reading frames (ORFs) at the following positions L1671, L1787, and L1923. In addition downstream of the rnl-U7 region group I introns were detected at positions L1971 and L2231, and a group II intron at L2059. A GIY-YIG type ORF was located within one mL1923 LAGLIDADG type ORF and a degenerated GIY-YIG ORF fused to a nad2 gene fragment was found in association with the mL1971 group I intron. The diversity of composite elements that appear to be sporadically distributed among closely related species of Ceratocystis illustrates the potential for homing endonucleases and their associated introns to invade new sites. Phylogenetic analysis showed that single motif LADGLIDADG ORFs related to the mL1923 ORFs have invaded the L1787 group II intron and the L1671 group I intron. Phylogenetic analysis of intron encoded single and double motif LAGLIDADG ORFs also showed that these ORFs transferred four times from group I into group II B1 type introns.     

Sectional limits and phylogenetic considerations in Carex section Ceratocystis (Cyperaceae)

Carex L. sectionCeratocystis Dumort. is distinct from the closely related sectionSpirostachyae (Drejer) L. Bailey. Differences in pistillate spike morphology, achene shape, and perigynium epidermis are augmented by chemical, cytological, and micromorphological evidence. Character compatibility analysis has been used to reconstruct the phylogenetic history of the sedges in sectionCeratocystis. The resulting network is shown to be robust when tested with cytological, ecological, and micromorphological data not included in its construction. Agmatoploidy has been the dominant process of chromosomal evolution in the section.     

Inheritance of phenotypic traits in the progeny of a Ceratocystis interspecific cross

Ceratocystis fimbriata is a fungal plant pathogen that causes black rot on Ipomoea batatas. Based on inoculation studies on numerous tree species, the pathogen is known to be host specific. The closely related species, Ceratocystis manginecans, causes severe wilt on a broad range of tree hosts, including Mangifera indica, Acacia mangium and other leguminous tree species. The genetic factors underlying the pathogenicity and host specificity of Ceratocystis species have rarely been investigated. In this study, an F₁ population of 70 recombinant progeny from a cross between C. fimbriata and C. manginecans was generated and the inheritance of various phenotypic traits was investigated. Results showed that colony colour, growth rate, asexual spore production and aggressiveness to I. batatas and A. mangium are all quantitative traits with high levels of heritability. However, conidia production and aggressiveness appeared to be regulated by a small number of genes. No correlation could be found between aggressiveness and other phenotypic traits, suggesting that these are inherited independently. This is the first study to consider genetic inheritance of pathogenicity and host specificity in Ceratocystis species and the results will contribute, in future, to the identification of quantitative trait loci and candidate genes associated with the traits investigated.     

Ceratocystis species, including two new species associated with nitidulid beetles, on eucalypts in Australia

The genus Ceratocystis includes important fungal pathogens of trees, including Eucalyptus spp. Ironically, very little is known regarding the diversity or biology of Ceratocystis species on Eucalyptus species in Australia, where most of these trees are native. The aim of this study was to survey for Ceratocystis spp., and their possible insect associates, on eucalypts in Australia and thus to establish a foundation of knowledge regarding these fungi on the continent. Collections were made in three states of Australia from wounds on trees, as well as from nitidulid beetles associated with these wounds. Ceratocystis spp. were identified based on morphology and multigene sequence comparisons. Of the 54 isolates obtained, two previously unknown species of Ceratocystis were found and these are described here as Ceratocystis corymbiicola sp. nov. and Ceratocystis tyalla sp. nov. Furthermore, the distribution of Ceratocystis pirilliformis is expanded to include Eucalyptus spp. in Tasmania.     

Successful restoration of Metrosideros polymorpha (ʻōhiʻa) is possible in forest sites with active Rapid ‘Ōhiʻa Death infections

Rapid ?ōhi?a Death (ROD), caused by the fungal pathogen Ceratocystis, is killing large numbers of ?ōhi?a trees (Metrosideros polymorpha) in Hawai?i. ?ōhi?a are a dominant tree in Hawaiian forests, have a range that goes from arid to wet forest climates, and are important for endangered species habitat and ecosystem function. To test whether actively planting ?ōhi?a seedlings is a viable restoration strategy in areas with high ROD mortality, we planted ?ōhi?a in a ROD‐affected forest and crossed this with weeding and fencing treatments to compare ROD mortality to other stressors. We also tested for viable Ceratocystis spores in soils around planting areas. We found that seedlings were more likely to die in unweeded and unfenced treatments than controls. Although viable Ceratocystis spores were found in soil, none of the 41 dead seedlings tested positive for Ceratocystis. This indicates that competition from exotic plants and exotic feral ungulate damage are more likely to kill seedlings than ROD within the first year after planting.     

Ceratocystidaceae exhibit high levels of recombination at the mating-type (MAT) locus

Mating is central to many fungal life cycles and is controlled by genes at the mating-type (MAT) locus. These genes determine whether the fungus will be self-sterile (heterothallic) or self-fertile (homothallic). Species in the ascomycete family Ceratocystidaceae have different mating strategies, making them interesting to consider with regards to their MAT loci. The aim of this study was to compare the composition of the MAT locus flanking regions in 11 species of Ceratocystidaceae representing four genera. Genome assemblies for each species were examined to identify the MAT locus and determine the structure of the flanking regions. Large contigs containing the MAT locus were then functionally annotated and analysed for the presence of transposable elements. Genes typically flanking the MAT locus in sordariomycetes were found to be highly conserved in the Ceratocystidaceae. The different genera in the Ceratocystidaceae displayed little synteny outside of the immediate MAT locus flanking genes. Even though species ofCeratocystis did not show much synteny outside of the immediate MAT locus flanking genes, species of Huntiella and Endoconidiophora were comparatively syntenic. Due to the high number of transposable elements present in Ceratocystis MAT flanking regions, we hypothesise that Ceratocystis species may have undergone recombination in this region.     

Immunochemical studies on L-rhamno-D-mannans of sporothrix schenckii and related fungi by use of rabbit and human antisera

Antisera were prepared in rabbits against the human pathogenic yeast Sporothrix schenckii (strain 1099.12) grown at two different temperatures (25° and 37°). Precipitation and inhibition data showed that the former serum had a specificity directed against α-L-Rhap-(1→2)-α-L-Rhap-(1→3)-D-Man-(1→ determinants, whereas the latter had a broad specificity in which α-L-rhamnosyl or α-L-Rhap-(1→3)-D-Man-was the immunodominant structure. These results are consistent with data on the structures of the L-rhamno-D-mannans isolated from the organism grown at the two different temperatures. Human sera from patients with sporotrichosis were shown to have different specificities resembling the specificities developed in the rabbits. The rabbit antisera were also used to examine the cross-reactivity with L-rhamno-D-mannans from species of the genus Ceratocystis, which is reputed to include the ascigerous (perfect) state of S. schenckii. Polysaccharides from four species of Ceratocystis grown at 25° reacted with the antisera in a manner resembling that of the L-rhamno-D-mannan from S. schenckii grown at 37°. This is in accord with earlier data that showed that only S. schenckii, of the species studied, produces a polysaccharide with large amounts of α-L-Rhap-(1→2)-α-L-Rhap-(1→ side-chains when grown at 25°.     

DEVELOPMENT OF THE ASCOCARP OF CERATOCYSTIS ULMI

Rosinski , Martin A. (U. Maine, Orono.) Development of the ascocarp of Ceratocystis ulmi. Amer. Jour. Bot. 48(4): 285–293. Illus. 1961.—A study of the development of the perithecium of Ceratocystis ulmi was conducted using classic histological techniques. This study revealed the presence of a singular combination of primitive and advanced characteristics. The perithecium possesses a simple centrum made up only of ascogenous hyphae and small, spherical asci, but croziers are formed prior to ascus formation, and the ascogenous hyphae are arranged in a hymenium. Since development of C. ulmi compares closely with most other accounts of development in other members of the genus Ceratocystis, it appears that Ceratocystis is a good taxon. In addition, because of its intermediate nature and because Ceratocystis is the type genus of the family Ophiostomataceae, this family should be placed in a separate order, the Ophiostomatales.     

Diversity and evolution of polyketide biosynthesis gene clusters in the Ceratocystidaceae

Polyketides are secondary metabolites with diverse biological activities. Polyketide synthases (PKS) are often encoded from genes clustered in the same genomic region. Functional analyses and genomic studies show that most fungi are capable of producing a repertoire of polyketides. We considered the potential of Ceratocystidaceae for producing polyketides using a comparative genomics approach. Our aims were to identify the putative polyketide biosynthesis gene clusters, to characterize them and predict the types of polyketide compounds they might produce. We used sequences from nineteen species in the genera, Ceratocystis, Endoconidiophora, Davidsoniella, Huntiella, Thielaviopsis and Bretziella, to identify and characterize PKS gene clusters, by employing a range of bioinformatics and phylogenetic tools. We showed that the genomes contained putative clusters containing a non-reducing type I PKS and a type III PKS. Phylogenetic analyses suggested that these genes were already present in the ancestor of the Ceratocystidaceae. By contrast, the various reducing type I PKS-containing clusters identified in these genomes appeared to have distinct evolutionary origins. Although one of the identified clusters potentially allows for the production of melanin, their functional characterization will undoubtedly reveal many novel and important compounds implicated in the biology of the Ceratocystidaceae.     

The oldest stylophoran echinoderm: a new <Emphasis Type="Italic">Ceratocystis</Emphasis> from the Middle Cambrian of Germany

A new stylophoran (Ceratocystis prosthiakida sp. nov.) from the early Middle Cambrian of the Franconian Forest (north-east Bavaria, Germany) is described with the aid of computed tomography, a powerful means of visualizing fossil anatomy. Investigation in this manner enabled previously inaccessible, buried parts of the fossil to be imaged and studied. In addition, a procedure for digitally correcting plate articulations was implemented. Ceratocystis prosthiakida sp. nov. possesses a thin anterior right spine which clearly differentiates it from other species of Ceratocystis. Similar to other Ceratocystis species, C. prosthiakida sp. nov. lived in storm-dominated shallow marine conditions. The species is from the Galgenberg Formation, which has an early Agdzian age in the unified West Gondwanan chronostratigraphy. As a result, it represents the oldest stylophoran known to date.     

Molecular and morphological evidence for a new species from South Africa: Carex rainbowii (Cyperaceae)

Carex rainbowii (Cariceae, Cyperaceae), a new species from the Drakensberg mountains (KwaZulu-Natal province), is described and illustrated. It was found in the shady understory of the Afromontane forest in the Cathedral Peak area. An additional, nearby population was also identified based on previously collected herbarium material. Morphological and molecular (cpDNA 5′trnK intron and nrDNA ITS and ETS sequences) data were used to evaluate the taxonomic status of these populations and shed light on their systematic placement. Our data strongly support their taxonomic identity and inclusion in Carex sect. Sylvaticae. The new species can be readily distinguished from other related taxa mainly by the frequently androgynecandrous terminal spike, dense female spikes, hyaline glumes, as well as by some quantitative features. This finding implies a considerable biogeographic disjunction from the mainly Eurasian-North African range of the remaining species of sect. Sylvaticae, a pattern also found in the related sections Ceratocystis, Rhynchocystis and Spirostachyae. Comments are provided on previous misidentifications of C. rainbowii as the closely related Carex sylvatica. Data pertinent to the conservation status of the species are provided.     

Diseases Severity,Genetic Variation,and Pathogenicity of Ceratocystis Wilt on Lansium domesticum in South Sumatra,Indonesia

Ceratocystis wilt disease has caused significant mortality in duku (Lansium domesticum) since 2014 and has now spread to all districts in South Sumatra, Indonesia. Recently, 16 isolates from duku representing populations from various districts in South Sumatra were isolated. Analysis for the morphological characteristic of the isolate showed that the population has a uniform morphology. Genetic analysis based on internal transcribed spacer (ITS) and β-tubulin sequences verified that the population has being dominated by the ITS5 haplotype of Ceratocystis fimbriata and a new ITS group, the ITS7b haplotype that was localized in Musi Banyuasin. Both haplotypes were highly pathogenic to duku. Inoculation tests on various forest and agroforestry plant hosts showed that both haplotypes were highly pathogenic to Acacia mangium, moderately pathogenic to Acacia carsicarpa, Eucalyptus urophylla, and Melaleuca cajuputi, but weakly pathogenic to Dyera costulata, Hevea brasiliensis, and Alstonia scholaris. Therefore, this pathogen becomes a serious threat to Indonesia’s biodiversity due to its ability to infect forest and agroforestry plants, especially the indigenous ones.     

Contrasting carbon metabolism in saprotrophic and pathogenic microascalean fungi from Protea trees

Protea-associated Knoxdaviesia species grow on decaying inflorescences, yet are closely related to plant pathogens such as Ceratocystis albifundus. C. albifundus also infects Protea, but occupies a distinct niche. We investigated substrate utilization in two Knoxdaviesia saprotrophs, a generalist and a specialist, and the pathogen C. albifundus by integrating phenome and whole-genome data. On shared substrates, the generalist grew slightly better than its specialist counterpart, alluding to how it has maintained its Protea host range. C. albifundus grew on few substrates and had limited cell wall-degrading enzymes. It did not utilize sucrose, but may prefer soluble oligosaccharides. Nectar monosaccharides are likely important carbon sources for early colonizing Knoxdaviesia species. Once the inflorescence ages, they could switch to degrading cell wall components. C. albifundus likely uses its limited cell wall-degrading arsenal to gain access to plant cells and exploit internal resources. Overall, carbon metabolism and gene content in three related fungi reflected their ecological adaptations.     

Genomic analysis of the aggressive tree pathogen Ceratocystis albifundus

The overall goal of this study was to determine whether the genome of an important plant pathogen in Africa, Ceratocystis albifundus, is structured into subgenomic compartments, and if so, to establish how these compartments are distributed across the genome. For this purpose, the publicly available genome of C. albifundus was complemented with the genome sequences for four additional isolates using the Illumina HiSeq platform. In addition, a reference genome for one of the individuals was assembled using both PacBio and Illumina HiSeq technologies. Our results showed a high degree of synteny between the five genomes, although several regions lacked detectable long-range synteny. These regions were associated with the presence of accessory genes, lower genetic similarity, variation in read-map depth, as well as transposable elements and genes associated with host-pathogen interactions (e.g. effectors and CAZymes). Such patterns are regarded as hallmarks of accelerated evolution, particularly of accessory subgenomic compartments in fungal pathogens. Our findings thus showed that the genome of C. albifundus is made-up of core and accessory subgenomic compartments, which is an important step towards characterizing its pangenome. This study also highlights the value of comparative genomics for understanding mechanisms that may underly and influence the biology and evolution of pathogens.     

Degradation and detoxification of softwood extractives by sapstain fungi

Wood extractives (resin) cause pitch deposition problems and effluent toxicity in pulp and papermaking. The ability of six sapstaining fungi to degrade and detoxify extractive constituents in Scots pine sapwood was examined, and the results were compared with those obtained with the commercial depitching fungus Cartapip (Ophiostoma piliferum). Pestalotiopsis crassiuscula and O. piliferum were the best strains and they provided high reductions of total resin (50–60% in 6 weeks). Both strains were highly effective in the degradation of individual extractive components including triglycerides, diglycerides and free fatty acids. Although all strains displayed moderate to high pitch degradation, their detoxifying capacity was limited. Two important exceptions were Ceratocystis deltoideospora and O. piliferum that caused a 11–14-fold decrease in toxicity (Microtox bioassay). These results indicate the potential of wood pretreatment with the selected sapstain fungi for minimizing pitch problems and decreasing effluent toxicity in pulping.     

Development of Perithecia and Reproductive Structures in Two Species of Ceratocystis

Two species of the fungus Ceratocystis are described which havebeen isolated from beetle galleries in Japanese larch. The detaileddevelopment of reproductive structures is described in each,confirming the work of Varitchak in the case of C. piceae. C.autographa is diagnosed as a new heterothallic species.