Pre-invasion assessment on African invasive grasses revealed five new species of ergot fungi, Claviceps section Pusillae.

Ergot, the genus Claviceps comprises several deeply diverged lineages, recently classified as sections. Among them, the section Pusillae, is the most speciose, with a centre of distribution in Africa but occurring worldwide, often as a consequence of its invasive potential. This section includes the most severe plant pathogens such as Claviceps africana and C. gigantea, responsible for toxicoses and a significant reduction in the seed yields of Sorghum and Zea. In this study we surveyed ergot diversity in South Africa, focusing on grasses native to this region, but known for their high potential of invasiveness. The revision based on molecular and phenotypic markers revealed 16 species, with a high proportion of undescribed diversity, confirming Africa as a hot spot for this section. Five new species, Claviceps tulasnei, Claviceps eulaliae, Claviceps hypertheliae, Claviceps fredericksoniae and Claviceps arundinellae were described from Setaria, Eulalia, Hyperthelia, Miscanthus and Arundinella respectively. Claviceps texensis infecting Cenchrus, previously only identified from the same host in Texas, USA, was confirmed to be present in Africa, which is assumed to be its primary area of distribution. In addition, the host grass genus Anthephora is newly reported as a host of Claviceps digitariae. The most of the taxa were negligible concerning alkaloid production, with the exception of C. fredericksoniae, which is a sister of potent alkaloid producer C. africana, and produces mainly DH-ergosine, together with traces of DH-ergocornine. The host/parasite associations within Pusillae section is very narrow, suggesting that co-speciation is the major speciation driver in this group. Host grasses of the described species are already recognised invasive species and their ovarial parasites need to be monitored. This is highlighted by the fact that all Pusillae produced air-borne secondary conidia, which is autapomorphy of this section and considered to be important for their invasive abilities.

Evolutionary history of ergot with a new infrageneric classification (Hypocreales: Clavicipitaceae: Claviceps).

The ergot, genus Claviceps, comprises approximately 60 species of specialised ovarial grass parasites famous for the production of food toxins and pharmaceutics. Although the ergot has been known for centuries, its evolution have not been resolved yet. Our approach combining multilocus phylogeny, molecular dating and the study of ecological, morphological and metabolic features shows that Claviceps originated in South America in the Palaeocene on a common ancestor of BEP (subfamilies Bambusoideae, Ehrhartoideae, Pooideae) and PACMAD (subfamilies Panicoideae, Aristidoideae, Chloridoideae, Micrairoideae, Arundinoideae, Danthonioideae) grasses. Four clades described here as sections diverged during the Paleocene and Eocene. Since Claviceps are parasitic fungi with a close relationship with their host plants, their evolution is influenced by interactions with the new hosts, either by the spread to a new continent or the radiation of the host plants. Three of the sections possess very narrow host ranges and biogeographical distributions and have relatively low toxicity. On the contrary, the section Claviceps, comprising the rye ergot, C. purpurea, is unique in all aspects. Fungi in this section of North American origin have spread all over the world and infect grasses in all subfamilies as well as sedges, and it is the only section synthesising toxic ergopeptines and secalonic acids. The evolutionary success of the Claviceps section members can be explained by high toxin presence, serving as feeding deterrents and playing a role in their protective mutualism with host plants. Closely related taxa Neoclaviceps monostipa and Cepsiclava phalaridis were combined into the genus Aciculosporium.

Ergot species of the Claviceps purpurea group from South Africa.

Results of a survey and study of the Claviceps purpurea group of species in South Africa are being presented and five new species are described. Morphological descriptions are based on the anamorphs and four nuclear genetic loci. Claviceps fimbristylidis sp. nov. on Fimbristylis complanata was discovered wide-spread across five provinces of the country associated with water and represents the fourth Claviceps species recorded from the Cyperaceae. Claviceps monticola sp. nov. is described from Brachypodium flexum growing in mountain forests in Mpumalanga Province, as well as the northern Drakensberg southwards into the Eastern Cape Province. Claviceps pazoutovae sp. nov. is recorded from Stipa dregeana var. dregeana and Ehrharta erecta var. erecta, also associated with these mountain ranges. Claviceps macroura sp. nov. is recorded from Cenchrus macrourus from the Eastern Cape and Claviceps capensis sp. nov. from Ehrharta villosa var. villosa is recorded from the Western Cape Province. Claviceps cyperi, only recorded from South Africa is included in the study. Ergot alkaloid profiles of all species are provided and showed similarity to C. purpurea. Only C. cyperi and in lesser degree C. capensis, C. macroura, and C. pazoutovae produced ergot alkaloids in clinically significant amounts. Several reported species infect invasive grass species, native to South Africa, and thus represent potentially invasive species.

Delimitation of cryptic species inside Claviceps purpurea.

Claviceps purpurea is an ovarian parasite infecting grasses (Poaceae) including cereals and forage plants. This fungus produces toxic alkaloids and consumption of contaminated grains can cause ergotism in humans and other mammals. Recent molecular genetics studies have indicated that it included three cryptic species (G1, G2, G3). In this study, reproductive isolation amongst these groups and among material from Phragmites and Molinia was tested using gene flow statistics for five polymorphic loci, and to support these data, phylogenetic affiliations based on gene trees and a multigene phylogeny were used. The four recognized species are characterized based on morphology and host spectrum and formal taxonomic names are proposed. Claviceps purpurea sensu stricto (G1 group) represents a typical rye ergot, but infects various other grasses. Typical hosts of Claviceps humidiphila (new name for G2 species), like Phalaris arundinacea, belong to grasses preferring humid locations. Claviceps spartinae (G3) is specific to chloridoid grasses from salt barches. The material from Phragmites and Molinia can be authenticated with the species Claviceps microcephala for which the new name Claviceps arundinis is proposed here. The divergence time between species was estimated and the tools for species identification are discussed.

Liberomyces gen. nov. with two new species of endophytic coelomycetes from broadleaf trees.

During a study of endophytic and saprotrophic fungi in the sapwood and phloem of broadleaf trees (Salix alba, Quercus robur, Ulmus laevis, Alnus glutinosa, Betula pendula) fungi belonging to an anamorphic coelomycetous genus not attributable to a described taxon were detected and isolated in pure culture. The new genus, Liberomyces, with two species, L. saliciphilus and L. macrosporus, is described. Both species have subglobose conidiomata containing holoblastic sympodial conidiogenous cells. The conidiomata dehisce irregularly or by ostiole and secrete a slimy suspension of conidia. The conidia are hyaline, narrowly allantoid with a typically curved distal end. In L. macrosporus simultaneous production of synanamorph with thin filamentous conidia was observed occasionally. The genus has no known teleomorph. Related sequences in the public databases belong to endophytes of angiosperms. Phylogenetic analysis revealed a position close to the Xylariales (Sordariomycetes), but family and order affiliation remained unclear.

The ergot alkaloid gene cluster: functional analyses and evolutionary aspects.

Ergot alkaloids and their derivatives have been traditionally used as therapeutic agents in migraine, blood pressure regulation and help in childbirth and abortion. Their production in submerse culture is a long established biotechnological process. Ergot alkaloids are produced mainly by members of the genus Claviceps, with Claviceps purpurea as best investigated species concerning the biochemistry of ergot alkaloid synthesis (EAS). Genes encoding enzymes involved in EAS have been shown to be clustered; functional analyses of EAS cluster genes have allowed to assign specific functions to several gene products. Various Claviceps species differ with respect to their host specificity and their alkaloid content; comparison of the ergot alkaloid clusters in these species (and of clavine alkaloid clusters in other genera) yields interesting insights into the evolution of cluster structure. This review focuses on recently published and also yet unpublished data on the structure and evolution of the EAS gene cluster and on the function and regulation of cluster genes. These analyses have also significant biotechnological implications: the characterization of non-ribosomal peptide synthetases (NRPS) involved in the synthesis of the peptide moiety of ergopeptines opened interesting perspectives for the synthesis of ergot alkaloids; on the other hand, defined mutants could be generated producing interesting intermediates or only single peptide alkaloids (instead of the alkaloid mixtures usually produced by industrial strains).

Liquid chromatography-tandem mass spectrometry characterization of ergocristam degradation products.

The UPLC method with diode array UV detection was developed for qualitative determination of ergocristine and ergocristam including degradation products. The mechanism of the ergocristam disruptive reaction was described based on MS/MS characterization of ammonolytic product, N-(d-lysergyl)-l-valinamide (A1) and two methanolytic products, methyl ester of N-(d-lysergyl)-l-valine (M2), and N-[N-(d-lysergyl)-l-valyl]-l-phenylalanyl-d-prolyl methyl ester (M1). The influence of extraction conditions on epimerization and degradation of ergocristine and ergocristam was tested and conditions for reproducible decomposition of ergocristam were found. The presented method could potentially be applied for ergot alkaloids determination in sclerotia, fermentation broth, mycelium, and possibly contaminated food products, i.e. corn, flour, bread, etc., and feeding stuffs containing ungrounded cereals.

Claviceps nigricans and Claviceps grohii: their alkaloids and phylogenetic placement.

Claviceps purpurea, C. grohii, C. zizaniae, C. cyperi, and C. nigricans are closely related ergot fungi and form a monophyletic clade inside the genus Claviceps. Analysis of alkaloid content in C. nigricans sclerotia using UPLC detected ergocristine (1), ergosine (2), alpha-ergocryptine (3), and ergocristam (4). Alkaloids 1, 3, and 4 were found in the sclerotia of C. grohii. The content of 4 in the mixture of alkaloids from C. nigricans and C. grohii (over 8% and over 20%, respectively) was unusually high. Submerged shaken cultures of C. nigricans produced no alkaloids, whereas C. grohii culture formed small amounts (15 mg L (-1)) of extracellular clavines and 1. In the previously used HPLC method the ergocristam degradation product could have been obscured by the ergosine peak. Therefore sclerotia of a C. purpurea habitat-specific population G2 with the dominant production of 1 and 2 have been reanalyzed, but no 4 was detected. The phylogeny of the C. purpurea-related species group is discussed with regard to alkaloid-specific nonribosomal peptide synthetase duplication leading to the production of two main ergopeptines instead of a single product.

Geosmithia fungi are highly diverse and consistent bark beetle associates: evidence from their community structure in temperate Europe.

Geosmithia spp. (Ascomycota: Hypocreales) are little-studied, dry-spored fungi that occur in galleries built by many phloeophagous bark beetles. This study mapped the distribution and environmental preferences of Geosmithia species occurring in galleries of temperate European bark beetles. One hundred seven host tree samples of 16 tree species infested with 23 subcortical insect species were collected from across Europe during the years 1997-2005. Over 600 Geosmithia isolates from the beetles were sorted into 17 operational taxonomic units (OTUs) based on their phenotype similarity and phylogeny of internal transcribed spacer (ITS) region of rDNA (ITS1-5.8S-ITS2). The OTUs represent six known species and eight undescribed taxa. Ninety-two samples infested with subcortical insects were characterized by the presence/absence of OTUs and the similarity among the samples was evaluated. Geographically distant populations of the same beetle species host relatively uniform Geosmithia communities across large geographic areas (ranging from southern Bulgaria to the Czech Republic). This suggests effective dispersal of Geosmithia spp. by bark beetles. Clustering of similar samples in ordination analysis is correlated predominantly with the isolation source (bark beetles and their respective feeding plant), but not with their geographical origin. The composition of the Geosmithia OTU community of each bark beetle species depends on the degree of isolation of the species' niches. Thus, Geosmithia communities associated with regularly co-occurring bark beetle species are highly similar. The similarity decreases with decreasing frequency of beetle species' co-occurrence, a pattern resembling that of entomochoric ophiostomatoid fungi. These findings suggest that: 1) communities of Geosmithia spp. are vector-specific; 2) at least in some cases, the association between Geosmithia OTUs and bark beetles may have been very stable and symbioses are likely to be a fundamental factor in the speciation of Geosmithia fungi; and 3) that even nonsticky spores of Geosmithia are suitable for maintaining an insect-fungus association, contrary to previous hypotheses.

Host range and diversity of the genus Geosmithia (Ascomycota: Hypocreales) living in association with bark beetles in the Mediterranean area.

Geosmithia spp. (Ascomycota: Hypocreales) are dry-spored fungi that occur in galleries built by many phloeophagous bark beetles. This study mapped the diversity, host spectrum and area of distribution of Geosmithia spp. occurring in galleries of bark beetle species with a Mediterranean distribution. Eighty-six wood samples of 19 tree species infested by 18 subcortical insect species were collected from across the Mediterranean Basin during the years 2003-2006. Geosmithia spp. were found in 82 samples of angiosperms and two host trees from the family Juniperaceae infested by 14 bark beetles and the bostrichid Scobicia pustulata, suggesting that the association of Geosmithia and phloeophagous bark beetles is very widespread in the Mediterranean. Geosmithia isolates were sorted into 13 operational taxonomic units (OTUs) based on their phenotype similarity and phylogeny of their ITS regions of rDNA (ITS1-5.8S-ITS2). The OTUs represent five known species (G. flava, G. langdonii, G. lavendula, G. pallida, G. putterillii) and seven undescribed taxa. Most of the bark beetles were associated with on average 1-2.5 OTUs per sample. G. lavendula, considered very uncommon in nature, was found as a common associate of bark beetles. Six out of 13 OTUs were found to be distributed in the Mediterranean but not in neighbouring areas of temperate Europe suggesting that Geosmithia spp. have a geographically limited distribution, probably due to their dependency on the geographically limited area of their vectors. The proportion of generalists and specialists among Geosmithia spp. was smaller compared with data from temperate Europe. A possible explanation is the effective dispersal of Geosmithia by polyphagous bostrichids across the niches defined by mutually exclusive bark beetles.

Daldinia decipiens and Entonaema cinnabarina as fungal symbionts of Xiphydria wood wasps.

The identity of symbiotic fungi associated with the Xiphydria spp. wood wasps was investigated using DNA analysis. The fungi were isolated from the mycangia of adult females of X. camelus, X. prolongata and X. longicollis reared from colonized logs of Alnus glutinosa, Salix alba and Quercus robur, respectively. Sequences of rDNA and beta-tubulin were obtained. Phylogenetic analysis based on the NJ method showed that the isolates from X. camelus clustered with Daldinia decipiens, whereas those of X. prolongata belonged to Entonaema cinnabarina. In X. longicollis, both symbiotic fungi (D. decipiens and E. cinnabarina) have been found. Morphological characteristics of the anamorphs are presented. In cultures of D. decipiens, sympodial holoblastic, as well as annelidic, conidiation was observed. So far, fruit bodies of D. decipiens have only been recorded from Betula spp. whereas the host spectrum of its wasp vectors covers predominantly oaks and alders. Fruiting bodies of E. cinnabarina do not occur in Central Europe. This is the first report of Entonaema as a symbiotic fungus of siricid wood wasps.

Amorosia littoralis gen. sp. nov., a new genus and species name for the scorpinone and caffeine-producing hyphomycete from the littoral zone in The Bahamas.

The new generic and species name Amorosia littoralis gen. sp. nov. is introduced for the conidial dematiaceous hyphomycete isolated from the littoral zone in The Bahamas and reported in 2001 to produce the novel aza-anthraquinone scorpinone, and also caffeine. No satisfactory generic placement was found at the time, but subsequent morphological and molecular investigations reveal that a new generic name is required. The new genus has some similarity to several fungi described in Trichocladium, but differs substantially from the type species of that genus in the form of the conidia and the lack of ornamentation. BLAST studies using the 18S and 28S rDNA gene sequences place the new genus in the Sporormiaceae. In addition to the morphological studies, an ultrastructural examination of the conspicuous porate septa of hyphae showed that they do not belong to a basidiomycete.

Analysis of Claviceps africana and C. sorghi from India using AFLPs, EF-1alpha gene intron 4, and beta-tubulin gene intron 3.

Isolates of Claviceps causing ergot on sorghum in India were analysed by AFLP analysis, and by analysis of DNA sequences of the EF-1alpha gene intron 4 and beta-tubulin gene intron 3 region. Of 89 isolates assayed from six states in India, four were determined to be C. sorghi, and the rest C. africana. A relatively low level of genetic diversity was observed within the Indian C. africana population. No evidence of genetic exchange between C. africana and C. sorghi was observed in either AFLP or DNA sequence analysis. Phylogenetic analysis was conducted using DNA sequences from 14 different Claviceps species. A multigene phylogeny based on the EF-1alpha gene intron 4, the beta-tubulin gene intron 3 region, and rDNA showed that C. sorghi grouped most closely with C. gigantea and C. africana. Although the Claviceps species we analysed were closely related, they colonize hosts that are taxonomically very distinct suggesting that there is no direct coevolution of Claviceps with its hosts.

Morphological and molecular characterisation of Geosmithia putterillii, G. pallida comb. nov. and G. flava sp. nov., associated with subcorticolous insects.

Geosmithia putterillii is an anamorphic fungus with connections to bark beetles. Genetic variability of 89 isolates traditionally grouped in G. putterillii and G. lavendula isolated from different geographical regions from subcorticolous insects and from other unspecific substrata was assessed using RAPD, sequencing of the ITS region (ITS1-5.8SrDNA-ITS2) and morphological characters. RAPD analysis revealed eight distinct groups. One group was represented by G. lavendula type strain and showed no relations to any other isolate. Five RAPD-types with similar ITS sequences and phenotype were related to the ex-type strain of Penicillium pallidum (generally given as a synonym of G. putterillii). Because of unique phylogenetic position and a phenotype markedly different from G. putterillii, the new combination G. pallida is made here. For another group of isolates formerly identified as G. putterillii the new species G. fiava is described based on a characteristic RAPD-type, a unique ITS sequences and a different phenotype. These newly recognized species are stable in culture and with worldwide distribution.

Pleomorphic conidiation in Claviceps.

Types of asexual sporulation in 17 Claviceps species and the closely related Corallocytostroma ornicopreoides were revised in relation to the phylogeny of clavicipitaceous fungi. We observed: (1) enteroblastic conidiation from branched phialidic conidiophores typical of the genus (anamorph Sphacelia) in all species including Corallocytostroma; (2) widespread and often sequential formation of terminal holoblastic secondary conidia on tapering hyphae arising from sphacelial macroconidia: and (3) in addition to sphacelial conidiation, sympodial holoblastic conidiation of the Ephelis-type in cultures of C. zizaniae and in both the culture and sphacelial tissue of C. citrina. Secondary conidiation was not found in C. purpurea, C. citrina and C. sorghicola. During sphacelial fructification, most species produced macroconidia and microconidia. Only macroconidia formed in planta underwent secondary conidiation whereas microconidia did not germinate at all. In C. phalaridis, the formation of holoblastic 2-3 celled appendaged conidia was observed, similar to that of Aciculosporium and Neoclaviceps. In dendrograms based on ITS1-5.8S rDNA-ITS2 sequences, genera and species with appendaged conidia grouped on a highly supported clade with ancestral Corallocytostroma. The clade was placed inside a group of tropical species of Claviceps, without any relationship to Balansiae.

Ergot alkaloids produced by submerged cultures of Claviceps zizaniae.

Two ergopeptine alkaloids, alpha-ergocryptine (1) and its C(8) epimer alpha-ergocryptinine, have been isolated from the mycelium and fermentation broth of submerged cultures of Claviceps zizaniae CCM 8240. The structure of 1 was determined by HPLC/positive ion APCI MS and NMR analysis. Alkaloid concentrations of 10 microg/mL in 14-day-old fermentation broth and 1 mg/g of dry mycelium mass were found. These results are of considerable biotechnological interest since these were the only detectable alkaloids produced. Toxicity of naturally occurring sclerotia of C. zizaniae cannot be excluded.

Rediscovery of Claviceps sorghi (Ascomycotina: Clavicipitaceae) in India.

During last twenty years, endemic Claviceps sorghi was largely replaced in India by an introduced species Claviceps africana. Recently, C. sorghi was found and isolated from Sorghum bicolor (Gulbarga, Karnataka). Macroconidia were smaller than in the type description, 7.6-14.5 (avg. 11.4) x 3.8-6.1 (avg. 4.8) microm; microconidia were rounded to oval, 2.5-3 microm in diameter. When plated, only the macroconidia underwent secondary conidiation. Sporulation was maintained only in cultures grown on sucrose-asparagine medium T2. Mycelium was reduced to sparse hyphae with numerous short conidiophores soon obscured by yeastlike conidial mass. RAPD patterns of isolates GUL, MH74 (Maharashtra), NAP7 and NAP5 (Andhra Pradesh) with 6 primers were almost identical and distinct from those of C. africana. Sequence of rDNA containing ITS1-5.8S-ITS2 region for isolate GUL (AJ306621) corresponded to that of the authentic material of C. sorghi (AJ242869) confirming thus the presence of C. sorghi in Central India.

Relations Among Sorghum Ergot Isolates from the Americas, Africa, India, and Australia.

Sorghum ergot, initially restricted to Asia and Africa, was recently found in the Americas and Australia. Three species causing the disease have been reported: Claviceps sorghi in India, C. sorghicola in Japan, and C. africana in all ergot-positive countries. The objective of our study was to study the intraspecific variation in C. africana isolates in the Americas, Africa, India, and Australia. We confirmed C. africana, C. sorghi, and C. sorghicola as different species using differences in nucleotide sequences of internal transcribed spacer 1 and 5.8S rDNA regions. Sequences of this region obtained from the representative American, Indian, and Australian isolates of C. africana were identical. In addition, random amplified polymorphic DNA (RAPD) banding patterns of sorghum ergot pathogen isolates from the United States, Mexico, Puerto Rico, Bolivia, Australia, and India were evaluated with nearly 100 primers. A total of 65 primers gave identical patterns for all isolates, which confirmed that all were C. africana. The identity of RAPD pattern and rDNA sequence of Indian isolates with those of C. africana confirmed that the species is now present in India. Only 20 primers gave small pattern differences and 7 of them were used for routine testing. All of the American isolates were identical and three isolates of the same type were also found in South Africa, suggesting Africa as the origin of the invasion clone in the Americas. Australian and Indian isolates were distinguishable by a single band difference; therefore, migration from the Asian region to Australia is suspected. Another distinct group was found in Africa. Cluster analysis of the informative bands revealed that the American and African group are on the same moderately (69%) supported clade. Isolates from Australia and India belonged to another clade.