Two new Ceratocystis species associated with mango disease in Brazil

Mangifera indica, a disease known as mango blight, murcha or seca da mangueira in Brazil, is caused by the canker wilt pathogen Ceratocystis fimbriata sensu lato. It is also closely associated with infestation by the non-native wood-boring beetle Hypocryphalus mangiferae (Coleoptera: Scolytinae). The aim of this study was to characterize Ceratocystis isolates obtained from diseased mango trees in Brazil. Identification was based on sequence data from ITS1+5.8S+ITS2 rDNA, part of the Beta-tubulin 1 gene, and part of the Transcription Elongation Factor 1-alpha gene. The Brazilian isolates grouped in two well defined and unique clades within C. fimbriata s.l. These were also distinct from C. manginecans, which causes a similar disease associated with H. mangiferae in Oman and Pakistan. Based on sequence comparisons and morphological characteristics, isolates representing the two phylogenetic clades are described as C. mangicola sp. nov. and C. mangivora sp. nov.The National Research Foundation (NRF), members of the Tree Protection Co-operative Programme (TPCP), the THRIP initiative of the Department of Trade and Industry, and the Department of Science and Technology (DST)/NRF Centre of Excellence in Tree Health Biotechnology (CTHB).http://www.mycotaxon.com/nf201

Microevolution of Serial Clinical Isolates of Cryptococcus neoformans var. grubii and C. gattii

We thank the Broad Institute Sequencing Platform for generating the Illumina sequences. We thank Chen-Hsin Yu for helping on the data processing of the phenotypic tests. We acknowledge the South African National Institute for Communicable Diseases’ GERMS-SA surveillance network through which these isolates were originally collected. This project has been funded in whole or in part by the following U.S. Health and Human Services grants from the National Institute of Allergy and Infectious Diseases: U19 AI110818 (Broad Institute), R01 AI93257 (J.R.P.), R01 AI73896 (J.R.P.), and R01 AI025783 (T.G.M.). R.A.F. was supported by the Wellcome Trust. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The content is solely our responsibility and does not necessarily represent the official views of the funders. The use of product names in this manuscript does not imply their endorsement by the U.S. Department of Health and Human Services. The findings and conclusions in this article are those of the authors and do not necessarily represent the views of the CDC.Peer reviewedPublisher PD

Taxonomy and population biology of selected Ceratocystis spp. with hat-shaped ascospores

This dissertation represents a study on Ceratocystis spp. with hat-shaped ascospores. Previously only six species of Ceratocystis with this spore form were known. These include C. fimbriata, C. pirilliformis, C. albofundus, C. moniliformis, C. moniliformopsis and C. acericola. In this study, we have discovered and tentatively described three new species with hat-shaped ascospores. One of these group with the larger C. fimbriata clade, while the other two reside within the larger C. coerulescens clade. Chapter one provides a concise summary of the literature pertaining to the genus Ceratocystis. The intention of the chapter is to introduce readers to this important genus of plant pathogens and to provide a background regarding their taxonomy, ecology, biochemistry and variation in species. Emphasis is placed on the distinction of Ceratocystis from Ophiostoma, as ell as on those Ceratocystis spp. with hat-shaped ascospores that negatively impact upon plantation forestry species. This chapter shows how Ceratocystis spp. associated with hardwood species in commercial forestry plantations have increased in number and it provides the background for research presented in the following five chapters. In the second chapter of this dissertation, a new species of Ceratocystis was discovered amongst isolates from the Himalayan mountain range of Bhutan. This fungus, in association with the bark beetle Ips schmutzenhoferi, is responsible for large-scale deaths of Himalayan spruce trees in Bhutan. The fungus is morphologically very similar to C. moniliformis and C. moniliformopsis, but differences in culture morphology, survival at different incubation temperatures and DNA sequence data based on three different gene regions supported the fact that this is a unique species. The fungus has thus been tentatively described as Ceratocystis bhutanensis prov. nom. In chapter 3, I consider isolates of a Ceratocystis sp. recently discovered associated with dying clove trees in Northern Sulawesi, Indonesia. The fungus was found at a very high level of incidence, but was at first identified as C. fimbriata, based on morphological characteristics. Differences were observed in cultures of this fungus when they were compared with C. fimbriata especially in terms of colony colour and growth at different temperatures. Morphological differences were also observed when the clove fungus was compared with C. fimbriata isolates. When three different DNA gene regions were sequenced and compared, it was clear that this fungus represents a new species. The fungus is, therefore, tentatively described as Ceratocystis polychrome prov. nom. in this dissertation. Ceratocystis polychrome prov. nom. Isolates obtained from cloves in Sulawesi displayed three distinctly different culture morphologies. In Chapter 4 of this dissertation we used DNA sequence data and microsatellite markers to consider whether these differences could be observed at the molecular level. Comparisons of sequence data for the ITS region gave no distinction between any of the morphological groups. A total of 50 isolates were studies using microsatellites markers developed for C. fimbriata. No distinction could be obtained between isolates representing the three different culture morphological groups. The 50 isolates were subsequently treated as one population in further analyses. With the aid of the microsatellite markers, it was shown that this population probably originated from Sulawesi and that it benefits form sexual outcrossing. In chapter five of this dissertation, a study was undertaken to consider the taxonomic status of C. moniliformis. Consideratble variation has been noted in different descriptions of this species. It also has a very wide host and geographic distribution raising speculation that C. moniliformis represents a species complex rather than a single taxon. Based on morphological and DNA sequence data from three gene regions, isolated from Sumatra were described as a new species, which we have tentatively named C. tribiliformis prov. nom. The other C. moniliformis isolates were all the same, despite the fact that they originated from a wide range of hosts and areas. The fungus correctly bearing the name C. moniliformis, C. moniliformis sensu stricto, therefore does not seem to tepresent a species complex. Species such as the closely related C. tribiliformis prov. nom., C bhutanensis prov. nom., C. omanensis prov. nom. and C. moniliformopsis all belong to the larger C. moniliformis sensu lato group, and all have hat-shaped ascospores, conical spines on the ascomatal bases, disc-shaped bases to the ascomatal necks and are phylogenetically closely related to C. moniliformis. Studies presented in this dissertation provide considerable new knowledge regarding various Ceratocystis spp. with hat-shaped ascospores. Three new species are described and I have also been able to show that C. moniliformis sensu stricto is monophyletic. Two of the species (C. tribiliformis prov. nom. and C. bhutanensis nom. prov.) group within the larger C. coerulescens clade while C. polychrome prov. nom. groups within the larger C. fimbriata clade. Studies in this dissertation have also improved our knowledge of the identity of several species previously incorrectly identified as either C. moniliformis or C. fimbriata. What has clearly emerged from this dissertation is the need for a monograph of Ceratocystis to include all new species and to thoroughly consider the population biology and ecology of all species.Dissertation (MSc)--University of Pretoria, 2008.Microbiology and Plant PathologyMScUnrestricte

Phylogenetic relationships and taxonomy of species in ceratocystis sensu lato

Ceratocystis is a genus of Ascomycete fungi residing in the family Ceratocystidaceae (Order: Microascales; Class: Sordariomycetes). Species of Ceratocystis are best-known as woundinfecting fungi, which are mostly carried by insects. Little is known regarding the basis of the relationship between insects and Ceratocystis spp., but in some cases it might extend to levels of mutualism. The taxonomic history of the genus is complex and has, during the course of more than 100 years, been intertwined with several other genera. However, in recent years, the resolution arising from DNA sequence comparisons has resulted in the recognition of at least four major groups within Ceratocystis. major groups within Ceratocystis. Ceratocystis species in the broad sense (including Ambrosiella) represent ecologically diverse assemblages, including four very clearly defined groups. One of these groups, which I refer to as the C. coerulescens sensu lato (s.l.) group, which mainly cause blue-stain in timber, is the only group that is known to have a very close association with conifer-infesting bark beetles. Perhaps the best-known group is the C. fimbriata s.l. assemblage of species, which includes a large number of serious pathogens of angiosperms, mostly trees. This group also includes nonpathogenic wound-infecting species. There are no specific insect vectors of these species but they produce fruity aromas, which attract a wide range of insects such as flies, ants, mites and nitidulid beetles that aid in their dissemination to fresh wounds. The majority of the species in the C. fimbriata s.l. group are primary pathogens causing cankers that can girdle and eventually kill the affected areas. The third group of fungi in the broadly defined Ceratocystis s.l. is the C. moniliformis s.l. group. This group includes only saprophytes that, similar to species in C. fimbriata s.l., produce fruity odours and they are thus vectored by opportunistic insects including flies and nitidulid beetles. Ambrosiella spp. are broadly included in the assemblage accommodating Ceratocystis spp. These fungi have no known sexual state and they live in obligate symbioses with ambrosia beetles (Scolytinae). Ceratocystis and its relatives have had a complex taxonomic history ever since the genus was first described by Halstead in 1890. Much of the confusion regarding their taxonomy has arisen from a dependence on morphology for classification and the fact that these and other fungi have undergone convergent evolution related to their association with insects. Until the late 1990s, genera and species in this group were described based only on morphology, although the importance of various morphological features was strongly debated. However, between 1950- 1999, a turning point arose in the way that these fungi were recognised. This change emerged first from the inclusion of various chemical approaches and then later the application of molecular techniques to identify them. The most dramatic changes have come with the widespread application of DNA sequence comparisons, which were first applied to the group in the early 1990’s. This made it possible to define higher-order relationships and brought the first clear evidence that species in Ceratocystis and Ophiostoma were phylogenetically unrelated. A complete taxonomic history of this group is provided in the timeline presented in Table 1. This thesis deals primarily with two groups in the broadly defined Ceratocystis. These are species in the C. moniliformis s.l. and the C. fimbriata s.l. complexes, but with a considerably greater focus on the C. fimbriata s.l. complex. Morphologically, species in the two complexes are easy to distinguish from each other even though they share some common characteristics. Species in both complexes have hat-shaped ascospores and both have the anamorphic characteristics typical of species in the genus Thielaviopsis. The ascomatal bases are mostly globose to sub-globose with elongated necks. There are two main features that species in the C. moniliformis s.l. complex have that are not observed in the C. fimbriata s.l. complex. One is a very distinct “collar-like” structure at the base of the ascomatal necks of species in the C. moniliformis s.l. complex. These “collar-like” structures result in the necks being fragile and they dislodge from the ascomatal bases very easily. The other distinct characteristic is that these fungi have conical spines on their ascomatal bases, giving the structures a textured appearance. This is in contrast to the ascomatal bases in the C. fimbriata complex that are smooth. Species in the C. moniliformis s.l. complex are non-pathogenic and cause only some sap-stain in the host tissues. The C. fimbriata s.l. complex includes a large number of primary pathogens, mainly of trees but also including root crops. A phylogenetic tree including all species in the C. fimbriata s.l and C. moniliformis s.l. complexes is presented in Figure 1-3. In generating this tree, three gene regions i.e. the Internal Transcribed Spacer Region including the 5.8S rRNA operon (ITS), part of the the Beta-Tubulin 1 (BT) gene and part of the Transcription Elongation Factor 1 alpha (TEF) gene have been combined to represent species in the two complexes. Both Bootstrap as well as Bayesian analyses were run to obtain confidence intervals. More detailed methods are presented in the legends to the trees. Chapter one of this thesis presents a review of the groups in Ceratocystis. With the aid of DNA sequence comparisons, three phylogenetically distinct groups are identified. These groups are characterised by three well-known species each now defined as a species complex i.e. the Ceratocystis coerulescens complex, the C. moniliformis s.l. complex and the C. fimbriata s.l. complex. Species of Ambrosiella are also recognised as representing a discrete and related group. These three species complexes are not only differentiated based on DNA sequence comparison but have distinct morphological features that distinguish them from each other. They are also defined by having very distinct ecological roles in nature. Chapter 2 specifically reviews the key literature dealing with the taxonomy of species in the C. fimbriata complex. This group is defined by Ceratocystis fimbriata s.s., which was first described as a pathogen causing black rot on sweet potato. Subsequent to the emergence of DNA sequencing techniques to define species, many new species in the C. fimbriata s.l. complex have been defined and described. Chapters three and four of this thesis deal with a species of Ceratocystis collected from dying Mango trees in Oman. A very serious disease known as Mango Sudden Decline disease was first observed in Oman and has recently been subjected to intensive investigation. This led to a recognition that a species related to C. fimbriata might be responsible for the disease of Mangifera indica. As part of the process of identifying the causal agent of Mango Sudden Decline, two Ceratocystis spp. were described. One is the previously described and nonpathogenic C. omanensis residing in the C. moniliformis s.l. complex and the other is C. manginecans, a virulent pathogen residing in the C. fimbriata s.l. complex (Chapter 4). Chapters five through eleven of this thesis include the descriptions of new species in the C. fimbriata complex that have emerged over a seven year period. These descriptions have all relied strongly on DNA sequence based phylogenetic inference as well as morphology. It is important to recognise, however, that the morphological features are very similar in most species and that differences rely on the size ranges of key features in this group. The genus Ceratocystis represents a complex of many species and is set to be sub-divided into at least three discrete genera, not including Ambrosiella, which is also closely related and already well-defined. Studies in this thesis began at a time when it became possible to define species of Ceratocystis based strongly on a phylogenetic concept. Thus, while morphological and ecological features were taken into account, there has been a heavy reliance on DNA sequence comparisons. It is likely that in coming years, new gene regions will be recognised that will allow a refinement of genus and species boundaries. Furthermore, having access to whole-genome sequencing will add deeply to our understanding of taxonomic relationships between these important and fascinating fungi. It is my hope that the foundation provided by the studies incorporated in this thesis will be useful as this new wave of study emerges.Thesis (PhD)--University of Pretoria, 2013.gm2013Geneticsunrestricte

Four new Ceratocystis spp. associated with wounds on Eucalyptus, Schizolobium and Terminalia trees in Ecuador

Species of Ceratocystis commonly infect wounds on trees. In this study, artificially induced wounds were made on the stems of Eucalyptus, Schizolobium and Terminalia trees in Ecuadorian lowland forests, in an effort to determine the presence of Ceratocystis spp. in that environment. Species belonging to the C. fimbriata sensu lato (s.l.) complex and others in the C. moniliformis s.l. complex were collected. Phylogenetic analyses for both major groups in Ceratocystis using three gene regions (ITS, β-tubulin and EF1-α) revealed three distinct clades in the C. fimbriata s.l. complex and two in the C. moniliformis s.l. complex. Isolates in the three clades representing the C. fimbriata s.l. complex represent morphologically distinct species that are described here as C. curvata sp. nov., C. ecuadoriana sp. nov. and C. diversiconidia sp. nov. Isolates in one of the two clades in the C. moniliformis species complex represented C. moniliformis sensu stricto (s.s.) and the other was of a species with a distinct morphology that is described here as C. sublaevis sp. nov.National Research Foundation (NRF), members of the Tree Protection Co-operative Programme (TPCP), the THRIP initiative of the Department of Trade and Industry, the Department of Science and Technology (DST)/NRF Centre of Excellence in Tree Health Biotechnology (CTHB

New Ceratocystis species infecting coffee, cacao, citrus and native trees in Colombia

Ceratocystis fimbriata sensu lato includes a large number of plant and especially tree pathogens. In Colombia, isolates of this fungus cause a serious cankerstain disease on coffee as well as other fruit trees. Large collections of these isolates have been shown to occur in two distinct phylogenetic lineages based on ITS sequence comparisons. The aim of this study was to compare representatives of these two groups of isolates from coffee, citrus, cacao and native trees in Colombia, based on morphology and DNA-sequences for three gene regions. Host-specificity of the fungus was also considered. Representatives of the two groups of isolates were morphologically distinct and could be distinguished based on DNA sequence comparisons. They are also distinct from other species in the C. fimbriata sensu lato species complex and the sweet potato pathogen C. fimbriata sensu stricto and are provided with the names C. colombiana sp. nov and C. papillata sp. nov. There was no evidence for host-specificity amongst isolates of these two fungi that collectively represent a serious threat to coffee production in Colombia

Pruning quality affects infection of Acacia mangium and A. crassicarpa by Ceratocystis acaciivora and Lasiodiplodia theobromae

Pruning (singling) is a common silvicultural practice in commercial Acacia plantations because these trees tend to have multiple stems. The wounds resulting from pruning are susceptible to infection by pathogens. Ceratocystis acaciivora and Lasiodiplodia theobromae have recently been shown to be important pathogens of A. mangium in Indonesia, where they are commonly associated with wounds on trees. The aim of this study was to determine the impact of different wound types on infection of A. mangium and A. crassicarpa by these two pathogens. Isolates of C. acaciivora and L. theobromae, found to be the most pathogenic in a prior study, were used to inoculate pruning wounds. Results showed that pruning conducted in a manner to reduce stem damage, resulted in lower levels of fungal infection. Where pruning resulted in tearing of the bark, there were greater levels of infection and disease occurred even without artificial inoculation.Inoculation of pruning wounds on A. mangium and A. crassicarpa showed that both fungi have the potential to cause disease. However, C. acaciivora was most pathogenic. Results of this study showed conclusively that careful pruning will result in lower levels of disease in young A. mangium and A. crassicarpa plantations in Indonesia.The Tree Protection Co-operative Programme (TPCP) and the South African National Research Foundation are thanked for financial support to undertake this study. The first author also recognizes the support of PT Riau Andalan Pulp and Paper for support to conduct post graduate studies in South Africa.http://www.nisc.co.za/journals?id=11nf201

Ceratocystis fimbriatomima, a new species in the C. fimbriata sensu lato complex isolated from Eucalyptus trees in Venezuela

Species of Ceratocystis represent a group of important plant pathogens as well as saprobes that occur, primarily on woody substrates. The number of species in Ceratocystis has increased substantially in recent years, particularly as DNA-based methods have allowed for the recognition of cryptic taxa. The aim of this study was to identify isolates of a Ceratocystis sp. collected from freshly cut stumps of Eucalyptus trees in Venezuela. This was carried out using morphological comparisons with similar fungi as well as DNA sequence comparisons for the Internal Transcribed Spacer regions 1 and 2 including the 5.8S rDNA operon, part of the Beta-tubulin gene and part of the Transcription Elongation Factor 1-alpha gene region. Characteristics of the fungus in culture and its morphology resembled most species in the C. fimbriata sensu lato species complex. Microscopically, the fungus was most similar to C. fimbriata sensu stricto. Based on phylogenetic analyses, it was distinct from other species of Ceratocystis sensu lato having C. manginecans as its closest relative. The Ceratocystis sp. from Eucalyptus in Venezuela clearly represents a distinct taxon for which the name C. fimbriatomima sp. nov. is provided

Insect associates of Ceratocystis albifundus and patterns of association in a native savanna ecosystem in South Africa

Species of Ceratocystis Ellis and Halstead s.l. include important plant pathogens such as C. albifundus Morris, De Beer, and M. J. Wingfield that causes a serious wilt disease of non-native, plantation-grown Acacia mearnsii De Wild. trees in Africa. The aim of this study was to identify the insects associated with C. albifundus in South Africa and to consider the means by which the pathogen spreads. Insects were collected weekly for 77 wk in a native ecosystem using modified pitfall traps. Trapped insects were identified, and fungi were isolated using carrot baiting and by plating them onto malt extract agar. Fungi were identified using morphological characteristics and DNA sequence comparisons. Three different nitidulid (Coleoptera: Nitidulidae) beetles, Brachypeplus depressus Erichson, Carpophilus bisignatus Boheman, and Ca. hemipterus L, were collected, of which the most common were the Carpophilus spp. Two Ceratocystis spp., namely C. albifundus and C. oblonga R. N. Heath and Jolanda Roux, were isolated from all three insect species. Insect numbers and fungal isolates decreased significantly in the colder months of the year. Of the two Ceratocystis spp., C. oblonga was most abundant, occurring on 0.5% of the Carpophilus spp. C. albifundus was isolated from 1.1% of the Brachypeplus individuals and from 0.01% of the Carpophilus individuals. This study presents the first record of insects associated with C. albifundus and C. oblonga and provides an indication of environmental influences on fungal and insect populations, which could contribute to future disease management

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